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Sample records for human whole-blood mononuclear

  1. Real-time cytotoxicity assays in human whole blood.

    PubMed

    Hsiao, Ching-Wen; Lo, Yen-Ting; Liu, Hong; Hsiao, Sonny C

    2014-01-01

    A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex vivo condition. PMID:25406660

  2. Real-time Cytotoxicity Assays in Human Whole Blood

    PubMed Central

    Liu, Hong; Hsiao, Sonny C.

    2014-01-01

    A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex vivo condition. PMID:25406660

  3. Hemolyzed, lyophilized bovine blood for quality control of lead determination of human whole blood

    SciTech Connect

    Subramanian, K.S.; Meranger, J.C.; Connor, J.

    1985-09-01

    The determination of Pb in human whole blood is perhaps the most common application of biological monitoring in occupational and environmental health. However, the lack of an adequate reference material has prevented a thorough evaluation of current analytical methods for Pb in blood and hence the evaluation of data generated by using such methods. As a result the concentration of Pb in human whole blood is not accurately known. In this paper the authors report the preparation and analysis of two freeze-dried bovine whole blood reference specimens containing two different levels of Pb.

  4. A Simple Method for Human Whole Blood Microcultures and Its Application in Radiation Biodosimetry.

    PubMed

    Yan, Guo; Wen, Zhang; Chaoxian, Gao; Changye, Hui; Xueqin, Yang; Xinyue, Yang; Zhimin, Li

    2015-10-01

    The scoring of the cytokinesis-block micronucleus and dicentric chromosomes in human peripheral blood lymphocytes is used as a dosimeter of radiation exposure. A detailed methodology is presented for human whole blood microculture for cytogenetic analysis. The technique described yields more than sufficient numbers of mitotic lymphocytes for analyzing micronuclei and chromosome aberrations following exposure to radiation. PMID:26313591

  5. Abnormal Whole Blood Thrombi in Humans with Inherited Platelet Receptor Defects

    PubMed Central

    Castellino, Francis J.; Liang, Zhong; Davis, Patrick K.; Balsara, Rashna D.; Musunuru, Harsha; Donahue, Deborah L.; Smith, Denise L.; Sandoval-Cooper, Mayra J.; Ploplis, Victoria A.; Walsh, Mark

    2012-01-01

    To delineate the critical features of platelets required for formation and stability of thrombi, thromboelastography and platelet aggregation measurements were employed on whole blood of normal patients and of those with Bernard-Soulier Syndrome (BSS) and Glanzmann’s Thrombasthenia (GT). We found that separation of platelet activation, as assessed by platelet aggregation, from that needed to form viscoelastic stable whole blood thrombi, occurred. In normal human blood, ristocetin and collagen aggregated platelets, but did not induce strong viscoelastic thrombi. However, ADP, arachidonic acid, thrombin, and protease-activated-receptor-1 and -4 agonists, stimulated both processes. During this study, we identified the genetic basis of a very rare double heterozygous GP1b deficiency in a BSS patient, along with a new homozygous GP1b inactivating mutation in another BSS patient. In BSS whole blood, ADP responsiveness, as measured by thrombus strength, was diminished, while ADP-induced platelet aggregation was normal. Further, the platelets of 3 additional GT patients showed very weak whole blood platelet aggregation toward the above agonists and provided whole blood thrombi of very low viscoelastic strength. These results indicate that measurements of platelet counts and platelet aggregability do not necessarily correlate with generation of stable thrombi, a potentially significant feature in patient clinical outcomes. PMID:23300803

  6. Purification of human genomic DNA from whole blood using sodium perchlorate in place of phenol.

    PubMed

    Johns, M B; Paulus-Thomas, J E

    1989-08-01

    We have developed a new, rapid method for the extraction of human genomic DNA from whole blood samples. Traditionally, genomic DNA has been extracted from blood by overnight proteinase K digestion of lysed peripheral lymphocytes followed by phenol/chloroform extraction. In addition to being time consuming, the use of phenol involves inherent risks due to the toxic nature of the reagent. Our method for the extraction of DNA from whole blood uses sodium perchlorate and chloroform instead of phenol with a significant time savings realized as well as fewer hazards to the technician. Furthermore, DNA prepared by this new method is an excellent substrate for restriction endonuclease digestion and Southern hybridization analysis.

  7. 21 CFR 640.1 - Whole Blood.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Whole Blood. 640.1 Section 640.1 Food and Drugs... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Whole Blood § 640.1 Whole Blood. The proper name of this product shall be Whole Blood. Whole Blood is defined as blood collected from human donors for...

  8. 21 CFR 640.1 - Whole Blood.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Whole Blood. 640.1 Section 640.1 Food and Drugs... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Whole Blood § 640.1 Whole Blood. The proper name of this product shall be Whole Blood. Whole Blood is defined as blood collected from human donors for...

  9. 21 CFR 640.1 - Whole Blood.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Whole Blood. 640.1 Section 640.1 Food and Drugs... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Whole Blood § 640.1 Whole Blood. The proper name of this product shall be Whole Blood. Whole Blood is defined as blood collected from human donors for...

  10. 21 CFR 640.1 - Whole Blood.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Whole Blood. 640.1 Section 640.1 Food and Drugs... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Whole Blood § 640.1 Whole Blood. The proper name of this product shall be Whole Blood. Whole Blood is defined as blood collected from human donors for...

  11. 21 CFR 640.1 - Whole Blood.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Whole Blood. 640.1 Section 640.1 Food and Drugs... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Whole Blood § 640.1 Whole Blood. The proper name of this product shall be Whole Blood. Whole Blood is defined as blood collected from human donors for...

  12. Extraction of human genomic DNA from whole blood using a magnetic microsphere method.

    PubMed

    Gong, Rui; Li, Shengying

    2014-01-01

    With the rapid development of molecular biology and the life sciences, magnetic extraction is a simple, automatic, and highly efficient method for separating biological molecules, performing immunoassays, and other applications. Human blood is an ideal source of human genomic DNA. Extracting genomic DNA by traditional methods is time-consuming, and phenol and chloroform are toxic reagents that endanger health. Therefore, it is necessary to find a more convenient and efficient method for obtaining human genomic DNA. In this study, we developed urea-formaldehyde resin magnetic microspheres and magnetic silica microspheres for extraction of human genomic DNA. First, a magnetic microsphere suspension was prepared and used to extract genomic DNA from fresh whole blood, frozen blood, dried blood, and trace blood. Second, DNA content and purity were measured by agarose electrophoresis and ultraviolet spectrophotometry. The human genomic DNA extracted from whole blood was then subjected to polymerase chain reaction analysis to further confirm its quality. The results of this study lay a good foundation for future research and development of a high-throughput and rapid extraction method for extracting genomic DNA from various types of blood samples.

  13. Viable capture and release of cancer cells in human whole blood

    NASA Astrophysics Data System (ADS)

    Doh, Il; Yoo, Hwan-il; Cho, Young-Ho; Lee, Jinseon; Kwan Kim, Hong; Kim, Jhingook

    2012-07-01

    We present viable cancer cell isolation devices utilizing the physical properties of cells. The tapered slit structure is proposed to isolate cancer cells from blood cells and collect them by reversed flow. From the experimental study using the spiked cancer cells in human whole blood, we verified the capability of the present cancer cell isolation chip in terms of capture efficiency, viability, and release rate. The viable cancer cells obtained from the present chip can be used for the further applications of cancer diagnosis, treatment monitoring, and new target drug development for cancer stem cells.

  14. Cytogenetic and oxidative alterations after exposure of cultured human whole blood cells to lithium metaborate dehydrate.

    PubMed

    Çelikezen, Fatih Çağlar; Toğar, Başak; Özgeriş, Fatma Betül; İzgi, Mehmet Sait; Türkez, Hasan

    2016-08-01

    Boron compounds have an ability of supporting antioxidant properties in human and animal tissues. Lithium metaborate dihydrate (LiBO2·2H2O; LMD) is commonly used in nonlinear optic materials, cellular phones and pagers. But, there are limited data on the genotoxic and antioxidant effects of LMD in cultured human whole blood cells. The aim of this study was to evaluate for the genotoxicity and antioxidant/oxidant activity of LMD on human whole blood lymphocytes (n = 5). LMD was applied at various concentrations (0-1,280 µg/ml) to cultured blood samples. Antioxidant/oxidant activity was evaluated by measuring the total oxidant status (TOS) and total antioxidant capacity levels. Micronuclei and chromosomal aberration tests were used in genotoxicity studies. Our results clearly revealed that all tested concentrations of LMD were found to be non-genotoxic when compared to that of the control group. In addition, LMD exhibited antioxidant activities at low concentrations. In addition the TOS levels were not changed at all concentrations of LMD. Consequently, our results clearly demonstrated that LMD is non-genotoxic and it has an important antioxidant potential in vitro.

  15. Heat shock inhibits lipopolysaccharide-induced tissue factor activity in human whole blood

    PubMed Central

    Sucker, Christoph; Zacharowski, Kai; Thielmann, Matthias; Hartmann, Matthias

    2007-01-01

    Background During gram-negative sepsis, lipopolysaccharide (LPS) induces tissue factor expression on monocytes. The resulting disseminated intravascular coagulation leads to tissue ischemia and worsens the prognosis of septic patients. There are indications, that fever reduces the mortality of sepsis, the effect on tissue factor activity on monocytes is unknown. Therefore, we investigated whether heat shock modulates LPS-induced tissue factor activity in human blood. Methods Whole blood samples and leukocyte suspensions, respectively, from healthy probands (n = 12) were incubated with LPS for 2 hours under heat shock conditions (43°C) or control conditions (37°C), respectively. Subsequent to further 3 hours of incubation at 37°C the clotting time, a measure of tissue factor expression, was determined. Cell integrity was verified by trypan blue exclusion test and FACS analysis. Results Incubation of whole blood samples with LPS for 5 hours at normothermia resulted in a significant shortening of clotting time from 357 ± 108 sec to 82 ± 8 sec compared to samples incubated without LPS (n = 12; p < 0.05). This LPS effect was mediated by tissue factor, as inhibition with active site-inhibited factor VIIa (ASIS) abolished the effect of LPS on clotting time. Blockade of protein synthesis using cycloheximide demonstrated that LPS exerted its procoagulatory effect via an induction of tissue factor expression. Upon heat shock treatment, the LPS effect was blunted: clotting times were 312 ± 66 s in absence of LPS and 277 ± 65 s in presence of LPS (n = 8; p > 0.05). Similarly, heat shock treatment of leukocyte suspensions abolished the LPS-induced tissue factor activity. Clotting time was 73 ± 31 s, when cells were treated with LPS (100 ng/mL) under normothermic conditions, and 301 ± 118 s, when treated with LPS (100 ng/mL) and heat shock (n = 8, p < 0.05). Control experiments excluded cell damage as a potential cause of the observed heat shock effect. Conclusion Heat

  16. Analyzing Illumina Gene Expression Microarray Data Obtained From Human Whole Blood Cell and Blood Monocyte Samples.

    PubMed

    Teumer, Alexander; Schurmann, Claudia; Schillert, Arne; Schramm, Katharina; Ziegler, Andreas; Prokisch, Holger

    2016-01-01

    Microarray profiling of gene expression is widely applied to studies in molecular biology and functional genomics. Experimental and technical variations make not only the statistical analysis of single studies but also meta-analyses of different studies very challenging. Here, we describe the analytical steps required to substantially reduce the variations of gene expression data without affecting true effect sizes. A software pipeline has been established using gene expression data from a total of 3358 whole blood cell and blood monocyte samples, all from three German population-based cohorts, measured on the Illumina HumanHT-12 v3 BeadChip array. In summary, adjustment for a few selected technical factors greatly improved reliability of gene expression analyses. Such adjustments are particularly required for meta-analyses of different studies. PMID:26614070

  17. The relationship between the iron isotopic composition of human whole blood and iron status parameters.

    PubMed

    Van Heghe, Lana; Delanghe, Joris; Van Vlierberghe, Hans; Vanhaecke, Frank

    2013-11-01

    As the iron status of an individual cannot be adequately assessed on the basis of the (total) Fe concentration in whole blood or serum, in medicine a number of parameters, such as the serum concentrations of ferritin, transferrin and soluble transferrin receptor and the transferrin saturation, are routinely determined instead. As previous research has shown that also the isotopic composition of Fe in blood and tissues is dependent on the metabolism, the present study assessed whether Fe isotopic composition in whole blood provides information as to an individual's iron status. Fe isotopic analysis of whole blood samples from a reference population (healthy volunteers) was carried out using multi-collector ICP-mass spectrometry (after chromatographic target element isolation) and the results obtained were investigated by statistical means as to their potential relation with the iron status parameters conventionally used in medicine. A low δ(56)Fe value was demonstrated to coincide with high iron status and a high δ(56)Fe value with low iron status, thus reflecting the response of the body to this iron status in terms of iron uptake, distribution between blood and stores and mobilization of storage iron. In a second phase, the iron isotopic composition in blood from patients treated for hemochromatosis type I and from patients with anemia of chronic disease (ACD) was determined. The results for hemochromatosis patients plotted with the values of low iron status, while those for ACD patients plotted with the values of high iron status. By taking a closer look at the aberrant iron metabolism that comes with these diseases, it can be seen that the patient samples confirm the conclusions drawn for the reference population. Patients with hemochromatosis type I have a strongly upregulated iron uptake, like healthy individuals with low iron status. The metabolism of patients suffering from ACD tries to remove iron from the circulation by downregulating the iron uptake

  18. Human mononuclear phagocyte system reunited.

    PubMed

    Haniffa, Muzlifah; Bigley, Venetia; Collin, Matthew

    2015-05-01

    The human mononuclear phagocyte network comprises dendritic cells (DCs), monocytes and macrophages with a range of immune functions including antigen presentation linking innate and adaptive immunity. A number of DC, monocyte and macrophage subsets have been described in lymphoid and non-lymphoid tissues of mouse and human, with increased understanding of their distinct functional properties and genetic and cellular pathways of development. More recently, through comparative biology studies, a unified nomenclature of mononuclear phagocytes has begun to emerge with the identification of homologous subsets in several species. In this review, we discuss the current classification of human mononuclear phagocytes and the parallel organization of this network in the mouse. We also review the genetic control and developmental pathway of human mononuclear phagocytes and the immunological functions of the distinct subsets in health and inflammation.

  19. Screening vaccine formulations for biological activity using fresh human whole blood.

    PubMed

    Brookes, Roger H; Hakimi, Jalil; Ha, Yukyung; Aboutorabian, Sepideh; Ausar, Salvador F; Hasija, Manvi; Smith, Steven G; Todryk, Stephen M; Dockrell, Hazel M; Rahman, Nausheen

    2014-01-01

    Understanding the relevant biological activity of any pharmaceutical formulation destined for human use is crucial. For vaccine-based formulations, activity must reflect the expected immune response, while for non-vaccine therapeutic agents, such as monoclonal antibodies, a lack of immune response to the formulation is desired. During early formulation development, various biochemical and biophysical characteristics can be monitored in a high-throughput screening (HTS) format. However, it remains impractical and arguably unethical to screen samples in this way for immunological functionality in animal models. Furthermore, data for immunological functionality lag formulation design by months, making it cumbersome to relate back to formulations in real-time. It is also likely that animal testing may not accurately reflect the response in humans. For a more effective formulation screen, a human whole blood (hWB) approach can be used to assess immunological functionality. The functional activity relates directly to the human immune response to a complete formulation (adjuvant/antigen) and includes adjuvant response, antigen response, adjuvant-modulated antigen response, stability, and potentially safety. The following commentary discusses the hWB approach as a valuable new tool to de-risk manufacture, formulation design, and clinical progression. PMID:24401565

  20. The increased sensitivity of human cytomegalovirus (HCMV) PCR quantitation in whole blood affects reproductive rate (Ro) measurement.

    PubMed

    Gurtler, Volker; Mayall, Barrie C; Wang, Jenny; Ghaly-Derias, Shahbano

    2014-02-01

    In order to determine the effect of the increase in sensitivity of HCMV detection in whole blood compared to plasma on reproductive rate (Ro) measurement, an optimized human cytomegalovirus (HCMV) quantitative PCR assay was developed. The results presented in this study are summarized by the following three methodological improvements: (i) at values below the limit of quantitation (LOQ) of 60copies/ml, determination of HCMV load was more sensitive with whole blood than plasma, (ii) for the determination of viral load, whole blood was more sensitive than plasma below 1000copies/ml but little difference was observed above 1000copies/ml and (iii) the measurement of "Reproductive Rate" can be affected by imprecise measurement of HCMV viral load in either plasma or whole blood compartments depending on whether samples were taken from patients on antiviral treatment or from patients where HCMV load was rising. Taken together this study provides methodological improvements suggesting that below HCMV viral load levels of 1000copies/ml (1640IU/ml) both plasma and whole blood should be tested.

  1. Assessment of whole blood thrombosis in a microfluidic device lined by fixed human endothelium.

    PubMed

    Jain, Abhishek; van der Meer, Andries D; Papa, Anne-Laure; Barrile, Riccardo; Lai, Angela; Schlechter, Benjamin L; Otieno, Monicah A; Louden, Calvert S; Hamilton, Geraldine A; Michelson, Alan D; Frelinger, Andrew L; Ingber, Donald E

    2016-08-01

    The vascular endothelium and shear stress are critical determinants of physiological hemostasis and platelet function in vivo, yet current diagnostic and monitoring devices do not fully incorporate endothelial function under flow in their assessment and, therefore, they can be unreliable and inaccurate. It is challenging to include the endothelium in assays for clinical laboratories or point-of-care settings because living cell cultures are not sufficiently robust. Here, we describe a microfluidic device that is lined by a human endothelium that is chemically fixed, but still retains its ability to modulate hemostasis under continuous flow in vitro even after few days of storage. This device lined with a fixed endothelium supports formation of platelet-rich thrombi in the presence of physiological shear, similar to a living arterial vessel. We demonstrate the potential clinical value of this device by showing that thrombus formation and platelet function can be measured within minutes using a small volume (0.5 mL) of whole blood taken from subjects receiving antiplatelet medications. The inclusion of a fixed endothelial microvessel will lead to biomimetic analytical devices that can potentially be used for diagnostics and point-of-care applications. PMID:27464497

  2. Immunosuppressive Interactions among Calcium Channel Antagonists and Selected Corticosteroids and Macrolides Using Human whole Blood Lymphocytes

    PubMed Central

    Chow, Fung-Sing; Jusko, William J.

    2014-01-01

    Summary The immunosuppressive interactions of calcium channel antagonists [diltiazem (Dil), verapamil (Ver) and nifedipine (Nif)], with corticosteroids [methylprednisolone (Mpl), prednisolone (Prd)], and macrolides [tacrolimus (Tac) and sirolnnus (Sir)] were examined in human whole blood lymphocyte cultures. Gender-related differences in responses in the interactions between these drug classes were studied using blood from 6 males and 6 females. The nature and intensity of interactions were determined using an extended Loewe additivity model. All immunosuppressants exhibited higher potency than the calcium channel antagonists with mean IC50 values of: Dil (mM)Ver (mM)Nif (mM)Mpl (nM)Prd (nM)Tac (nM)Sir (nM)Male13541.921312.118.6150327Female11431.847.44.68.8111106 Gender-related differences in responses to Mpl and Prd were observed while the others were not significant. Additive interactions were found among calcium channel antagonists and corticosteroids. Significant synergistic interactions were observed between calcium channel antagonists and tacrolimus and sirolimus, although these are unlikely to be of clinical importance. These studies demonstrate diverse drug interactions in the examination of an important array of immunosuppressant drug combinations. PMID:15681895

  3. Identification of common genetic variants controlling transcript isoform variation in human whole blood.

    PubMed

    Zhang, Xiaoling; Joehanes, Roby; Chen, Brian H; Huan, Tianxiao; Ying, Saixia; Munson, Peter J; Johnson, Andrew D; Levy, Daniel; O'Donnell, Christopher J

    2015-04-01

    An understanding of the genetic variation underlying transcript splicing is essential to dissect the molecular mechanisms of common disease. The available evidence from splicing quantitative trait locus (sQTL) studies has been limited to small samples. We performed genome-wide screening to identify SNPs that might control mRNA splicing in whole blood collected from 5,257 Framingham Heart Study participants. We identified 572,333 cis sQTLs involving 2,650 unique genes. Many sQTL-associated genes (40%) undergo alternative splicing. Using the National Human Genome Research Institute (NHGRI) genome-wide association study (GWAS) catalog, we determined that 528 unique sQTLs were significantly enriched for 8,845 SNPs associated with traits in previous GWAS. In particular, we found 395 (4.5%) GWAS SNPs with evidence of cis sQTLs but not gene-level cis expression quantitative trait loci (eQTLs), suggesting that sQTL analysis could provide additional insights into the functional mechanism underlying GWAS results. Our findings provide an informative sQTL resource for further characterizing the potential functional roles of SNPs that control transcript isoforms relevant to common diseases.

  4. World-to-digital-microfluidic interface enabling extraction and purification of RNA from human whole blood.

    PubMed

    Jebrail, Mais J; Sinha, Anupama; Vellucci, Samantha; Renzi, Ronald F; Ambriz, Cesar; Gondhalekar, Carmen; Schoeniger, Joseph S; Patel, Kamlesh D; Branda, Steven S

    2014-04-15

    Digital microfluidics (DMF) is a powerful technique for simple and precise manipulation of microscale droplets of fluid. This technique enables processing and analysis of a wide variety of samples and reagents and has proven useful in a broad range of chemical, biological, and medical applications. Handling of "real-world" samples has been a challenge, however, because typically their volumes are greater than those easily accommodated by DMF devices and contain analytes of interest at low concentration. To address this challenge, we have developed a novel "world-to-DMF" interface in which an integrated companion module drives the large-volume sample through a 10 μL droplet region on the DMF device, enabling magnet-mediated recovery of bead-bound analytes onto the device as they pass through the region. To demonstrate its utility, we use this system for extraction of RNA from human whole blood lysates (110-380 μL) and further purification in microscale volumes (5-15 μL) on the DMF device itself. Processing by the system was >2-fold faster and consumed 12-fold less reagents, yet produced RNA yields and quality fully comparable to conventional preparations and supporting qRT-PCR and RNA-Seq analyses. The world-to-DMF system is designed for flexibility in accommodating different sample types and volumes, as well as for facile integration of additional modules to enable execution of more complex protocols for sample processing and analysis. As the first technology of its kind, this innovation represents an important step forward for DMF, further enhancing its utility for a wide range of applications. PMID:24479881

  5. Comparison of human whole blood, plasma, and serum matrices for the determination of perfluorooctanesulfonate (PFOS), perfluorooctanoate (PFOA), and other fluorochemicals

    SciTech Connect

    Ehresman, David J.; Froehlich, John W.; Olsen, Geary W. . E-mail: gwolsen@mmm.com; Chang, Shu-Ching; Butenhoff, John L.

    2007-02-15

    Interest in human exposure to perfluorinated acids, including perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), perfluorooctanesulfonate (PFOS), and perfluorooctanoate (PFOA) has led to their measurement in whole blood, plasma and serum. Comparison of measurements in these different blood-based matrices, however, has not been rigorously investigated to allow for across-matrix comparisons. This research evaluated concentrations of PFBS, PFHS, PFOS, and PFOA in whole blood collected in heparin (lithium) and ethylenediamine tetraacetic acid (EDTA), plasma samples collected in heparin and EDTA, and serum (from whole blood allowed to clot). Blood samples were collected from 18 voluntary participants employed at 3M Company. Solid phase extraction methods were used for all analytical sample preparations, and analyses were completed using high-pressure liquid chromatography/tandem mass spectrometry methods. Serum concentrations ranged from: limit of quantitation (LOQ, 5 ng/mL) to 25 ng/mL for PFBS; LOQ (5 ng/mL) to 75 ng/mL for PFHS; LOQ (5 ng/mL) to 880 ng/mL for PFOS; and LOQ (5 or 10 ng/mL) to 7320 ng/mL for PFOA. Values less than the LOQ were not included in the statistical analyses of the mean of the ratios of individual values for the matrices. PFBS was not quantifiable in most samples. Serum to plasma ratios for PFHS, PFOS, and PFOA were 1:1 and this ratio was independent of the level of concentrations measured. Serum or plasma to whole blood ratios, regardless of the anticoagulant used, approximated 2:1. The difference between plasma and serum and whole blood corresponded to volume displacement by red blood cells, suggesting that the fluorochemicals are not found intracellularly or attached to the red blood cells.

  6. Raman and SERS recognition of β-carotene and haemoglobin fingerprints in human whole blood

    NASA Astrophysics Data System (ADS)

    Casella, Michele; Lucotti, Andrea; Tommasini, Matteo; Bedoni, Marzia; Forvi, Elena; Gramatica, Furio; Zerbi, Giuseppe

    2011-09-01

    The present work reports on Raman and Surface Enhanced Raman Scattering (SERS) vibrational fingerprints of β-carotene and haemoglobin in fresh whole blood (i.e. right after blood test) with different laser excitations, i.e. visible (514 nm) and near-infrared (NIR, 785 nm). The use of colloidal silver nanoparticles significantly increases the Raman signal, thus providing a clear SERS spectrum of blood. The collected spectra have been examined and marker bands of β-carotene and of the haem prosthetic group of haemoglobin have been found. In particular, the fundamental features of β-carotene (514 nm excitation), blood proteins and haem molecules (785 nm excitation) were recognized and assigned. Moreover haemoglobin SERS signals can be identified and related with its oxygenation state (oxy-haemoglobin). The data reported show the prospects of Raman and SERS techniques to detect important bio-molecules in a whole blood sample with no pre-treatment.

  7. Raman and SERS recognition of β-carotene and haemoglobin fingerprints in human whole blood.

    PubMed

    Casella, Michele; Lucotti, Andrea; Tommasini, Matteo; Bedoni, Marzia; Forvi, Elena; Gramatica, Furio; Zerbi, Giuseppe

    2011-09-01

    The present work reports on Raman and Surface Enhanced Raman Scattering (SERS) vibrational fingerprints of β-carotene and haemoglobin in fresh whole blood (i.e. right after blood test) with different laser excitations, i.e. visible (514 nm) and near-infrared (NIR, 785 nm). The use of colloidal silver nanoparticles significantly increases the Raman signal, thus providing a clear SERS spectrum of blood. The collected spectra have been examined and marker bands of β-carotene and of the haem prosthetic group of haemoglobin have been found. In particular, the fundamental features of β-carotene (514 nm excitation), blood proteins and haem molecules (785 nm excitation) were recognized and assigned. Moreover haemoglobin SERS signals can be identified and related with its oxygenation state (oxy-haemoglobin). The data reported show the prospects of Raman and SERS techniques to detect important bio-molecules in a whole blood sample with no pre-treatment.

  8. Pilot clinical study to investigate the human whole blood spectrum characteristics in the sub-THz region

    NASA Astrophysics Data System (ADS)

    Tseng, Tzu-Fang; You, Borwen; Gao, Hao-Cheng; Wang, Tzung-Dau; Sun, Chi-Kuang

    2015-04-01

    We have conducted a pilot clinical study to not only investigate the THz spectra of ex-vivo fresh human whole blood of 28 patients following 8-hours fasting guideline, but also to find out the critical blood ingredients of which the concentration dominantly affects those THz spectra. A great difference between the THz absorption properties of human blood among different people was observed, while the difference can be up to ~15% of the averaged absorption coefficient of the 28 samples. Our pilot clinical study indicates that triglyceride and red blood cell were two dominant factors to have significant clinically defined negative correlation to the sub-THz absorption coefficients.

  9. Leucocytes isolated from simply frozen whole blood can be used in human biomonitoring for DNA damage measurement with the comet assay.

    PubMed

    Akor-Dewu, Maryam B; El Yamani, Naouale; Bilyk, Olena; Holtung, Linda; Tjelle, Torunn E; Blomhoff, Rune; Collins, Andrew R

    2014-04-01

    Preservation of human blood cells for DNA damage analysis with the comet assay conventionally involves the isolation of mononuclear cells by centrifugation, suspension in freezing medium and slow freezing to -80 °C-a laborious process. A recent publication (Al-Salmani et al. Free Rad Biol Med 2011; 51: 719-725) describes a simple method in which small volumes of whole blood are frozen to -20 or -80 °C; on subsequent thawing, the comet assay is performed, with no indication of elevated DNA strand breakage resulting from the rapid freezing. However, leucocytes in whole blood (whether fresh or frozen) are abnormally resistant to damage by H2 O2 , and so a common test of antioxidant status (resistance to strand breakage by H2 O2 ) cannot be used. We have refined this method by separating the leucocytes from the thawed blood; we find that, after three washes, the cells respond normally to H2 O2 . In addition, we have measured specific endogenous base damage (oxidized purines) in the isolated leucocytes, using the enzyme formamidopyrimidine DNA glycosylase. In a study of blood samples from 10 subjects, H2 O2 sensitivity and endogenous damage-both reflecting the antioxidant status of the cells-correlated significantly. This modified approach to sample collection and storage is particularly applicable when the available volume of blood is limited and has great potential in biomonitoring and ecogenotoxicology studies where samples are obtained in the field or at sites remote from the testing laboratory.

  10. Hemocompatible control of sulfobetaine-grafted polypropylene fibrous membranes in human whole blood via plasma-induced surface zwitterionization.

    PubMed

    Chen, Sheng-Han; Chang, Yung; Lee, Kueir-Rarn; Wei, Ta-Chin; Higuchi, Akon; Ho, Feng-Ming; Tsou, Chia-Chun; Ho, Hsin-Tsung; Lai, Juin-Yih

    2012-12-21

    In this work, the hemocompatibility of zwitterionic polypropylene (PP) fibrous membranes with varying grafting coverage of poly(sulfobetaine methacrylate) (PSBMA) via plasma-induced surface polymerization was studied. Charge neutrality of PSBMA-grafted layers on PP membrane surfaces was controlled by the low-pressure and atmospheric plasma treatment in this study. The effects of grafting composition, surface hydrophilicity, and hydration capability on blood compatibility of the membranes were determined. Protein adsorption onto the different PSBMA-grafted PP membranes from human fibrinogen solutions was measured by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. Blood platelet adhesion and plasma clotting time measurements from a recalcified platelet-rich plasma solution were used to determine if platelet activation depends on the charge bias of the grafted PSBMA layer. The charge bias of PSBMA layer deviated from the electrical balance of positively and negatively charged moieties can be well-controlled via atmospheric plasma-induced interfacial zwitterionization and was further tested with human whole blood. The optimized PSBMA surface graft layer in overall charge neutrality has a high hydration capability and keeps its original blood-inert property of antifouling, anticoagulant, and antithrmbogenic activities when it comes into contact with human blood. This work suggests that the hemocompatible nature of grafted PSBMA polymers by controlling grafting quality via atmospheric plasma treatment gives a great potential in the surface zwitterionization of hydrophobic membranes for use in human whole blood. PMID:23181727

  11. Enhanced differentiation of human osteoblasts on Ti surfaces pre-treated with human whole blood.

    PubMed

    Kopf, Brigitte S; Schipanski, Angela; Rottmar, Markus; Berner, Simon; Maniura-Weber, Katharina

    2015-06-01

    Early and effective integration of a metal implant into bone tissue is of crucial importance for its long-term stability. While different material properties including surface roughness and wettability but also initial blood-implant surface interaction are known to influence this osseointegration, implications of the latter process are still poorly understood. In this study, early interaction between blood and the implant surface and how this affects the mechanism of osseointegration were investigated. For this, blood coagulation on a micro-roughened hydrophobic titanium (Ti) surface (SLA-H(phob)) and on a hydrophilic micro-roughened Ti surface with nanostructures (SLActive-H(phil)NS), as well as the effects of whole human blood pre-incubation of these two surfaces on the differentiation potential of primary human bone cells (HBC) was assessed. Interestingly, pre-incubation with blood resulted in a dense fibrin network over the entire surface on SLActive-H(phil)NS but only in single patches of fibrin and small isolated fibre complexes on SLA-H(phob). On SLActive-H(phil)NS, the number of HBCs attaching to the fibrin network was greatly increased and the cells displayed enhanced cell contact to the fibrin network. Notably, HBCs displayed increased expression of the osteogenic marker proteins alkaline phosphatase and collagen-I when cultivated on both surfaces upon blood pre-incubation. Additionally, blood pre-treatment promoted an earlier and enhanced mineralization of HBCs cultivated on SLActive-H(phil)NS compared to SLA-H(phob). The results presented in this study therefore suggest that blood pre-incubation of implant surfaces mimics a more physiological situation, eventually providing a more predictive in vitro model for the evaluation of novel bone implant surfaces.

  12. Multiple enzyme-doped thread-based microfluidic system for blood urea nitrogen and glucose detection in human whole blood

    PubMed Central

    Yang, Yu-An

    2015-01-01

    This research presents a multiple enzyme-doped thread-based microfluidic system for blood urea nitrogen (BUN) and glucose detection in human whole blood. A novel enzyme-doped thread coated with a thin polyvinylchloride (PVC) membrane is produced for on-site electrochemical detection of urea and glucose in whole blood. Multiple enzymes can be directly applied to the thread without delicate pretreatment or a surface modification process prior to sealing the thread with PVC membrane. Results indicate that the developed device exhibits a good linear dynamic range for detecting urea and glucose in concentrations from 0.1 mM–10.0 mM (R2 = 0.9850) and 0.1 mM–13.0 mM (R2 = 0.9668), which is suitable for adoption in detecting the concentrations of blood urea nitrogen (BUN, 1.78–7.12 mM) and glucose (3.89–6.11 mM) in serum. The detection result also shows that the developed thread-based microfluidic system can successfully separate and detect the ions, BUN, and glucose in blood. The calculated concentrations of BUN and glucose ante cibum (glucose before meal) in the whole blood sample are 3.98 mM and 4.94 mM, respectively. The developed thread-based microfluidic system provides a simple yet high performance for clinical diagnostics. PMID:25825613

  13. Development of a Modular Assay for Detailed Immunophenotyping of Peripheral Human Whole Blood Samples by Multicolor Flow Cytometry

    PubMed Central

    Rühle, Paul F.; Fietkau, Rainer; Gaipl, Udo S.; Frey, Benjamin

    2016-01-01

    The monitoring of immune cells gained great significance in prognosis and prediction of therapy responses. For analyzing blood samples, the multicolor flow cytometry has become the method of choice as it combines high specificity on single cell level with multiple parameters and high throughput. Here, we present a modular assay for the detailed immunophenotyping of blood (DIoB) that was optimized for an easy and direct application in whole blood samples. The DIoB assay characterizes 34 immune cell subsets that circulate the peripheral blood including all major immune cells such as T cells, B cells, natural killer (NK) cells, monocytes, dendritic cells (DCs), neutrophils, eosinophils, and basophils. In addition, it evaluates their functional state and a few non-leukocytes that also have been associated with the outcome of cancer therapy. This DIoB assay allows a longitudinal and close-meshed monitoring of a detailed immune status in patients requiring only 2.0 mL of peripheral blood and it is not restricted to peripheral blood mononuclear cells. It is currently applied for the immune monitoring of patients with glioblastoma multiforme (IMMO-GLIO-01 trial, NCT02022384), pancreatic cancer (CONKO-007 trial, NCT01827553), and head and neck cancer (DIREKHT trial, NCT02528955) and might pave the way for immune biomarker identification for prediction and prognosis of therapy outcome. PMID:27529227

  14. Cellular defense system gene expression profiling of human whole blood: opportunities to predict health benefits in response to diet.

    PubMed

    Drew, Janice E

    2012-07-01

    Diet is a critical factor in the maintenance of human cellular defense systems, immunity, inflammation, redox regulation, metabolism, and DNA repair that ensure optimal health and reduce disease risk. Assessment of dietary modulation of cellular defense systems in humans has been limited due to difficulties in accessing target tissues. Notably, peripheral blood gene expression profiles associated with nonhematologic disease are detectable. Coupled with recent innovations in gene expression technologies, gene expression profiling of human blood to determine predictive markers associated with health status and dietary modulation is now a feasible prospect for nutrition scientists. This review focuses on cellular defense system gene expression profiling of human whole blood and the opportunities this presents, using recent technological advances, to predict health status and benefits conferred by diet. PMID:22797985

  15. West Nile virus adheres to human red blood cells in whole blood.

    PubMed

    Rios, Maria; Daniel, Sylvester; Chancey, Caren; Hewlett, Indira K; Stramer, Susan L

    2007-07-15

    Background. West Nile virus (WNV) is endemic in the United States. It is transmissible by blood transfusion, and the nation's blood supply is currently screened for WNV. Documented transmission of WNV infection through red blood cell (RBC) units in which the plasma co-component had a low viral load could be explained, in at least 1 instance, by cell-association of WNV; in this case, the RBC unit was released as negative by minipool nucleic acid testing (NAT) performed on plasma but was intermittently NAT-positive when subsequently tested as an individual sample. We hypothesized that a proportion of WNV bound to blood cells and was not measured by NAT performed on plasma samples. We have investigated whether WNV binds to RBCs, leading to reduction of WNV RNA detection by NAT performed on plasma samples.Methods. Equal volumes of leukoreduced RBCs and their corresponding plasma components from 20 blood donors with NAT results that were positive for WNV were tested in 5 replicates by reverse-transcriptase polymerase chain reaction TaqMan for WNV. In addition, aliquots from 8 of the RBC units were tested by infectivity assays using Vero cells.Results. The reverse-transcriptase polymerase chain reaction TaqMan assay showed that the viral load in the RBC components exceeded that in the corresponding plasma units by 1 order of magnitude. In addition, viruses associated with the RBCs were infectious in Vero cell cultures.Conclusions. These observations reinforce the notion that extraction of viral RNA from whole blood could improve assay sensitivity for blood donor screening and further reduce the residual risk of WNV transmission through transfusion.

  16. Tumor necrosis factor-alpha (TNF-alpha) concentrations from whole blood cultures correlate with isolated peripheral blood mononuclear cell cultures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many cellular immune assays are impractical because they require labor-intensive isolation of cells from their natural environment. The objectives of this study were to determine the relationship between cell culture supernatant TNF-alpha from isolated peripheral blood mononuclear cells (PBMC) and w...

  17. Pilot clinical study to investigate the human whole blood spectrum characteristics in the sub-THz region.

    PubMed

    Tseng, Tzu-Fang; You, Borwen; Gao, Hao-Cheng; Wang, Tzung-Dau; Sun, Chi-Kuang

    2015-04-01

    We have conducted a pilot clinical study to not only investigate the sub-THz spectra of ex-vivo fresh human whole blood of 28 patients following 8-hours fasting guideline, but also to find out the critical blood ingredients of which the concentration dominantly affects those sub-THz spectra. A great difference between the sub-THz absorption properties of human blood among different people was observed, while the difference can be up to ~15% of the averaged absorption coefficient of the 28 samples. Our pilot clinical study indicates that triglycerides and the number of red blood cells were two dominant factors to have significant negative correlation to the sub-THz absorption coefficients. PMID:25968774

  18. Microfluidic assay for precise measurements of mouse, rat, and human neutrophil chemotaxis in whole-blood droplets.

    PubMed

    Jones, Caroline N; Hoang, Anh N; Martel, Joseph M; Dimisko, Laurie; Mikkola, Amy; Inoue, Yoshitaka; Kuriyama, Naohide; Yamada, Marina; Hamza, Bashar; Kaneki, Masao; Warren, H Shaw; Brown, Diane E; Irimia, Daniel

    2016-07-01

    Animal models of human disease differ in innate immune responses to stress, pathogens, or injury. Precise neutrophil phenotype measurements could facilitate interspecies comparisons. However, such phenotype comparisons could not be performed accurately with the use of current assays, as they require the separation of neutrophils from blood using species-specific protocols, and they introduce distinct artifacts. Here, we report a microfluidic technology that enables robust characterization of neutrophil migratory phenotypes in a manner independent of the donor species and performed directly in a droplet of whole blood. The assay relies on the particular ability of neutrophils to deform actively during chemotaxis through microscale channels that block the advance of other blood cells. Neutrophil migration is measured directly in blood, in the presence of other blood cells and serum factors. Our measurements reveal important differences among migration counts, velocity, and directionality among neutrophils from 2 common mouse strains, rats, and humans. PMID:26819316

  19. Effect of prenylated flavonoids and chalcones isolated from Artocarpus species on platelet aggregation in human whole blood.

    PubMed

    Jantan, Ibrahim; Mohd Yasin, Yusyila H; Jamil, Shajarahtunnur; Sirat, Hasnah; Basar, Norazah

    2010-07-01

    Five prenylflavonoids and two prenylchalcones from Artocarpus lowii King, A. scortechinii King and A. teysmanii Miq., and acetylated derivatives of cycloheterophyllin and artonin E were investigated for their ability to inhibit arachidonic acid (AA), collagen and adenosine diphosphate (ADP)-induced platelet aggregation in human whole blood by using an electrical impedance method. Among the tested compounds, only cycloheterophyllin inhibited AA-induced platelet aggregation with an IC(50) value of 100.9 microM. It also showed strong inhibition against ADP-induced aggregation, with an IC(50) value of 57.1 microM. Isobavachalcone, 2',4'-dihydroxy-4-methoxy-3'-prenyldihydrochalcone, cycloartobiloxanthone, artonin E and artonin E triacetate showed selective inhibition against ADP-induced aggregation, with IC(50) values ranging from 55.3 to 192.0 microM, but did not show such effect against other inducers.

  20. EFFECTS OF STORAGE, RNA EXTRACTION, GENECHIP TYPE, AND DONOR SEX ON GENE EXPRESSION PROFILING OF HUMAN WHOLE BLOOD

    EPA Science Inventory

    Background: Gene expression profiling of whole blood may be useful for monitoring toxicological exposure and for diagnosis and monitoring of various diseases. Several methods are available that can be used to transport, store, and extract RNA from whole blood, but it is not clear...

  1. Molecularly imprinted solid-phase extraction for the selective determination of methamphetamine, amphetamine, and methylenedioxyphenylalkylamine designer drugs in human whole blood by gas chromatography-mass spectrometry.

    PubMed

    Kumazawa, Takeshi; Hasegawa, Chika; Hara, Kenji; Uchigasaki, Seisaku; Lee, Xiao-Pen; Seno, Hiroshi; Suzuki, Osamu; Sato, Keizo

    2012-03-01

    A novel method is described for the extraction of methamphetamine, amphetamine, and methylenedioxyphenylalkylamine designer drugs, such as 3,4-methylenedioxy-methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine, and 3,4-(methylenedioxyphenyl)-2-butanamine, from human whole blood using molecularly imprinted solid-phase extraction as highly selective sample clean-up technique. Whole blood samples were diluted with 10 mmol/L ammonium acetate (pH 8.6) and applied to a SupelMIP-Amphetamine molecularly imprinted solid-phase extraction cartridge. The cartridge was then washed to eliminate interferences, and the amphetamines of interest were eluted with formic acid/methanol (1:100, v/v). After derivatization with trifluoroacetic anhydride, the analytes were quantified using gas chromatography-mass spectrometry. Recoveries of the seven amphetamines spiked into whole blood were 89.1-102%. The limits of quantification for each compound in 200 μL of whole blood were between 0.25 and 1.0 ng. The maximum intra- and inter-day coefficients of variation were 9.96 and 13.8%, respectively. The results show that methamphetamine, amphetamine, and methylenedioxyphenylalkyl-amine designer drugs can be efficiently extracted from crude biological samples such as whole blood by molecularly imprinted solid-phase extraction with good reproducibility. This extraction method will be useful for the pretreatment of human samples before gas chromatography-mass spectrometry.

  2. THz Exposure of Whole Blood for the Study of Biological Effects on Human Lymphocytes.

    PubMed

    Scarfì, M R; Romanò, M; Di Pietro, R; Zeni, O; Doria, A; Gallerano, G P; Giovenale, E; Messina, G; Lai, A; Campurra, G; Coniglio, D; D'Arienzo, M

    2003-06-01

    The aim of the present study is toinvestigate the genotoxic effect of THzradiation in human peripheral bloodlymphocytes following 20 minutes exposureto 1 mW average power Free Electron Laserradiation in the frequency range 120-140GHz. For this purpose 9 healthy donors wereemployed and cytokinesis block techniquewas applied to study micronucleusfrequency and cell proliferation. Theresults obtained indicate that all theelectromagnetic conditions adopted so far do not alter the investigated parameters,suggesting absence of direct chromosomaldamage and alteration of cell cyclekinetics (two tailed paired Student's test:p> 0.05 in all cases). PMID:23345833

  3. Monitoring of glucose, salt and pure water in human whole blood: An in vitro study.

    PubMed

    Imran, Muhammad; Ullah, Hafeez; Akhtar, Munir; Sial, Muhammad Aslam; Ahmed, Ejaz; Durr-E-Sabeeh; Ahmad, Mukhtar; Hussain, Fayyaz

    2016-07-01

    Designing and implementation of non-invasive methods for glucose monitoring in blood is main focus of biomedical scientists to provide a relief from skin puncturing of diabete patient. The objective of this research work is to investigate the shape deformations and the aggregation of red blood cells (RBCs) in the human blood after addition of three different analytes i) (0mM-400mM: Range) of glucose (C(6)H(12)O(6)), ii) (0mM-400mM: range) of pure salt (NaCl) and iii) (0mM- 350mM: range) of pure water (H(2)O). We have observed that the changes in the shape of individual cells from biconcave discs to spherical shapes and eventually the lysis of the cells at optimum concentration of glucose, salts and pure water. This demonstration also provides a base line to facilitate diabetes during partial diagnosis and monitoring of the glucose levels qualitatively both in research laboratories and clinical environment. PMID:27393437

  4. MnO2-induced synthesis of fluorescent polydopamine nanoparticles for reduced glutathione sensing in human whole blood.

    PubMed

    Kong, Xiang-Juan; Wu, Shuang; Chen, Ting-Ting; Yu, Ru-Qin; Chu, Xia

    2016-08-25

    Polydopamine (PDA) nanoparticles, as a kind of popular polymer material, have attracted a great deal of attention from various areas including materials science, biomedicine, energy, environmental science and so on owing to their striking physicochemical properties. Herein, we reported for the first time the synthesis of intrinsic fluorescent PDA nanoparticles using MnO2 as an oxidant. In the presence of MnO2, dopamine was quickly oxidized into its quinone derivative, and autopolymerized into fluorescent PDA nanoparticles. Using fluorescent PDA nanoparticles as a fluorescence signal indicator, we further established a cost-effective sensor for rapid, sensitive and selective sensing of reduced glutathione (GSH) based on the redox reaction between MnO2 and GSH, and the key role of MnO2 in the formation of fluorescent PDA nanoparticles. GSH has the capability of reducing MnO2 into Mn(2+), which inhibited the formation of the fluorescent PDA nanoparticles. Thus, the concentration of GSH was directly related to the decreased fluorescence signal intensity of the PDA nanoparticles. The sensor showed good sensing performance for GSH detection with high sensitivity and desirable selectivity over other potential interfering species. Additionally, the sensor exhibited excellent practical applications for GSH detection in human whole blood samples, which presents potential applications in biological detection and clinical diagnosis. PMID:27511888

  5. Flow cytofluorometric assay of human whole blood leukocyte DNA degradation in response to Yersinia pestis and Staphylococcus aureus

    NASA Astrophysics Data System (ADS)

    Kravtsov, Alexander L.; Grebenyukova, Tatyana P.; Bobyleva, Elena V.; Golovko, Elena M.; Malyukova, Tatyana A.; Lyapin, Mikhail N.; Kostyukova, Tatyana A.; Yezhov, Igor N.; Kuznetsov, Oleg S.

    2001-05-01

    Human leukocytes containing less than 2C DNA per cell (damaged or dead cells) were detected and quantified by flow cytometry and DNA-specific staining with ethidium bromide and mithramycin in whole blood infected with Staphylococcus aureus or Yersinia pestis. Addition of live S. aureus to the blood (100 microbe cells per one leukocyte) resulted in rapid degradation of leukocyte DNA within 3 to 6 hours of incubation at 37 degree(s)C. However, only about 50 percent cells were damaged and the leukocytes with the intact genetic apparatus could be found in the blood for a period up to 24 hours. The leukocyte injury was preceded by an increase of DNA per cell content (as compared to the normal one) that was likely to be connected with the active phagocytosis of S. aureus by granulocytes (2C DNA of diploid phagocytes plus the all bacterial DNA absorbed). In response to the same dose of actively growing (at 37 degree(s)C) virulent Y. pestis cells, no increase in DNA content per cell could be observed in the human blood leukocytes. The process of the leukocyte DNA degradation started after a 6-hour incubation, and between 18 to 24 hours of incubation about 90 percent leukocytes (phagocytes and lymphocytes) lost their specific DNA fluorescence. These results demonstrated a high potential of flow cytometry in comparative analysis in vitro of the leukocyte DNA degradation process in human blood in response to bacteria with various pathogenic properties. They agree with the modern idea of an apoptotic mechanism of immunosuppression in plague.

  6. Effects of Montelukast on free radical production in whole blood and isolated human polymorphonuclear neutrophils (PMNs) in asthmatic children

    PubMed Central

    Al Saadi, Muslim M.; Meo, Sultan Ayoub; Mustafa, Ali; Shafi, Ahmed; Tuwajri, Ali S. Al

    2011-01-01

    Montelukast is a highly selective leukotriene-receptor antagonist (LTRA). It is widely used in the treatment of bronchial asthma, primarily as an adjunct to corticosteroids. Reactive oxygen species (ROSs) play an important role in the pathogenesis of asthma and oxidative stress contributing to the initiation and worsening of inflammatory respiratory disorders, such as asthma. Antioxidant drugs may have a role in minimizing or preventing damage in asthmatic children. The aim of this study was to assess the antioxidant effect of montelukast on the production of free radicals in the whole blood and polymorphonuclear neutrophils (PMNs) in asthmatic children. A group of 48 (38 males and 10 females), apparently healthy asthmatic children were recruited with ages ranging between 6 and 14 years. In asthmatic children, base line (premedication) and post medication free radicals activity in the whole blood and polymorphonuclear neutrophils (PMNs) was determined by measuring chemiluminescence (CL) response through chemiluminescence luminometer. Free radical productions were significantly decreased in the whole blood, when stimulated with Phorbol Myristate Acetate (p < 0.04) and Opsonised Zymosan (p < 0.05). The free radicals were also significantly decreased in isolated polymorphonuclear neutrophils (PMNs) when stimulated with Opsonised Zymosan (p < 0.05) after the post medication treatment of montelukast in asthmatic children. Montelukast decreased the reactive oxygen species production, both in the whole blood as well as isolated PMNs in asthmatic children. PMID:23960762

  7. Quantitation of the enantiomers of tramadol and its three main metabolites in human whole blood using LC-MS/MS.

    PubMed

    Haage, Pernilla; Kronstrand, Robert; Carlsson, Björn; Kugelberg, Fredrik C; Josefsson, Martin

    2016-02-01

    The analgesic drug tramadol and its metabolites are chiral compounds, with the (+)- and (-)-enantiomers showing different pharmacological and toxicological effects. This novel enantioselective method, based on LC-MS/MS in reversed phase mode, enabled measurement of the parent compound and its three main metabolites O-desmethyltramadol, N-desmethyltramadol and N,O-didesmethyltramadol simultaneously. Whole blood samples of 0.5g were fortified with internal standards (tramadol-(13)C-D3 and O-desmethyl-cis-tramadol-D6) and extracted under basic conditions (pH 11) by liquid-liquid extraction. Chromatography was performed on a chiral alpha-1-acid glycoprotein (AGP) column preceded by an AGP guard column. The mobile phase consisted of 0.8% acetonitrile and 99.2% ammonium acetate (20mM, pH 7.2). A post-column infusion with 0.05% formic acid in acetonitrile was used to enhance sensitivity. Quantitation as well as enantiomeric ratio measurements were covered by quality controls. Validation parameters for all eight enantiomers included selectivity (high), matrix effects (no ion suppression/enhancement), calibration model (linear, weight 1/X(2), in the range of 0.25-250ng/g), limit of quantitation (0.125-0.50ng/g), repeatability (2-6%) and intermediate precision (2-7%), accuracy (83-114%), dilution integrity (98-115%), carry over (not exceeding 0.07%) and stability (stable in blood and extract). The method was applied to blood samples from a healthy volunteer administrated a single 100mg dose and to a case sample concerning an impaired driver, which confirmed its applicability in human pharmacokinetic studies as well as in toxicological and forensic investigations. PMID:26625281

  8. Optical and spectroscopic properties of human whole blood and plasma with and without Y₂O₃ and Nd³⁺:Y₂O₃ nanoparticles.

    PubMed

    Barrera, Frederick J; Yust, Brian; Mimun, Lawrence C; Nash, Kelly L; Tsin, Andrew T; Sardar, Dhiraj K

    2013-11-01

    The optical properties of human whole blood and blood plasma with and without Y₂O₃ and Nd³⁺:Y₂O₃ nanoparticles are characterized in the near infrared region at 808 nm using a double integrating sphere technique. Using experimentally measured quantities of diffuse reflectance and diffuse transmittance, a computational analysis was conducted utilizing the Kubelka-Munk, the Inverse Adding Doubling, and Magic Light Kubelka-Munk and Monte Carlo Methods to determine optical properties of the absorption and scattering coefficients. Room temperature absorption and emission spectra were also acquired of Nd³⁺:Y₂O₃ nanoparticles elucidating their utility as biological markers. The emission spectra of Nd³⁺:Y₂O₃ were taken by exciting the nanoparticles before and after entering the whole blood sample. The emission from the ⁴F(3/2) → ⁴I(11/2) manifold transition of Nd³⁺:Y₂O₃ nanoparticles readily propagates through the blood sample at excitation of 808 nm and exhibits a shift in relative intensities of the peaks due to differences in scattering. At 808 nm, in both whole blood and plasma samples, a direct relationship was found with absorption coefficient and Y₂O₃ nanoparticle concentration. Results for the whole blood indicate a small inverse relationship with Y₂O₃ nanoparticle concentration and scattering coefficient and in contrast a direct relation for the plasma.

  9. Transcriptome Analysis of Neisseria meningitidis in Human Whole Blood and Mutagenesis Studies Identify Virulence Factors Involved in Blood Survival

    PubMed Central

    Del Tordello, Elena; Seib, Kate L.; Francois, Patrice; Rappuoli, Rino; Pizza, Mariagrazia; Serruto, Davide

    2011-01-01

    During infection Neisseria meningitidis (Nm) encounters multiple environments within the host, which makes rapid adaptation a crucial factor for meningococcal survival. Despite the importance of invasion into the bloodstream in the meningococcal disease process, little is known about how Nm adapts to permit survival and growth in blood. To address this, we performed a time-course transcriptome analysis using an ex vivo model of human whole blood infection. We observed that Nm alters the expression of ≈30% of ORFs of the genome and major dynamic changes were observed in the expression of transcriptional regulators, transport and binding proteins, energy metabolism, and surface-exposed virulence factors. In particular, we found that the gene encoding the regulator Fur, as well as all genes encoding iron uptake systems, were significantly up-regulated. Analysis of regulated genes encoding for surface-exposed proteins involved in Nm pathogenesis allowed us to better understand mechanisms used to circumvent host defenses. During blood infection, Nm activates genes encoding for the factor H binding proteins, fHbp and NspA, genes encoding for detoxifying enzymes such as SodC, Kat and AniA, as well as several less characterized surface-exposed proteins that might have a role in blood survival. Through mutagenesis studies of a subset of up-regulated genes we were able to identify new proteins important for survival in human blood and also to identify additional roles of previously known virulence factors in aiding survival in blood. Nm mutant strains lacking the genes encoding the hypothetical protein NMB1483 and the surface-exposed proteins NalP, Mip and NspA, the Fur regulator, the transferrin binding protein TbpB, and the L-lactate permease LctP were sensitive to killing by human blood. This increased knowledge of how Nm responds to adaptation in blood could also be helpful to develop diagnostic and therapeutic strategies to control the devastating disease cause by

  10. Speciation of mercury in human whole blood by capillary gas chromatography with a microwave-induced plasma emission detector system following complexometric extraction and butylation.

    PubMed

    Bulska, E; Emteborg, H; Baxter, D C; Frech, W; Ellingsen, D; Thomassen, Y

    1992-03-01

    Methyl- and inorganic mercury were extracted from human whole blood samples, as their diethyldithiocarbamate complexes, into toluene and butylated by using a Grignard reagent. The mercury species were then separated by gas chromatography (on a 12 m non-polar DB-1 capillary column) and detected by a microwave-induced plasma atomic emission spectrometric (GC-MPD) system. The accuracy and precision of the proposed method were established by the analysis of Seronorm lyophilized human whole blood standards for methyl- and inorganic mercury. No statistical difference (t-test) between the sum of these two species determined by the GC-MPD based method and the recommended total mercury concentrations in the Seronorm samples was observed. Results for the determination of methyl- and inorganic mercury in 60 controls and 90 previously occupationally exposed (to inorganic mercury) workers are presented to illustrate the practical utility of the proposed method. No significantly elevated inorganic mercury concentrations between the two groups were evident.

  11. Antithrombin III, but not C1 esterase inhibitor reduces inflammatory response in lipopolysaccharide-stimulated human monocytes in an ex-vivo whole blood setting.

    PubMed

    Kellner, Patrick; Nestler, Frank; Leimert, Anja; Bucher, Michael; Czeslick, Elke; Sablotzki, Armin; Raspè, Christoph

    2014-12-01

    In order to examine the immunomodulatory effects of antithrombin III (AT-III) and C1 esterase inhibitor (C1-INH) in human monocytes, we investigated the intracellular expression of interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α in an ex-vivo laboratory study in a whole blood setting. Heparinized whole blood samples from 23 healthy male and female volunteers (mean age: 27±7years) were pre-incubated with clinically relevant concentrations of AT-III (n=11) and C1-INH (n=12), then stimulated with 0.2 ng/mL lipopolysaccharide (LPS) for 3h. After phenotyping CD14⁺ monocytes, intracellular expression of IL-6, IL-8, and TNF-α was assessed using flow cytometry. In addition, 12 whole blood samples (AT-III and C1-INH, n=6 each) were examined using hirudin for anticoagulation; all samples were processed in the same way. To exclude cytotoxicity effects, 7-amino-actinomycin D and Nonidet P40 staining were used to investigate probes. This study is the first to demonstrate the influence of C1-INH and AT-III on the monocytic inflammatory response in a whole blood setting, which mimics the optimal physiological setting. Cells treated with AT-III exhibited significant downregulation of the proportion of gated CD14⁺ monocytes for IL-6 and IL-8, in a dose-dependent manner; downregulation for TNF-α did not reach statistical significance. There were no significant effects on mean fluorescence intensity (MFI). In contrast, C1-INH did not significantly reduce the proportion of gated CD14⁺ monocytes or the MFI regarding IL-6, TNF-α, and IL-8. When using hirudin for anticoagulation, no difference in the anti-inflammatory properties of AT-III and C1-INH in monocytes occurs. Taken together, in contrast to TNF-α, IL-6 and IL-8 were significantly downregulated in monocytes in an ex-vivo setting of human whole blood when treated with AT-III. This finding implicates monocytes as an important point of action regarding the anti-inflammatory properties of AT-III in sepsis. C1

  12. Glutamine and alanine-induced differential expression of intracellular IL-6, IL-8, and TNF-α in LPS-stimulated monocytes in human whole-blood.

    PubMed

    Raspé, C; Czeslick, E; Weimann, A; Schinke, C; Leimert, A; Kellner, P; Simm, A; Bucher, M; Sablotzki, A

    2013-04-01

    To investigate the effects of the commonly-used immunomodulators l-glutamine, l-alanine, and the combination of both l-alanyl-l-glutamine (Dipeptamin(®)) on intracellular expression of IL-6, IL-8, and TNF-α during endotoxemia, lipopolysaccharide (LPS)-stimulated human monocytes in a whole blood system were investigated by flow cytometry. Whole blood of twenty-seven healthy volunteers was stimulated with LPS and incubated with three different amino acid solutions (1. l-glutamine, 2. l-alanine, 3. l-alanyl-l-glutamine, each concentration 2 mM, 5 mM, incubation time 3 h). CD14(+) monocytes were phenotyped in whole-blood and intracellular expression of cytokines was assessed by flow cytometry. Our investigations showed for the first time in whole blood probes, imitating best physiologically present cellular interactions, that l-glutamine caused a dose-independent inhibitory effect on IL-6 and TNF-α production in human monocytes stimulated with LPS. However, l-alanine had contrary effects on IL-6 expression, significantly upregulating expression of IL-6 in LPS-treated monocytes. The impact of l-alanine on the expression of TNF-α was comparable with glutamine. Neither amino acid was able to affect IL-8 production in LPS-stimulated monocytes. The combination of both did not influence significantly IL-6 and IL-8 expression in monocytes during endotoxemia, however strongly reduced TNF-α production. For the regulation of TNF-α, l-glutamine, l-alanine and the combination of both show a congruent and exponentiated downregulating effect during endotoxemia, for the modulation of IL-6, l-glutamine and l-alanine featured opposite regulation leading to a canceling impact of each other when recombining both amino acids.

  13. Pipette tip solid-phase extraction and gas chromatography - mass spectrometry for the determination of methamphetamine and amphetamine in human whole blood.

    PubMed

    Hasegawa, Chika; Kumazawa, Takeshi; Lee, Xiao-Pen; Marumo, Akemi; Shinmen, Natsuko; Seno, Hiroshi; Sato, Keizo

    2007-09-01

    Methamphetamine and amphetamine were extracted from human whole blood samples using pipette tip solid-phase extraction (SPE) with MonoTip C(18) tips, on which C(18)-bonded monolithic silica gel was fixed. Human whole blood (0.1 mL) containing methamphetamine and amphetamine, with N-methylbenzylamine as an internal standard, was mixed with 0.4 mL of distilled water and 50 microL of 5 M sodium hydroxide solution. After centrifugation, the supernatant was extracted to the C(18) phase of the tip (pipette tip volume, 200 microL) by 25 repeated aspirating/dispensing cycles using a manual micropipettor. Analytes retained in the C(18) phase were eluted with methanol by five repeated aspirating/dispensing cycles. After derivatization with trifluoroacetic anhydride, analytes were measured by gas chromatography - mass spectrometry with selected ion monitoring in the positive-ion electron impact mode. Recoveries of methamphetamine and amphetamine spiked into whole blood were more than 87.6 and 81.7%, respectively. Regression equations for methamphetamine and amphetamine showed excellent linearity in the range of 0.5-100 ng/0.1 mL. The limits of detection for methamphetamine and amphetamine were 0.15 and 0.11 ng/0.1 mL, respectively. Intra- and interday coefficients of variation for both stimulants were not greater than 9.6 and 13.8%, respectively. The determination of methamphetamine and amphetamine in autopsy whole blood samples is presented, and was shown to validate the present methodology.

  14. 21 CFR 640.6 - Modifications of Whole Blood.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Modifications of Whole Blood. 640.6 Section 640.6...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Whole Blood § 640.6 Modifications of Whole Blood. Upon approval by the Director, Center for Biologics Evaluation and Research, of a supplement...

  15. Simple Real-Time PCR and Amplicon Sequencing Method for Identification of Plasmodium Species in Human Whole Blood.

    PubMed

    Lefterova, Martina I; Budvytiene, Indre; Sandlund, Johanna; Färnert, Anna; Banaei, Niaz

    2015-07-01

    Malaria is the leading identifiable cause of fever in returning travelers. Accurate Plasmodium species identification has therapy implications for P. vivax and P. ovale, which have dormant liver stages requiring primaquine. Compared to microscopy, nucleic acid tests have improved specificity for species identification and higher sensitivity for mixed infections. Here, we describe a SYBR green-based real-time PCR assay for Plasmodium species identification from whole blood, which uses a panel of reactions to detect species-specific non-18S rRNA gene targets. A pan-Plasmodium 18S rRNA target is also amplified to allow species identification or confirmation by sequencing if necessary. An evaluation of assay accuracy, performed on 76 clinical samples (56 positives using thin smear microscopy as the reference method and 20 negatives), demonstrated clinical sensitivities of 95.2% for P. falciparum (20/21 positives detected) and 100% for the Plasmodium genus (52/52), P. vivax (20/20), P. ovale (9/9), and P. malariae (6/6). The sensitivity of the P. knowlesi-specific PCR was evaluated using spiked whole blood samples (100% [10/10 detected]). The specificities of the real-time PCR primers were 94.2% for P. vivax (49/52) and 100% for P. falciparum (51/51), P. ovale (62/62), P. malariae (69/69), and P. knowlesi (52/52). Thirty-three specimens were used to test species identification by sequencing the pan-Plasmodium 18S rRNA PCR product, with correct identification in all cases. The real-time PCR assay also identified two samples with mixed P. falciparum and P. ovale infection, which was confirmed by sequencing. The assay described here can be integrated into a malaria testing algorithm in low-prevalence areas, allowing definitive Plasmodium species identification shortly after malaria diagnosis by microscopy.

  16. Detection of total and A1c-glycosylated hemoglobin in human whole blood using sandwich immunoassays on polydimethylsiloxane-based antibody microarrays.

    PubMed

    Chen, Huang-Han; Wu, Chih-Hsing; Tsai, Mei-Ling; Huang, Yi-Jing; Chen, Shu-Hui

    2012-10-16

    The percentage of glycosylated hemoglobin A1c (%GHbA1c) in human whole blood indicates the average plasma glucose concentration over a prolonged period of time and is used to diagnose diabetes. However, detecting GHbA1c in the whole blood using immunoassays has limited detection sensitivity due to its low percentage in total hemoglobin (tHb) and interference from various glycan moieties in the sample. We have developed a sandwich immunoassay using an antibody microarray on a polydimethylsiloxane (PDMS) substrate modified with fluorinated compounds to detect tHb and glycosylated hemoglobin A1c (GHbA1c) in human whole blood without sample pretreatment. A polyclonal antibody against hemoglobin (Hb) immobilized on PDMS is used as a common capture probe to enrich all forms of Hb followed by detection via monoclonal anti-Hb and specific monoclonal anti-GHbA1c antibodies for tHb and GHbA1c detection, respectively. This method prevents the use of glycan binding molecules and dramatically reduces the background interference, yielding a detection limit of 3.58 ng/mL for tHb and 0.20 ng/mL for GHbA1c. The fluorinated modification on PDMS is superior to the glass substrate and eliminates the need for the blocking step which is required in commercial enzyme linked immunosorbent assay (ELISA) kits. Moreover, the detection sensitivity for GHbA1c is 4-5 orders of magnitude higher, but the required sample amount is 25 times less than the commercial method. On the basis of patient sample data, a good linear correlation between %GHbA1c values determined by our method and the certified high performance liquid chromatography (HPLC) standard method is shown with R(2) > 0.98, indicating the great promise of the developed method for clinical applications.

  17. Micropallet arrays for the capture, isolation and culture of circulating tumor cells from whole blood of mice engrafted with primary human pancreatic adenocarcinoma.

    PubMed

    Gach, Philip C; Attayek, Peter J; Whittlesey, Rebecca L; Yeh, Jen Jen; Allbritton, Nancy L

    2014-04-15

    Circulating tumor cells (CTCs) are important biomarkers of cancer progression and metastatic potential. The rarity of CTCs in peripheral blood has driven the development of technologies to isolate these tumor cells with high specificity; however, there are limited techniques available for isolating target CTCs following enumeration. A strategy is described to capture and isolate viable tumor cells from whole blood using an array of releasable microstructures termed micropallets. Specific capture of nucleated cells or cells expressing epithelial cell adhesion molecules (EpCAM) was achieved by functionalizing micropallet surfaces with either fibronectin, Matrigel or anti-EpCAM antibody. Surface grafting of poly(acrylic acid) followed by covalent binding of protein A/G enabled efficient capture of EpCAM antibody on the micropallet surface. MCF-7 cells, a human breast adenocarcinoma, were retained on the array surface with 90±8% efficiency when using an anti-EpCAM-coated array. To demonstrate the efficiency of tumor cell retention on micropallet arrays in the presence of blood, MCF-7 cells were mixed into whole blood and added to small arrays (71 mm(2)) coated with fibronectin, Matrigel or anti-EpCAM. These approaches achieved MCF-7 cell capture from ≤10 µL of whole blood with efficiencies greater than 85%. Furthermore, MCF-7 cells intermixed with 1 mL blood and loaded onto large arrays (7171 mm(2)) were captured with high efficiencies (≥97%), could be isolated from the array by a laser-based approach and were demonstrated to yield a high rate of colony formation (≥85%) after removal from the array. Clinical utility of this technology was shown through the capture, isolation and successful culture of CTCs from the blood of mice engrafted with primary human pancreatic tumors. Direct capture and isolation of living tumor cells from blood followed by analysis or culture will be a valuable tool for cancer cell characterization.

  18. Gene expression profiling of Gram-negative bacteria-induced inflammation in human whole blood: The role of complement and CD14-mediated innate immune response.

    PubMed

    Lau, Corinna; Olstad, Ole Kristoffer; Holden, Marit; Nygård, Ståle; Fure, Hilde; Lappegård, Knut Tore; Brekke, Ole-Lars; Espevik, Terje; Hovig, Eivind; Mollnes, Tom Eirik

    2015-09-01

    Non-sterile pathogen-induced sepsis and sterile inflammation like in trauma or ischemia-reperfusion injury may both coincide with the life threatening systemic inflammatory response syndrome and multi-organ failure. Consequently, there is an urgent need for specific biomarkers in order to distinguish sepsis from sterile conditions. The overall aim of this study was to uncover putative sepsis biomarkers and biomarker pathways, as well as to test the efficacy of combined inhibition of innate immunity key players complement and Toll-like receptor co-receptor CD14 as a possible therapeutic regimen for sepsis. We performed whole blood gene expression analyses using microarray in order to profile Gram-negative bacteria-induced inflammatory responses in an ex vivo human whole blood model. The experiments were performed in the presence or absence of inhibitors of complement proteins (C3 and CD88 (C5a receptor 1)) and CD14, alone or in combination. In addition, we used blood from a C5-deficient donor. Anti-coagulated whole blood was challenged with heat-inactivated Escherichia coli for 2 h, total RNA was isolated and microarray analyses were performed on the Affymetrix GeneChip Gene 1.0 ST Array platform. The initial experiments were performed in duplicates using blood from two healthy donors. C5-deficiency is very rare, and only one donor could be recruited. In order to increase statistical power, a technical replicate of the C5-deficient samples was run. Subsequently, log2-transformed intensities were processed by robust multichip analysis and filtered using a threshold of four. In total, 73 microarray chips were run and analyzed. The normalized and filtered raw data have been deposited in NCBI's Gene Expression Omnibus (GEO) and are accessible with GEO Series accession number GSE55537. Linear models for microarray data were applied to estimate fold changes between data sets and the respective multiple testing adjusted p-values (FDR q-values). The interpretation of the

  19. Data on gene and protein expression changes induced by apabetalone (RVX-208) in ex vivo treated human whole blood and primary hepatocytes.

    PubMed

    Wasiak, Sylwia; Gilham, Dean; Tsujikawa, Laura M; Halliday, Christopher; Norek, Karen; Patel, Reena G; McLure, Kevin G; Young, Peter R; Gordon, Allan; Kulikowski, Ewelina; Johansson, Jan; Sweeney, Michael; Wong, Norman C

    2016-09-01

    Apabetalone (RVX-208) inhibits the interaction between epigenetic regulators known as bromodomain and extraterminal (BET) proteins and acetyl-lysine marks on histone tails. Data presented here supports the manuscript published in Atherosclerosis "RVX-208, a BET-inhibitor for Treating Atherosclerotic Cardiovascular Disease, Raises ApoA-I/HDL and Represses Pathways that Contribute to Cardiovascular Disease" (Gilham et al., 2016) [1]. It shows that RVX-208 and a comparator BET inhibitor (BETi) JQ1 increase mRNA expression and production of apolipoprotein A-I (ApoA-I), the main protein component of high density lipoproteins, in primary human and African green monkey hepatocytes. In addition, reported here are gene expression changes from a microarray-based analysis of human whole blood and of primary human hepatocytes treated with RVX-208. PMID:27570805

  20. Stability of human immunodeficiency virus type 1 antibodies in whole blood dried on filter paper and stored under various tropical conditions in Kinshasa, Zaire.

    PubMed

    Behets, F; Kashamuka, M; Pappaioanou, M; Green, T A; Ryder, R W; Batter, V; George, J R; Hannon, W H; Quinn, T C

    1992-05-01

    The use of whole-blood spots on filter paper for the detection of antibody to human immunodeficiency virus type 1 (HIV-1) was evaluated during a 20-week period under a variety of storage environments simulating the harsh tropical field conditions in Kinshasa, Zaire. During the first 6 weeks of storage, all replicates of high- and low-titer HIV-1-positive reference samples remained positive by enzyme immunoassay and Western blotting (immunoblotting), and all replicates of HIV-1-negative samples remained negative under all storage conditions. However, hot and humid storage conditions for up to 20 weeks caused a progressive decline in enzyme immunoassay optical density ratio values, which was particularly noticeable in samples with a low HIV-1 antibody titer. Harsh tropical operational conditions did not cause any repeatedly false-positive results during the 20-week storage period. The use of gas-impermeable bags with desiccant for the storage of blood spots on filter paper improved the stability of HIV-1 antibody detection over time and is recommended for the storage of whole-blood spots on filter paper in harsh tropical field settings.

  1. Market study: Whole blood analyzer

    NASA Technical Reports Server (NTRS)

    1977-01-01

    A market survey was conducted to develop findings relative to the commercialization potential and key market factors of the whole blood analyzer which is being developed in conjunction with NASA's Space Shuttle Medical System.

  2. Nanotoxicity of poly(n-butylcyano-acrylate) nanoparticles at the blood-brain barrier, in human whole blood and in vivo.

    PubMed

    Kolter, Marise; Ott, Melanie; Hauer, Christian; Reimold, Isolde; Fricker, Gert

    2015-01-10

    Therapy of diseases of the central nervous system is a major challenge since drugs have to overcome the blood-brain barrier (BBB). A powerful strategy to enhance cerebral drug concentration is administration of drug-loaded poly(n-butylcyano-acrylate) (PBCA) nanoparticles coated with polysorbate 80 (PS80). This study evaluates the toxicity of PBCA-nanoparticles at the BBB, representing the target organ, the inflammatory response in human whole blood, as the site of administration and in a rat model in vivo. PBCA-nanoparticles were prepared by a mini-emulsion method and characterized concerning size, surface charge, shape and PS80-adsorption. The influence on metabolic activity, cell viability and integrity of the BBB was analyzed in an in vitro model of the BBB. In ex vivo experiments in human whole blood the release of 12 inflammatory cytokines was investigated. In addition, the inflammatory response was studied in vivo in rats and complemented with the analysis of different organ toxicity parameters. PBCA-nanoparticles showed time- and concentration-dependent effects on metabolic activity, cell viability and BBB integrity. No cell death or loss of metabolic activity was observed for nanoparticle-concentrations ≤500μg/ml up to 3h of treatment. Within 12 tested inflammatory cytokines, only interleukin-8 displayed a significant release after nanoparticle exposure in human blood. No severe inflammatory processes or organ damages were identified in rats in vivo. Thus, PBCA-nanoparticles are a promising drug delivery system to overcome the BBB since they showed hardly any cytotoxic or inflammatory effect at therapeutic concentrations and incubation times.

  3. Chronic exposure to aluminum, nickel, thallium and uranium and their relationship with essential elements in human whole blood and blood serum.

    PubMed

    Zeneli, Lulzim; Sekovanić, Ankica; Daci, Nexhat

    2015-01-01

    This study aimed to evaluate the influence of exposure to aluminum, nickel, thallium and uranium on the metabolism of essential elements in humans, as well as the relationship between uranium, thallium, nickel, and aluminum and essential elements (Ca, Mg, Zn, Se, Mn, Co, Cr, and Mo) in the whole blood and blood serum of healthy men who were occupationally exposed. This study included 97 healthy men, 31-64 years age, including 70 workers in a thermo power plant and 27 control subjects. The results showed that chronic, moderate exposure of trace elements (Al, Ni, Tl, and U) lead to decreased serum chromium (SCr) and blood molybdenum levels (BMo), whereas by the results achieved in terms of correlations between non-essential and essential elements, non-essential elements such as uranium, thallium, nickel, and aluminum, despite their concentration within the reference values, are strongly competitive with essential elements in biochemical processes.

  4. Characteristics of 2,4,5,2',4',5'-hexachlorobiphenyl distribution among lipoproteins in vitro. [Rats and human whole blood, plasma, and lipoprotein fractions

    SciTech Connect

    Vomachka, M.S.; Vodicnik, M.J.; Lech, J.J.

    1983-09-01

    The uptake, distribution, and transfer of 2,4,5,2',4',5'-hexachlorobiphenyl (6-CB) were examined in vitro with human and rat whole blood, plasma, and lipoprotein fractions. 6-CB distribution between plasma and erythrocytes as well as among lipoproteins was determined following sedimentation of erythrocytes and ultracentrifugal fractionation of plasma. In both rat and human whole blood, 70 to 75% of 6-CB partitioned into plasma and 25 to 30% into erythrocytes. The uptake of 6-CB into plasma was extremely rapid and the rate of uptake was found to be dependent upon temperature. The distribution of 6-CB among lipoproteins was relatively homogeneous with 20 to 30% being distributed in very low-density lipoproteins (VLDL, d . 0.95-1.006 g/ml), 15 to 20% in low-density lipoproteins (LDL, d . 1.006-1.063 g/ml), and 15 to 25% in high-density lipoproteins (HDL, d . 1.063-1.21 g/ml). Over 25% of 6-CB was found in the remaining bottom fraction. In addition, each isolated fraction when incubated alone with 6-CB was shown capable of uptake. The relative proportion of 6-CB among the lipoproteins was independent of the level taken up by plasma. 6-CB was also found to transfer among lipoproteins. This exchange of 6-CB proved to be dependent upon the concentrations of both protein and triacylglycerol in the incubations. Two proteins in the bottom fraction (Bf), albumin and a steroid binding globulin, were capable of competing with the lipoproteins for 6-CB uptake.

  5. CD14 and Complement Crosstalk and Largely Mediate the Transcriptional Response to Escherichia coli in Human Whole Blood as Revealed by DNA Microarray

    PubMed Central

    Lau, Corinna; Nygård, Ståle; Fure, Hilde; Olstad, Ole Kristoffer; Holden, Marit; Lappegård, Knut Tore; Brekke, Ole-Lars; Espevik, Terje; Hovig, Eivind; Mollnes, Tom Eirik

    2015-01-01

    Systemic inflammation like in sepsis is still lacking specific diagnostic markers and effective therapeutics. The first line of defense against intruding pathogens and endogenous damage signals is pattern recognition by e.g., complement and Toll-like receptors (TLR). Combined inhibition of a key complement component (C3 and C5) and TLR-co-receptor CD14 has been shown to attenuate certain systemic inflammatory responses. Using DNA microarray and gene annotation analyses, we aimed to decipher the effect of combined inhibition of C3 and CD14 on the transcriptional response to bacterial challenge in human whole blood. Importantly, combined inhibition reversed the transcriptional changes of 70% of the 2335 genes which significantly responded to heat-inactivated Escherichia coli by on average 80%. Single inhibition was less efficient (p<0.001) but revealed a suppressive effect of C3 on 21% of the responding genes which was partially counteracted by CD14. Furthermore, CD14 dependency of the Escherichia coli-induced response was increased in C5-deficient compared to C5-sufficient blood. The observed crucial distinct and synergistic roles for complement and CD14 on the transcriptional level correspond to their broad impact on the inflammatory response in human blood, and their combined inhibition may become inevitable in the early treatment of acute systemic inflammation. PMID:25706641

  6. Improved dialytic removal of protein-bound uraemic toxins with use of albumin binding competitors: an in vitro human whole blood study

    PubMed Central

    Tao, Xia; Thijssen, Stephan; Kotanko, Peter; Ho, Chih-Hu; Henrie, Michael; Stroup, Eric; Handelman, Garry

    2016-01-01

    Protein-bound uraemic toxins (PBUTs) cause various deleterious effects in end-stage kidney disease patients, because their removal by conventional haemodialysis (HD) is severely limited by their low free fraction in plasma. Here we provide an experimental validation of the concept that the HD dialytic removal of PBUTs can be significantly increased by extracorporeal infusion of PBUT binding competitors. The binding properties of indoxyl sulfate (IS), indole-3-acetic acid (IAA) and hippuric acid (HIPA) and their binding competitors, ibuprofen (IBU), furosemide (FUR) and tryptophan (TRP) were studied in uraemic plasma. The effect of binding competitor infusion on fractional removal of PBUT was then quantified in an ex vivo single-pass HD model using uraemic human whole blood. The infusion of a combination of IBU and FUR increased the fractional removal of IS from 6.4 ± 0.1 to 18.3 ± 0.4%. IAA removal rose from 16.8 ± 0.3 to 34.5 ± 0.7%. TRP infusion increased the removal of IS and IAA to 10.5 ± 0.1% and 27.1 ± 0.3%, respectively. Moderate effects were observed on HIPA removal. Pre-dialyzer infusion of PBUT binding competitors into the blood stream can increase the HD removal of PBUTs. This approach can potentially be applied in current HD settings. PMID:27001248

  7. Application of a new nanocarbonaceous sorbent in electromembrane surrounded solid phase microextraction for analysis of amphetamine and methamphetamine in human urine and whole blood.

    PubMed

    Rezazadeh, Maryam; Yamini, Yadollah; Seidi, Shahram

    2015-05-29

    Application of a new carbon-based sorbent was studied for the first time for extraction and quantification of amphetamine and methamphetamine as model analytes by means of electromembrane surrounded solid phase microextraction (EM-SPME). Since the basis of this microextraction method is adsorption of target analytes on the sorbent surface (after transferring across a supported liquid membrane) in an electrical field, the sorbent, which also performs the electrical potential, should have a conductive nature. On the other hand, using a synthesized fiber is a suitable solution to eliminate the interfering compounds existing in the fiber. To extract the model analytes from acidic sample solution through a thin layer of organic phase and into the aqueous acceptor phase and their final adsorption, 150V electrical potential was applied for 15min. Regardless of the high sample cleanup ability of the proposed method, which makes the analysis of complicated biological fluids possible, admissible extraction recoveries (9.0-18.8%) and suitable detection limits (less than 2.0ngmL(-1)) were obtained. Repeatability and reproducibility of the method were studied and intra- and inter-assay precisions were in the ranges of 2.0-7.3% and 7.5-12.5%, respectively. Coefficients of determination larger than 0.9964 were achieved by scrutinizing of the linearity up to 500ngmL(-1) and calibration curves were utilized for quantification of analytes of interest in human urine and whole blood samples.

  8. An integrated direct loop-mediated isothermal amplification microdevice incorporated with an immunochromatographic strip for bacteria detection in human whole blood and milk without a sample preparation step.

    PubMed

    Lee, Dohwan; Kim, Yong Tae; Lee, Jee Won; Kim, Do Hyun; Seo, Tae Seok

    2016-05-15

    We have developed an integrated direct loop-mediated isothermal amplification (Direct LAMP) microdevice incorporated with an immunochromatographic strip (ICS) to identify bacteria contaminated in real samples. The Direct LAMP is a novel isothermal DNA amplification technique which does not require thermal cycling steps as well as any sample preparation steps such as cell lysis and DNA extraction for amplifying specific target genes. In addition, the resultant amplicons were colorimetrically detected on the ICS, thereby enabling the entire genetic analysis process to be simplified. The two functional units (Direct LAMP and ICS) were integrated on a single device without use of the tedious and complicated microvalve and tubing systems. The utilization of a slidable plate allows us to manipulate the fluidic control in the microchannels manually and the sequential operation of the Direct LAMP and ICS detection could be performed by switching the slidable plate to each functional unit. Thus, the combination of the direct isothermal amplification without any sample preparation and thermal cycling steps, the ICS based amplicon detection by naked eyes, and the slidable plate to eliminate the microvalves in the integrated microdevice would be an ideal platform for point-of-care DNA diaganotics. On the integrated Direct LAMP-ICS microdevice, we could analyze Staphylococcus aureus (S. aureus) and Escherichia coli O157:H7 (E. coli O157:H7) contaminated in human whole blood or milk at a single-cell level within 1h.

  9. Improved dialytic removal of protein-bound uraemic toxins with use of albumin binding competitors: an in vitro human whole blood study.

    PubMed

    Tao, Xia; Thijssen, Stephan; Kotanko, Peter; Ho, Chih-Hu; Henrie, Michael; Stroup, Eric; Handelman, Garry

    2016-03-22

    Protein-bound uraemic toxins (PBUTs) cause various deleterious effects in end-stage kidney disease patients, because their removal by conventional haemodialysis (HD) is severely limited by their low free fraction in plasma. Here we provide an experimental validation of the concept that the HD dialytic removal of PBUTs can be significantly increased by extracorporeal infusion of PBUT binding competitors. The binding properties of indoxyl sulfate (IS), indole-3-acetic acid (IAA) and hippuric acid (HIPA) and their binding competitors, ibuprofen (IBU), furosemide (FUR) and tryptophan (TRP) were studied in uraemic plasma. The effect of binding competitor infusion on fractional removal of PBUT was then quantified in an ex vivo single-pass HD model using uraemic human whole blood. The infusion of a combination of IBU and FUR increased the fractional removal of IS from 6.4 ± 0.1 to 18.3 ± 0.4%. IAA removal rose from 16.8 ± 0.3 to 34.5 ± 0.7%. TRP infusion increased the removal of IS and IAA to 10.5 ± 0.1% and 27.1 ± 0.3%, respectively. Moderate effects were observed on HIPA removal. Pre-dialyzer infusion of PBUT binding competitors into the blood stream can increase the HD removal of PBUTs. This approach can potentially be applied in current HD settings.

  10. Rapid, point-of-care extraction of human immunodeficiency virus type 1 proviral DNA from whole blood for detection by real-time PCR.

    PubMed

    Jangam, Sujit R; Yamada, Douglas H; McFall, Sally M; Kelso, David M

    2009-08-01

    PCR detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA is the method recommended for use for the diagnosis of HIV-1 infection in infants in limited-resource settings. Currently, testing must be performed in central laboratories, which are usually located some distance from health care facilities. While the collection and transportation of samples, such as dried blood spots, has improved test accessibility, the results are often not returned for several weeks. To enable PCR to be performed at the point of care while the mothers wait, we have developed a vertical filtration method that uses a separation membrane and an absorbent pad to extract cellular DNA from whole blood in less than 2 min. Cells are trapped in the separation membrane as the specimen is collected, and then a lysis buffer is added. The membrane retains the DNA, while the buffer washes away PCR inhibitors, which get wicked into the absorbent blotter pad. The membrane containing the entrapped DNA is then added to the PCR mixture without further purification. The method demonstrates a high degree of reproducibility and analytical sensitivity and allows the quantification of as few as 20 copies of HIV-1 proviral DNA from 100 microl of blood. In a blinded study with 182 longitudinal samples from infants (ages, 0 to 72 weeks) obtained from the Women and Infants Transmission Study, our assay demonstrated a sensitivity of 99% and a specificity of 100%. PMID:19644129

  11. Activity of the de novo engineered antimicrobial peptide WLBU2 against Pseudomonas aeruginosa in human serum and whole blood: implications for systemic applications.

    PubMed

    Deslouches, Berthony; Islam, Kazi; Craigo, Jodi K; Paranjape, Shruti M; Montelaro, Ronald C; Mietzner, Timothy A

    2005-08-01

    Cationic amphipathic peptides have been extensively investigated as a potential source of new antimicrobials that can complement current antibiotic regimens in the face of emerging drug-resistant bacteria. However, the suppression of antimicrobial activity under certain biologically relevant conditions (e.g., serum and physiological salt concentrations) has hampered efforts to develop safe and effective antimicrobial peptides for clinical use. We have analyzed the activity and selectivity of the human peptide LL37 and the de novo engineered antimicrobial peptide WLBU2 in several biologically relevant conditions. The host-derived synthetic peptide LL37 displayed high activity against Pseudomonas aeruginosa but demonstrated staphylococcus-specific sensitivity to NaCl concentrations varying from 50 to 300 mM. Moreover, LL37 potency was variably suppressed in the presence of 1 to 6 mM Mg(2+) and Ca(2+) ions. In contrast, WLBU2 maintained its activity in NaCl and physiologic serum concentrations of Mg(2+) and Ca(2+). WLBU2 is able to kill P. aeruginosa (10(6) CFU/ml) in human serum, with a minimum bactericidal concentration of <9 microM. Conversely, LL37 is inactive in the presence of human serum. Bacterial killing kinetic assays in serum revealed that WLBU2 achieved complete bacterial killing in 20 min. Consistent with these results was the ability of WLBU2 (15 to 20 microM) to eradicate bacteria from ex vivo samples of whole blood. The selectivity of WLBU2 was further demonstrated by its ability to specifically eliminate P. aeruginosa in coculture with human monocytes or skin fibroblasts without detectable adverse effects to the host cells. Finally, WLBU2 displayed potent efficacy against P. aeruginosa in an intraperitoneal infection model using female Swiss Webster mice. These results establish a potential application of WLBU2 in the treatment of bacterial sepsis.

  12. Blood cell oxidative stress precedes hemolysis in whole blood-liver slice co-cultures of rat, dog, and human tissues

    SciTech Connect

    Vickers, Alison E.M.; Sinclair, John R.; Fisher, Robyn L.; Morris, Stephen R.; Way, William

    2010-05-01

    A novel in vitro model to investigate time-dependent and concentration-dependent responses in blood cells and hemolytic events is studied for rat, dog, and human tissues. Whole blood is co-cultured with a precision-cut liver slice. Methimazole (MMI) was selected as a reference compound, since metabolism of its imidazole thione moiety is linked with hematologic disorders and hepatotoxicity. An oxidative stress response occurred in all three species, marked by a decline in blood GSH levels by 24 h that progressed, and preceded hemolysis, which occurred at high MMI concentrations in the presence of a liver slice with rat (>= 1000 muM at 48 h) and human tissues (>= 1000 muM at 48 h, >= 750 muM at 72 h) but not dog. Human blood-only cultures exhibited a decline of GSH levels but minimal to no hemolysis. The up-regulation of liver genes for heme degradation (Hmox1 and Prdx1), iron cellular transport (Slc40a1), and GSH synthesis and utilization (mGST1 and Gclc) were early markers of the oxidative stress response. The up-regulation of the Kupffer cell lectin Lgals3 gene expression indicated a response to damaged red blood cells, and Hp (haptoglobin) up-regulation is indicative of increased hemoglobin uptake. Up-regulation of liver IL-6 and IL-8 gene expression suggested an activation of an inflammatory response by liver endothelial cells. In summary, MMI exposure led to an oxidative stress response in blood cells, and an up-regulation of liver genes involved with oxidative stress and heme homeostasis, which was clearly separate and preceded frank hemolysis.

  13. Simultaneous LC-MS/MS determination of JWH-210, RCS-4, ∆(9)-tetrahydrocannabinol, and their main metabolites in pig and human serum, whole blood, and urine for comparing pharmacokinetic data.

    PubMed

    Schaefer, Nadine; Kettner, Mattias; Laschke, Matthias W; Schlote, Julia; Peters, Benjamin; Bregel, Dietmar; Menger, Michael D; Maurer, Hans H; Ewald, Andreas H; Schmidt, Peter H

    2015-05-01

    A series of new synthetic cannabinoids (SC) has been consumed without any toxicological testing. For example, pharmacokinetic data have to be collected from forensic toxicological case work and/or animal studies. To develop a corresponding model for assessing such data, samples of controlled pig studies with two selected SC (JWH-210, RCS-4) and, as reference, ∆(9)-tetrahydrocannabinol (THC) should be analyzed as well as those of human cases. Therefore, a method for determination of JWH-210, RCS-4, THC, and their main metabolites in pig and human serum, whole blood, and urine samples is presented. Specimens were analyzed by liquid-chromatography tandem mass spectrometry and multiple-reaction monitoring with three transitions per compound. Full validation was carried out for the pig specimens and cross-validation for the human specimens concerning precision and bias. For the pig studies, the limits of detection were between 0.05 and 0.50 ng/mL in serum and whole blood and between 0.05 and 1.0 ng/mL in urine, the lower limits of quantification between 0.25 and 1.0 ng/mL in serum and 0.50 and 2.0 ng/mL in whole blood and urine, and the intra- and interday precision values lower than 15% and bias values within ±15%. The applicability was tested with samples taken from a pharmacokinetic pilot study with pigs following intravenous administration of a mixture of 200 μg/kg body mass dose each of JWH-210, RCS-4, and THC. The cross-validation data for human serum, whole blood, and urine showed that this approach should also be suitable for human specimens, e.g., of clinical or forensic cases. PMID:25772567

  14. Analysis of Human Serum and Whole Blood for Mineral Content by ICP-MS and ICP-OES: Development of a Mineralomics Method

    PubMed Central

    Harrington, James M.; Young, Daniel J.; Essader, Amal S.; Sumner, Susan J.; Levine, Keith E.

    2014-01-01

    Minerals are inorganic compounds that are essential to the support of a variety of biological functions. Understanding the range and variability of the content of these minerals in biological samples can provide insight into the relationships between mineral content and the health of individuals. In particular, abnormal mineral content may serve as an indicator of illness. The development of robust, reliable analytical methods for the determination of the mineral content of biological samples is essential to developing biological models for understanding the relationship between minerals and illnesses. This manuscript describes a method for the analysis of the mineral content of small volumes of serum and whole blood samples from healthy individuals. Interday and intraday precision for the mineral content of the blood (250 μl) and serum (250 μl) samples was measured for eight essential minerals, sodium (Na), calcium (Ca), magnesium (Mg), potassium (K), iron (Fe), zinc (Zn), copper (Cu), and selenium (Se) by plasma spectrometric methods and ranged from 0.635 – 10.1% relative standard deviation (RSD) for serum and 0.348 – 5.98% for whole blood. A comparison of the determined ranges for ten serum samples and six whole blood samples provided good agreement with literature reference ranges. The results demonstrate that the digestion and analysis methods can be used to reliably measure the content of these minerals, and potentially to add other minerals. PMID:24917052

  15. Analysis of human serum and whole blood for mineral content by ICP-MS and ICP-OES: development of a mineralomics method.

    PubMed

    Harrington, James M; Young, Daniel J; Essader, Amal S; Sumner, Susan J; Levine, Keith E

    2014-07-01

    Minerals are inorganic compounds that are essential to the support of a variety of biological functions. Understanding the range and variability of the content of these minerals in biological samples can provide insight into the relationships between mineral content and the health of individuals. In particular, abnormal mineral content may serve as an indicator of illness. The development of robust, reliable analytical methods for the determination of the mineral content of biological samples is essential to developing biological models for understanding the relationship between minerals and illnesses. This paper describes a method for the analysis of the mineral content of small volumes of serum and whole blood samples from healthy individuals. Interday and intraday precision for the mineral content of the blood (250 μL) and serum (250 μL) samples was measured for eight essential minerals--sodium (Na), calcium (Ca), magnesium (Mg), potassium (K), iron (Fe), zinc (Zn), copper (Cu), and selenium (Se)--by plasma spectrometric methods and ranged from 0.635 to 10.1% relative standard deviation (RSD) for serum and 0.348-5.98% for whole blood. A comparison of the determined ranges for ten serum samples and six whole blood samples provided good agreement with literature reference ranges. The results demonstrate that the digestion and analysis methods can be used to reliably measure the content of these minerals and potentially of other minerals.

  16. Melatonin synthesis in human colostrum mononuclear cells enhances dectin-1-mediated phagocytosis by mononuclear cells.

    PubMed

    Pires-Lapa, Marco A; Tamura, Eduardo K; Salustiano, Eugenia M A; Markus, Regina P

    2013-10-01

    Many cells in the organism besides pinealocytes, synthesize melatonin. Here, we evaluate both the mechanism of zymosan-induced melatonin synthesis and its autocrine effect in human colostral mononuclear cells. The synthesis of melatonin was induced by activation of the transcription factor nuclear factor kappa B (NF-κB), as either the blockade of the proteasome or the binding of NF-κB to DNA inhibits zymosan-induced melatonin synthesis. As observed in RAW 264.7 lineage cells, the dimer involved is RelA/c-Rel. Melatonin plays a direct role in mononuclear cell activity, increasing zymosan-induced phagocytosis by stimulating MT2 melatonin receptors and increasing the expression of dectin-1. This role was confirmed by the blockade of melatonin receptors using the competitive antagonist luzindole and the MT2 -selective partial agonist 4P-PDOT. In summary, we show that melatonin produced by immune-competent cells acts in an autocrine manner, enhancing the clearance of pathogens by increasing phagocyte efficiency. Given that these cells are present in human colostrum for 4 or 5 days after birth, this mechanism may be relevant for the protection of infant health.

  17. Highly sensitive analysis of methamphetamine and amphetamine in human whole blood using headspace solid-phase microextraction and gas chromatography-mass spectrometry.

    PubMed

    Okajima, K; Namera, A; Yashiki, M; Tsukue, I; Kojima, T

    2001-02-01

    A simple and highly sensitive method for analysis of derivatized methamphetamine (MA) and amphetamine (AM) in whole blood was developed using headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry electron impact ionization selected ion monitoring (GC-MS-EI-SIM). A whole blood sample, deuterated-MA (d(5)-MA), as an internal standard (IS), tri-n-propylamine and pentafluorobenzyl bromide were placed in a vial. The vial was heated and stirred at 90 degrees C for 30min. Then the extraction fiber of the SPME was exposed at 90 degrees C for 30min in the headspace of the vial while being stirred. The derivatives adsorbed on the fiber were desorbed by exposing the fiber in the injection port of a GC-MS. The calibration curves showed linearity in the range of 0.5-1000ng/g for both MA and AM. The time for analysis was about 80min per sample. In addition, this proposed method was applied to two autopsy cases where MA ingestion was suspected. In one case, MA and AM concentrations in the mixed left and right heart blood were 165 and 36.9ng/g, respectively. In the other case, MA and AM concentrations were 1.79 and 0.119 microg/g in the left heart blood, and 1.27 and 0.074 microg/g in the right heart blood, respectively.

  18. Microfluidic immunomagnetic cell separation from whole blood.

    PubMed

    Bhuvanendran Nair Gourikutty, Sajay; Chang, Chia-Pin; Puiu, Poenar Daniel

    2016-02-01

    Immunomagnetic-based separation has become a viable technique for the separation of cells and biomolecules. Here we report on the design and analysis of a simple and efficient microfluidic device for high throughput and high efficiency capture of cells tagged with magnetic particles. This is made possible by using a microfluidic chip integrated with customized arrays of permanent magnets capable of creating large magnetic field gradients, which determine the effective capturing of the tagged cells. This method is based on manipulating the cells which are under the influence of a combination of magnetic and fluid dynamic forces in a fluid under laminar flow through a microfluidic chip. A finite element analysis (FEA) model is developed to analyze the cell separation process and predict its behavior, which is validated subsequently by the experimental results. The magnetic field gradients created by various arrangements of magnetic arrays have been simulated using FEA and the influence of these field gradients on cell separation has been studied with the design of our microfluidic chip. The proof-of-concept for the proposed technique is demonstrated by capturing white blood cells (WBCs) from whole human blood. CD45-conjugated magnetic particles were added into whole blood samples to label WBCs and the mixture was flown through our microfluidic device to separate the labeled cells. After the separation process, the remaining WBCs in the elute were counted to determine the capture efficiency, and it was found that more than 99.9% WBCs have been successfully separated from whole blood. The proposed design can be used for positive selection as well as for negative enrichment of rare cells. PMID:26773879

  19. Determination of tumor cell procoagulant activity by Sonoclot analysis in whole blood.

    PubMed

    Amirkhosravi, A; Biggerstaff, J P; Warnes, G; Francis, D A; Francis, J L

    1996-12-01

    Coagulation activation in cancer may be due to expression of procoagulant activity (PCA) by tumor cells. Some PCA activate coagulation, while others influence platelet aggregation. Thus, clotting time assessments of PCA may not reflect the potential for hypercoagulability. We therefore studied the effect of various malignant and non-malignant cells on whole blood coagulation using the Sonoclot Analyzer. Five human (HT29 colon, J82 bladder, MCF-7 breast, BT-474 breast and A375 malignant melanoma) and three rodent (MC28, WEHI-164 and Neuro2a) cell lines were used. Rat thymocytes and peritoneal macrophages and human endotoxin-stimulated mononuclear cells and umbilical vein endothelial cells (HUVEC) were used as non-malignant controls. All tumor cells markedly shortened the recalcification time of citrated blood and the most potent (HT29 and J82) also increased clot rate (P < 0.01). The breast cancer cells MCF-7 and BT-474 expressed only weak PCA and did not affect clotting rate. None of the non-malignant cells significantly affected clotting time or rate in whole blood. J82 and HT29 cells grown in serum-rich media shortened the recalcification time of both normal and FVII-deficient plasmas. However, cells grown in serum-free conditions had significantly less PCA in FVII-deficient plasma. We conclude that the Sonoclot Analyzer is useful for determining cellular PCA; significant PCA is a feature of malignant cells and cells grown in medium containing serum supplements cannot be reliably evaluated for PCA.

  20. Evaluation of whole blood coagulation process by optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Xu, Xiangqun; Lin, Jia

    2010-11-01

    This study was to investigate the feasibility of using optical coherence tomography (OCT) to evaluate whole blood coagulation process. Attenuation coefficients and 1/e light penetration depth (D1/e) against time of human whole blood during in vitro clot formation under static were measured from the OCT profiles of reflectance vs depth. The results obtained clearly showed that the optical parameters are able to identify three stages during the in vitro blood clotting process. It is concluded that D1/e measured by OCT is a potential parameter to quantify and follow the liquid-gel transition of blood during clotting.

  1. High level of IFN-γ released from whole blood of human tuberculosis infections following stimulation with Rv2073c of Mycobacterium tuberculosis.

    PubMed

    Tan, Kun; Zhang, Jingyan; Teng, Xindong; Liang, Jinping; Wang, Xiaochun; Yuan, Xuefeng; Tian, Maopeng; Fan, Xionglin

    2015-07-01

    More efficacious and specific biomarkers are urgently needed for better control of tuberculosis (TB), the second leading infectious cause of mortality worldwide. The region of difference 9 (RD9) presents the genome of the causative pathogen Mycobacterium tuberculosis rather than other species of the genus Mycobacterium, which might be promising targets for specific diagnosis, vaccine development and pathogenesis. In this study, two proteins Rv2073c and Rv2074, encoded by the RD9 were expressed and purified from Escherichia coli system. Following stimulation with both proteins, the levels of IFN-γ secreted by T cells from a total of 49 whole blood samples obtained from clinically diagnosed active TB patients, patients with latent TB infections (LTBIs), and healthy donors, were compared with those of the incubation with recombinant fusion protein of CFP21 and MPT64 (rCM). Our results demonstrated that only Rv2073c could induce a higher level of IFN-γ in TB infections than healthy controls and there was a positive correlation between Rv2073c- and rCM-specific IFN-γ levels in TB infections and healthy donors, respectively. These findings indicate that Rv2073c might be a promising antigen for specific diagnostic reagents and vaccine candidates of TB.

  2. Effect of erythrocytes and prostacyclin production in the effect of fructose and sorbitol on platelet activation in human whole blood in vitro.

    PubMed

    De la Cruz, J P; Maximo, M A; Blanco, E; Moreno, A; Sánchez de la Cuesta, F

    1997-06-15

    We analyzed the in vitro effects of sorbitol and fructose on platelet function. Sorbitol and fructose increased platelet aggregation induced with adenosine diphosphate (ADP) or collagen in whole blood, but had no effect in platelet-rich plasma. The concentration that increased basal aggregation by 50% with ADP as the inducer was 12.89 +/- 1.55 mmol/L for fructose, and 18.99 +/- 2.01 mmol/L for sorbitol. When collagen was the inducer, these concentrations were 15.02 +/- 0.98 mmol/L for fructose, and 12.94 +/- 1.57 mmol/L for sorbitol. Both sugars increased, in a concentration-dependent way, the proaggregatory effect of erythrocytes, and erythrocyte uptake of adenosine. Time to uptake of 50% adenosine was 2.1 +/- 0.3 min in control samples, 0.14 +/- 0.01 min in the presence of fructose, and 0.23 +/- 0.03 min with sorbitol. Both sugars reduced vascular prostacyclin synthesis, with 50% inhibitory concentrations of 26.48 +/- 1.97 mmol/L for fructose, and 39.53 +/- 2.81 mmol/L for sorbitol. Both sugars also increased arterial lipid peroxidation by 30% (sorbitol) and 23% (fructose). We conclude that these two sugars enhance platelet function and disrupt the thromboxane/prostacyclin ratio.

  3. Screening of drugs of abuse and toxic compounds in human whole blood using online solid-phase extraction and high-performance liquid chromatography with time-of-flight mass spectrometry.

    PubMed

    Teng, Xiaomei; Liang, Chen; Wang, Rong; Sun, Tao; Rao, Yulan; Ni, Chunfang; Zeng, Libo; Xiong, Lingjuan; Li, Yuan; Zhang, Yurong

    2015-01-01

    A novel method for the screening of 151 drugs of abuse and toxic compounds in human whole blood has been developed and validated by online solid-phase extraction with liquid chromatography coupled to time-of-flight mass spectrometry. Analytes were extracted and separated by using a fully automated online solid-phase extraction liquid chromatography system with total chromatographic run time of 26 min. Time-of-flight mass spectrometry screening of 151 drugs of abuse and toxic compounds was performed in a full-scan (m/z 50-800) mode using an MS(E) acquisition of molecular ions and fragment ions data at two collision energies (one was 6 eV and another one was in the range of 5-45 eV). The compounds were identified based on retention times and exact mass of molecular ions and fragment ions. The limit of detection ranged from 1 to 100 ng/mL and the recovery of the method ranged from 6.3 to 163.5%. This method is proved to be a valuable screening method allowing fast and specific identification of drugs in human whole blood.

  4. Parnaparin, a low-molecular-weight heparin, prevents P-selectin-dependent formation of platelet-leukocyte aggregates in human whole blood.

    PubMed

    Maugeri, Norma; Di Fabio, Giovannina; Barbanti, Miriam; de Gaetano, Giovanni; Donati, Maria Benedetta; Cerletti, Chiara

    2007-06-01

    Parnaparin, a low-molecular-weight heparin (LMWH), prevents platelet activation and interaction with polymorphonuclear leukocyte (PMN) in a washed cell system. The in-vitro effect of parnaparin was studied here on platelet-PMN aggregates formed with more physiologic approaches in whole blood, in parallel with unfractionated heparin and enoxaparin, another LMWH. Citrated blood from healthy subjects was stimulated: i) from passage through the "Platelet Function Analyzer" (PFA-100), a device that exposes blood to standardized high shear flow through collagen/ADP cartridges; ii) by collagen and ADP (2 and 50 mug/ml, respectively) added in combination under stirring in an aggregometer cuvette; iii) with recombinant Tissue Factor, to generate thrombin concentrations able to activate platelets without inducing blood clotting, or iv) the Thrombin Receptor Activating Peptide-6 (TRAP-6). Platelet P-selectin and platelet-PMN aggregates were measured by flow cytometry upon stimulation of blood. Fibrinogen binding to platelets and markers of PMN activation were also detected. Platelet P-selectin expression and platelet-PMN aggregate formation were induced in all four activation conditions tested. Parnaparin prevented in a concentration-dependent manner (0.3-0.8 IUaXa/ml) the expression of P-selectin and the formation of mixed aggregates, while the two reference heparin preparations had a much weaker effect. Platelet fibrinogen binding and PMN activation markers (fibrinogen binding, CD11b and CD40) were also prevented by parnaparin. These data extend in more physiological systems of platelet activation, the anti-inflammatory profile of parnaparin, previously reported in washed cells. The greater effect of parnaparin, as compared to the reference heparins, could be due to chemico-physical differences possibly unrelated to their anticoagulant effect. PMID:17549299

  5. Quantitative, single-step dual measurement of hemoglobin A1c and total hemoglobin in human whole blood using a gold sandwich immunochromatographic assay for personalized medicine.

    PubMed

    Ang, Shu Hwang; Rambeli, Musalman; Thevarajah, T Malathi; Alias, Yatimah Binti; Khor, Sook Mei

    2016-04-15

    We describe a gold nanoparticle-based sandwich immunoassay for the dual detection and measurement of hemoglobin A1c (HbA1c) and total hemoglobin in the whole blood (without pretreatment) in a single step for personalized medicine. The optimized antibody-functionalized gold nanoparticles immunoreact simultaneously with HbA1c and total hemoglobin to form a sandwich at distinctive test lines to transduce visible signals. The applicability of this method as a personal management tool was demonstrated by establishing a calibration curve to relate % HbA1c, a useful value for type 2 diabetes management, to the signal ratio of captured HbA1c to all other forms of hemoglobin. The platform showed excellent selectivity (100%) toward HbA1c at distinctive test lines when challenged with HbA0, glycated HbA0 and HbA2. The reproducibility of the measurement was good (6.02%) owing to the dual measurement of HbA1c and total hemoglobin. A blood sample stability test revealed that the quantitative measurement of % HbA1c was consistent and no false-positive results were detected. Also, this method distinguished the blood sample with elevated HbF from the normal samples and the variants. The findings of this study highlight the potential of a lateral flow immunosensor as a simple, inexpensive, consistent, and convenient strategy for the dual measurement of HbA1c and total Hb to provide useful % HbA1c values for better on-site diabetes care.

  6. Experimental hypercalcaemia and whole blood clotting

    PubMed Central

    Hilgard, P.

    1973-01-01

    Experimental hypercalcaemia was induced in rats by (1) transplantation of the solid Walker 256 tumour, and (2) intraperitoneal injections of calcium gluconate. Whole blood clotting was studied by means of thromboelastography and whole blood clotting times in polystyrene and glass test tubes. At serum calcium levels between 10·3 and 11·5 m-equiv/l a slight delay in clot formation was found which was reversible by the addition of EDTA to whole blood. Acute, calcium-gluconate-induced hypercalcaemia, however, leads to a significant shortening of the clotting time in the polystyrene tube and to a lesser degree in the glass tube. Maximal factor XII activation in vitro with ellagic acid levels the difference of clotting times again. From these experiments it is concluded that acute hypercalcaemia induces a hypercoagulable state, possibly by partial contact activation, and thus may favour thrombus formation in vivo. PMID:4200324

  7. Whole blood filterability in elderly obese women.

    PubMed

    Gelmini, G; Butturini, L; Cucinotta, D; Delsignore, R; Coiro, V

    1987-01-01

    In order to establish whether obesity alters whole blood filterability, the corrected whole blood filtration (VRBC) was measured in 54 elderly obese women (mean age +/- SE = 67 +/- 2 years) without (n = 15) or with associated cardiovascular risk factors such as impaired glucose tolerance (IGT) (n = 11), non-insulin dependent diabetes mellitus (NIDDM) (n = 14) or hypertension (n = 14). Twenty-two age matched women with normal body weight participated as controls. VRBC values were similar in normal controls and obese women with normal glucose tolerance (NGT), whereas they were significantly lower in obese subjects with hypertension, NIDDM or IGT. When subjects with normal and impaired glucose tolerance were combined, a significant negative correlation was found between glucose incretory areas during OGTT and VRBC values. These data demonstrate that obesity per se does not alter whole blood filterability; furthermore, our results indicate that this modification is a precocious and sensitive index of altered glucose metabolism.

  8. Differences in iron concentration in whole blood of animal models using NAA

    NASA Astrophysics Data System (ADS)

    Bahovschi, V.; Zamboni, C. B.; Lopes Silva, L. F. F.; Metairon, S.; Medeiros, I. M. M. A.

    2015-07-01

    In this study Neutron Activation Analysis technique (NAA) was applied to determine Fe concentration in whole blood samples of several animal models such as: mice (Mus musculus), Golden Hamster (Mesocricetus auratus), Wistar rats, Albinic Rabbits of New Zealand, Golden Retriever dogs and Crioulabreed horses. These results were compared with human whole blood estimation to check their similarities.

  9. Piperine inhibits cytokine production by human peripheral blood mononuclear cells.

    PubMed

    Chuchawankul, S; Khorana, N; Poovorawan, Y

    2012-01-01

    Piperine, an amide isolated from Piper species (Piperaceae), has been reported to exhibit central nervous system depression, anti-pyretic and anti-inflammatory activity. Immunomodulatory and anti-tumor activity of piperine has been demonstrated in mouse carcinomas. However, there is little information available concerning the effect of piperine on humans. We evaluated the immunopharmacological activity of this compound in human immune cells. Human peripheral blood mononuclear cells (PBMCs) were exposed to piperine, and cell proliferation was determined by the MTS assay. Piperine significantly inhibited phytohemagglutinin-stimulated human PBMC proliferation after exposure for 72 h. This compound inhibited PBMC activity, with an IC(50) of 100.73 ± 11.16 μg/mL. Production of interleukin-2 (IL-2) and interferon-γ (IFN-γ) was measured using an ELISA assay and RT-PCR. Piperine inhibited IL-2 and IFN-γ production in the PBMCs. RT-PCR data indicated that IL-2 and IFN-γ mRNA expression in PBMCs is suppressed by piperine. This compound significantly inhibited the production of these two cytokines by activated PBMCs in a dose-dependent manner. In conclusion, piperine appears to have potential as an immunomodulatory agent for immune system suppression. PMID:22535397

  10. A single meal containing raw, crushed garlic influences expression of immunity- and cancer-related genes in whole blood of humans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Preclinical and epidemiological studies suggest that garlic intake is inversely associated with the progression of cancer and cardiovascular disease. To probe mechanisms of garlic action in humans, we conducted a randomized crossover feeding trial in which 17 volunteers consumed a garlic-containing...

  11. Determination of tumor cell procoagulant activity by Sonoclot analysis in whole blood.

    PubMed

    Amirkhosravi, A; Biggerstaff, J P; Warnes, G; Francis, D A; Francis, J L

    1996-12-01

    Coagulation activation in cancer may be due to expression of procoagulant activity (PCA) by tumor cells. Some PCA activate coagulation, while others influence platelet aggregation. Thus, clotting time assessments of PCA may not reflect the potential for hypercoagulability. We therefore studied the effect of various malignant and non-malignant cells on whole blood coagulation using the Sonoclot Analyzer. Five human (HT29 colon, J82 bladder, MCF-7 breast, BT-474 breast and A375 malignant melanoma) and three rodent (MC28, WEHI-164 and Neuro2a) cell lines were used. Rat thymocytes and peritoneal macrophages and human endotoxin-stimulated mononuclear cells and umbilical vein endothelial cells (HUVEC) were used as non-malignant controls. All tumor cells markedly shortened the recalcification time of citrated blood and the most potent (HT29 and J82) also increased clot rate (P < 0.01). The breast cancer cells MCF-7 and BT-474 expressed only weak PCA and did not affect clotting rate. None of the non-malignant cells significantly affected clotting time or rate in whole blood. J82 and HT29 cells grown in serum-rich media shortened the recalcification time of both normal and FVII-deficient plasmas. However, cells grown in serum-free conditions had significantly less PCA in FVII-deficient plasma. We conclude that the Sonoclot Analyzer is useful for determining cellular PCA; significant PCA is a feature of malignant cells and cells grown in medium containing serum supplements cannot be reliably evaluated for PCA. PMID:8948059

  12. Simultaneous congener-specific determination of selected organochlorine compounds and nitro musks in human whole blood samples by solid-phase extraction and capillary gas chromatography with electron capture detection.

    PubMed

    Bauer, Maret; Hilpert, Felix; Arnold, Norbert; Pfisterer, Jacobus; Jonat, Walter; Kruse, Hermann

    2005-03-01

    Chlororganic compounds like pesticides or polychlorinated biphenyls (PCB) and nitro musks are environmental contaminants, which remain public health concerns because of their persistence in humans and their toxicological properties. Many of these substances are associated with endocrine dysfunction or with carcinogenicity. Therefore, a simple method using solid-phase extraction followed by capillary gas chromatography with electron capture detection for the simultaneous determination of both organochlorines and nitro musks in human whole blood samples has been developed. Recovery rates of 13 PCB congeners and of 7 pesticides ranged from 67.5% to 100.4% and from 81.1% to 110.5%, respectively. Recoveries of the 5 nitro musks were consistent and ranged from 90.2% to 98.8%. The accuracy for organochlorines and nitro musks varied from 6.3% to 8.6%. Method detection limits ranged from 0.02 microg/L to 0.11 microg/L for the organochlorines and from 0.04 microg/L to 0.08 microg/L for the nitro musks. The method has a high sensitivity with a low detection limit even in slightly contaminated human blood samples. The time and technical effort is small, so the method is feasible for epidemiological studies with regard to the impact of organochlorines and nitro musks on certain diseases.

  13. Sample preparation on polymeric solid phase extraction sorbents for liquid chromatographic-tandem mass spectrometric analysis of human whole blood--a study on a number of beta-agonists and beta-antagonists.

    PubMed

    Josefsson, Martin; Sabanovic, Alma

    2006-07-01

    sample preparation was evaluated by ESI ion-suppression studies by post column infusion of the target analyte. An ethanol zinc sulphate aq mixture was found to be more effective than acetonitrile, methanol or ethanol for PP of human whole blood samples. Beside suppression by salts in the front peak, only limited suppression from other artefacts such as more lipophilic compounds was found late in the chromatograms. Some tendency though to concentrate more lipophilic artefacts on the Oasis sorbents was seen. These findings show that the Oasis MCX sorbent is well suited for sample preparation of beta-agonists and beta-antagonists from human whole blood if the objective is to cover a great number of the analytes in the same assay. PMID:16600255

  14. Mechanism and role of MCP-1 upregulation upon chikungunya virus infection in human peripheral blood mononuclear cells

    PubMed Central

    Ruiz Silva, Mariana; van der Ende-Metselaar, Heidi; Mulder, H. Lie; Smit, Jolanda M.; Rodenhuis-Zybert, Izabela A.

    2016-01-01

    Monocyte chemoattractant protein-1 (MCP-1/CCL2)-mediated migration of monocytes is essential for immunological surveillance of tissues. During chikungunya virus (CHIKV) infection however, excessive production of MCP-1 has been linked to disease pathogenesis. High MCP-1 serum levels are detected during the viremic phase of CHIKV infection and correlate with the virus titre. In vitro CHIKV infection was also shown to stimulate MCP-1 production in whole blood; yet the role and the mechanism of MCP-1 production upon infection of human peripheral blood mononuclear cells remain unknown. Here we found that active CHIKV infection stimulated production of MCP-1 in monocytes. Importantly however, we found that communication with other leukocytes is crucial to yield MCP-1 by monocytes upon CHIKV infection. Indeed, blocking interferon-α/β receptor or the JAK1/JAK2 signalling downstream of the receptor abolished CHIKV-mediated MCP-1 production. Additionally, we show that despite the apparent correlation between IFN type I, CHIKV replication and MCP-1, modulating the levels of the chemokine did not influence CHIKV infection. In summary, our data disclose the complexity of MCP-1 regulation upon CHIKV infection and point to a crucial role of IFNβ in the chemokine secretion. We propose that balance between these soluble factors is imperative for an appropriate host response to CHIKV infection. PMID:27558873

  15. Mechanism and role of MCP-1 upregulation upon chikungunya virus infection in human peripheral blood mononuclear cells.

    PubMed

    Ruiz Silva, Mariana; van der Ende-Metselaar, Heidi; Mulder, H Lie; Smit, Jolanda M; Rodenhuis-Zybert, Izabela A

    2016-01-01

    Monocyte chemoattractant protein-1 (MCP-1/CCL2)-mediated migration of monocytes is essential for immunological surveillance of tissues. During chikungunya virus (CHIKV) infection however, excessive production of MCP-1 has been linked to disease pathogenesis. High MCP-1 serum levels are detected during the viremic phase of CHIKV infection and correlate with the virus titre. In vitro CHIKV infection was also shown to stimulate MCP-1 production in whole blood; yet the role and the mechanism of MCP-1 production upon infection of human peripheral blood mononuclear cells remain unknown. Here we found that active CHIKV infection stimulated production of MCP-1 in monocytes. Importantly however, we found that communication with other leukocytes is crucial to yield MCP-1 by monocytes upon CHIKV infection. Indeed, blocking interferon-α/β receptor or the JAK1/JAK2 signalling downstream of the receptor abolished CHIKV-mediated MCP-1 production. Additionally, we show that despite the apparent correlation between IFN type I, CHIKV replication and MCP-1, modulating the levels of the chemokine did not influence CHIKV infection. In summary, our data disclose the complexity of MCP-1 regulation upon CHIKV infection and point to a crucial role of IFNβ in the chemokine secretion. We propose that balance between these soluble factors is imperative for an appropriate host response to CHIKV infection. PMID:27558873

  16. Separation of large quantities of mononuclear cells from human blood using a blood processor.

    PubMed

    Beaujean, F; Gourdin, M F; Farcet, J P; Le Forestier, C; Bouguet, J; Reyes, F; Duedari, N

    1985-01-01

    A blood processor (IBM 2991) was used to separate lymphocytes from large volumes of blood. The procedure included the centrifugation of 200 ml whole blood on a density gradient. The results of this procedure were compared with those obtained with a manual procedure. Mononuclear cell (MNC) viability was preserved well in the two methods. But with the processor, recovery of MNC was better (63.5 +/- 2.5%) than with manual separation (26.5 +/- 4.1%). Monoclonal antibodies were used to identify the various cell subsets in the MNC fractions. No particular cell selection was observed when MNC fractions were obtained by the separator. In conclusion, the use of a cell separator provided an efficient technique for rapid isolation of large quantities of lymphocytes. PMID:3872489

  17. Identification of tuberculosis-associated proteins in whole blood supernatant

    PubMed Central

    2011-01-01

    Background Biological parameters are useful tools for understanding and monitoring complicated disease processes. In this study, we attempted to identify proteins associated with active pulmonary tuberculosis (TB) using a proteomic approach. Methods To assess TB-associated changes in the composition of human proteins, whole blood supernatants were collected from patients with active TB and healthy control subjects. Two-dimensional difference gel electrophoresis (2D-DIGE) was performed to analyze proteins with high molecular weights (approximately >20 kDa). Baseline protein levels were initially compared between patients with active TB and control subjects. Possible changes of protein patterns in active TB were also compared ex vivo between whole blood samples incubated with Mycobacterium tuberculosis (Mtb)-specific antigens (stimulated condition) and under unstimulated conditions. Immunoblot and enzyme-linked immunosorbent assays (ELISA) were performed to confirm differences in identified proteins. Results Under the baseline condition, we found that the levels of retinol-binding protein 4 (RBP4), fetuin-A (also called α-HS-glycoprotein), and vitamin D-binding protein differed between patients with active TB and control subjects on 2D gels. Immunoblotting results confirmed differential expression of RBP4 and fetuin-A. ELISA results further confirmed significantly lower levels of these two proteins in samples from patients with active TB than in control subjects (P < 0.0001). Mtb-specific antigen stimulation ex vivo altered clusterin expression in whole blood samples collected from patients with active TB. Conclusions We identified TB-associated proteins in whole blood supernatants. The dynamics of protein expression during disease progression may improve our understanding of the pathogenesis of TB. PMID:21418657

  18. Improved Whole-Blood-Staining Device

    NASA Technical Reports Server (NTRS)

    Sams, Clarence F.; Crucian, Brian; Paul, Bonnie; Melton, Shannon; Guess, Terry

    2012-01-01

    Dramatic improvements have been made in NASA s Whole Blood Staining Device (WBSD) since it was last described in "Whole-Blood-Staining Device," NASA Tech Briefs, Vol. 23, No. 10 (October 1999), page 64. The new system has a longer shelf life, a simpler and more effective operational procedure, improved interface with instrumentation, and shorter processing time. More specifically, the improvements have targeted bag and locking clip materials, sampling ports, and air pocket prevention. The WBSD stains whole blood collected during spaceflight for subsequent flow cytometric analysis. In short, the main device stains white blood cells by use of monoclonal antibodies conjugated to various fluorochromes, followed by lysing and fixing of the cells by use of a commercial reagent that has been diluted according to NASA safety standards. This system is compact, robust, and does not require electric power, precise mixing, or precise incubation times. Figure 1 depicts the present improved version for staining applications, which is a poly(tetrafluoroethylene) bag with a Luer-lock port and plastic locking clips. An InterLink (or equivalent) intravenous- injection port screws into the Luer-lock port. The inflatable/collapsible nature of the bag facilitates loading and helps to minimize the amount of air trapped in the fully loaded bag. Some additional uses have been identified for the device beyond whole blood staining. The WBSD has been configured for functional assays that require culture of live cells by housing sterile culture media, mitogens, and fixatives prior to use [Figure 2(a)]. Simple injection of whole blood allows cell-stimulation culture to be performed in reduced gravity conditions, and product stabilization prior to storage, while protecting astronauts from liquid biohazardous materials. Also, the improved WBSD has reconstituted powdered injectable antibiotics by mixing them with diluent liquids [Figure 2(b)]. Although such mixing can readily be performed on

  19. Bioenergetic analysis of human peripheral blood mononuclear cells.

    PubMed

    Jones, N; Piasecka, J; Bryant, A H; Jones, R H; Skibinski, D O F; Francis, Nigel J; Thornton, C A

    2015-10-01

    Leucocytes respond rapidly to pathogenic and other insults, with responses ranging from cytokine production to migration and phagocytosis. These are bioenergetically expensive, and increased glycolytic flux provides adenosine triphosphate (ATP) rapidly to support these essential functions. However, much of this work is from animal studies. To understand more clearly the relative role of glycolysis and oxidative phosphorylation in human leucocytes, especially their utility in a translational research setting, we undertook a study of human peripheral blood mononuclear cells (MNCs) bioenergetics. Glycolysis was essential during lipopolysaccharide (LPS)-mediated interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF)-α production, as 2-deoxy-D-glucose decreased significantly the output of all three cytokines. After optimizing cell numbers and the concentrations of all activators and inhibitors, oxidative phosphorylation and glycolysis profiles of fresh and cryopreserved/resuscitated MNCs were determined to explore the utility of MNCs for determining the bioenergetics health profile in multiple clinical settings. While the LPS-induced cytokine response did not differ significantly between fresh and resuscitated cells from the same donors, cryopreservation/resuscitation significantly affected mainly some measures of oxidative phosphorylation, but also glycolysis. Bioenergetics analysis of human MNCs provides a quick, effective means to measure the bioenergetics health index of many individuals, but cryopreserved cells are not suitable for such an analysis. The translational utility of this approach was tested by comparing MNCs of pregnant and non-pregnant women to reveal increased bioenergetics health index with pregnancy but significantly reduced basal glycolysis and glycolytic capacity. More detailed analysis of discrete leucocyte populations would be required to understand the relative roles of glycolysis and oxidative phosphorylation during inflammation and

  20. Bioenergetic analysis of human peripheral blood mononuclear cells

    PubMed Central

    Jones, N; Piasecka, J; Bryant, A H; Jones, R H; Skibinski, D O F; Francis, Nigel J; Thornton, C A

    2015-01-01

    Leucocytes respond rapidly to pathogenic and other insults, with responses ranging from cytokine production to migration and phagocytosis. These are bioenergetically expensive, and increased glycolytic flux provides adenosine triphosphate (ATP) rapidly to support these essential functions. However, much of this work is from animal studies. To understand more clearly the relative role of glycolysis and oxidative phosphorylation in human leucocytes, especially their utility in a translational research setting, we undertook a study of human peripheral blood mononuclear cells (MNCs) bioenergetics. Glycolysis was essential during lipopolysaccharide (LPS)-mediated interleukin (IL)−1β, IL-6 and tumour necrosis factor (TNF)-α production, as 2-deoxy-D-glucose decreased significantly the output of all three cytokines. After optimizing cell numbers and the concentrations of all activators and inhibitors, oxidative phosphorylation and glycolysis profiles of fresh and cryopreserved/resuscitated MNCs were determined to explore the utility of MNCs for determining the bioenergetics health profile in multiple clinical settings. While the LPS-induced cytokine response did not differ significantly between fresh and resuscitated cells from the same donors, cryopreservation/resuscitation significantly affected mainly some measures of oxidative phosphorylation, but also glycolysis. Bioenergetics analysis of human MNCs provides a quick, effective means to measure the bioenergetics health index of many individuals, but cryopreserved cells are not suitable for such an analysis. The translational utility of this approach was tested by comparing MNCs of pregnant and non-pregnant women to reveal increased bioenergetics health index with pregnancy but significantly reduced basal glycolysis and glycolytic capacity. More detailed analysis of discrete leucocyte populations would be required to understand the relative roles of glycolysis and oxidative phosphorylation during inflammation and

  1. Determination of ecgonine and seven other cocaine metabolites in human urine and whole blood by ultra-high-pressure liquid chromatography-quadrupole time-of-flight mass spectrometry.

    PubMed

    Xiong, Lingjuan; Wang, Rong; Liang, Chen; Cao, Fangqi; Rao, Yulan; Wang, Xin; Zeng, Libo; Ni, Chunfang; Ye, Haiying; Zhang, Yurong

    2013-12-01

    Ecgonine is suggested to be a promising marker of cocaine (COC) ingestion. A combined mass spectrometry (MS) and tandem MS (MS/MS) method was developed to simultaneously determine ecgonine and seven other metabolites of cocaine in human urine and whole blood with ultra-high-pressure liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. The compounds were extracted from as little as 100 μL of sample by solid-phase extraction with a 96-well μElution solid-phase extraction plate. The protonated molecules or fragment ions at accurate mass acquired in MS mode were used to quantify specific analytes, following by dedicated MS/MS identification. The assay was linear in the range from 5 to 50-100 ng/mL for urine samples, except for ecgonine methyl ester (10-200 ng/mL) and ecgonine (40-400 ng/mL), and was linear from 1-2 to 50 ng/mL for whole blood samples, except for ecgonine methyl ester (20-1,000 ng/mL) and ecgonine (40-2,000 ng/mL). The correlation coefficients were all greater than 0.99. The limits of detection ranged from 0.2 to 16 ng/mL, and the lower limits of quantification ranged from 1 to 40 ng/mL. The repeatability and intermediate precision were 18.1% or less. The accuracy was in the range from 80.0 to 122.9%, process efficiencies were in the range from 8.6 to 177.4%, matrix effects were in the range from 28.7 to 171.0%, and extraction recoveries were in the range from 41.0 to 114.3%, except for ecgonine (12.8% and 9.3% at low and high concentrations, respectively). This method was highly sensitive in comparison with previously published methods. The validated method was successfully applied to the analysis of real samples derived from forensic cases, and the results verified that, on the basis of data from four positive samples, ecgonine is a promising marker of cocaine ingestion.

  2. Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells Using Sendai Virus.

    PubMed

    Soares, Filipa A C; Pedersen, Roger A; Vallier, Ludovic

    2016-01-01

    This protocol describes the efficient isolation of peripheral blood mononuclear cells from circulating blood via density gradient centrifugation and subsequent generation of integration-free human induced pluripotent stem cells. Peripheral blood mononuclear cells are cultured for 9 days to allow expansion of the erythroblast population. The erythroblasts are then used to derive human induced pluripotent stem cells using Sendai viral vectors, each expressing one of the four reprogramming factors Oct4, Sox2, Klf4, and c-Myc.

  3. 21 CFR 864.7500 - Whole blood hemoglobin assays.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Whole blood hemoglobin assays. 864.7500 Section... blood hemoglobin assays. (a) Identification. A whole blood hemoglobin assay is a device consisting or... hemoglobin content of whole blood for the detection of anemia. This generic device category does not...

  4. 21 CFR 864.7500 - Whole blood hemoglobin assays.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Whole blood hemoglobin assays. 864.7500 Section... blood hemoglobin assays. (a) Identification. A whole blood hemoglobin assay is a device consisting or... hemoglobin content of whole blood for the detection of anemia. This generic device category does not...

  5. 21 CFR 864.7500 - Whole blood hemoglobin assays.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Whole blood hemoglobin assays. 864.7500 Section... blood hemoglobin assays. (a) Identification. A whole blood hemoglobin assay is a device consisting or... hemoglobin content of whole blood for the detection of anemia. This generic device category does not...

  6. 21 CFR 864.7500 - Whole blood hemoglobin assays.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Whole blood hemoglobin assays. 864.7500 Section... blood hemoglobin assays. (a) Identification. A whole blood hemoglobin assay is a device consisting or... hemoglobin content of whole blood for the detection of anemia. This generic device category does not...

  7. 21 CFR 864.7500 - Whole blood hemoglobin assays.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Whole blood hemoglobin assays. 864.7500 Section... blood hemoglobin assays. (a) Identification. A whole blood hemoglobin assay is a device consisting or... hemoglobin content of whole blood for the detection of anemia. This generic device category does not...

  8. Platelet lysate from whole blood-derived pooled platelet concentrates and apheresis-derived platelet concentrates for the isolation and expansion of human bone marrow mesenchymal stromal cells: production process, content and identification of active components

    PubMed Central

    Fekete, Natalie; Gadelorge, Mélanie; Fürst, Daniel; Maurer, Caroline; Dausend, Julia; Fleury-Cappellesso, Sandrine; Mailänder, Volker; Lotfi, Ramin; Ignatius, Anita; Sensebé, Luc; Bourin, Philippe; Schrezenmeier, Hubert; Rojewski, Markus Thomas

    2012-01-01

    Background aims The clinical use of human mesenchymal stromal cells (MSC) requires ex vivo expansion in media containing supplements such as fetal bovine serum or, alternatively, human platelet lysate (PL). Methods Platelet concentrates were frozen, quarantine stored, thawed and sterile filtered to obtain PL. PL content and its effect on fibroblast-colony-forming unit (CFU-F) formation, MSC proliferation and large-scale expansion were studied. Results PL contained high levels of basic fibroblast growth factor (bFGF), soluble CD40L (sCD40L), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), platelet-derived growth factor AA (PDGF-AA), platelet-derived growth factor AB/BB (PDGF-AB/BB), chemokine (C-C) ligand 5 (CCL5; RANTES) transforming growth factor-β1 (TGF-β1) and chemokine (C-X-C) ligand 1/2/3 (GRO), with low batch-to-batch variability, and most were stable for up to 14 days. Inhibition of PDGF-BB and bFGF decreased MSC proliferation by about 20% and 50%, respectively. The strongest inhibition (about 75%) was observed with a combination of anti-bFGF + anti-PDGF-BB and anti-bFGF + anti-TGF-β1 + anti-PDGF-BB. Interestingly, various combinations of recombinant PDGF-BB, bFGF and TGF-β1 were not sufficient to promote cell proliferation. PL from whole blood-derived pooled platelet concentrates and apheresis platelet concentrates did not differ significantly in their growth-promoting activity on MSC. Conclusions PL enhances MSC proliferation and can be regarded as a safe tool for MSC expansion for clinical purposes. \\in particular, PDGF-BB and bFGF are essential components for the growth-promoting effect of PL, but are not sufficient for MSC proliferation. PMID:22296115

  9. Human and Mouse Mononuclear Phagocyte Networks: A Tale of Two Species?

    PubMed Central

    Reynolds, Gary; Haniffa, Muzlifah

    2015-01-01

    Dendritic cells (DCs), monocytes, and macrophages are a heterogeneous population of mononuclear phagocytes that are involved in antigen processing and presentation to initiate and regulate immune responses to pathogens, vaccines, tumor, and tolerance to self. In addition to their afferent sentinel function, DCs and macrophages are also critical as effectors and coordinators of inflammation and homeostasis in peripheral tissues. Harnessing DCs and macrophages for therapeutic purposes has major implications for infectious disease, vaccination, transplantation, tolerance induction, inflammation, and cancer immunotherapy. There has been a paradigm shift in our understanding of the developmental origin and function of the cellular constituents of the mononuclear phagocyte system. Significant progress has been made in tandem in both human and mouse mononuclear phagocyte biology. This progress has been accelerated by comparative biology analysis between mouse and human, which has proved to be an exceptionally fruitful strategy to harmonize findings across species. Such analyses have provided unexpected insights and facilitated productive reciprocal and iterative processes to inform our understanding of human and mouse mononuclear phagocytes. In this review, we discuss the strategies, power, and utility of comparative biology approaches to integrate recent advances in human and mouse mononuclear phagocyte biology and its potential to drive forward clinical translation of this knowledge. We also present a functional framework on the parallel organization of human and mouse mononuclear phagocyte networks. PMID:26124761

  10. Whole Blood RNA as a Source of Transcript-Based Nutrition- and Metabolic Health-Related Biomarkers

    PubMed Central

    Petrov, Petar D.; Bonet, M. Luisa; Reynés, Bárbara; Oliver, Paula; Palou, Andreu; Ribot, Joan

    2016-01-01

    Blood cells are receiving an increasing attention as an easily accessible source of transcript-based biomarkers. We studied the feasibility of using mouse whole blood RNA in this context. Several paradigms were studied: (i) metabolism-related transcripts known to be affected in rat tissues and peripheral blood mononuclear cells (PBMC) by fasting and upon the development of high fat diet (HFD)-induced overweight were assessed in whole blood RNA of fasted rats and mice and of HFD-fed mice; (ii) retinoic acid (RA)-responsive genes in tissues were assessed in whole blood RNA of control and RA-treated mice; (iii) lipid metabolism-related transcripts previously identified in PBMC as potential biomarkers of metabolic health in a rat model were assessed in whole blood in an independent model, namely retinoblastoma haploinsufficient (Rb+/-) mice. Blood was collected and stored in RNAlater® at -80°C until analysis of selected transcripts by real-time RT-PCR. Comparable changes with fasting were detected in the expression of lipid metabolism-related genes when RNA from either PBMC or whole blood of rats or mice was used. HFD-induced excess body weight and fat mass associated with expected changes in the expression of metabolism-related genes in whole blood of mice. Changes in gene expression in whole blood of RA-treated mice reproduced known transcriptional actions of RA in hepatocytes and adipocytes. Reduced expression of Fasn, Lrp1, Rxrb and Sorl1 could be validated as early biomarkers of metabolic health in young Rb+/- mice using whole blood RNA. Altogether, these results support the use of whole blood RNA in studies aimed at identifying blood transcript-based biomarkers of nutritional/metabolic status or metabolic health. Results also support reduced expression of Fasn, Lrp1, Rxrb and Sorl1 in blood cells at young age as potential biomarkers of metabolic robustness. PMID:27163124

  11. Whole Blood RNA as a Source of Transcript-Based Nutrition- and Metabolic Health-Related Biomarkers.

    PubMed

    Petrov, Petar D; Bonet, M Luisa; Reynés, Bárbara; Oliver, Paula; Palou, Andreu; Ribot, Joan

    2016-01-01

    Blood cells are receiving an increasing attention as an easily accessible source of transcript-based biomarkers. We studied the feasibility of using mouse whole blood RNA in this context. Several paradigms were studied: (i) metabolism-related transcripts known to be affected in rat tissues and peripheral blood mononuclear cells (PBMC) by fasting and upon the development of high fat diet (HFD)-induced overweight were assessed in whole blood RNA of fasted rats and mice and of HFD-fed mice; (ii) retinoic acid (RA)-responsive genes in tissues were assessed in whole blood RNA of control and RA-treated mice; (iii) lipid metabolism-related transcripts previously identified in PBMC as potential biomarkers of metabolic health in a rat model were assessed in whole blood in an independent model, namely retinoblastoma haploinsufficient (Rb+/-) mice. Blood was collected and stored in RNAlater® at -80°C until analysis of selected transcripts by real-time RT-PCR. Comparable changes with fasting were detected in the expression of lipid metabolism-related genes when RNA from either PBMC or whole blood of rats or mice was used. HFD-induced excess body weight and fat mass associated with expected changes in the expression of metabolism-related genes in whole blood of mice. Changes in gene expression in whole blood of RA-treated mice reproduced known transcriptional actions of RA in hepatocytes and adipocytes. Reduced expression of Fasn, Lrp1, Rxrb and Sorl1 could be validated as early biomarkers of metabolic health in young Rb+/- mice using whole blood RNA. Altogether, these results support the use of whole blood RNA in studies aimed at identifying blood transcript-based biomarkers of nutritional/metabolic status or metabolic health. Results also support reduced expression of Fasn, Lrp1, Rxrb and Sorl1 in blood cells at young age as potential biomarkers of metabolic robustness. PMID:27163124

  12. Whole blood EBV-DNA predicts outcome in diffuse large B-cell lymphoma.

    PubMed

    Tisi, Maria Chiara; Cupelli, Elisa; Santangelo, Rosaria; Maiolo, Elena; Alma, Eleonora; Giachelia, Manuela; Martini, Maurizio; Bellesi, Silvia; D'Alò, Francesco; Voso, Maria Teresa; Pompili, Maurizio; Leone, Giuseppe; Larocca, Luigi Maria; Hohaus, Stefan

    2016-01-01

    An association between Epstein-Barr Virus (EBV) infection and lymphoproliferative diseases has been reported with EBV + diffuse large B cell-lymphoma (DLBCL) of the elderly described as a distinct entity. In a cohort of 218 human immunodeficiency virus (HIV)-negative patients with diffuse large B-cell lymphomas, we detected EBV-DNA in 25% of whole blood (WB) samples at diagnosis. Presence and viral load in WB, mononuclear cells or plasma did not predict the presence of EBV in the tumor biopsy. Positive Hepatitis C virus (HCV) serology was associated with a higher frequency of EBV in WB. Patients with EBV-DNA in WB had a significantly shorter progression-free (p = 0.02) and overall survival (p = 0.05) after immunochemotherapy with R-CHOP (Rituximab, Cyclophosphamide, Doxorubicin, Vincristine, Prednisolone). We conclude that detection of EBV in WB is not a surrogate marker for EBV-association in diffuse large B-cell lymphoma, however it associates with worse outcome.

  13. Simple and inexpensive quantification of ammonia in whole blood.

    PubMed

    Ayyub, Omar B; Behrens, Adam M; Heligman, Brian T; Natoli, Mary E; Ayoub, Joseph J; Cunningham, Gary; Summar, Marshall; Kofinas, Peter

    2015-01-01

    Quantification of ammonia in whole blood has applications in the diagnosis and management of many hepatic diseases, including cirrhosis and rare urea cycle disorders, amounting to more than 5 million patients in the United States. Current techniques for ammonia measurement suffer from limited range, poor resolution, false positives or large, complex sensor set-ups. Here we demonstrate a technique utilizing inexpensive reagents and simple methods for quantifying ammonia in 100 μL of whole blood. The sensor comprises a modified form of the indophenol reaction, which resists sources of destructive interference in blood, in conjunction with a cation-exchange membrane. The presented sensing scheme is selective against other amine containing molecules such as amino acids and has a shelf life of at least 50 days. Additionally, the resulting system has high sensitivity and allows for the accurate reliable quantification of ammonia in whole human blood samples at a minimum range of 25 to 500 μM, which is clinically for rare hyperammonemic disorders and liver disease. Furthermore, concentrations of 50 and 100 μM ammonia could be reliably discerned with p = 0.0001.

  14. Whole blood: the future of traumatic hemorrhagic shock resuscitation.

    PubMed

    Murdock, Alan D; Berséus, Olle; Hervig, Tor; Strandenes, Geir; Lunde, Turid Helen

    2014-05-01

    Toward the end of World War I and during World War II, whole-blood transfusions were the primary agent in the treatment of military traumatic hemorrhage. However, after World War II, the fractionation of whole blood into its components became widely accepted and replaced whole-blood transfusion to better accommodate specific blood deficiencies, logistics, and financial reasons. This transition occurred with very few clinical trials to determine which patient populations or scenarios would or would not benefit from the change. A smaller population of patients with trauma hemorrhage will require massive transfusion (>10 U packed red blood cells in 24 h) occurring in 3% to 5% of civilian and 10% of military traumas. Advocates for hemostatic resuscitation have turned toward a ratio-balanced component therapy using packed red blood cells-fresh frozen plasma-platelet concentration in a 1:1:1 ratio due to whole-blood limited availability. However, this "reconstituted" whole blood is associated with a significantly anemic, thrombocytopenic, and coagulopathic product compared with whole blood. In addition, several recent military studies suggest a survival advantage of early use of whole blood, but the safety concerns have limited is widespread civilian use. Based on extensive military experience as well as recent published literature, low-titer leukocyte reduced cold-store type O whole blood carries low adverse risks and maintains its hemostatic properties for up to 21 days. A prospective randomized trial comparing whole blood versus ratio balanced component therapy is proposed with rationale provided. PMID:24662782

  15. Induction of autoantibody-producing cells after the coculture of haptenated and normal human mononuclear leukocytes.

    PubMed

    Pisko, E J; Foster, S L; Turner, R A

    1981-10-01

    The coculture of normal human peripheral blood mononuclear leukocytes (PBL) and autologous mononuclear leukocytes coupled to the trinitrophenyl (TNP) hapten (TNP-PBL) was found to induce a polyclonal activation of antibody-producing cells. The polyclonal activation of antibody-producing cells was demonstrated by detecting the induction of cells producing antibody to sheep red blood cells using a complement-dependent, direct, hemolytic plaque-forming cell (PFC) assay. A ratio of four normal to one haptenated mononuclear leukocyte was found to be optimal for inducing the polyclonal activation of antibody-producing cell in these cultures. The plaque-forming cells assay in these experiments utilized monolayers of indicator red cells. Further evidence for the polyclonal induction of antibody-producing cells by TNP-PBL was provided by demonstrating PFC on monolayers of not only sheep red blood cells, but also autologous human red cells, bromelain-treated autologous red cells, TNP-coupled human and sheep red cells, and human autologous red cells coupled to human heat-aggregated IgG with chromic chloride. Thus cells secreting antibody to TNP, human red cells, and human IgG were induced. Anti-IgG and anti-human red cell-producing cells were first detected on Day 2 of culture and were still present on Day 9. Mononuclear leukocytes altered by chemical haptenation polyclonally stimulate normal mononuclear leukocytes to become antibody-producing cells. This polyclonal stimulation of antibody-producing cells includes cells producing antibodies to human IgG and human autologous red blood cells suggesting that autoantibody-producing cells are induced.

  16. Separation of granulocytes from whole blood by leukoadhesion, phase 1

    NASA Technical Reports Server (NTRS)

    1976-01-01

    Capillary glass tubes are investigated for the separation and retrieval of large quantities of viable granulocytes and monocytes from whole blood on a continuous basis from a single donor. This effort represented the feasibility demonstration of a three phase program for development of a capillary tube cell separation device. The activity included the analysis and parametric laboratory testing with subscale models required to design a prototype device. Capillary tubes 40 cm long with a nominal 0.030 cm internal diameter yielded the highest total process efficiency. Recovery efficiencies as high as 89% of the adhering cell population were obtained. Granulocyte phagocytosis of latex particles indicated approximately 90% viability. Monocytes recovered from the separation column retained their capability to stimulate human bone marrow colony growth, as demonstrated in an in vitro cell culture assay.

  17. Development and validation of a liquid chromatography-tandem mass spectrometry method for the identification and quantification of JWH-018, JWH-073, JWH-019, and JWH-250 in human whole blood.

    PubMed

    Kacinko, Sherri L; Xu, Allan; Homan, Joseph W; McMullin, Matthew M; Warrington, Donna M; Logan, Barry K

    2011-09-01

    A sensitive and specific method for the quantification of JWH-018, JWH-073, and JWH-250 and the qualitative identification of JWH-019 in whole blood was developed and validated. Samples fortified with JWH-018-d₉ and JWH-073-d₉ underwent liquid-liquid extraction and were analyzed by liquid chromatography-positive ion electrospray ionization-tandem mass spectrometry. Two transitions were monitored for all analytes except JWH-250, for which there was only one available transition. JWH-019 did not meet the stringent requirements for quantitative analysis, and thus this method is only appropriate for the qualitative identification of this compound in whole blood. The linear range was 0.1-20 μg/L for all quantitative analytes. The maximum average within and between-run imprecision was 7.9% and 10.2%, respectively, and all controls quantified within 8.2% of target concentrations. Process efficiency, a measurement that takes into effect extraction efficiency and matrix effect, was ≥ 32.0% for all quantitative analytes; similar results were obtained for the deuterated internal standards. All analytes were stable at room, refrigerated, and frozen temperatures for at least 30 days. The method was used to quantify JWH-018 and JWH-073 in a blood specimen collected from a person known to have used an herbal incense blend containing these substances.

  18. Pharmacologic immunosuppression of mononuclear phagocyte phagocytosis by caffeine.

    PubMed

    Steck, Ryan P; Hill, Spencer L; Weagel, Evita G; Weber, K Scott; Robison, Richard A; O'Neill, Kim L

    2015-12-01

    Caffeine is the most widely used neurostimulant in the world. There is considerable debate on its effect on immune cells as it has been shown to antagonize adenosine receptors (ARs), which mediate an anti-inflammatory switch in activated immune cells. A second target is phosphodiesterase, where it acts as an inhibitor. If the primary effect of caffeine on mononuclear phagocytes were to antagonize ARs we would expect cells exposed to caffeine to have a prolonged proinflammatory response. The aim of this study was to investigate the effects and mechanism of action of caffeine in mononuclear phagocytes. Human mononuclear phagocytes were separated from whole blood and pretreated with protein kinase A inhibitor (PKA) and then exposed to micromolar physiological concentrations of caffeine. Phagocytosis and phagocytosis exhaustion were quantified using flow cytometry. Treatments were analyzed and compared to controls, using a beta regression controlling for factors of age, gender, caffeine intake, and exercise. We found that caffeine suppresses phagocytosis at micromolar physiological concentrations. This suppression was prevented when mononuclear phagocytes were pretreated with PKA inhibitor, suggesting that caffeine's phagocytic suppression may be due to its function as a phosphodiesterase inhibitor, pushing cells towards an anti-inflammatory response. Additionally, these effects are altered by regular caffeine intake and fitness level, emphasizing that tolerance and immune robustness are important factors in mononuclear phagocyte activation. These results demonstrate that caffeine may be acting as a phosphodiesterase inhibitor and suppressing phagocytosis in mononuclear phagocytes by promoting an anti-inflammatory response.

  19. Adjusting MtDNA Quantification in Whole Blood for Peripheral Blood Platelet and Leukocyte Counts

    PubMed Central

    Gonzalez-Lazaro, Monica; Moreno-Loshuertos, Raquel; Fernandez-Silva, Patricio; Enriquez, Jose Antonio; Laclaustra, Martin

    2016-01-01

    Alterations of mitochondrial DNA copy number (mtDNAcn) in the blood (mitochondrial to nuclear DNA ratio) appear associated with several systemic diseases, including primary mitochondrial disorders, carcinogenesis, and hematologic diseases. Measuring mtDNAcn in DNA extracted from whole blood (WB) instead of from peripheral blood mononuclear cells or buffy coat may yield different results due to mitochondrial DNA present in platelets. The aim of this work is to quantify the contribution of platelets to mtDNAcn in whole blood [mtDNAcn(WB)] and to propose a correction formula to estimate leukocytes' mtDNAcn [mtDNAcn(L)] from mtDNAcn(WB). Blood samples from 10 healthy adults were combined with platelet-enriched plasma and saline solution to produce artificial blood preparations. Aliquots of each sample were combined with five different platelet concentrations. In 46 of these blood preparations, mtDNAcn was measured by qPCR. MtDNAcn(WB) increased 1.07 (95%CI 0.86, 1.29; p<0.001) per 1000 platelets present in the preparation. We proved that leukocyte count should also be taken into account as mtDNAcn(WB) was inversely associated with leukocyte count; it increased 1.10 (95%CI 0.95, 1.25, p<0.001) per unit increase of the ratio between platelet and leukocyte counts. If hematological measurements are available, subtracting 1.10 the platelets/leukocyte ratio from mtDNAcn(WB) may serve as an estimation for mtDNAcn(L). Both platelet and leukocyte counts in the sample are important sources of variation if comparing mtDNAcn among groups of patients when mtDNAcn is measured in DNA extracted from whole blood. Not taking the platelet/leukocyte ratio into account in whole blood measurements, may lead to overestimation and misclassification if interpreted as leukocytes' mtDNAcn. PMID:27736919

  20. Whole blood is the sample matrix of choice for monitoring systemic triclocarban levels.

    PubMed

    Schebb, Nils Helge; Ahn, Ki Chang; Dong, Hua; Gee, Shirley J; Hammock, Bruce D

    2012-05-01

    The antibacterial triclocarban (TCC) concentrates in the cellular fraction of blood. Consequently, plasma levels are at least two-fold lower than the TCC amount present in blood. Utilizing whole blood sampling, a low but significant absorption of TCC from soap during showering is demonstrated for a small group of human subjects. PMID:22273184

  1. Fresh Whole Blood Transfusion: Military and Civilian Implications.

    PubMed

    Goforth, Carl W; Tranberg, John W; Boyer, Phillip; Silvestri, Peter J

    2016-06-01

    Uncontrolled hemorrhage and exsanguination are the leading cause of preventable death, and resuscitative therapy is a critical component for survival. In various combinations, fresh whole blood, blood components, colloids, and crystalloids have all been staples of trauma care. The use of fresh whole blood is a well-established military practice that has saved the lives of thousands of American and coalition military personnel. Civilian use of fresh whole blood is far less established owing to the wide availability of individual blood components. However, this highly tailored blood supply is vulnerable to both natural and man-made disasters. In the event of such disruption, such as a major hurricane, it may be necessary for civilian hospitals to rapidly enact a fresh whole blood program. Therefore, the aim of this article is to review the current use of blood therapy for trauma resuscitation, the US military's approach to fresh whole blood, and how maintaining a civilian capacity for fresh whole blood collection in the event of future man-made and natural disasters is key to promoting survival from trauma. PMID:27252101

  2. Relationship between plasma cholesterol levels and cholesterol esterification in isolated human mononuclear cells

    SciTech Connect

    Dallongeville, J.; Davignon, J.; Lussier-Cacan, S. )

    1990-01-01

    The authors studied the relationship between plasma lipoprotein concentrations and cholesterol esterification in freshly isolated human mononuclear cells from 27 normolipidemic and 32 hyperlipidemic individuals. Cells were either incubated for 5 hours with radiolabeled oleate immediately after isolation or were preincubated for 18 hours in the presence of exogenous cholesterol, and then incubated with ({sup 14}C)sodium-oleate-albumin complex. In the absence of exogenous cholesterol, control and hypercholesterolemic subjects had similarly low values of intracellular cholesterol esterification. In the presence of exogenous cholesterol, both hypertriglyceridemic and hypercholesterolemic subjects had higher cholesterol esterification than controls. There was a significant correlation between the rate of cholesterol esterification and plasma total cholesterol. These results suggest that plasma cholesterol levels may regulate mononuclear cell intra-cellular cholesterol esterification in humans.

  3. Immunotoxicity Assessment of Rice-Derived Recombinant Human Serum Albumin Using Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Fu, Kai; Cheng, Qin; Liu, Zhenwei; Chen, Zhen; Wang, Yan; Ruan, Honggang; Zhou, Lu; Xiong, Jie; Xiao, Ruijing; Liu, Shengwu; Zhang, Qiuping; Yang, Daichang

    2014-01-01

    Human serum albumin (HSA) is extensively used in clinics to treat a variety of diseases, such as hypoproteinemia, hemorrhagic shock, serious burn injuries, cirrhotic ascites and fetal erythroblastosis. To address supply shortages and high safety risks from limited human donors, we recently developed recombinant technology to produce HSA from rice endosperm. To assess the risk potential of HSA derived from Oryza sativa (OsrHSA) before a First-in-human (FIH) trial, we compared OsrHSA and plasma-derived HSA (pHSA), evaluating the potential for an immune reaction and toxicity using human peripheral blood mononuclear cells (PBMCs). The results indicated that neither OsrHSA nor pHSA stimulated T cell proliferation at 1x and 5x dosages. We also found no significant differences in the profiles of the CD4+ and CD8+ T cell subsets between OsrHSA- and pHSA-treated cells. Furthermore, the results showed that there were no significant differences between OsrHSA and pHSA in the production of cytokines such as interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-10 and IL-4. Our results demonstrated that OsrHSA has equivalent immunotoxicity to pHSA when using the PBMC model. Moreover, this ex vivo system could provide an alternative approach to predict potential risks in novel biopharmaceutical development. PMID:25099245

  4. Different responses of human mononuclear phagocyte populations to Mycobacterium tuberculosis.

    PubMed

    Duque, Camilo; Arroyo, Leonar; Ortega, Héctor; Montúfar, Franco; Ortíz, Blanca; Rojas, Mauricio; Barrera, Luis F

    2014-03-01

    Mycobacterium tuberculosis (Mtb) infects different populations of macrophages. Alveolar macrophages (AMs) are initially infected, and their response may contribute to controlling Mtb infection and dissemination. However, Mtb infection may disseminate to other tissues, infecting a wide variety of macrophages. Given the difficulty in obtaining AMs, monocyte-derived macrophages (MDMs) are used to model macrophage-mycobacteria interactions in humans. However, the response of other tissue macrophages to Mtb infection has been poorly explored. We have compared MDMs, AMs and splenic human macrophages (SMs) for their in vitro capacity to control Mtb growth, cytokine production, and induction of cell death in response to Mtb H37Rv, and the Colombian isolate UT205, and to the virulence factor ESAT-6. Significant differences in the magnitude of cell death and cytokine production depending mainly on the Mtb strain were observed; however, no major differences in the mycobacteriostatic/mycobacteriocidal activity were detected among the macrophage populations. Infection with the clinical isolate UT205 was associated with an increased cell death with membrane damage, particularly in IFNγ-treated SMs and H37Rv induced a higher production of cytokines compared to UT205. These results are concordant with the interpretation of a differential response to Mtb infection mainly depending upon the strain of Mtb.

  5. A Metabolomics Study of Retrospective Forensic Data from Whole Blood Samples of Humans Exposed to 3,4-Methylenedioxymethamphetamine: A New Approach for Identifying Drug Metabolites and Changes in Metabolism Related to Drug Consumption.

    PubMed

    Nielsen, Kirstine L; Telving, Rasmus; Andreasen, Mette F; Hasselstrøm, Jørgen B; Johannsen, Mogens

    2016-02-01

    The illicit drug 3,4-methylenedioxymethamphetamine (MDMA) has profound physiological cerebral, cardiac, and hepatic effects that are reflected in the blood. Screening of blood for MDMA and other narcotics are routinely performed in forensics analysis using ultra-performance liquid chromatography with high-resolution time-of-flight mass spectrometry (UPLC-HR-TOFMS). The aim of this study was to investigate whether such UPLC-HR-TOFMS data collected over a two-year period could be used for untargeted metabolomics to determine MDMA metabolites as well as endogenous changes related to drug response and toxicology. Whole blood samples from living Danish drivers' positive for MDMA in different concentrations were compared to negative control samples using various statistical methods. The untargeted identification of known MDMA metabolites was used to validate the methods. The results further revealed changes of several acylcarnitines, adenosine monophosphate, adenosine, inosine, thiomorpholine 3-carboxylate, tryptophan, S-adenosyl-l-homocysteine (SAH), and lysophospatidylcholine (lysoPC) species in response to MDMA. These endogenous metabolites could be implicated in an increased energy demand and mechanisms related to the serotonergic syndrome as well as drug induced neurotoxicity. The findings showed that it was possible to extract meaningful results from retrospective UPLC-HR-TOFMS screening data for metabolic profiling in relation to drug metabolism, endogenous physiological effects, and toxicology. PMID:26705142

  6. Metals in air pollution particles decrease whole-blood coagulation time.

    PubMed

    Sangani, Rahul G; Soukup, Joleen M; Ghio, Andrew J

    2010-07-01

    The mechanism underlying procoagulative effects of air pollution particle exposure is not known. The authors tested the postulate that (1) the water-soluble components of an air pollution particle could affect whole-blood coagulation time and (2) metals included in this fraction were responsible for this effect. Exposure to the water-soluble fraction of particulate matter (PM), at doses as low as 50 ng/ml original particle, significantly diminished the whole-blood coagulation time. Inclusion of deferoxamine prolonged coagulation time following the exposures to the water-soluble fraction, whereas equivalent doses of ferroxamine had no effect. Except for nickel, all metal sulfates shortened the whole-blood coagulation time. Iron and zinc were two metals with the greatest capacity to reduce the coagulation time, with an effect observed at 10 ng/ml. Finally, in contrast to the anticoagulants citrate and EDTA, their iron complexes were found to be procoagulative. The authors conclude that metals in the water-soluble fraction of air pollution particles decrease whole-blood coagulation time. These metals can potentially contribute to procoagulative effects observed following human exposures to air pollution particles.

  7. Differences in the influence of the interaction between acetylsalicylic acid and salicylic acid on platelet function in whole blood and isolated platelets: influence of neutrophils.

    PubMed

    González-Correa, J A; Muñoz-Marín, J; López-Villodres, J A; Navas, M D; Guerrero, A; Torres, J A; De La Cruz, J P

    2007-08-01

    The aim of this study was to characterize the influence of the interaction between acetylsalicylic acid (ASA) and salicylic acid (SA) on the inhibition by ASA of platelet aggregation in platelets isolated from whole blood, and to determine whether leukocytes influence this pharmacological interaction. This in vitro study was done in human blood from which we prepared samples of whole blood, platelet-rich plasma (PRP), PRP plus mononuclear leukocytes, and PRP plus neutrophils. The variables recorded were maximum platelet aggregation intensity, thromboxane B2 (TxB2) production, and nitric oxide (NO) production (N=10 different samples in each type of experiment). Different concentrations of ASA and SA were incubated with all samples. In PRP, the concentration of ASA that inhibited maximum aggregation by 50% (IC50) (281+/-16microM) increased with increasing SA concentration to a maximum of more than 2mM when 500microM SA was used. In whole blood, the IC50 for ASA (24.9+/-1.2microM) decreased with decreasing SA concentrations to 7.9+/-0.8microM with 50microM SA and 15.6+/-0.9microM with 125microM SA, and increased to 46.2+/-2.6microM with 250microM SA and 96.3+/-7.2microM with 500microM SA. In experiments with PRP+neutrophils the IC50 of ASA increased in the presence of all concentrations of SA. The antagonistic interactions were also reflected in the changes in TxB2 production in all samples. In samples of neutrophils incubated with ASA, the curve for NO production was shifted to the right, a finding that paralleled the changes in platelet aggregation. In conclusion, the influence of the interaction between ASA and its metabolite SA on platelet aggregation difference depending on the type of sample, and was antagonistic in PRP but partially agonistic in whole blood. Nitric oxide synthesis showed an additive effect of the two compounds.

  8. Measurement and Comparison of Organic Compound Concentrations in Plasma, Whole Blood, and Dried Blood Spot Samples

    PubMed Central

    Batterman, Stuart A.; Chernyak, Sergey; Su, Feng-Chiao

    2016-01-01

    The preferred sampling medium for measuring human exposures of persistent organic compounds (POPs) is blood, and relevant sample types include whole blood, plasma, and dried blood spots (DBS). Because information regarding the performance and comparability of measurements across these sample types is limited, it is difficult to compare across studies. This study evaluates the performance of POP measurements in plasma, whole blood and DBS, and presents the distribution coefficients needed to convert concentrations among the three sample types. Blood samples were collected from adult volunteers, along with demographic and smoking information, and analyzed by GC/MS for organochlorine pesticides (OCPs), chlorinated hydrocarbons (CHCs), polychlorinated biphenyls (PCBs), and brominated diphenyl ethers (PBDEs). Regression models were used to evaluate the relationships between the sample types and possible effects of personal covariates. Distribution coefficients also were calculated using physically-based models. Across all compounds, concentrations in plasma were consistently the highest; concentrations in whole blood and DBS samples were comparable. Distribution coefficients for plasma to whole blood concentrations ranged from 1.74 to 2.26 for pesticides/CHCs, averaged 1.69 ± 0.06 for the PCBs, and averaged 1.65 ± 0.03 for the PBDEs. Regression models closely fit most chemicals (R2 > 0.80), and whole blood and DBS samples generally showed very good agreement. Distribution coefficients estimated using biologically-based models were near one and did not explain the observed distribution. Among the study population, median concentrations of several pesticides/CHCs and PBDEs exceeded levels reported in the 2007–2008 National Health and Nutrition Examination Survey, while levels of other OCPs and PBDEs were comparable or lower. Race and smoking status appeared to slightly affect plasma/blood concentration ratios for several POPs. The experimentally

  9. The removal of leukocytes and platelets from whole blood.

    PubMed

    Beutler, E; West, C; Blume, K G

    1976-08-01

    Various methods for the removal of leukocytes from whole blood have been compared and a new technique has been devised. This procedure consists of passing whole blood through a bed composed of microcrystalline cellulose and alpha-cellulose. The method is rapid, reliable, removes over 99.75 per cent of the leukocytes from blood, and does not seem selectively to retain reticulocytes or to release a significant proportion of leukocyte enzymes. Most of the platelets are also removed from anticoagulated blood, and platelet-free red cells can be obtained by passing defibrinated blood over the microcrystalline cellulose-alpha-cellulose bed.

  10. Proteinase-activated receptors induce nonoxidative, antimicrobial peptides and increased antimicrobial activity in human mononuclear phagocytes.

    PubMed

    Lippuner, Nadine; Morell, Bernhard; Schaffner, Andreas; Schaer, Dominik J

    2007-02-01

    As thrombin and SFLLRNPNDKYEPF (SFLLRN-14), a synthetic ligand, mainly of the proteinase-activated receptor-1 (PAR-1), induce in monocytes the synthesis and secretion of chemokines, the PAR pathway can be viewed as a mononuclear phagocyte-activating principle. Classically, antimicrobial activity of mononuclear phagocytes is the measure for activation. Here, we investigated whether thrombin or SFLLRN-14 increases the antimicrobial activity of human monocytes and compared these effects to those of IFN-gamma. Furthermore, we measured the effects of these agents on the secretion of reactive oxygen intermediates and the antimicrobial activity of acid peptide extracts from monocytes. Human monocytes were exposed to maximally active concentrations of thrombin, SFLLRN-14, and IFN-gamma. Human monocytes treated with thrombin or SFLLRN-14 and then challenged with Salmonella enterica serovar typhimurium, including its attenuated mutant phoP, or Listeria monocytogenes killed, within 3 h, significantly more bacteria than control cells, an effect comparable with or surpassing the effect of IFN-gamma. This finding establishes the proteinase-PAR pathway as a potent, alternate activation pathway of mononuclear phagocytes. Thrombin and SFLLRN-14 had no significant effects on the amount of H(2)O(2) secreted by monocytes. This was in contrast to IFN-gamma, which as expected, increased the secretion of H(2)O(2) by approximately fourfold. Thrombin and SFLLRN-14, but not IFN-gamma, however, significantly increased the antimicrobial activity of acid peptide extracts of monocytes in a radial diffusion assay. Taken together, these findings suggest that IFN-gamma and thrombin differentially regulate oxidative and nonoxidative killing systems of human monocytes. PMID:17095611

  11. A high-throughput assay of NK cell activity in whole blood and its clinical application

    SciTech Connect

    Lee, Saet-byul; Cha, Junhoe; Kim, Im-kyung; Yoon, Joo Chun; Lee, Hyo Joon; Park, Sang Woo; Cho, Sunjung; Youn, Dong-Ye; Lee, Heyja; Lee, Choong Hwan; Lee, Jae Myun; Lee, Kang Young; Kim, Jongsun

    2014-03-14

    Graphical abstract: - Highlights: • We demonstrated a simple assay of NK cell activity from whole blood. • The measurement of secreted IFN-γ from NK cell enables high-throughput screening. • The NKA assay was validated by clinical results of colorectal cancer patients. - Abstract: Natural killer (NK) cells are lymphocytes of the innate immune system and have the ability to kill tumor cells and virus-infected cells without prior sensitization. Malignant tumors and viruses have developed, however, strategies to suppress NK cells to escape from their responses. Thus, the evaluation of NK cell activity (NKA) could be invaluable to estimate the status and the outcome of cancers, viral infections, and immune-mediated diseases. Established methods that measure NKA, such as {sup 51}Cr release assay and CD107a degranulation assay, may be used to determine NK cell function, but they are complicated and time-consuming because they require isolation of peripheral blood mononuclear cells (PBMC) or NK cells. In some cases these assays require hazardous material such as radioactive isotopes. To overcome these difficulties, we developed a simple assay that uses whole blood instead of PBMC or isolated NK cells. This novel assay is suitable for high-throughput screening and the monitoring of diseases, because it employs serum of ex vivo stimulated whole blood to detect interferon (IFN)-γ secreted from NK cells as an indicator of NKA. After the stimulation of NK cells, the determination of IFNγ concentration in serum samples by enzyme-linked immunosorbent assay (ELISA) provided a swift, uncomplicated, and high-throughput assay of NKA ex vivo. The NKA results microsatellite stable (MSS) colorectal cancer patients was showed significantly lower NKA, 263.6 ± 54.5 pg/mL compared with healthy subjects, 867.5 ± 50.2 pg/mL (p value <0.0001). Therefore, the NKA could be utilized as a supportive diagnostic marker for microsatellite stable (MSS) colorectal cancer.

  12. Measurement of peripheral B cell subpopulations in common variable immunodeficiency (CVID) using a whole blood method

    PubMed Central

    Ferry, B L; Jones, J; Bateman, E A; Woodham, N; Warnatz, K; Schlesier, M; Misbah, S A; Peter, H H; Chapel, H M

    2005-01-01

    Recent reports have described reduced populations of CD27+ memory B cells and increased percentages of undifferentiated B cells in peripheral blood of patients with common variable immunodeficiency (CVID). This work has prompted two attempts to classify CVID based on rapid flow cytometric quantification of peripheral blood memory B cells and immature B cells. Evidence to support the hypothesis that such in vitro B cell classification systems correlate with clinical subtypes of CVID is being sought. For the classification to be useful in routine diagnosis, it is important that the flow cytometric method can be used without prior separation of peripheral blood mononuclear cells (PBMC). We have examined 23 CVID patients and 24 controls, using both PBMC and whole blood, and find an excellent correlation between these methods. The reproducibility of the method was excellent. We classified the CVID patients by all three of the existing classifications, including secretion of immunoglobulin by B cells in vitro as described by Bryant, as well as the more recent flow cytometric classification methods. Only one patient changed classification as a result of using whole blood. PMID:15932516

  13. Metals in airpollution particles decrease whole blood coagulation time

    EPA Science Inventory

    The mechanism underlying the pro-coagulative effect of air pollution particle exposure is not known. We tested the postulate that 1) the soluble fraction ofan air pollution particle can affect whole blood coagulation time and 2) metals included in the soluble fraction are respons...

  14. Antibody Conjugated Magnetic Iron Oxide Nanoparticles for Cancer Cell Separation in Fresh Whole Blood

    PubMed Central

    Xu, Hengyi; Aguilar, Zoraida P.; Yang, Lily; Kuang, Min; Duan, Hongwei; Xiong, Yonghua; Wei, Hua; Wang, Andrew

    2011-01-01

    A highly efficient process using iron oxide magnetic nanoparticles (IO)-based immunomagnetic separation of tumor cells from fresh whole blood has been developed. The process involved polymer coated 30 nm IO that was modified with antibodies (Ab) against human epithelial growth factor receptor 2 (anti-HER2 or anti-HER2/neu) forming IO-Ab. HER2 is a cell membrane protein that is over expressed in several types of human cancer cells. Using a HER2/neu over expressing human breast cancer cell line, SK-BR3, as a model cell, the IO-Ab was used to separate 73.6 % (with a maximum capture of 84%) of SK-BR3 cells that were spiked in 1 mL of fresh human whole blood. The IO-Ab preferentially bound to SK-BR3 cells over normal cells found in blood due to the high level of HER2/neu receptor on the cancer cells unlike the normal cell surfaces. The results showed that the nanosized magnetic nanoparticles exhibited an enrichment factor (cancer cells over normal cells) of 1:10,000,000 in a magnetic field (with gradient of 100 T/m) through the binding of IO-Ab on the cell surface that resulted in the preferential capture of the cancer cells. This research holds promise for efficient separation of circulating cancer cells in fresh whole blood. PMID:21920599

  15. Dengue-2 virus infection of human mononuclear cell lines and establishment of persistent infections.

    PubMed

    Kurane, I; Kontny, U; Janus, J; Ennis, F A

    1990-01-01

    Twenty three human mononuclear cell lines including ten myelomonocytic cell lines, eight B cell lines and five T cell lines, were examined to determine whether they could be infected with dengue-2 virus. All the cell lines were infected with dengue-2 virus as determined by immunofluorescent staining and by virus titration of culture supernatant fluids. K562, Jiyoye and Jurkat, respectively, showed the highest percentage of infected cells of these myelomonocytic, B and T cell lines. Antibody to dengue-2 virus at subneutralizing concentrations augmented dengue-2 virus infection of myelomonocytic cell lines, but not of B cell lines or of T cell lines. Persistent dengue-2 virus infection was established using a myelomonocytic cell line (K562), a B cell line (Raji), and a T cell line (HSB-2). These cell lines maintained a high percentage (more than 70%) of dengue-2 virus antigen-positive cells for at least 25 weeks. Very low titers of infectious dengue-2 virus were detected in the culture supernatant fluids of the persistently infected cells. Dengue-2 virus antigen-positive Raji cell clones were established from persistently-infected Raji cells using limiting dilutions and all of the cells in these clones were dengue-2 virus antigen-positive. These findings demonstrate that a variety of human mononuclear cell lines can be infected with dengue-2 virus and may be useful as models for the analysis of dengue virus-human cell interactions in dengue virus infections.

  16. 21 CFR 864.7140 - Activated whole blood clotting time tests.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Activated whole blood clotting time tests. 864....7140 Activated whole blood clotting time tests. (a) Identification. An activated whole blood clotting... pulmonary embolism by measuring the coagulation time of whole blood. (b) Classification. Class...

  17. 21 CFR 864.7140 - Activated whole blood clotting time tests.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Activated whole blood clotting time tests. 864....7140 Activated whole blood clotting time tests. (a) Identification. An activated whole blood clotting... pulmonary embolism by measuring the coagulation time of whole blood. (b) Classification. Class...

  18. 21 CFR 864.7140 - Activated whole blood clotting time tests.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Activated whole blood clotting time tests. 864....7140 Activated whole blood clotting time tests. (a) Identification. An activated whole blood clotting... pulmonary embolism by measuring the coagulation time of whole blood. (b) Classification. Class...

  19. 21 CFR 864.7140 - Activated whole blood clotting time tests.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Activated whole blood clotting time tests. 864....7140 Activated whole blood clotting time tests. (a) Identification. An activated whole blood clotting... pulmonary embolism by measuring the coagulation time of whole blood. (b) Classification. Class...

  20. 21 CFR 864.7140 - Activated whole blood clotting time tests.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Activated whole blood clotting time tests. 864....7140 Activated whole blood clotting time tests. (a) Identification. An activated whole blood clotting... pulmonary embolism by measuring the coagulation time of whole blood. (b) Classification. Class...

  1. Metabolic effects of microwave radiation and convection heating on human mononuclear leukocytes

    SciTech Connect

    Kiel, J.L.; Wong, L.S.; Erwin, D.N.

    1986-01-01

    The effects of microwave radiation (2450 MHz, continuous wave, mean specific absorption rate of 103.5 +/- 4.2 W/kg) and convection heating on the nonphosphorylating oxidative metabolism of human peripheral mononuclear leukocytes (96% lymphocytes, 4% monocytes) at 37 degrees C were investigated. Metabolic activity, determined by chemiluminescence (CL) of cells challenged with luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) linked to bovine serum albumin, was detected with a brightness photometer. A significant stimulation after microwave exposure (p less than 0.005) over total CL of matched 37 degrees C incubator controls was observed. A similar degree of stimulation compared to incubator controls was also detected after sham treatment. There was no significant difference between changes in total CL or stimulation indices of the microwave and sham exposed groups. It appears that exposure to microwave radiation, under normothermic (37 +/- 0.03 degrees C) conditions, has no effect on the oxidative metabolic activity of human peripheral mononuclear leukocytes. However, the significant differences between microwave or sham exposed cells and their respective incubator controls occurred because the temperature of the incubator controls did not exceed 35.9 degrees C and this temperature required 39 minutes to reach from 22 degrees C. Slow heating of incubator controls must be accounted for in thermal and radiofrequency radiation studies in vitro.

  2. Pro-angiogenic Cell Colonies Grown In Vitro from Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Mavromatis, Kreton; Sutcliffe, Diane; Joseph, Giji; Alexander, R. Wayne; Waller, Edmund K.; Quyyumi, Arshed A.; Taylor, W. Robert

    2014-01-01

    Although multiple culture assays have been designed to identify “endothelial progenitor cells” (EPCs), the phenotype of cells grown in culture often remains undefined. We sought to define and characterize the pro-angiogenic cell population within human peripheral blood mononuclear cells. Mononuclear cells were isolated from peripheral blood and grown under angiogenic conditions for 7 days. Formed colonies (CFU-As) were identified and analyzed for proliferation, mRNA and surface antigen expression, tube-forming ability and chromosomal content. Colonies were composed of a heterogeneous group of cells expressing the leukocyte antigens CD45, CD14, and CD3, as well as the endothelial proteins vascular endothelial (VE) cadherin, von Willebrand's Factor (vWF), CD31 and endothelial nitric oxide synthase (eNOS). Colony cells expressed increased levels of pro-angiogenic growth factors, and they formed tubes in Matrigel. In comparison with colonies from the CFU-Hill assay, our assay resulted in a greater number of colonies (19±9 vs. 13±7; p<0.0001) with a substantial number of cells expressing an endothelial phenotype (20.2±7.4% vs. 2.2±1.2% expressing eNOS, p=0006). Chromosomal analysis indicated the colony cells were bone marrow-derived. We, therefore, describe a colony forming unit assay that measures bone marrow-derived circulating mononuclear cells with the capacity to proliferate and mature into proangiogenic leukocytic and endothelial-like cells. This assay, therefore, reflects circulating, bone marrow-derived pro-angiogenic activity. PMID:22904201

  3. Globin mRNA reduction for whole-blood transcriptome sequencing

    PubMed Central

    Krjutškov, Kaarel; Koel, Mariann; Roost, Anne Mari; Katayama, Shintaro; Einarsdottir, Elisabet; Jouhilahti, Eeva-Mari; Söderhäll, Cilla; Jaakma, Ülle; Plaas, Mario; Vesterlund, Liselotte; Lohi, Hannes; Salumets, Andres; Kere, Juha

    2016-01-01

    The transcriptome analysis of whole-blood RNA by sequencing holds promise for the identification and tracking of biomarkers; however, the high globin mRNA (gmRNA) content of erythrocytes hampers whole-blood and buffy coat analyses. We introduce a novel gmRNA locking assay (GlobinLock, GL) as a robust and simple gmRNA reduction tool to preserve RNA quality, save time and cost. GL consists of a pair of gmRNA-specific oligonucleotides in RNA initial denaturation buffer that is effective immediately after RNA denaturation and adds only ten minutes of incubation to the whole cDNA synthesis procedure when compared to non-blood RNA analysis. We show that GL is fully effective not only for human samples but also for mouse and rat, and so far incompletely studied cow, dog and zebrafish. PMID:27515369

  4. Globin mRNA reduction for whole-blood transcriptome sequencing.

    PubMed

    Krjutškov, Kaarel; Koel, Mariann; Roost, Anne Mari; Katayama, Shintaro; Einarsdottir, Elisabet; Jouhilahti, Eeva-Mari; Söderhäll, Cilla; Jaakma, Ülle; Plaas, Mario; Vesterlund, Liselotte; Lohi, Hannes; Salumets, Andres; Kere, Juha

    2016-01-01

    The transcriptome analysis of whole-blood RNA by sequencing holds promise for the identification and tracking of biomarkers; however, the high globin mRNA (gmRNA) content of erythrocytes hampers whole-blood and buffy coat analyses. We introduce a novel gmRNA locking assay (GlobinLock, GL) as a robust and simple gmRNA reduction tool to preserve RNA quality, save time and cost. GL consists of a pair of gmRNA-specific oligonucleotides in RNA initial denaturation buffer that is effective immediately after RNA denaturation and adds only ten minutes of incubation to the whole cDNA synthesis procedure when compared to non-blood RNA analysis. We show that GL is fully effective not only for human samples but also for mouse and rat, and so far incompletely studied cow, dog and zebrafish. PMID:27515369

  5. Gene-expression analysis of clozapine treatment in whole blood of patients with psychosis

    PubMed Central

    Harrison, Rebecca N.S.; Murray, Robin M.; Lee, Sang Hyuck; Paya Cano, Jose; Dempster, David; Curtis, Charles J.; Dima, Danai; Gaughran, Fiona; Breen, Gerome

    2016-01-01

    Objectives Clozapine is an atypical antipsychotic primarily prescribed for treatment-resistant schizophrenia. We tested the specific effect of clozapine versus other drug treatments on whole-blood gene expression in a sample of patients with psychosis from the UK. Methods A total of 186 baseline whole-blood samples from individuals receiving treatment for established psychosis were analysed for gene expression on Illumina HumanHT-12.v4 BeadChips. After standard quality-control procedures, 152 samples remained, including 55 from individuals receiving clozapine. In a within-case study design, weighted gene correlation network analysis was used to identify modules of coexpressed genes. The influence of mood stabilizers, lithium carbonate/lithium citrate and sodium valproate was studied to identify their possible roles as confounders. Results Individuals receiving clozapine as their only antipsychotic (clozapine monotherapy) had a nominal association with one gene-expression module, whereas no significant change in gene expression was found for other drugs. Conclusion Overall, this study does not provide evidence that clozapine treatment induces medium to large different gene-expression patterns in human whole blood versus other antipsychotic treatments. This does not rule out the possibility of smaller effects as observed for other common antipsychotic treatments. PMID:27315048

  6. Receptor expression and responsiveness of human peripheral blood mononuclear cells to a human cytomegalovirus encoded CC chemokine.

    PubMed

    Zheng, Qi; Xu, Jun; Gao, Huihui; Tao, Ran; Li, Wei; Shang, Shiqiang; Gu, Weizhong

    2015-01-01

    Human cytomegalovirus is a ubiquitous pathogen that infects the majority of the world's population. After long period of time co-evolving with human being, this pathogen has developed several strategies to evade host immune surveillance. One of the major trick is encoding homologous to those of the host organism or stealing host cellular genes that have significant functions in immune system. To date, we have found several viral immune analogous which include G protein coupled receptor, class I major histocompatibility complex and chemokine. Chemokine is a small group of molecules which is defined by the presence of four cysteines in highly conserved region. The four kinds of chemokines (C, CC, CXC, and CX3C) are classified based on the arrangement of 1 or 2 N-terminal cysteine residues. UL128 protein is one of the analogous that encoded by human cytomegalovirus that has similar amino acid sequences to the human CC chemokine. It has been proved to be one of the essential particles that involved in human cytomegalovirus entry into epithelial/endothelial cells as well as macrophages. It is also the target of potent neutralizing antibodies in human cytomegalovirus-seropositive individuals. We had demonstrated the chemotactic trait of UL128 protein in our previous study. Recombinant UL128 in vitro has the ability to attract monocytes to the infection region and enhances peripheral blood mononuclear cell proliferation by activating the MAPK/ERK signaling pathway. However, the way that this viral encoded chemokine interacting with peripheral blood mononuclear cells and the detailed mechanism that involving the virus entry into host cells keeps unknown. Here we performed in vitro investigation into the effects of UL128 protein on peripheral blood mononuclear cell's activation and receptor binding, which may help us further understand the immunomodulatory function of UL128 protein as well as human cytomegalovirus diffusion mechanism.

  7. Rapid measurement of fibrinogen concentration in whole blood using a steel ball coagulometer

    PubMed Central

    Schlimp, Christoph J.; Khadem, Anna; Klotz, Anton; Solomon, Cristina; Hochleitner, Gerald; Ponschab, Martin; Redl, Heinz; Schöchl, Herbert

    2015-01-01

    BACKGROUND Fibrinogen plays a key role in hemostasis and is the first coagulation factor to reach critical levels in bleeding patients. Current European guidelines on the management of traumatic or perioperative bleeding recommend fibrinogen supplementation at specific threshold levels. Whole blood viscoelastic tests provide fast evaluation of fibrin deficits. Fast measurement of plasma fibrinogen concentration is not yet available. We investigated a method to rapidly determine whole blood fibrinogen concentration using standard Clauss assays and a steel ball coagulometer and provide an estimate of the “plasma-equivalent” fibrinogen concentration within minutes by adjustment of the measured whole blood fibrinogen concentration with a quickly measureable hemoglobin-derived hematocrit. METHODS The feasibility of this approach was tested with a Clauss assay using multiple porcine fresh blood samples obtained during in vivo bleeding, hemodilution, and after treatment with hemostatic therapy. Two different Clauss assays were then tested using multiple human volunteers’ blood samples diluted in vitro and supplemented with fibrinogen concentrate. Comparative measurements with fibrin-based thromboelastometry tests were performed. RESULTS Regression and Bland-Altman analyses of derived “plasma-equivalent” fibrinogen and measured plasma fibrinogen concentration was excellent in porcine and human blood samples, especially in the ranges relevant to traumatic or perioperative bleeding. CONCLUSION Fast whole blood fibrinogen measurements could be considered as an alternative to plasma fibrinogen measurement for acute bleeding management in trauma and perioperative care settings. Further studies are needed to prove this concept and determine the turnaround times for its clinical application in emergency departments and operating theaters. PMID:25742256

  8. Release of gelatinase and superoxide from human mononuclear phagocytes in response to particulate Tamm Horsfall protein.

    PubMed Central

    Thomas, D. B.; Davies, M.; Williams, J. D.

    1993-01-01

    This study describes the in vitro activation of human mononuclear phagocytes by particulate Tamm Horsfall protein (THP). Peripheral blood monocytes phagocytosed THP particles with the accompanying release of superoxide radicals, N-acetyl-beta-D-glucosaminidase, and neutral metalloproteinase. Immunoprecipitation and substrate gel analysis identified the neutral proteinase as a 95-kd gelatinase. A comparison with other particulate ligands highlighted the specificity of the response to THP and showed that the magnitude of the response was comparable with that obtained with lipopolysaccharide (100 micrograms/ml). Parallel studies using peritoneal macrophages resulted in a similar pattern of enzyme release and reactive oxygen species synthesis. THP has been implicated in the pathogenesis of tubulointerstitial nephritis associated with reflux nephropathy. The present study indicates that an inflammatory response initiated by a neutrophil-THP interaction may be extended into a chronic phase via the activation of mononuclear phagocytes. The subsequent release of reactive oxygen metabolites and proteinases may contribute to the tissue damage and fibrosis associated with chronic immune-mediated tubulointerstitial nephritis. Images Figure 4 Figure 5 Figure 6 PMID:8380953

  9. Particulate matter induces prothrombotic microparticle shedding by human mononuclear and endothelial cells.

    PubMed

    Neri, Tommaso; Pergoli, Laura; Petrini, Silvia; Gravendonk, Lotte; Balia, Cristina; Scalise, Valentina; Amoruso, Angela; Pedrinelli, Roberto; Paggiaro, Pierluigi; Bollati, Valentina; Celi, Alessandro

    2016-04-01

    Particulate airborne pollution is associated with increased cardiopulmonary morbidity. Microparticles are extracellular vesicles shed by cells upon activation or apoptosis involved in physiological processes such as coagulation and inflammation, including airway inflammation. We investigated the hypothesis that particulate matter causes the shedding of microparticles by human mononuclear and endothelial cells. Cells, isolated from the blood and the umbilical cords of normal donors, were cultured in the presence of particulate from a standard reference. Microparticles were assessed in the supernatant as phosphatidylserine concentration. Microparticle-associated tissue factor was assessed by an one-stage clotting assay. Nanosight technology was used to evaluate microparticle size distribution. Particulate matter induces a dose- and time- dependent, rapid (1h) increase in microparticle generation in both cells. These microparticles express functional tissue factor. Particulate matter increases intracellular calcium concentration and phospholipase C inhibition reduces microparticle generation. Nanosight analysis confirmed that upon exposure to particulate matter both cells express particles with a size range consistent with the definition of microparticles (50-1000 nm). Exposure of mononuclear and endothelial cells to particulate matter upregulates the generation of microparticles at least partially mediated by calcium mobilization. This observation might provide a further link between airborne pollution and cardiopulmonary morbidity. PMID:26876346

  10. Particulate matter induces prothrombotic microparticle shedding by human mononuclear and endothelial cells.

    PubMed

    Neri, Tommaso; Pergoli, Laura; Petrini, Silvia; Gravendonk, Lotte; Balia, Cristina; Scalise, Valentina; Amoruso, Angela; Pedrinelli, Roberto; Paggiaro, Pierluigi; Bollati, Valentina; Celi, Alessandro

    2016-04-01

    Particulate airborne pollution is associated with increased cardiopulmonary morbidity. Microparticles are extracellular vesicles shed by cells upon activation or apoptosis involved in physiological processes such as coagulation and inflammation, including airway inflammation. We investigated the hypothesis that particulate matter causes the shedding of microparticles by human mononuclear and endothelial cells. Cells, isolated from the blood and the umbilical cords of normal donors, were cultured in the presence of particulate from a standard reference. Microparticles were assessed in the supernatant as phosphatidylserine concentration. Microparticle-associated tissue factor was assessed by an one-stage clotting assay. Nanosight technology was used to evaluate microparticle size distribution. Particulate matter induces a dose- and time- dependent, rapid (1h) increase in microparticle generation in both cells. These microparticles express functional tissue factor. Particulate matter increases intracellular calcium concentration and phospholipase C inhibition reduces microparticle generation. Nanosight analysis confirmed that upon exposure to particulate matter both cells express particles with a size range consistent with the definition of microparticles (50-1000 nm). Exposure of mononuclear and endothelial cells to particulate matter upregulates the generation of microparticles at least partially mediated by calcium mobilization. This observation might provide a further link between airborne pollution and cardiopulmonary morbidity.

  11. Adhesion molecules on the endothelium and mononuclear cells in human atherosclerotic lesions.

    PubMed Central

    van der Wal, A. C.; Das, P. K.; Tigges, A. J.; Becker, A. E.

    1992-01-01

    Atherosclerotic lesions show features of a cell-mediated immune inflammatory process. From this viewpoint, the potential role of arterial endothelium in the recruitment of mononuclear cells (T lymphocytes and macrophages) was studied. The endothelium of diffuse intimal thickening (DIT) and atheromatous plaques (AP) in human coronary arteries and abdominal aortas was characterized for the expression of adhesion molecules ELAM-1, ICAM-1, and the major histocompatibility complex (MHC) class II antigens HLA-DR/DP. A marked increase in expression of ICAM-1 and ELAM-1, and to a lesser extent HLA-DR/DP was observed on endothelial cells that were adjacent to subendothelial infiltrates of T lymphocytes (CD3+, CD11a+, HLA-DR/DP+) and macrophages (CD14+, CD11a+, CD11c+, HLA-DR/DP+). This contrasted with a lower or absent expression of these activation markers at sites without prominent inflammatory cell infiltrates. These findings could be demonstrated in DIT as well as in AP. The observations suggest that cytokines produced by the subintimal infiltrates may activate the endothelium in a similar way as is observed in the microvasculature at sites of immune inflammation. The expression of these activation markers in the microvasculature is associated with enhanced leukocyte adhesion, permeability for macromolecules, and procoagulant activity, features known to occur also in early experimental atherosclerosis. The findings therefore support the concept that arterial endothelium plays an active role in the recruitment of mononuclear cells in atherosclerotic lesions. Images Figure 1 PMID:1281621

  12. Whole blood metal ion measurement reproducibility between different laboratories.

    PubMed

    Rahmé, Michel; Lavigne, Martin; Barry, Janie; Cirtiu, Ciprian Mihai; Bélanger, Patrick; Vendittoli, Pascal-André

    2014-11-01

    Monitoring patients' metal ion blood concentrations can be useful in cases of problematic metal on metal hip implants. Our objective was to evaluate the reproducibility of metal ion level values measured by two different laboratories. Whole blood samples were collected in 46 patients with metal on metal hip arthroplasty. For each patients, two whole blood samples were collected and analyzed by two laboratories. Laboratory 1 had higher results than laboratory 2. There was a clinically significant absolute difference between the two laboratories, above the predetermined threshold, 35% of Cr samples and 38% of Co samples. All laboratories do not use the same technologies for their measurements. Therefore, decision to revise a metal on metal hip arthroplasty should rely on metal ion trends and have to be done in the same laboratory.

  13. Platelet concentrates, from whole blood or collected by apheresis?

    PubMed

    van der Meer, Pieter F

    2013-04-01

    Platelet concentrates can be isolated from donated whole blood with the platelet-rich plasma-method or the buffy coat-method. Alternatively, platelets can be obtained by apheresis, harvesting the platelets but returning all other cells to the donor. The quality and characteristics of platelets during storage are affected by a number of factors, such as anticoagulant, centrifugation and processing after collection, and pre- or post storage pooling, but when comparing literature on the various methods, most differences balance out. To have sufficient platelets to treat an adult patient, whole-blood-derived platelet concentrates need pooling of multiple donations, thereby increasing the risk of infectious agent transmission at least two-fold as compared with apheresis units. Allo immunization rates, acute reaction rates, and transfusion related acute lung injury rates are not different. Apheresis donation procedures have fewer adverse events. All these factors need to be considered and weighed when selecting a method of platelet collection for a blood center.

  14. Velocity measurements in whole blood using acoustic resolution photoacoustic Doppler.

    PubMed

    Brunker, Joanna; Beard, Paul

    2016-07-01

    Acoustic resolution photoacoustic Doppler velocimetry promises to overcome the spatial resolution and depth penetration limitations of current blood flow measuring methods. Despite successful implementation using blood-mimicking fluids, measurements in blood have proved challenging, thus preventing in vivo application. A common explanation for this difficulty is that whole blood is insufficiently heterogeneous relative to detector frequencies of tens of MHz compatible with deep tissue photoacoustic measurements. Through rigorous experimental measurements we provide new insight that refutes this assertion. We show for the first time that, by careful choice of the detector frequency and field-of-view, and by employing novel signal processing methods, it is possible to make velocity measurements in whole blood using transducers with frequencies in the tens of MHz range. These findings have important implications for the prospects of making deep tissue measurements of blood flow relevant to the study of microcirculatory abnormalities associated with cancer, diabetes, atherosclerosis and other conditions. PMID:27446707

  15. Velocity measurements in whole blood using acoustic resolution photoacoustic Doppler

    PubMed Central

    Brunker, Joanna; Beard, Paul

    2016-01-01

    Acoustic resolution photoacoustic Doppler velocimetry promises to overcome the spatial resolution and depth penetration limitations of current blood flow measuring methods. Despite successful implementation using blood-mimicking fluids, measurements in blood have proved challenging, thus preventing in vivo application. A common explanation for this difficulty is that whole blood is insufficiently heterogeneous relative to detector frequencies of tens of MHz compatible with deep tissue photoacoustic measurements. Through rigorous experimental measurements we provide new insight that refutes this assertion. We show for the first time that, by careful choice of the detector frequency and field-of-view, and by employing novel signal processing methods, it is possible to make velocity measurements in whole blood using transducers with frequencies in the tens of MHz range. These findings have important implications for the prospects of making deep tissue measurements of blood flow relevant to the study of microcirculatory abnormalities associated with cancer, diabetes, atherosclerosis and other conditions. PMID:27446707

  16. Productive Infection of Human Peripheral Blood Mononuclear Cells by Feline Immunodeficiency Virus: Implications for Vector Development

    PubMed Central

    Johnston, James; Power, Christopher

    1999-01-01

    Feline immunodeficiency virus (FIV) is a lentivirus causing immune suppression and neurological disease in cats. Like primate lentiviruses, FIV utilizes the chemokine receptor CXCR4 for infection. In addition, FIV gene expression has been demonstrated in immortalized human cell lines. To investigate the extent and mechanism by which FIV infected primary and immortalized human cell lines, we compared the infectivity of two FIV strains, V1CSF and Petaluma, after cell-free infection. FIV genome was detected in infected human peripheral blood mononuclear cells (PBMC) and macrophages at 21 and 14 days postinfection, respectively. Flow cytometry analysis of FIV-infected human PBMC indicated that antibodies to FIV p24 recognized 12% of the cells. Antibodies binding the CCR3 chemokine receptor maximally inhibited infection of human PBMC by both FIV strains compared to antibodies to CXCR4 or CCR5. Reverse transcriptase levels increased in FIV-infected human PBMC, with detection of viral titers of 101.3 to 102.1 50% tissue culture infective doses/106 cells depending on the FIV strain examined. Cell death in human PBMC infected with either FIV strain was significantly elevated relative to uninfected control cultures. These findings indicate that FIV can productively infect primary human cell lines and that viral strain specificity should be considered in the development of an FIV vector for gene therapy. PMID:9971834

  17. Whole blood viscosity of preterm infants - differences to term neonates.

    PubMed

    Kuss, N; Bauknecht, E; Felbinger, C; Gehm, J; Gehm, L; Pöschl, J; Ruef, P

    2015-01-01

    Determination of shear stresses at given shear rates with the LS 300 allows approximation of flow curves by mathematical models and to calculate viscosities of non-Newtonian fluids across the entire physiological shear range. Particular in preterm neonates, which in comparison to term neonates and adults have a substantially lower mean arterial blood pressure (MAP), rheological blood properties play a decisive role to maintain the perfusion. Whole blood viscosity was measured in umbilical cord blood taken from 34 preterm neonates using the LS 300 viscometer. In addition aggregation index, plasma viscosity, hematocrit and red blood cell (RBC) deformability was determined. The highest quality of approximation of the flow curve of whole blood was achieved by the method of Ostwald. Shear stresses of whole blood of preterm neonates were significantly lower compared to term neonates at similar shear rates (velocity range 6.16 s-1-50 s-1). With hematocrit calculated to 0.40 the related exponent (n) of the viscosity of preterm neonates (by Ostwald) showed a significant lower exponent (n) (0.71 ± 0.07) than term neonates (0.76 ± 0.06). The highest quality of approximation of the flow curve of plasma was achieved with the model of Ostwald in preterm neonates as well as in term neonates. The viscosity of plasma determined by Newton was lower for preterm neonates (0.89 ± 0.19) than for term neonates (1.04 ± 0.16). Concurrent to term neonates the whole blood of preterm neonates showed a very low aggregation index (term neonates 2.97 ± 2.10; preterm neonates 2.37 ± 1.32) and preterm neonates showed higher RBC deformability than term neonates. Because of the physiologically lower MAP and the particular viscous properties of neonatal blood special attention should be given when treating neonates with conventional blood products derived from adult donors. PMID:26444619

  18. Whole blood analysis of kwashiorkor cases by PIXE

    NASA Astrophysics Data System (ADS)

    Spoelstra, B.; Sellschop, J. P. F.; Annegarn, H. J.; Renan, M. J.

    1981-03-01

    A particle-induced X-ray emission (PIXE) analysis of whole blood has been performed on samples supplied by a hospital. Of these samples 45 were from kwashiorkor cases and 37 were from a control group. The relative concentrations determined for the elements Cl, K, Ca, Fe, Cu and Zn indicated significant deficiencies in all these elements for the kwashiorkor group. The statistical treatment of the results is discussed as well as the suitability of PIXE for this work.

  19. Whole blood analysis rotor assembly having removable cellular sedimentation bowl

    DOEpatents

    Burtis, C.A.; Johnson, W.F.

    1975-08-26

    A rotor assembly for performing photometric analyses using whole blood samples is described. Following static loading of a gross blood sample within a centrally located, removable, cell sedimentation bowl, the red blood cells in the gross sample are centrifugally separated from the plasma, the plasm displaced from the sedimentation bowl, and measured subvolumes of plasma distributed to respective sample analysis cuvettes positioned in an annular array about the rotor periphery. Means for adding reagents to the respective cuvettes are also described. (auth)

  20. Analysis of cyanide in whole blood of dosed cathartids

    USGS Publications Warehouse

    Krynitsky, A.J.; Wiemeyer, Stanley N.; Hill, E.F.; Carpenter, J.W.

    1986-01-01

    A gas-liquid chromatographic method was modified to quantify both unmetabolized ('free') and metabolized ('bound', i.e., thiocyanates) cyanides. The methods for both are efficient and sensitive to 0.05 ppm. Repeated freezing and thawing of whole blood from treated cathartids caused an initial increase in free cyanide concentrations, followed by a gradual decline to a plateau. Bound cyanide concentrations declined after repeated freezing and thawing.

  1. Molecular recognition of HER-1 in whole-blood samples.

    PubMed

    Moldoveanu, Iuliana; Stanciu Gavan, Camelia; Stefan-van Staden, Raluca-Ioana

    2014-11-01

    Multimode sensing was proposed for molecular screening and recognition of HER-1 in whole blood. The tools used for molecular recognition were platforms based on nanostructured materials such as the complex of Mn(III) with meso-tetra (4-carboxyphenyl) porphyrin, and maltodextrin (dextrose equivalence between 4 and 7), immobilized in diamond paste, graphite paste or C60 fullerene paste. The identification of HER-1 in whole-blood samples, at molecular level, is performed using stochastic mode and is followed by the quantification of it using stochastic and differential pulse voltammetry modes. HER-1 can be identified in the concentration range between 280 fg/ml and 4.86 ng/ml using stochastic mode, this making possible the early detection of cancers such as gastrointestinal, pancreatic and lung cancers. The recovery tests performed using whole-blood samples proved that the platforms can be used for identification and quantification of HER-1 with high sensitivity and reliability in such samples, these making them good molecular screening tools.

  2. Molecular recognition of HER-1 in whole-blood samples.

    PubMed

    Moldoveanu, Iuliana; Stanciu Gavan, Camelia; Stefan-van Staden, Raluca-Ioana

    2014-11-01

    Multimode sensing was proposed for molecular screening and recognition of HER-1 in whole blood. The tools used for molecular recognition were platforms based on nanostructured materials such as the complex of Mn(III) with meso-tetra (4-carboxyphenyl) porphyrin, and maltodextrin (dextrose equivalence between 4 and 7), immobilized in diamond paste, graphite paste or C60 fullerene paste. The identification of HER-1 in whole-blood samples, at molecular level, is performed using stochastic mode and is followed by the quantification of it using stochastic and differential pulse voltammetry modes. HER-1 can be identified in the concentration range between 280 fg/ml and 4.86 ng/ml using stochastic mode, this making possible the early detection of cancers such as gastrointestinal, pancreatic and lung cancers. The recovery tests performed using whole-blood samples proved that the platforms can be used for identification and quantification of HER-1 with high sensitivity and reliability in such samples, these making them good molecular screening tools. PMID:25277089

  3. A field-deployable device for the rapid detection of cyanide poisoning in whole blood

    NASA Astrophysics Data System (ADS)

    Boehringer, Hans; Tong, Winnie; Chung, Roy; Boss, Gerry; O'Farrell, Brendan

    2012-06-01

    Feasibility of a field-deployable device for the rapid and early diagnosis of cyanide poisoning in whole blood using the spectral shift of the vitamin B12 precursor cobinamide upon binding with cyanide as an indicator is being assessed. Cyanide is an extremely potent and rapid acting poison with as little as 50 mg fatal to humans. Cyanide poisoning has been recognized as a threat from smoke inhalation and potentially through weapons of mass destruction. Currently, no portable rapid tests for the detection of cyanide in whole blood are available. Cobinamide has an extremely high affinity for cyanide and captures hemoglobin associated cyanide from red blood cells. Upon binding of cyanide, cobinamide undergoes a spectral shift that can be measured with a spectrophotometer. We have combined the unique cyanide-binding properties of cobinamide with blood separation technology, sample transport and a detection system, and are developing a rapid, field deployable, disposable device which will deliver an intuitive result to a first responder, allowing for rapid response to exposure events. Feasibility of the cobinamide-Cyanide chemistry in a rapid test using a whole blood sample from a finger-stick has been demonstrated with an assay time from sample collection to a valid result of under 5 minutes. Data showing the efficacy of the diagnostic method and initial device design concepts will be shown.

  4. Human cord blood-derived mononuclear cell transplantation for viral encephalitis-associated cognitive impairment: a case report

    PubMed Central

    2013-01-01

    Introduction Herpes simplex virus is the most common cause of sporadic viral encephalitis. Cognitive impairments persist in most patients who survive herpes simplex virus-caused encephalitis after undergoing currently available treatments. This is the first report on the development of human cord blood-derived mononuclear cell transplantation as a new treatment intervention to improve the prognosis of sequelae of viral encephalitis. Case presentation An 11-year-old Han Chinese boy developed sequelae of viral encephalitis with cognitive, mental and motor impairments in the 8 months following routine treatments. Since receiving allogeneic cord blood-derived mononuclear cell transplantation combined with comprehensive rehabilitation therapies 7 years ago, the patient’s health has significantly improved and remained stable. Conclusions Human cord blood-derived mononuclear cell transplantation may be a potential therapeutic strategy for treating the neuropsychiatric and neurobehavioral sequelae of viral encephalitis. PMID:23835552

  5. Human mononuclear cell function after 4 C storage during 1-G and microgravity conditions of spaceflight

    NASA Technical Reports Server (NTRS)

    Meehan, Richard; Taylor, Gerald; Lionetti, Fabian; Neale, Laurie; Curren, Tim

    1989-01-01

    To investigate the possibility of restoring immune competence of crewmembers during a prolonged spaceflight by infusions of autologous blood components, the effect of storage at 4 C aboard Space Shuttle Columbia (Mission 61-c) on the activity of human peripheral blood mononuclear cells (PBMNCs), stored as leukocyte concentrates in autologous plasa, was investigated. The results of preflight storage at 4 C demonstrated a progressive daily loss in mitogen-stimulated protein synthesis, and thymidine uptake, as well as a progressive reduction in the percentage of PBMNCs expressing cell-surface phenotype markers. The ability of PBMNCs stored at 4 C for 8 d in Columbia's middeck, to become activated and proliferate in vitro was similar to that of cells that remained for 7 d on ground.

  6. The effects of teriflunomide on lymphocyte subpopulations in human peripheral blood mononuclear cells in vitro.

    PubMed

    Li, Li; Liu, Jingchun; Delohery, Thomas; Zhang, Donghui; Arendt, Christopher; Jones, Catherine

    2013-12-15

    Teriflunomide is an inhibitor of dihydro-orotate dehydrogenase (DHODH), and is hypothesized to ameliorate multiple sclerosis by reducing proliferation of stimulated lymphocytes. We investigated teriflunomide's effects on proliferation, activation, survival, and function of stimulated human peripheral blood mononuclear cell subsets in vitro. Teriflunomide had little/no impact on lymphocyte activation but exerted significant dose-dependent inhibition of T- and B-cell proliferation, which was uridine-reversible (DHODH-dependent). Viability analyses showed no teriflunomide-associated cytotoxicity. Teriflunomide significantly decreased release of several pro-inflammatory cytokines from activated monocytes in a DHODH-independent fashion. In conclusion, teriflunomide acts on multiple immune cell types and processes via DHODH-dependent and independent mechanisms. PMID:24182769

  7. Grass immunotherapy induces inhibition of allergen-specific human peripheral blood mononuclear cell proliferation.

    PubMed

    Baskar, S; Hamilton, R G; Norman, P S; Ansari, A A

    1997-02-01

    The peripheral blood mononuclear cells (PBMC) from humans allergic to grass pollens (GR+ subjects) show strong in vitro proliferative responses to purified allergens from Lolium perenne pollen Lol p 1, and to a lesser extent to Lol p 2 and Lol p 3. By contrast, PBMC from grass allergic patients undergoing immunotherapy (GR + IT subjects) exhibit a very poor Lol p-specific proliferative response, similar to that observed in nongrass allergic subjects (GR-subjects). Unlike GR-subjects, both GR+ and GR + IT subjects have high levels of antigen-specific serum IgG and IgE antibodies to Lol p 1, Lol p 2 and Lol p 3. While GR+ subjects exhibit a significant correlation between antigen-specific serum antibody and PBMC responses, GR + IT subjects do not show a correlation between the two responses. The possible mechanisms by which immunotherapy may modulate allergen-specific T cell proliferative response are discussed.

  8. The effect of catechol on human peripheral blood mononuclear cells (in vitro study).

    PubMed

    Bukowska, Bożena; Michałowicz, Jaromir; Marczak, Agnieszka

    2015-01-01

    Catechol also known as pyrocatechol or 1,2-dihydroxybenzene is formed endogenously in the organism from neurotransmitters including adrenaline, noradrenaline, and dopamine. It is also a metabolite of many drugs like DOPA, isoproterenol or aspirin and it is also formed in the environment during transformation of various xenobiotics. We evaluated in vitro the effect of catechol on the structure and function of human peripheral blood mononuclear cells (PBMCs). The cells were incubated with xenobiotic at concentration range from 2 to 500μg/mL for 1h. Human blood mononuclear cells were obtained from leucocyte-platelet buffy coat taken from healthy donors in the Blood Bank of Łódź, Poland. Using flow cytometry we have evaluated necrotic, apoptotic and morphological changes in PBMCs incubated with catechol. Moreover, we have estimated changes in reactive oxygen species (ROS) formation, protein carbonylation and lipid peroxidation in the cells studied. The compound studied provoked necrotic (from 250μg/mL), apoptotic (from 100μg/mL), and morphological changes (from 250μg/mL) in the incubated cells. We have also noted that catechol decreased H2DCF oxidation at 2 and 10μg/mL but at higher concentrations of 250 and 500μg/mL it caused statistically significant increase in the oxidation of this probe. We also observed an increase in lipid peroxidation (from 250μg/mL) and protein carbonylation (from 50μg/mL) of PBMCs. It was observed that catechol only at high concentrations was capable of inducing changes in PBMCs. The obtained results clearly showed that catechol may induce change in PBMCs only in the caste of poisoning with this compound. PMID:25528409

  9. Novel Fc gamma receptor I family gene products in human mononuclear cells.

    PubMed Central

    Porges, A J; Redecha, P B; Doebele, R; Pan, L C; Salmon, J E; Kimberly, R P

    1992-01-01

    Unlike the human Fc gamma RII and Fc gamma RIII families, which exhibit considerable diversity at both the nucleic acid and protein levels, the human Fc gamma RI family has only a single recognized product expressed as a 70-kD cell surface receptor with high affinity for monomeric IgG (hFc gamma RIa1). Using both polymerase chain reaction-based amplification and Northern hybridization, we document multiple interferon-gamma-inducible hFc gamma RI RNA transcripts in human mononuclear cells and neutrophils. The sequences of two of these Fc gamma RI related transcripts indicate that they are alternatively spliced products of a second Fc gamma RI family gene, termed Fc gamma RIB. The cDNA derived from the larger of these transcripts, termed hFc gamma RIb1, encodes a surface molecule that is not recognized by Fc gamma RI specific monoclonal antibodies when transfected into COS-7 cells. Unlike the interferon-gamma-inducible hFc gamma RIA gene product, hFc gamma RIb1 does not bind monomeric IgG with high affinity. However, both hFc gamma RIa1 and hFc gamma RIb1 do bind aggregated human IgG. Previously unrecognized diversity within the hFc gamma RI family includes an interferon-gamma-inducible, putative low affinity Fc gamma receptor that may play an important role in the mechanism by which Fc gamma receptors participate in the humoral immune response. Images PMID:1430234

  10. High-throughput acoustic separation of platelets from whole blood.

    PubMed

    Chen, Yuchao; Wu, Mengxi; Ren, Liqiang; Liu, Jiayang; Whitley, Pamela H; Wang, Lin; Huang, Tony Jun

    2016-09-21

    Platelets contain growth factors which are important in biomedical and clinical applications. In this work, we present an acoustic separation device for high-throughput, non-invasive platelet isolation. In particular, we separated platelets from whole blood at a 10 mL min(-1) throughput, which is three orders of magnitude greater than that of existing acoustic-based platelet separation techniques. Without sample dilution, we observed more than 80% RBC/WBC removal and platelet recovery. High throughput, high separation efficiency, and biocompatibility make this device useful for many clinical applications. PMID:27477388

  11. High-throughput acoustic separation of platelets from whole blood.

    PubMed

    Chen, Yuchao; Wu, Mengxi; Ren, Liqiang; Liu, Jiayang; Whitley, Pamela H; Wang, Lin; Huang, Tony Jun

    2016-09-21

    Platelets contain growth factors which are important in biomedical and clinical applications. In this work, we present an acoustic separation device for high-throughput, non-invasive platelet isolation. In particular, we separated platelets from whole blood at a 10 mL min(-1) throughput, which is three orders of magnitude greater than that of existing acoustic-based platelet separation techniques. Without sample dilution, we observed more than 80% RBC/WBC removal and platelet recovery. High throughput, high separation efficiency, and biocompatibility make this device useful for many clinical applications.

  12. [Detection of stimulator-induced cytotoxicity of human mononuclear cells in short-term culture].

    PubMed

    von Baehr, R; Timm, M; Fröbe, I

    1983-01-01

    A new test modification for the detection of stimulant-induced cytotoxicity of human mononuclear blood cells is described. Papain-treated human erythrocytes were used as indicator cells. The effector cells were lymphocytes. The ratio of target cells to effector cells was 10/1. Haemoglobin as a marker of lysis of the erythrocytes, released in the supernatant, was measured quantitatively in form of its pseudoperoxidase-activity. PHA, ConA and tannic acid were ascertained and tested as stimulants of cytotoxicity. The reaction was inhibitable by anti-human-lymphocyte-globulin. The test conditions were optimized in regard to incubation time, -temperature, -vessels, culture medium and target cells. The technique is easy to manipulate, has only slight pretensions to the equipment of the laboratory and appears to be very effective. We recommend to apply this method of stimulant-induced cytotoxicity within the detection of the immune state, especially in the progress of immunopathological diseases and the analysis of efficiency of immunosuppressive therapy. PMID:6224413

  13. Influence of calorie reduction on DNA repair capacity of human peripheral blood mononuclear cells.

    PubMed

    Matt, Katja; Burger, Katharina; Gebhard, Daniel; Bergemann, Jörg

    2016-03-01

    Caloric restrictive feeding prolongs the lifespan of a variety of model organisms like rodents and invertebrates. It has been shown that caloric restriction reduces age-related as well as overall-mortality, reduces oxidative stress and influences DNA repair ability positively. There are numerous studies underlining this, but fewer studies involving humans exist. To contribute to a better understanding of the correlation of calorie reduction and DNA repair in humans, we adapted the host cell reactivation assay to an application with human peripheral blood mononuclear cells. Furthermore, we used this reliable and reproducible assay to research the influence of a special kind of calorie reduction, namely F. X. Mayr therapy, on DNA repair capacity. We found a positive effect in all persons with low pre-existing DNA repair capacity. In individuals with normal pre-existing DNA repair capacity, no effect on DNA repair capacity was detectable. Decline of DNA repair, accumulation of oxidative DNA damages, mitochondrial dysfunction, telomere shortening as well as caloric intake are widely thought to contribute to aging. With regard to that, our results can be considered as a strong indication that calorie reduction may support DNA repair processes and thus contribute to a healthier aging.

  14. TiO2 nanoparticles and bulk material stimulate human peripheral blood mononuclear cells☆

    PubMed Central

    Becker, Kathrin; Schroecksnadel, Sebastian; Geisler, Simon; Carriere, Marie; Gostner, Johanna M.; Schennach, Harald; Herlin, Nathalie; Fuchs, Dietmar

    2014-01-01

    Nanomaterials are increasingly produced and used throughout recent years. Consequently the probability of exposure to nanoparticles has risen. Because of their small 1–100 nm size, the physicochemical properties of nanomaterials may differ from standard bulk materials and may pose a threat to human health. Only little is known about the effects of nanoparticles on the human immune system. In this study, we investigated the effects of TiO2 nanoparticles and bulk material in the in vitro model of human peripheral blood mononuclear cells (PBMC) and cytokine-induced neopterin formation and tryptophan breakdown was monitored. Both biochemical processes are closely related to the course of diseases like infections, atherogenesis and neurodegeneration. OCTi60 (25 nm diameter) TiO2 nanoparticles and bulk material increased neopterin production in unstimulated PBMC and stimulated cells significantly, the effects were stronger for OCTi60 compared to bulk material, while P25 TiO2 (25 nm diameter) nanoparticles had only little influence. No effect of TiO2 nanoparticles on tryptophan breakdown was detected in unstimulated cells, whereas in stimulated cells, IDO activity and IFN-γ production were suppressed but only at the highest concentrations tested. Because neopterin was stimulated and tryptophan breakdown was suppressed in parallel, data suggests that the total effect of particles would be strongly pro-inflammatory. PMID:24361406

  15. [Distribution of chemical elements in whole blood and plasma].

    PubMed

    Barashkov, G K; Zaĭtseva, L I; Kondakhchan, M A; Konstantinova, E A

    2003-01-01

    The distribution factor (Fd) of 35 elements of plasma and whole blood in 26 healthy men and women was detected by ICP-OES. Usilig this parameter the elements were subdivided in 3 pools. 9 of them have Fd higher than 1.5 ("elements of plasma"-Ag, Ca, Cu, In, Li, Na, Se, Si, Sr); 6 have lower than 0.5 ("elements of blood cells"-Fe, K, Mn, Ni, V, Zn), other 20-about 1 ("blood elements"). Fd of all elements depends on ionic radius. Elements of 2nd sub-groups of all groups of Mendeleev's periodic table ("heavy metals") depend on the similar law: "with growing of ionic radius the concentration of elements in plasma enhances". In alkaline metals Fd depends on the opposite law:" with growing of ionic radius of alkaline metal the quantity of elements in blood cells enhance". Dependence of Fd on the value of atomic mass in periods or in exterior electronic cloud (s-, p-, d-, f-) was not established. The table of distribution of all detected elements in whole blood in relation to 8 macroelements (Ca, Mg, K, Na, S, P, Fe, Zn,) is presented, as a basic diagnostic criteria in metal-ligand homeostasis disturbance.

  16. Microemboli Characterization in Whole Blood Medium by Laser Scattering Method

    NASA Astrophysics Data System (ADS)

    Kim, Jungkuk

    The current light scattering method to detect the thromboemboli generated inside blood vessels or by direct contact of biomaterials has been a powerful method because of its advantages: continuous real time measurement, non-ionizing radiation, practically unlimited aggregate size distribution, no disturbance of blood flow, and low cost. But this method only detects the number and size of thromboemboli that is assumed to be known. If there are air bubbles or foreign particles, this method can not distinguish them from the thromboemboli. An improved light scattering detection method that can characterize emboli, air bubbles, or any foreign particles, their number and size and also their nature, will be proposed by finding coefficients of a Legendre polynomial expansion of phase functions for a microembolus, air bubble, or a foreign particle. The scattered light distribution in whole blood medium is obtained by deriving and solving a multiple order scattering transport approximation of the radiative transport equation. This approximation will be derived on the basis of the fact that blood cells are highly anisotropic scatterers and applied to detect and characterize the microemboli mentioned above. Also a more practical method is devised. This method uses three different scattering angles that produce maximum differences in scattering phase function of microemboli according to their nature. This method is tested for a cylindrical whole blood medium with several kinds of known inhomogeneous particles: polystyrene spheres, air bubbies, and clots. The results produced an excellent agreement with theoretical calculations.

  17. Bovine colostrum modulates immune activation cascades in human peripheral blood mononuclear cells in vitro.

    PubMed

    Jenny, Marcel; Pedersen, Ninfa R; Hidayat, Budi J; Schennach, Harald; Fuchs, Dietmar

    2010-04-01

    Bovine colostrum (BC) is the thick yellow fluid a lactating cow gives to a suckling calf during its first days of life to support the growth of the calf and prevent gastrointestinal infections until the calf has synthesized its own active immune defense system. BC contains a complex system of immune factors and has a long history of use in traditional medicine. In an approach to evaluate the effects of bovine colostrum (BC) on the T-cell/macrophage interplay, we investigated and compared the capacity of BC containing low and high amounts of lactose and lactoferrin to modulate tryptophan degradation and neopterin formation in unstimulated and mitogen-stimulated human peripheral blood mononuclear cells (PBMC). The present study shows significant immunomodulatory effects of these BC preparations in human PBMC, either by enhancing or suppressing the occurrence of a Th-1 type immune response. The amount of lactose present in BC seems to diminish the activity of BC in our test system, since BC with higher amounts of lactose attenuated the stimulatory as well as the suppressive activity of BC. PMID:20518274

  18. Effects of rare earth elements on telomerase activity and apoptosis of human peripheral blood mononuclear cells.

    PubMed

    Yu, Li; Dai, Yucheng; Yuan, Zhaokang; Li, Jie

    2007-04-01

    To study the effects of rare earth exposure on human telomerase and apoptosis of mononuclear cells from human peripheral blood (PBMNCs). The blood contents of 15 rare earth elements, including La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, and Y, were measured by inductively coupled plasma-mass spectrometry. Telomeric repeat amplification protocol assay and flow cytometer analysis were carried out to analyze the telomerase activity and apoptosis of PBMNCs, respectively. The total content of rare earth elements in the blood showed significant differences between the exposed group and the control group. The rare earth exposure increased the telomerase activity and the percentages of cells in the S-phase and the G2/M phase in PBMNCs, but it had no effect on the apoptotic rate of PBMNCs. Under the exposure to lower concentrations of rare earth elements, the telomerase activity of PBMNCs in the exposed group was higher than that of the control group, and there was no effect on the apoptotic rate of PBMNCs, but promoted the diploid DNA replication and increased the percentages of G2/M- and S-phase cells.

  19. In vitro transdifferentiation of human peripheral blood mononuclear cells to photoreceptor-like cells

    PubMed Central

    Komuta, Yukari; Ishii, Toshiyuki; Kaneda, Makoto; Ueda, Yasuji; Miyamoto, Kiyoko; Toyoda, Masashi; Umezawa, Akihiro

    2016-01-01

    ABSTRACT Direct reprogramming is a promising, simple and low-cost approach to generate target cells from somatic cells without using induced pluripotent stem cells. Recently, peripheral blood mononuclear cells (PBMCs) have attracted considerable attention as a somatic cell source for reprogramming. As a cell source, PBMCs have an advantage over dermal fibroblasts with respect to the ease of collecting tissues. Based on our studies involving generation of photosensitive photoreceptor cells from human iris cells and human dermal fibroblasts by transduction of photoreceptor-related transcription factors via retrovirus vectors, we transduced these transcription factors into PBMCs via Sendai virus vectors. We found that retinal disease-related genes were efficiently detected in CRX-transduced cells, most of which are crucial to photoreceptor functions. In functional studies, a light-induced inward current was detected in some CRX-transduced cells. Moreover, by modification of the culture conditions including additional transduction of RAX1 and NEUROD1, we found a greater variety of retinal disease-related genes than that observed in CRX-transduced PBMCs. These data suggest that CRX acts as a master control gene for reprogramming PBMCs into photoreceptor-like cells and that our induced photoreceptor-like cells might contribute to individualized drug screening and disease modeling of inherited retinal degeneration. PMID:27170256

  20. Dynamic changes in the proteome of human peripheral blood mononuclear cells with low dose ionizing radiation.

    PubMed

    Nishad, S; Ghosh, Anu

    2016-02-01

    Humans are continually exposed to ionizing radiation from natural as well as anthropogenic sources. Though biological effects of high dose radiation exposures have been well accepted, studies on low-to-moderate dose exposures (in the range of 50-500 mGy) have been strongly debated even as researchers continue to search for elusive 'radiation signatures' in humans. Proteins are considered as dynamic functional players that drive cellular responses. However, there is little proteomic information available in context of human exposure to ionizing radiation. In this study, we determined differential expressed proteins in G0 peripheral blood mononuclear cells (PBMCs) from healthy individuals 1h and 4h after 'ex vivo' exposure with two radiation doses (300 mGy and 1 Gy). Twenty-three proteins were found to be significantly altered in irradiated cells when compared to sham irradiated cells with fold change ± 1.5-fold (p ≤ 0.05), with only three proteins showing ≥ 2.5-fold change, either with dose or with time. Mass spectrometry analyses identified redox sensor protein, chloride intracellular channel protein 1 (CLIC-1), the antioxidant protein, peroxiredoxin-6 and the pro-survival molecular chaperone 78 KDa glucose regulated protein (GRP78) among the 23 modulated proteins. The mean coefficient of variation (CV) for the twenty-three radiation responsive protein spots was found to be 33.7% for 300 mGy and 48.3% for 1 Gy. We thus, conclude that the radiation proteomic response of G0 human PBMCs, which are in the resting stage of the cell cycle, involves moderate upregulation of protective mechanisms, with low inter-individual variability. This study will help further our understanding of cellular effects of low dose acute radiation in humans and contribute toward differential biomarker discovery. PMID:26921016

  1. Ultrasonic monitoring of droplets' evaporation: Application to human whole blood.

    PubMed

    Laux, D; Ferrandis, J Y; Brutin, D

    2016-09-01

    During a colloidal droplet evaporation, a sol-gel transition can be observed and is described by the desiccation time τD and the gelation time τG. These characteristic times, which can be linked to viscoelastic properties of the droplet and to its composition, are classically rated by analysis of mass droplet evolution during evaporation. Even if monitoring mass evolution versus time seems straightforward, this approach is very sensitive to environmental conditions (vibrations, air flow…) as mass has to be evaluated very accurately using ultra-sensitive weighing scales. In this study we investigated the potentialities of ultrasonic shear reflectometry to assess τD and τG in a simple and reliable manner. In order to validate this approach, our study has focused on blood droplets evaporation on which a great deal of work has recently been published. Desiccation and gelation times measured with shear ultrasonic reflectometry have been perfectly correlated to values obtained from mass versus time analysis. This ultrasonic method which is not very sensitive to environmental perturbations is therefore very interesting to monitor the drying of blood droplets in a simple manner and is more generally suitable for complex fluid droplets evaporation investigation. PMID:27150753

  2. Ultrasonic monitoring of droplets' evaporation: Application to human whole blood.

    PubMed

    Laux, D; Ferrandis, J Y; Brutin, D

    2016-09-01

    During a colloidal droplet evaporation, a sol-gel transition can be observed and is described by the desiccation time τD and the gelation time τG. These characteristic times, which can be linked to viscoelastic properties of the droplet and to its composition, are classically rated by analysis of mass droplet evolution during evaporation. Even if monitoring mass evolution versus time seems straightforward, this approach is very sensitive to environmental conditions (vibrations, air flow…) as mass has to be evaluated very accurately using ultra-sensitive weighing scales. In this study we investigated the potentialities of ultrasonic shear reflectometry to assess τD and τG in a simple and reliable manner. In order to validate this approach, our study has focused on blood droplets evaporation on which a great deal of work has recently been published. Desiccation and gelation times measured with shear ultrasonic reflectometry have been perfectly correlated to values obtained from mass versus time analysis. This ultrasonic method which is not very sensitive to environmental perturbations is therefore very interesting to monitor the drying of blood droplets in a simple manner and is more generally suitable for complex fluid droplets evaporation investigation.

  3. Novel humanized anti-CD3 antibodies induce a predominantly immunoregulatory profile in human peripheral blood mononuclear cells.

    PubMed

    Silva, Hernandez M; Vieira, Pedro M M M; Costa, Patricia L N; Pimentel, Bárbara M S; Moro, Ana M; Kalil, Jorge; Maranhão, Andrea Q; Coelho, Verônica; Brigido, Marcelo M

    2009-08-15

    Strategies to minimize the immunogenicity and toxicity of murine anti-CD3 antibodies (e.g. OKT3) are of special interest for organ transplantation and for the treatment of autoimmune diseases. In the present work, we have developed two humanized anti-CD3 antibodies. These molecules were shown to bind to human CD3, though less efficiently, and display less mitogenic activity than OKT3. These results prompted us to investigate whether this reduced mitogenic potential was associated with the development of anti-inflammatory properties. Indeed, in peripheral blood mononuclear cells (PBMCs), the humanized antibody versions induced a predominantly anti-inflammatory cytokine profile, in contrast with the pro-inflammatory profile induced by OKT3. Neither OKT3 nor the humanized versions induced the expression of IL-4, IL-2 or TGF-beta. Both humanized antibodies induced significantly lower production of IFN-gamma and IL-5 and slightly higher production of IL-10 than OKT3. This immunomodulatory profile was most evident by the 80-fold higher ratio of IL-10/IFN-gamma production in PBMCs cultured in the presence of the humanized antibodies, compared to those stimulated with OKT3. Furthermore, these humanized anti-CD3 antibodies induced a late FOXP3 gene expression while OKT3 led to a more transient expression of FOXP3. Taken our results, we suggest that these humanized anti-CD3 antibodies may promote the development of T cells with immunoregulatory activity.

  4. Fresh whole blood transfusion capability for Special Operations Forces

    PubMed Central

    Beckett, Maj Andrew; Callum, Jeannie; da Luz, Luis Teodoro; Schmid, Joanne; Funk, Christopher; Glassberg, Col Elon; Tien, Col Homer

    2015-01-01

    Summary Fresh whole blood (FWB) transfusion is an option for providing volume and oxygen carrying capacity to bleeding Special Operations soldiers who are injured in an austere environment and who are far from a regular blood bank. Retrospective data from recent conflicts in Iraq and Afghanistan show an association between the use of FWB and survival. We reviewed the literature to document the issues surrounding FWB transfusion to Special Operations soldiers in the austere environment and surveyed the literature regarding best practice guidelines for and patient outcomes after FWB transfusions. Most literature regarding FWB transfusion is retrospective or historical. There is limited prospective evidence currently to change transfusion practice in tertiary care facilities, but FWB remains an option in the austere setting. PMID:26100776

  5. Fresh whole blood transfusion capability for Special Operations Forces.

    PubMed

    Beckett, Andrew; Callum, Jeannie; da Luz, Luis Teodoro; Schmid, Joanne; Funk, Christopher; Glassberg, Elon; Tien, Homer

    2015-06-01

    Fresh whole blood (FWB) transfusion is an option for providing volume and oxygen carrying capacity to bleeding Special Operations soldiers who are injured in an austere environment and who are far from a regular blood bank. Retrospective data from recent conflicts in Iraq and Afghanistan show an association between the use of FWB and survival. We reviewed the literature to document the issues surrounding FWB transfusion to Special Operations soldiers in the austere environment and surveyed the literature regarding best practice guidelines for and patient outcomes after FWB transfusions. Most literature regarding FWB transfusion is retrospective or historical. There is limited prospective evidence currently to change transfusion practice in tertiary care facilities, but FWB remains an option in the austere setting. PMID:26100776

  6. Age and metabolic risk factors associated with oxidatively damaged DNA in human peripheral blood mononuclear cells.

    PubMed

    Løhr, Mille; Jensen, Annie; Eriksen, Louise; Grønbæk, Morten; Loft, Steffen; Møller, Peter

    2015-02-20

    Aging is associated with oxidative stress-generated damage to DNA and this could be related to metabolic disturbances. This study investigated the association between levels of oxidatively damaged DNA in peripheral blood mononuclear cells (PBMCs) and metabolic risk factors in 1,019 subjects, aged 18-93 years. DNA damage was analyzed as strand breaks by the comet assay and levels of formamidopyrimidine (FPG-) and human 8-oxoguanine DNA glycosylase 1 (hOGG1)-sensitive sites There was an association between age and levels of FPG-sensitive sites for women, but not for men. The same tendency was observed for the level of hOGG1-sensitive sites, whereas there was no association with the level of strand breaks. The effect of age on oxidatively damaged DNA in women disappeared in multivariate models, which showed robust positive associations between DNA damage and plasma levels of triglycerides, cholesterol and glycosylated hemoglobin (HbA1c). In the group of men, there were significant positive associations between alcohol intake, HbA1c and FPG-sensitive sites in multivariate analysis. The levels of metabolic risk factors were positively associated with age, yet only few subjects fulfilled all metabolic syndrome criteria. In summary, positive associations between age and levels of oxidatively damaged DNA appeared mediated by age-related increases in metabolic risk factors. PMID:25650665

  7. Analysis of cytotoxic effects of silver nanoclusters on human peripheral blood mononuclear cells 'in vitro'.

    PubMed

    Orta-García, Sandra Teresa; Plascencia-Villa, Germán; Ochoa-Martínez, Angeles Catalina; Ruiz-Vera, Tania; Pérez-Vázquez, Francisco Javier; Velázquez-Salazar, J Jesús; Yacamán, Miguel José; Navarro-Contreras, Hugo Ricardo; Pérez-Maldonado, Iván N

    2015-10-01

    The antimicrobial properties of silver nanoparticles (AgNPs) have made these particles one of the most used nanomaterials in consumer products. Therefore, an understanding of the interactions (unwanted toxicity) between nanoparticles and human cells is of significant interest. The aim of this study was to assess the in vitro cytotoxicity effects of silver nanoclusters (AgNC, < 2 nm diameter) on peripheral blood mononuclear cells (PBMC). Using flow cytometry and comet assay methods, we demonstrate that exposure of PBMC to AgNC induced intracellular reactive oxygen species (ROS) generation, DNA damage and apoptosis at 3, 6 and 12 h, with a dose-dependent response (0.1, 1, 3, 5 and 30 µg ml(-1)). Advanced electron microscopy imaging of complete and ultrathin-sections of PBMC confirmed the cytotoxic effects and cell damage caused by AgNC. The present study showed that AgNC produced without coating agents induced significant cytotoxic effects on PBMC owing to their high aspect ratio and active surface area, even at much lower concentrations (<1 µg ml(-1)) than those applied in previous studies, resembling what would occur under real exposure conditions to nanosilver-functionalized consumer products.

  8. Effect of arsenic, cadmium and lead on the induction of apoptosis of normal human mononuclear cells

    PubMed Central

    DE LA FUENTE, H; PORTALES-PÉREZ, D; BARANDA, L; DÍAZ-BARRIGA, F; SAAVEDRA-ALANÍS, V; LAYSECA, E; GONZÁLEZ-AMARO, R

    2002-01-01

    The aim of this work was to investigate the effect of cadmium, lead and arsenic on the apoptosis of human immune cells. Peripheral blood mononuclear cells (MNC) were incubated with increasing concentrations of these metals and then cellular apoptosis was determined by flow cytometry and by DNA electrophoresis. We found that arsenic induced a significant level of apoptosis at 15 μm after 48h of incubation. Cadmium had a similar effect, but at higher concentrations (65 μm). In addition, cadmium exerted a cytotoxic effect on MNC that seemed to be independent of the induction of apoptosis. In contrast, concentrations of lead as high as 500 μm were nontoxic and did not induce a significant degree of apoptosis. Additional experiments showed that arsenic at concentrations as low as 1·0 μm had a significant pro-apoptotic effect when cells were cultured in the presence of this pollutant for more than 72. Non-T cells were more susceptible than T lymphocytes to the effect of arsenic and cadmium. Interestingly, MNC from children chronically exposed to arsenic showed a high basal rate of apoptosis and a diminished in vitro sensibility to this metalloid. Our results indicate that both arsenic and cadmium are able to induce apoptosis of lymphoid cells, and suggest that this phenomenon may contribute to their immunotoxic effect in vivo. PMID:12100024

  9. Pharmacokinetic properties of γ-hydroxybutyrate (GHB) in whole blood, serum, and urine.

    PubMed

    Brailsford, Alan D; Cowan, David A; Kicman, Andrew T

    2012-03-01

    Over the last 10-15 years, γ-hydroxybutyrate (GHB) and γ-butyrolactone have become increasingly popular "club drugs", but they have also gained attention as potential agents of drug-facilitated sexual assault (DFSA). Several studies have attempted to characterize GHB's pharmacokinetic properties in humans, and the aim of this paper is to build on this research with an emphasis on DFSA cases. A 25 mg/kg dose of GHB was given to 12 GHB-naïve volunteers (6 men and 6 women). Urine and blood samples (serum and whole blood) were collected and analyzed by gas chromatography-mass spectrometry following liquid-liquid extraction. The urinary T(max) was 1 h in 11 volunteers with a mean C(max) of 67.6 mg/L (32.6-161.3 mg/L). Urinary concentrations rapidly decreased to < 10 mg/L (interpretive limit) for 11 volunteers after just 4 h. Data derived from whole blood (mean C(max) = 48.0 mg/L, T(max) = 24.6 min) closely matched that from serum (mean C(max) = 59.4 mg/L, T(max) = 23.3 min), suggesting GHB is distributed into erythrocytes. All 12 volunteers had GHB concentrations of less than 5 mg/L in both whole blood and serum after 3 h. Results verify the rapid elimination of GHB and the limited retrospective power of a concentration-based approach to prove GHB administration in blood and urine and confirm that, in DFSA cases, samples should be collected as soon as possible.

  10. Preanalytical Conditions and DNA Isolation Methods Affect Telomere Length Quantification in Whole Blood

    PubMed Central

    Tolios, Alexander; Teupser, Daniel; Holdt, Lesca M.

    2015-01-01

    Telomeres are located at chromosome ends and their length (TL) has been associated with aging and human diseases such as cancer. Whole blood DNA is frequently used for TL measurements but the influence of preanalytical conditions and DNA isolation methods on TL quantification has not been thoroughly investigated. To evaluate potential preanalytical as well as methodological bias on TL, anonymized leftover EDTA-whole blood samples were pooled according to leukocyte counts and were incubated with and without actinomycin D to induce apoptosis as a prototype of sample degradation. DNA was isolated from fresh blood pools and after freezing at -80°C. Commercially available kits using beads (Invitrogen), spin columns (Qiagen, Macherey-Nagel and 5prime) or precipitation (Stratec/Invisorb) and a published isopropanol precipitation protocol (IPP) were used for DNA isolation. TL was assessed by qPCR, and normalized to the single copy reference gene 36B4 using two established single-plex and a new multiplex protocol. We show that the method of DNA isolation significantly affected TL (e.g. 1.86-fold longer TL when comparing IPP vs. Invitrogen). Sample degradation led to an average TL decrease of 22% when using all except for one DNA isolation method (5prime). Preanalytical storage conditions did not affect TL with exception of samples that were isolated with the 5prime kit, where a 27% increase in TL was observed after freezing. Finally, performance of the multiplex qPCR protocol was comparable to the single-plex assays, but showed superior time- and cost-effectiveness and required > 80% less DNA. Findings of the current study highlight the need for standardization of whole blood processing and DNA isolation in clinical study settings to avoid preanalytical bias of TL quantification and show that multiplex assays may improve TL/SCG measurements. PMID:26636575

  11. Filter Characteristics Influencing Circulating Tumor Cell Enrichment from Whole Blood

    PubMed Central

    Beck, Markus; Terstappen, Leon W. M. M.

    2013-01-01

    A variety of filters assays have been described to enrich circulating tumor cells (CTC) based on differences in physical characteristics of blood cells and CTC. In this study we evaluate different filter types to derive the properties of the ideal filter for CTC enrichment. Between 0.1 and 10 mL of whole blood spiked with cells from tumor cell lines were passed through silicon nitride microsieves, polymer track-etched filters and metal TEM grids with various pore sizes. The recovery and size of 9 different culture cell lines was determined and compared to the size of EpCAM+CK+CD45−DNA+ CTC from patients with metastatic breast, colorectal and prostate cancer. The 8 µm track-etched filter and the 5 µm microsieve had the best performance on MDA-231, PC3-9 and SKBR-3 cells, enriching >80% of cells from whole blood. TEM grids had poor recovery of ∼25%. Median diameter of cell lines ranged from 10.9–19.0 µm, compared to 13.1, 10.7, and 11.0 µm for breast, prostate and colorectal CTC, respectively. The 11.4 µm COLO-320 cell line had the lowest recovery of 17%. The ideal filter for CTC enrichment is constructed of a stiff, flat material, is inert to blood cells, has at least 100,000 regularly spaced 5 µm pores for 1 ml of blood with a ≤10% porosity. While cell size is an important factor in determining recovery, other factors must be involved as well. To evaluate a filtration procedure, cell lines with a median size of 11–13 µm should be used to challenge the system. PMID:23626725

  12. Filter characteristics influencing circulating tumor cell enrichment from whole blood.

    PubMed

    Coumans, Frank A W; van Dalum, Guus; Beck, Markus; Terstappen, Leon W M M

    2013-01-01

    A variety of filters assays have been described to enrich circulating tumor cells (CTC) based on differences in physical characteristics of blood cells and CTC. In this study we evaluate different filter types to derive the properties of the ideal filter for CTC enrichment. Between 0.1 and 10 mL of whole blood spiked with cells from tumor cell lines were passed through silicon nitride microsieves, polymer track-etched filters and metal TEM grids with various pore sizes. The recovery and size of 9 different culture cell lines was determined and compared to the size of EpCAM+CK+CD45-DNA+ CTC from patients with metastatic breast, colorectal and prostate cancer. The 8 µm track-etched filter and the 5 µm microsieve had the best performance on MDA-231, PC3-9 and SKBR-3 cells, enriching >80% of cells from whole blood. TEM grids had poor recovery of ∼25%. Median diameter of cell lines ranged from 10.9-19.0 µm, compared to 13.1, 10.7, and 11.0 µm for breast, prostate and colorectal CTC, respectively. The 11.4 µm COLO-320 cell line had the lowest recovery of 17%. The ideal filter for CTC enrichment is constructed of a stiff, flat material, is inert to blood cells, has at least 100,000 regularly spaced 5 µm pores for 1 ml of blood with a ≤10% porosity. While cell size is an important factor in determining recovery, other factors must be involved as well. To evaluate a filtration procedure, cell lines with a median size of 11-13 µm should be used to challenge the system.

  13. Demonstration and partial characterization of the interferon-gamma receptor on human mononuclear phagocytes.

    PubMed Central

    Celada, A; Allen, R; Esparza, I; Gray, P W; Schreiber, R D

    1985-01-01

    Radioiodinated recombinant human interferon-gamma (IFN gamma) bound to human monocytes, U937, and HL60 cells in a specific, saturable, and reversible manner. At 4 degrees C, the different cell types bound 3,000-7,000 molecules of IFN gamma, and binding was of comparable affinity (Ka = 4-12 X 10(8) M-1). No change in the receptor was observed after monocytes differentiated to macrophages or when the cell lines were pharmacologically induced to differentiate. The functional relevance of the receptor was validated by the demonstration that receptor occupancy correlated with induction of Fc receptors on U937. Binding studies using U937 permeabilized with digitonin showed that only 46% of the total receptor pool was expressed at the cell surface. The receptor appears to be a protein, since treatment of U937 with trypsin or pronase reduced 125I-IFN gamma binding by 87 and 95%, respectively. At 37 degrees C, ligand was internalized, since 32% of the cell-associated IFN gamma became resistant to trypsin stripping. Monocytes degraded 125I-IFN gamma into trichloroacetic acid-soluble counts at 37 degrees C but not at 4 degrees C, at an approximate rate of 5,000 molecules/cell per h. The receptor was partially characterized by SDS-polyacrylamide gel electrophoresis analysis of purified U937 membranes that had been incubated with 125I-IFN gamma. After cross-linking, the receptor-ligand complex migrated as a broad band that displayed an Mr of 104,000 +/- 18,000 at the top and 84,000 +/- 6,000 at the bottom. These results thereby define and partially characterize the IFN gamma receptor of human mononuclear phagocytes. Images PMID:2934408

  14. Autologous red blood cells potentiate antibody synthesis by unfractionated human mononuclear cell cultures.

    PubMed

    Rugeles, M T; La Via, M; Goust, J M; Kilpatrick, J M; Hyman, B; Virella, G

    1987-08-01

    We have tried to determine the most favourable conditions for the in vitro induction of specific antibody (Ab) responses to tetanus toxoid (TT) and keyhole limpet haemocyanin (KLH). Human peripheral blood mononuclear cells (PBMNC) were obtained from normal volunteers and stimulated with PWM, TT, KLH, and mixtures of PWM and antigens in the presence or absence of autologous red blood cells (RBC) (1:50 ratio of PBMNC/RBC). The cultures were harvested on day 11; immunoglobulins were determined immunonephelometrically and Ab levels by ELISA with human antibodies used for calibration. While anti-TT responses were easy to induce with PBMNC from recently boosted individuals, the production of anti-TT from PBMNC obtained from non-recently boosted individuals was only possible when PBMNC were stimulated with TT and PWM in the presence of autologous RBC. Similarly, anti-KLH responses were easier to induce with PBMNC from an immune donor; maximal response was observed after stimulation with PWM + KLH in the presence of autologous RBC. Stimulation of primary anti-KLH responses with PBMNC from non-immune donors was only successful when the cells were stimulated with KLH + PWM in the presence of autologous RBC. The potentiation of human B-cell responses with autologous RBC can be abrogated by pretreatment of PBMNC with anti-CD2 antibodies and is associated with increased expression of IL-2 receptors and increased production of gamma interferon (IFN-gamma). However, addition of IFN-gamma in different doses and at different times to PWM-stimulated PBMNC cultures was not as effective as addition of RBC in enhancing the production of immunoglobulin and antibody. PMID:3114872

  15. Persistent Alterations of Gene Expression Profiling of Human Peripheral Blood Mononuclear Cells From Smokers

    PubMed Central

    Weng, Daniel Y.; Chen, Jinguo; Taslim, Cenny; Hsu, Ping-Ching; Marian, Catalin; David, Sean P.; Loffredo, Christopher A.; Shields, Peter G.

    2016-01-01

    The number of validated biomarkers of tobacco smoke exposure is limited, and none exist for tobacco-related cancer. Additional biomarkers for smoke, effects on cellular systems in vivo are needed to improve early detection of lung cancer, and to assist the Food and Drug Administration in regulating exposures to tobacco products. We assessed the effects of smoking on the gene expression using human cell cultures and blood from a cross-sectional study. We profiled global transcriptional changes in cultured smokers’ peripheral blood mononuclear cells (PBMCs) treated with cigarette smoke condensate (CSC) in vitro (n = 7) and from well-characterized smokers’ blood (n = 36). ANOVA with adjustment for covariates and Pearson correlation were used for statistical analysis in this study. CSC in vitro altered the expression of 1 178 genes (177 genes with > 1.5-fold-change) at P < 0.05. In vivo, PBMCs of heavy and light smokers differed for 614 genes (29 with > 1.5-fold-change) at P < 0.05 (309 remaining significant after adjustment for age, race, and gender). Forty-one genes were persistently altered both in vitro and in vivo, 22 having the same expression pattern reported for non-small cell lung cancer. Our data provides evidence that persistent alterations of gene expression in vitro and in vivo may relate to carcinogenic effects of cigarette smoke, and the identified genes may serve as potential biomarkers for cancer. The use of an in vitro model to corroborate results from human studies provides a novel way to understand human exposure and effect. PMID:26294040

  16. HCG-Activated Human Peripheral Blood Mononuclear Cells (PBMC) Promote Trophoblast Cell Invasion

    PubMed Central

    Wang, Yaqin; Guo, Yue; Zhou, Danni; Xu, Mei; Ding, Jinli; Yang, Jing

    2015-01-01

    Successful embryo implantation and placentation depend on appropriate trophoblast invasion into the maternal endometrial stroma. Human chorionic gonadotropin (hCG) is one of the earliest embryo-derived secreted signals in the peripheral blood mononuclear cells (PBMC) that abundantly expresses hCG receptors. The aims of this study were to estimate the effect of human embryo–secreted hCG on PBMC function and investigate the role and underlying mechanisms of activated PBMC in trophoblast invasion. Blood samples were collected from women undergoing benign gynecological surgery during the mid-secretory phase. PBMC were isolated and stimulated with or without hCG for 0 or 24 h. Interleukin-1β (IL-1β) and leukemia inhibitory factor (LIF) expressions in PBMC were detected by enzyme-linked immunosorbent assay and real-time polymerase chain reaction (PCR). The JAR cell line served as a model for trophoblast cells and was divided into four groups: control, hCG only, PBMC only, and PBMC with hCG. JAR cell invasive and proliferative abilities were detected by trans-well and CCK8 assays and matrix metalloproteinase (MMP)-2 (MMP-2), MMP-9, vascular endothelial growth factor (VEGF), tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 expressions in JAR cells were detected by western blotting and real-time PCR analysis. We found that hCG can remarkably promote IL-1β and LIF promotion in PBMC after 24-h culture. PBMC activated by hCG significantly increased the number of invasive JAR cells in an invasion assay without affecting proliferation, and hCG-activated PBMC significantly increased MMP-2, MMP-9, and VEGF and decreased TIMP-1 and TIMP-2 expressions in JAR cells in a dose-dependent manner. This study demonstrated that hCG stimulates cytokine secretion in human PBMC and could stimulate trophoblast invasion. PMID:26087261

  17. Cytokine-like effects of prolactin in human mononuclear and polymorphonuclear leukocytes.

    PubMed

    Dogusan, Z; Hooghe, R; Verdood, P; Hooghe-Peters, E L

    2001-11-01

    Some biochemical events following the binding of prolactin (PRL) to its receptor in normal human leukocytes were investigated. PRL enhanced JAK2 phosphorylation in peripheral blood mononuclear cells (PBMC) but not in granulocytes. PRL also induced phosphorylation of Stat-5 in PBMC and Stat-1 in granulocytes. Subsequent binding of Stat-5- and of Stat-1-like molecules to a GAS responsive element from the beta-casein promoter was detected by EMSA. p38 MAPK (but not p42/p44 MAPK) was activated by PRL in both leukocyte populations. PRL induced iNOS and CIS mRNA expression in granulocytes. Increased expression of IRF-1 and SOCS-2 was observed in granulocytes and of SOCS-3 and iNOS in PBMC. Similar effects were obtained with ovine and human PRL. Antiserum to PRL reduced iNOS and IRF-1 expression induced by PRL in granulocytes and reduced iNOS expression in PBMC. Also, pretreatment of granulocytes with a p38 MAPK inhibitor (SB 203580) prevented in part PRL-induced iNOS and IRF-1 expression. In PBMC, the p38 inhibitor decreased PRL-induced iNOS gene expression. These results indicate that PRL-induced gene regulation in leukocytes requires the activation of at least two different pathways: the Stat and the MAP kinase pathways. Moreover, although PRL activates Stat in both leukocyte types, signal transduction is different in granulocytes and in PBMC. Most importantly, PRL modulates the expression of genes crucial to leukocyte function. The present findings reinforce the concept that PRL has "cytokine-like" activity in human leukocytes.

  18. Determination of bromadiolone in whole blood by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

    PubMed

    Jin, Mi-Cong; Ren, Yi-Ping; Xu, Xiao-Min; Chen, Xiao-Hong

    2007-08-24

    A rapid, sensitive and selective high-performance liquid chromatography tandem mass spectrometric method (HPLC/MS-MS) has been developed and validated for the determination of bromadiolone in whole blood using warfarin as an internal standard (IS). Bromadiolone was extracted from the whole blood samples by liquid-liquid extraction with ethyl acetate. Multiple-reaction monitoring (MRM) was used to detect bromadiolone and IS, using precursor --> product ion combinations at m/z 527 --> 465 and 307 --> 161, respectively. The calibration curve was linear (r2=0.998) in the concentration range of 0.5-100.0 ng/mL with a lower limit of quantification of 0.5 ng/mL in whole blood. Intra- and inter-day relative standard deviations (R.S.D.s) were less than 7.5 and 11.9%, respectively. Recoveries of bromadiolone ranged from 82.1 to 85.2%. This method is found to be determined trace bromadiolone in whole blood and can be used in the diagnosis of the poisoned human beings. PMID:17098388

  19. Treatment of Whole Blood With Riboflavin and UV Light: Impact on Malaria Parasite Viability and Whole Blood Storage

    PubMed Central

    Owusu-Ofori, Shirley; Kusi, Joseph; Owusu-Ofori, Alex; Freimanis, Graham; Olver, Christine; Martinez, Caitlyn R.; Wilkinson, Shilo; Mundt, Janna M.; Keil, Shawn D.; Goodrich, Raymond P.; Allain, Jean-Pierre

    2015-01-01

    ABSTRACT Background: Sub-Saharan African countries utilize whole blood (WB) to treat severe anemia secondary to severe blood loss or malaria on an emergency basis. In many areas with high prevalence of transfusion-transmissible agents, blood safety measures are insufficient. Pathogen reduction technology applied to WB might considerably improve blood safety. Methods: Whole blood from 40 different donors were treated with riboflavin and UV light (pathogen reduction technology) in order to inactivate malaria parasite replication. The extent of parasite inactivation was determined using quantitative polymerase chain reaction methods and was correlated to studies evaluating the replication of malaria parasites in culture. Products were also stored for 21 days at +4°C and monitored for cell quality throughout storage. Results: Plasmodium amplicon was present in 21 samples (>100 copies/mL), doubtful in four (10–100 genome equivalents [gEq]/mL), and negative in 15 U. The majority of asymptomatic parasitemic donors carried low parasite levels, with only six donors above 5,000 copies/mL (15%). After treatment with riboflavin and UV light, these six samples demonstrated a 0.5 to 1.2 log reduction in quantitative polymerase chain reaction amplification. This correlated to equal to or greater than 6.4 log reductions in infectivity. In treated WB units, cell quality parameters remained stable; however, plasma hemoglobin increased to 0.15 g/dL. All markers behaved similarly to published data for stored, untreated WB. Conclusions: Pathogen reduction technology treatment can inactivate malaria parasites in WB while maintaining adequate blood quality during posttreatment cold storage for 21 days. PMID:25423125

  20. On-chip Extraction of Intracellular Molecules in White Blood Cells from Whole Blood.

    PubMed

    Choi, Jongchan; Hyun, Ji-chul; Yang, Sung

    2015-10-14

    The extraction of virological markers in white blood cells (WBCs) from whole blood--without reagents, electricity, or instruments--is the most important first step for diagnostic testing of infectious diseases in resource-limited settings. Here we develop an integrated microfluidic chip that continuously separates WBCs from whole blood and mechanically ruptures them to extract intracellular proteins and nucleic acids for diagnostic purposes. The integrated chip is assembled with a device that separates WBCs by using differences in blood cell size and a mechanical cell lysis chip with ultra-sharp nanoblade arrays. We demonstrate the performance of the integrated device by quantitatively analyzing the levels of extracted intracellular proteins and genomic DNAs. Our results show that compared with a conventional method, the device yields 120% higher level of total protein amount and similar levels of gDNA (90.3%). To demonstrate its clinical application to human immunodeficiency virus (HIV) diagnostics, the developed chip was used to process blood samples containing HIV-infected cells. Based on PCR results, we demonstrate that the chip can extract HIV proviral DNAs from infected cells with a population as low as 10(2)/μl. These findings suggest that the developed device has potential application in point-of-care testing for infectious diseases in developing countries.

  1. Inorganic cobalt supplementation: prediction of cobalt levels in whole blood and urine using a biokinetic model.

    PubMed

    Unice, Kenneth M; Monnot, Andrew D; Gaffney, Shannon H; Tvermoes, Brooke E; Thuett, Kerry A; Paustenbach, Dennis J; Finley, Brent L

    2012-07-01

    Soluble cobalt (Co) supplements with recommended daily doses up to 1000 μg Co/day are increasingly being marketed to consumers interested in healthy living practices. For example, some athletes may consider using Co supplements as blood doping agents, as Co is known to stimulate erythropoesis. However, the distribution and excretion kinetics of ingested Co are understood in a limited fashion. We used a Co-specific biokinetic model to estimate whole blood and urine Co levels resulting from oral exposure or ingestion of Co in amounts exceeding typical dietary intake rates. Following 10 days of Co supplementation at a rate of 400 to 1000 μg/day, predicted adult Co concentrations range from 1.7 to 10 μg/L in whole blood, and from 20 to 120 μg/L in urine. Chronic supplementation (≥ 1 year) at a rate of 1000 μg Co/day is predicted to result in blood levels of 5.7 to 13 μg/L, and in urine levels from 65 to 150 μg/L. The model predictions are within those measured in humans following ingestion of known doses. The methodology presented in this paper can be used to predict urinary or blood Co levels following acute or chronic occupational incidental ingestion, medicinal therapy, supplemental intake, or other non-occupational exposures.

  2. Rac1 Regulates the NLRP3 Inflammasome Which Mediates IL-1beta Production in Chlamydophila pneumoniae Infected Human Mononuclear Cells

    PubMed Central

    Orlovski, Christine; Hocke, Andreas; Schmeck, Bernd; Hippenstiel, Stefan; N'Guessan, Philippe Dje; Suttorp, Norbert; Opitz, Bastian

    2012-01-01

    Chlamydophila pneumoniae causes acute respiratory tract infections and has been associated with development of asthma and atherosclerosis. The production of IL-1β, a key mediator of acute and chronic inflammation, is regulated on a transcriptional level and additionally on a posttranslational level by inflammasomes. In the present study we show that C. pneumoniae-infected human mononuclear cells produce IL-1β protein depending on an inflammasome consisting of NLRP3, the adapter protein ASC and caspase-1. We further found that the small GTPase Rac1 is activated in C. pneumoniae-infected cells. Importantly, studies with specific inhibitors as well as siRNA show that Rac1 regulates inflammasome activation in C. pneumoniae-infected cells. In conclusion, C. pneumoniae infection of mononuclear cells stimulates IL-1β production dependent on a NLRP3 inflammasome-mediated processing of proIL-1β which is controlled by Rac1. PMID:22276187

  3. Spontaneous cytotoxicity of human peripheral blood mononuclear cells for the lymphoblastoid cell line CCRF-CEM: augmentation by bacterial lipopolysaccharide.

    PubMed Central

    Schacter, B; Kleinhenz, M E; Edmonds, K; Ellner, J J

    1981-01-01

    Spontaneous cell-mediated cytotoxicity (SCMC) of human peripheral blood mononuclear cells for a poor target, CCRF-CEM, a lymphoblastoid cell line, was rapidly and markedly elevated by E. coli lipopolysaccharide (LPS). SCMC for K562, a myeloid cell line sensitive to SCMC, was only slightly elevated by LPS. The cytotoxicities of both adherent and non-adherent mononuclear cells for CCRF-CEM were elevated. A response to LPS was found in Fc gamma R-positive and Fc gamma R-negative T cells. LPS increased the binding of non-adherent cells to both targets, but analysis of the binding suggests that a subsequent step, either triggering of the cytotoxic mechanisms or susceptibility of the target was the basis for the increased SCMC. PMID:7039896

  4. Transient Canonical Wnt Stimulation Enriches Human Bone Marrow Mononuclear Cell Isolates for Osteoprogenitors

    PubMed Central

    Janeczek, Agnieszka A.; Tare, Rahul S.; Scarpa, Edoardo; Moreno‐Jimenez, Ines; Rowland, Caroline A.; Jenner, Dominic; Newman, Tracey A.; Oreffo, Richard O. C.

    2015-01-01

    Abstract Activation of the canonical Wnt signaling pathway is an attractive anabolic therapeutic strategy for bone. Emerging data suggest that activation of the Wnt signaling pathway promotes bone mineral accrual in osteoporotic patients. The effect of Wnt stimulation in fracture healing is less clear as Wnt signaling has both stimulatory and inhibitory effects on osteogenesis. Here, we tested the hypothesis that transient Wnt stimulation promotes the expansion and osteogenesis of a Wnt‐responsive stem cell population present in human bone marrow. Bone marrow mononuclear cells (BMMNCs) were isolated from patients undergoing hip arthroplasty and exposed to Wnt3A protein. The effect of Wnt pathway stimulation was determined by measuring the frequency of stem cells within the BMMNC populations by fluorescence‐activated cell sorting and colony forming unit fibroblast (CFU‐F) assays, before determining their osteogenic capacity in in vitro differentiation experiments. We found that putative skeletal stem cells in BMMNC isolates exhibited elevated Wnt pathway activity compared with the population as whole. Wnt stimulation resulted in an increase in the frequency of skeletal stem cells marked by the STRO‐1bright/Glycophorin A− phenotype. Osteogenesis was elevated in stromal cell populations arising from BMMNCs transiently stimulated by Wnt3A protein, but sustained stimulation inhibited osteogenesis in a concentration‐dependent manner. These results demonstrate that Wnt stimulation could be used as a therapeutic approach by transient targeting of stem cell populations during early fracture healing, but that inappropriate stimulation may prevent osteogenesis. Stem Cells 2016;34:418–430 PMID:26573091

  5. Lipopolysaccharide Stimulates Butyric Acid-Induced Apoptosis in Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Kurita-Ochiai, Tomoko; Fukushima, Kazuo; Ochiai, Kuniyasu

    1999-01-01

    We previously reported that butyric acid, an extracellular metabolite from periodontopathic bacteria, induced apoptosis in murine thymocytes, splenic T cells, and human Jurkat T cells. In this study, we examined the ability of butyric acid to induce apoptosis in peripheral blood mononuclear cells (PBMC) and the effect of bacterial lipopolysaccharide (LPS) on this apoptosis. Butyric acid significantly inhibited the anti-CD3 monoclonal antibody- and concanavalin A-induced proliferative responses in a dose-dependent fashion. This inhibition of PBMC growth by butyric acid depended on apoptosis in vitro. It was characterized by internucleosomal DNA digestion and revealed by gel electrophoresis followed by a colorimetric DNA fragmentation assay to occur in a concentration-dependent fashion. Butyric acid-induced PBMC apoptosis was accompanied by caspase-3 protease activity but not by caspase-1 protease activity. LPS potentiated butyric acid-induced PBMC apoptosis in a dose-dependent manner. Flow-cytometric analysis revealed that LPS increased the proportion of sub-G1 cells and the number of late-stage apoptotic cells induced by butyric acid. Annexin V binding experiments with fractionated subpopulations of PBMC in flow cytometory revealed that LPS accelerated the butyric acid-induced CD3+-T-cell apoptosis followed by similar levels of both CD4+- and CD8+-T-cell apoptosis. The addition of LPS to PBMC cultures did not cause DNA fragmentation, suggesting that LPS was unable to induce PBMC apoptosis directly. These data suggest that LPS, in combination with butyric acid, potentiates CD3+ PBMC T-cell apoptosis and plays a role in the apoptotic depletion of CD4+ and CD8+ cells. PMID:9864191

  6. A study of zearalenone cytotoxicity on human peripheral blood mononuclear cells.

    PubMed

    Vlata, Zaxarenia; Porichis, Filippos; Tzanakakis, George; Tsatsakis, Aristidis; Krambovitis, Elias

    2006-09-10

    The mycotoxin zearalenone (ZEA) is a common contaminant of all major cereal grains worldwide with estrogenic and anabolic activity. We investigated the in vitro cytopathic effects of ZEA on freshly isolated human peripheral blood mononuclear cells (PBMC) in relation to proliferation and cell death patterns of untreated and mitogen-activated cells. The higher concentration of 30microg/ml ZEA was found to totally inhibit T and B lymphocyte proliferation from the stimulation with phytohemagglutinin and pokeweed mitogen. The inhibitory effects of ZEA were further related to cell necrosis/apoptosis. Flow cytometry analysis showed a distinct necrotic effect on PBMC, irrespective of mitogen stimulation, whereas apoptotic activity was less evident. Necrosis was observed in both the lymphocyte and monocyte/granulocyte gates. Measurements of ZEA-induced intracellular calcium ion (Ca(2+)) mobilization showed an increase of both Ca(2+) levels and the number of cells with high Ca(2+) only in the monocyte/granulocyte gated cells. Using phenylmethyl sulfonyl fluoride (PMSF), a serine protease inhibitor, and ammonium chloride (NH(4)Cl), a lysosomal inhibitor, both associated with cell necrosis inhibition, we showed that PMSF at 0.05mM and NH(4)Cl at 1 and 10mM reduced the cytopathic effects induced by 30microg/ml ZEA, whereas apoptosis was less affected. Expose of PBMC to 1microg/ml ZEA did not alter the viability of the cells. Our results suggest that high ZEA concentrations in the blood may well exert cytotoxic effects that merit further investigation.

  7. Evidence that leishmania donovani utilizes a mannose receptor on human mononuclear phagocytes to establish intracellular parasitism

    SciTech Connect

    Wilson, M.E.; Pearson, R.D.

    1986-01-01

    The pathogenic protozoan Leishmania donovani must gain entrance into mononuclear phagocytes to successfully parasitize man. The parasite's extracellular promastigote stage is ingested by human peripheral blood monocytes or monocyte-derived macrophages in the absence of serum, in a manner characteristic of receptor-mediated endocytosis. Remarkable similarities have been found between the macrophage receptor(s) for promastigotes and a previously characterized eucaryotic receptor system, the mannose/fucose receptor (MFR), that mediates the binding of zymosan particles and mannose- or fucose-terminal glycoconjugates to macrophages. Ingestion of promastigotes by monocyte-derived macrophages was inhibited by several MFR ligands; that is mannan, mannose-BSA and fucose-BSA. In contrast, promastigote ingestion by monocytes was unaffected by MFR ligands. Furthermore, attachment of promastigotes to macrophages, assessed by using cytochalasin D to prevent phagocytosis, was reduced 49.8% by mannan. Reorientation of the MFR to the ventral surface of the cell was achieved by plating macrophages onto mannan-coated coverslips, reducing MFR activity on the exposed cell surface by 94% as assessed by binding of /sup 125/I-mannose-BSA. Under these conditions, ingestion of promastigotes was inhibited by 71.4%. Internalization of the MFR by exposure of macrophages to zymosan before infection with promastigotes resulted in a 62.3% decrease in parasite ingestion. Additionally, NH/sub 4/Cl decreased macrophage ingestion of promastigotes by 38.2%. Subinhibitory concentration of NH/sub 4/Cl (10 mM) and of mannan (0.25 mg/ml) together inhibited parsite ingestion by 76.4%.

  8. Immunological effects of the anti-programmed death-1 antibody on human peripheral blood mononuclear cells.

    PubMed

    Akiyama, Yasuto; Nonomura, Chizu; Kondou, Ryota; Miyata, Haruo; Ashizawa, Tadashi; Maeda, Chie; Mitsuya, Koichi; Hayashi, Nakamasa; Nakasu, Yoko; Yamaguchi, Ken

    2016-09-01

    Immune checkpoint antibody-mediated blockade has gained attention as a new cancer immunotherapy strategy. Accumulating evidence suggests that this therapy imparts a survival benefit to metastatic melanoma and non-small cell lung cancer patients. A substantial amount of data on immune checkpoint antibodies has been collected from clinical trials; however, the direct effect of the antibodies on human peripheral blood mononuclear cells (PBMCs) has not been exclusively investigated. In this study, we developed an anti-programmed death-1 (PD-1) antibody (with biosimilarity to nivolumab) and examined the effects of the antibody on PBMCs derived from cancer patients. Specifically, we investigated the effects of the anti-PD-1 antibody on proliferation, cytokine production, cytotoxic T lymphocytes (CTL) and regulatory T cells. These investigations yielded several important results. First, the anti-PD-1 antibody had no obvious effect on resting PBMCs; however, high levels of the anti-PD-1 antibody partly stimulated PBMC proliferation when accompanied by an anti-CD3 antibody. Second, the anti-PD-1 antibody restored the growth inhibition of anti-CD3 Ab-stimulated PBMCs mediated by PD-L1. Third, the anti-PD-1 antibody exhibited a moderate inhibitory effect on the induction of myeloid-derived suppressor cells (MDSCs) by anti-CD3 antibody stimulation. Additionally, the presence of the anti-PD-1 antibody promoted antigen-specific CTL induction, which suggests that combining anti-PD-1 antibody and conventional immunotherapy treatments may have beneficial effects. These results indicate that specific cellular immunological mechanisms are partly responsible for the antitumor effect exhibited by the anti-PD-1 antibody against advanced cancers in clinical trials. PMID:27573705

  9. Calcitriol stimulates prolactin expression in non-activated human peripheral blood mononuclear cells: breaking paradigms.

    PubMed

    Díaz, Lorenza; Martínez-Reza, Isela; García-Becerra, Rocío; González, Leticia; Larrea, Fernando; Méndez, Isabel

    2011-08-01

    Calcitriol, the hormonal form of vitamin D(3), exerts immunomodulatory effects through the vitamin D(3) receptor (VDR) and increases prolactin (PRL) expression in the pituitary and decidua. Nevertheless, the effects of calcitriol upon lymphocyte PRL have not been evaluated. Therefore, we investigated calcitriol effects upon PRL in resting and phytohemagglutinin-activated human peripheral blood mononuclear cells (PBMNC) and Jurkat T lymphoma cells. Immunoblots showed constitutive expression of the 50-kDa VDR species in activated PBMNC and Jurkat cells, while a 75-kDa species was recognized in both resting and activated-PBMNC. Only in resting PBMNC calcitriol significantly stimulated PRL expression in a dose-dependent manner. The positive control CYP24A1, a highly VDR-responsive gene, was stimulated by calcitriol, effect that was stronger in resting than in activated-PBMNC (P<0.05), and without effect in Jurkat cells. Calcitriol upregulation of PRL and CYP24A1 was significantly inhibited by the VDR antagonist TEI-9647. EMSA showed that resting PBMNC contain a protein that binds to DR3-type VDRE. Cell activation reduced basal CYP24A1 while induced CYP27B1, VDR and pregnane X receptor (PXR) expression. In summary, calcitriol stimulated PRL and CYP24A1 gene expression in quiescent lymphocytes through a VDR-mediated mechanism. Our results suggest that the 75-kDa VDR species could be participating in calcitriol-mediated effects, and that activation induces factors such as PXR that restrain VDR transcriptional processes. This study supports the presence of a functional VDR in quiescent lymphocytes, providing evidence to reevaluate the VDR paradigm that assumes that lymphocytes respond to calcitriol only after activation. Altogether, our results offer new insights into the mechanisms whereby PRL is regulated in immune cells.

  10. Disease-specific classification using deconvoluted whole blood gene expression.

    PubMed

    Wang, Li; Oh, William K; Zhu, Jun

    2016-01-01

    Blood-based biomarker assays have an advantage in being minimally invasive. Diagnostic and prognostic models built on peripheral blood gene expression have been reported for various types of disease. However, most of these studies focused on only one disease type, and failed to address whether the identified gene expression signature is disease-specific or more widely applicable across diseases. We conducted a meta-analysis of 46 whole blood gene expression datasets covering a wide range of diseases and physiological conditions. Our analysis uncovered a striking overlap of signature genes shared by multiple diseases, driven by an underlying common pattern of cell component change, specifically an increase in myeloid cells and decrease in lymphocytes. These observations reveal the necessity of building disease-specific classifiers that can distinguish different disease types as well as normal controls, and highlight the importance of cell component change in deriving blood gene expression based models. We developed a new strategy to develop blood-based disease-specific models by leveraging both cell component changes and cell molecular state changes, and demonstrate its superiority using independent datasets. PMID:27596246

  11. Portable microfluidic cytometer for whole blood cell analysis

    NASA Astrophysics Data System (ADS)

    Grafton, Meggie M.; Zordan, Michael D.; Chuang, Han-Sheng; Rajdev, Pooja; Reece, Lisa M.; Irazoqui, Pedro P.; Wereley, Steven T.; Byrnes, Ron; Todd, Paul; Leary, James F.

    2010-02-01

    Lab-on-a-chip (LOC) systems allow complex laboratory assays to be carried out on a single chip using less time, reagents, and manpower than traditional methods. There are many chips addressing PCR and other DNA assays, but few that address blood cell analysis. Blood analysis, particularly of the cellular component, is highly important in both medical and scientific fields. Traditionally blood samples require a vial of blood, then several processing steps to separate and stain the various components, followed by the preparations for each specific assay to be performed. A LOC system for blood cell analysis and sorting would be ideal. The microfluidic-based system we have developed requires a mere drop of blood to be introduced onto the chip. Once on chip, the blood is mixed with both fluorescent and magnetic labels. The lab-on-a-chip device then uses a syringe drive to push the cells through the chip, while a permanent magnet is positioned to pull the magnetically labeled white blood cells to a separate channel. The white blood cells, labeled with different color fluorescent quantum dots (Qdots) conjugated to antibodies against WBC subpopulations, are analyzed and counted, while a sampling of red blood cells is also counted in a separate channel. This device will be capable of processing whole blood samples on location in a matter of minutes and displaying the cell count and should eventually find use in neonatology, AIDS and remote site applications.

  12. Asymmetric dimethylarginine and whole blood viscosity in renal failure

    PubMed Central

    Hammes, Mary S.; Watson, Sydeaka; Coe, Frederic L.; Ahmed, Faris; Beltran, Emily; Dhar, Promila

    2015-01-01

    BACKGROUND Renal failure is a disease with accelerated atherosclerosis beginning with endothelial cell dysfunction. Factors affecting endothelial cell dysfunction include whole blood viscosity (WBV) and asymmetric dimethylarginine (ADMA). The relationship in controls and renal failure was determined. METHODS 51 subjects, 20 controls, 11 renal transplant recipients, 10 chronic kidney disease and 10 end-stage renal disease patients had blood samples drawn for WBV, Hematocrit, and ADMA. WBV was measured at various shear rates from 10 s−1 to 780 s−1 at 37°C. Hematocrit using CritSpin, and ADMA was assayed using an ELISA method. The significance between groups was compared by boxplots and analysis of variance. Linear relationships were shown by regression lines and correlation coefficients. RESULTS ADMA was elevated in all groups with renal failure when compared to controls (p < 0.05). Control subjects showed a positive correlation between ADMA and WBV, while those who received a renal transplant had a negative correlation (p < 0.05). The difference in ADMA comparing pre-dialysis to post-dialysis conditions was positive (p < 0.05). CONCLUSIONS The positive relationship between WBV and ADMA in controls is a novel finding and allows for comparison with other groups. This relationship is dramatically altered in renal failure. PMID:24840340

  13. Whole blood glucose analysis based on smartphone camera module.

    PubMed

    Devadhasan, Jasmine Pramila; Oh, Hyunhee; Choi, Cheol Soo; Kim, Sanghyo

    2015-11-01

    Complementary metal oxide semiconductor (CMOS) image sensors have received great attention for their high efficiency in biological applications. The present work describes a CMOS image sensor-based whole blood glucose monitoring system through a point-of-care (POC) approach. A simple poly-ethylene terephthalate (PET) chip was developed to carry out the enzyme kinetic reaction at various concentrations (110–586 mg∕dL) of mouse blood glucose. In this technique, assay reagent is immobilized onto amine functionalized silica (AFSiO2) nanoparticles as an electrostatic attraction in order to achieve glucose oxidation on the chip. The assay reagent immobilized AFSiO2 nanoparticles develop a semi-transparent reaction platform, which is technically a suitable chip to analyze by a camera module. The oxidized glucose then produces a green color according to the glucose concentration and is analyzed by the camera module as a photon detection technique; the photon number decreases when the glucose concentration increases. The combination of these components, the CMOS image sensor and enzyme immobilized PET film chip, constitute a compact, accurate, inexpensive, precise, digital, highly sensitive, specific, and optical glucose-sensing approach for POC diagnosis. PMID:26524683

  14. Filtration parameters influencing circulating tumor cell enrichment from whole blood.

    PubMed

    Coumans, Frank A W; van Dalum, Guus; Beck, Markus; Terstappen, Leon W M M

    2013-01-01

    Filtration can achieve circulating tumor cell (CTC) enrichment from blood. Key parameters such as flow-rate, applied pressure, and fixation, vary largely between assays and their influence is not well understood. Here, we used a filtration system, to monitor these parameters and determine their relationships. Whole blood, or its components, with and without spiked tumor cells were filtered through track-etched filters. We characterize cells passing through filter pores by their apparent viscosity; the viscosity of a fluid that would pass with the same flow. We measured a ratio of 5·10(4)∶10(2)∶1 for the apparent viscosities of 15 µm diameter MDA-231 cells, 10 µm white cells and 90 fl red cells passing through a 5 µm pore. Fixation increases the pressure needed to pass cells through 8 µm pores 25-fold and halves the recovery of spiked tumor cells. Filtration should be performed on unfixed samples at a pressure of ∼10 mbar for a 1 cm(2) track-etched filter with 5 µm pores. At this pressure MDA-231 cells move through the filter in 1 hour. If fixation is needed for sample preservation, a gentle fixative should be selected. The difference in apparent viscosity between CTC and blood cells is key in optimizing recovery of CTC.

  15. Filtration Parameters Influencing Circulating Tumor Cell Enrichment from Whole Blood

    PubMed Central

    Beck, Markus; Terstappen, Leon W. M. M.

    2013-01-01

    Filtration can achieve circulating tumor cell (CTC) enrichment from blood. Key parameters such as flow-rate, applied pressure, and fixation, vary largely between assays and their influence is not well understood. Here, we used a filtration system, to monitor these parameters and determine their relationships. Whole blood, or its components, with and without spiked tumor cells were filtered through track-etched filters. We characterize cells passing through filter pores by their apparent viscosity; the viscosity of a fluid that would pass with the same flow. We measured a ratio of 5·104∶102∶1 for the apparent viscosities of 15 µm diameter MDA-231 cells, 10 µm white cells and 90 fl red cells passing through a 5 µm pore. Fixation increases the pressure needed to pass cells through 8 µm pores 25-fold and halves the recovery of spiked tumor cells. Filtration should be performed on unfixed samples at a pressure of ∼10 mbar for a 1 cm2 track-etched filter with 5 µm pores. At this pressure MDA-231 cells move through the filter in 1 hour. If fixation is needed for sample preservation, a gentle fixative should be selected. The difference in apparent viscosity between CTC and blood cells is key in optimizing recovery of CTC. PMID:23658615

  16. Whole blood glucose analysis based on smartphone camera module

    NASA Astrophysics Data System (ADS)

    Devadhasan, Jasmine Pramila; Oh, Hyunhee; Choi, Cheol Soo; Kim, Sanghyo

    2015-11-01

    Complementary metal oxide semiconductor (CMOS) image sensors have received great attention for their high efficiency in biological applications. The present work describes a CMOS image sensor-based whole blood glucose monitoring system through a point-of-care (POC) approach. A simple poly-ethylene terephthalate (PET) chip was developed to carry out the enzyme kinetic reaction at various concentrations (110-586 mg/dL) of mouse blood glucose. In this technique, assay reagent is immobilized onto amine functionalized silica (AFSiO2) nanoparticles as an electrostatic attraction in order to achieve glucose oxidation on the chip. The assay reagent immobilized AFSiO2 nanoparticles develop a semi-transparent reaction platform, which is technically a suitable chip to analyze by a camera module. The oxidized glucose then produces a green color according to the glucose concentration and is analyzed by the camera module as a photon detection technique; the photon number decreases when the glucose concentration increases. The combination of these components, the CMOS image sensor and enzyme immobilized PET film chip, constitute a compact, accurate, inexpensive, precise, digital, highly sensitive, specific, and optical glucose-sensing approach for POC diagnosis.

  17. Disease-specific classification using deconvoluted whole blood gene expression

    PubMed Central

    Wang, Li; Oh, William K.; Zhu, Jun

    2016-01-01

    Blood-based biomarker assays have an advantage in being minimally invasive. Diagnostic and prognostic models built on peripheral blood gene expression have been reported for various types of disease. However, most of these studies focused on only one disease type, and failed to address whether the identified gene expression signature is disease-specific or more widely applicable across diseases. We conducted a meta-analysis of 46 whole blood gene expression datasets covering a wide range of diseases and physiological conditions. Our analysis uncovered a striking overlap of signature genes shared by multiple diseases, driven by an underlying common pattern of cell component change, specifically an increase in myeloid cells and decrease in lymphocytes. These observations reveal the necessity of building disease-specific classifiers that can distinguish different disease types as well as normal controls, and highlight the importance of cell component change in deriving blood gene expression based models. We developed a new strategy to develop blood-based disease-specific models by leveraging both cell component changes and cell molecular state changes, and demonstrate its superiority using independent datasets. PMID:27596246

  18. A simple screening test for fatty acid oxidation defects using whole-blood palmitate oxidation.

    PubMed

    Seargeant, L E; Balachandra, K; Mallory, C; Dilling, L A; Greenberg, C R

    1999-08-01

    We report that measurement of whole-blood palmitate oxidation is a rapid and inexpensive screening test for fatty acid oxidation defects. The assay has been adapted from published assays using cultured fibroblasts or isolated white blood cells. Micro whole-blood samples are incubated with tritiated palmitic acid as substrate. The tritiated water produced is proportional to the mitochondrial beta-oxidation of palmitic acid. Patients with confirmed beta-oxidation defects show low whole-blood palmitate oxidation.

  19. Effects of oral eicosapentaenoic acid versus docosahexaenoic acid on human peripheral blood mononuclear cell gene expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have beneficial effects on inflammation and cardiovascular disease (CVD). Our aim was to assess the effect of a six-week supplementation with either olive oil, EPA, or DHA on gene expression in peripheral blood mononuclear cells (...

  20. Lymphocyte transformation and interferon production in human mononuclear cell microcultures for assay of cellular immunity to herpes simplex virus.

    PubMed Central

    Haahr, S; Rasmussen, L; Merigan, T C

    1976-01-01

    Interferon production and transformation in response to herpes simplex virus antigen were studied in microcultures of human mononuclear cells. Mononuclear cells consisting of monocytes and both T and B lymphocytes were purified by Ficoll-Hypaque gradients. Lymphocytes, predominantly T with 5% B, were obtained by passage of buffy-coat cells through nylon fiber columns. For some experiments, autochthonous macrophages and column-purified lymphocytes were stimulated with herpesvirus antigen. The effect of specific antibody and cell concentration on reactivity is described. Crude and purified antigens were compared as cell culture stimulants. Significant differences in transformation and interferon were observed between donors with a history of herpes labialis and donors with no detectable antibody, both in cultures prepared by Ficoll-Hypaque gradients and by column purification of lymphocytes. Cultures from seronegative donors prepared by Ficoll-Hypaque gradients produced interferon but did not transform when stimulated by herpes simplex antigen. "Immune" interferon production, that is, type II as opposed to type I, occurred only with autochthonous macrophage and column-purified lymphocyte cultures. Interferon produced by Ficoll-Hypaque-purified mononuclear cultures was type I, and its production was unrelated to immune status. Similarly, column-purified lymphocytes responded to herpes simplex virus antigen with type I interferon if obtained from a seropositive donor. PMID:181328

  1. Transplantation of mononuclear cells from human umbilical cord blood promotes functional recovery after traumatic spinal cord injury in Wistar rats.

    PubMed

    Rodrigues, L P; Iglesias, D; Nicola, F C; Steffens, D; Valentim, L; Witczak, A; Zanatta, G; Achaval, M; Pranke, P; Netto, C A

    2012-01-01

    Cell transplantation is a promising experimental treatment for spinal cord injury. The aim of the present study was to evaluate the efficacy of mononuclear cells from human umbilical cord blood in promoting functional recovery when transplanted after a contusion spinal cord injury. Female Wistar rats (12 weeks old) were submitted to spinal injury with a MASCIS impactor and divided into 4 groups: control, surgical control, spinal cord injury, and one cell-treated lesion group. Mononuclear cells from umbilical cord blood of human male neonates were transplanted in two experiments: a) 1 h after surgery, into the injury site at a concentration of 5 x 10(6) cells diluted in 10 µL 0.9% NaCl (N = 8-10 per group); b) into the cisterna magna, 9 days after lesion at a concentration of 5 x 10(6) cells diluted in 150 µL 0.9% NaCl (N = 12-14 per group). The transplanted animals were immunosuppressed with cyclosporin-A (10 mg/kg per day). The BBB scale was used to evaluate motor behavior and the injury site was analyzed with immunofluorescent markers to label human transplanted cells, oligodendrocytes, neurons, and astrocytes. Spinal cord injury rats had 25% loss of cord tissue and cell treatment did not affect lesion extension. Transplanted cells survived in the injured area for 6 weeks after the procedure and both transplanted groups showed better motor recovery than the untreated ones (P < 0.05). The transplantation of mononuclear cells from human umbilical cord blood promoted functional recovery with no evidence of cell differentiation. PMID:22183246

  2. Transplantation of mononuclear cells from human umbilical cord blood promotes functional recovery after traumatic spinal cord injury in Wistar rats

    PubMed Central

    Rodrigues, L.P.; Iglesias, D.; Nicola, F.C.; Steffens, D.; Valentim, L.; Witczak, A.; Zanatta, G.; Achaval, M.; Pranke, P.; Netto, C.A.

    2011-01-01

    Cell transplantation is a promising experimental treatment for spinal cord injury. The aim of the present study was to evaluate the efficacy of mononuclear cells from human umbilical cord blood in promoting functional recovery when transplanted after a contusion spinal cord injury. Female Wistar rats (12 weeks old) were submitted to spinal injury with a MASCIS impactor and divided into 4 groups: control, surgical control, spinal cord injury, and one cell-treated lesion group. Mononuclear cells from umbilical cord blood of human male neonates were transplanted in two experiments: a) 1 h after surgery, into the injury site at a concentration of 5 x 106 cells diluted in 10 µL 0.9% NaCl (N = 8-10 per group); b) into the cisterna magna, 9 days after lesion at a concentration of 5 x 106 cells diluted in 150 µL 0.9% NaCl (N = 12-14 per group). The transplanted animals were immunosuppressed with cyclosporin-A (10 mg/kg per day). The BBB scale was used to evaluate motor behavior and the injury site was analyzed with immunofluorescent markers to label human transplanted cells, oligodendrocytes, neurons, and astrocytes. Spinal cord injury rats had 25% loss of cord tissue and cell treatment did not affect lesion extension. Transplanted cells survived in the injured area for 6 weeks after the procedure and both transplanted groups showed better motor recovery than the untreated ones (P < 0.05). The transplantation of mononuclear cells from human umbilical cord blood promoted functional recovery with no evidence of cell differentiation. PMID:22183246

  3. Adaptive Force Sonorheometry for Assessment of Whole Blood Coagulation

    PubMed Central

    Mauldin, F. William; Viola, Francesco; Hamer, Theresa C.; Ahmed, Eman M.; Crawford, Shawna B.; Haverstick, Doris M.; Lawrence, Michael B.; Walker, William F.

    2010-01-01

    Background: Viscoelastic diagnostics that monitor the hemostatic function of whole blood (WB), such as thromboelastography, have been developed with demonstrated clinical utility. By measuring the cumulative effects of all components of hemostasis, viscoelastic diagnostics have circumvented many of the challenges associated with more common tests of blood coagulation. Methods: We describe a new technology, called sonorheometry, that adaptively applies acoustic radiation force to assess coagulation function in WB. The repeatability (precision) of coagulation parameters was assessed using citrated WB samples. A reference range of coagulation parameters, along with corresponding measurements from prothrombin time (PT) and partial thromboplastin time (PTT), were obtained from WB samples of 20 healthy volunteers. In another study, sonorheometry monitored anticoagulation with heparin (0 – 5 IU/ml) and reversal from varied dosages of protamine (0 – 10 IU/ml) in heparinized WB (2 IU/ml). Results: Sonorheometry exhibited low CVs for parameters: clot initiation time (TC1), < 7%; clot stabilization time (TC2), < 6.5%; and clotting angle (θ), < 3.5%. Good correlation was observed between clotting times, TC1 and TC2, and PTT (r = 0.65 and 0.74 respectively; n=18). Linearity to heparin dosage was observed with average linearity r > 0.98 for all coagulation parameters. We observed maximum reversal of heparin anticoagulation at protamine to heparin ratios of 1.4:1 from TC1 (P=0.6) and 1.2:1 from θ (P=0.55). Conclusions: Sonorheometry is a non-contact method for precise assessment of WB coagulation. PMID:20096680

  4. Whole blood for hemostatic resuscitation of major bleeding.

    PubMed

    Spinella, Philip C; Pidcoke, Heather F; Strandenes, Geir; Hervig, Tor; Fisher, Andrew; Jenkins, Donald; Yazer, Mark; Stubbs, James; Murdock, Alan; Sailliol, Anne; Ness, Paul M; Cap, Andrew P

    2016-04-01

    Recent combat experience reignited interest in transfusing whole blood (WB) for patients with life-threatening bleeding. US Army data indicate that WB transfusion is associated with improved or comparable survival compared to resuscitation with blood components. These data complement randomized controlled trials that indicate that platelet (PLT)-containing blood products stored at 4°C have superior hemostatic function, based on reduced bleeding and improved functional measures of hemostasis, compared to PLT-containing blood products at 22°C. WB is rarely available in civilian hospitals and as a result is rarely transfused for patients with hemorrhagic shock. Recent developments suggest that impediments to WB availability can be overcome, specifically the misconceptions that WB must be ABO specific, that WB cannot be leukoreduced and maintain PLTs, and finally that cold storage causes loss of PLT function. Data indicate that the use of low anti-A and anti-B titer group O WB is safe as a universal donor, WB can be leukoreduced with PLT-sparing filters, and WB stored at 4°C retains PLT function during 15 days of storage. The understanding that these perceived barriers are not insurmountable will improve the availability of WB and facilitate its use. In addition, there are logistic and economic advantages of WB-based resuscitation compared to component therapy for hemorrhagic shock. The use of low-titer group O WB stored for up to 15 days at 4°C merits further study to compare its efficacy and safety with current resuscitation approaches for all patients with life-threatening bleeding. PMID:27100756

  5. Postprandial activation of metabolic and inflammatory signalling pathways in human peripheral mononuclear cells.

    PubMed

    Ehlers, Kerstin; Brand, Tina; Bangert, Adina; Hauner, Hans; Laumen, Helmut

    2014-06-28

    High-fat, high-carbohydrate (HFHC) meals induce an inflammatory response in mononuclear cells (MNC). Here, we studied the interaction between metabolic and inflammatory signalling pathways by the measurement of postprandial effects of three different test meals on intracellular Akt, S6 kinase (S6K)/mammalian target of rapamycin and NF-κB signalling in human MNC. We recruited six healthy, lean individuals. Each individual ingested three different meals in the morning separated by at least 3 d: a HFHC meal; an oral lipid-tolerance test meal; a healthy breakfast. Blood samples were obtained before and 1, 2, 4, 6 and 8 h after ingestion. Plasma insulin and IL-6 levels were measured. Intracellular metabolic and inflammatory signalling pathways were assessed by measuring the phosphorylation of Akt kinase and S6K, the degradation of inhibitory κB-α (IκB-α) protein and the DNA binding activity of NF-κB in MNC. mRNA expression levels of the Akt and NF-κB target genes Mn superoxide dismutase (MnSOD), CC-chemokine-receptor 5 (CCR5), intercellular adhesion molecule 1 (ICAM-1) and plasminogen activator inhibitor-1 (PAI-1) were measured by quantitative RT-PCR. We found a positive correlation of Akt phosphorylation with NF-κB activation (NF-κB binding activity: r 0·4500, P= 0·0003; IκB-α protein levels: r -0·5435, P< 0·0001), a negative correlation of plasma insulin levels with NF-κB binding activity (r -0·3993, P= 0·0016) and a positive correlation of plasma insulin levels with S6K activation (r 0·4786, P< 0·0001). The activation of Akt and pro-inflammatory NF-κB signalling was supported by the up-regulation of the respective target genes MnSOD and CCR5. In conclusion, the present data suggest a postprandial interaction between the metabolic and inflammatory signalling pathways Akt and NF-κB in MNC.

  6. Microfluidic isolation of leukocytes from whole blood for phenotype and gene expression analysis.

    PubMed

    Sethu, Palaniappan; Moldawer, Lyle L; Mindrinos, Michael N; Scumpia, Philip O; Tannahill, Cynthia L; Wilhelmy, Julie; Efron, Philip A; Brownstein, Bernard H; Tompkins, Ronald G; Toner, Mehmet

    2006-08-01

    Technologies that enable the isolation of cell subtypes from small samples of complex populations will greatly facilitate the implementation of proteomics and genomics to human diseases. Transcriptome analysis of blood requires the depletion of contaminating erythrocytes. We report an automated microfluidic device to rapidly deplete erythrocytes from whole blood via deionized water lysis and to collect enriched leukocytes for phenotype and genomic analyses. Starting with blood from healthy subjects, we demonstrate the utility of this microfluidic cassette and lysis protocol to prepare unstimulated leukocytes, and leukocytes stimulated ex vivo with Staphylococcal enterotoxin B, which mimics some of the cellular effects seen in patients with severe bacterial infections. Microarrays are used to assess the global gene expression response to enterotoxin B. The results demonstrate that this system can isolate unactivated leukocytes from small blood samples without any significant loss, which permits more information to be obtained from subsequent analysis, and will be readily applicable to clinical settings.

  7. The effect of pseudomonas exotoxin A on cytokine production in whole blood exposed to Pseudomonas aeruginosa.

    PubMed

    Schultz, M J; Speelman, P; Zaat, S A; Hack, C E; van Deventer, S J; van der Poll, T

    2000-11-01

    To determine the effect of Pseudomonas aeruginosa exotoxin A (P-ExA) on cytokine production, we studied cytokine release induced by heat-killed P. aeruginosa (HKPA) in human whole blood in the presence or absence of P-ExA. P-ExA (0.01-1 microgram ml(-1)) caused a dose-dependent decrease in HKPA-induced production of tumor necrosis factor alpha (TNF), interleukin (IL-) 10, IL-6 and IL-8 (all P<0.05). P-ExA-induced inhibition of IL-10, IL-6 and IL-8 release was not dependent on reduced TNF concentrations, since the relative attenuation of the production of these cytokines was similar in the presence or absence of a neutralizing anti-TNF antibody. The effect of P-ExA on cytokine production may offer a disadvantage to the host with respect to clearance of the infection.

  8. 9 CFR 147.3 - The stained-antigen, rapid, whole-blood test. 3

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false The stained-antigen, rapid, whole-blood test. 3 147.3 Section 147.3 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... Blood Testing Procedures § 147.3 The stained-antigen, rapid, whole-blood test. 3 3 The...

  9. 9 CFR 147.3 - The stained-antigen, rapid, whole-blood test. 3

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false The stained-antigen, rapid, whole-blood test. 3 147.3 Section 147.3 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... Blood Testing Procedures § 147.3 The stained-antigen, rapid, whole-blood test. 3 3 The...

  10. 9 CFR 147.3 - The stained-antigen, rapid, whole-blood test. 3

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false The stained-antigen, rapid, whole-blood test. 3 147.3 Section 147.3 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... Blood Testing Procedures § 147.3 The stained-antigen, rapid, whole-blood test. 3 3 The...

  11. 9 CFR 147.3 - The stained-antigen, rapid, whole-blood test. 3

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false The stained-antigen, rapid, whole-blood test. 3 147.3 Section 147.3 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... Blood Testing Procedures § 147.3 The stained-antigen, rapid, whole-blood test. 3 3 The...

  12. A successful transfusion in a tamandua (Tamandua tetradactyla) using both whole blood and blood replacement products.

    PubMed

    Raines, Janis A; Storms, Timothy

    2015-03-01

    A 10-yr-old tamandua (Tamandua tetradactyla) presented with severe lethargy and ataxia. Severe anemia (9% hematocrit) was diagnosed and ultimately resolved with the administration of a whole blood transfusion from conspecific and additional blood replacement products (Oxyglobin). This is the first described use of a whole blood transfusion in an edentate. PMID:25831593

  13. A successful transfusion in a tamandua (Tamandua tetradactyla) using both whole blood and blood replacement products.

    PubMed

    Raines, Janis A; Storms, Timothy

    2015-03-01

    A 10-yr-old tamandua (Tamandua tetradactyla) presented with severe lethargy and ataxia. Severe anemia (9% hematocrit) was diagnosed and ultimately resolved with the administration of a whole blood transfusion from conspecific and additional blood replacement products (Oxyglobin). This is the first described use of a whole blood transfusion in an edentate.

  14. Differentiation of human mononuclear phagocytes increases their innate response to Mycobacterium tuberculosis infection.

    PubMed

    Castaño, Diana; García, Luis F; Rojas, Mauricio

    2014-05-01

    The heterogeneity of mononuclear phagocytes, partially explained by cell differentiation, influences the activation of innate responses. It has been reported that Mycobacterium tuberculosis inhibits monocyte differentiation into either dendritic cells or macrophages. To evaluate whether the activation of effector mechanisms against M. tuberculosis differ between less and more differentiated mononuclear phagocytes, we compared monocytes differentiated in vitro for 24 h (MON24) and 120 h (MDM120) infected with M. tuberculosis H37Rv, H37Ra and the clinical isolate UT127 at different multiplicity of infection. MDM120 phagocytosed more M. tuberculosis, inhibited mycobacterial growth and did not die in response to the infection, compared with MON24. In contrast, MON24 become Annexin V and Propidium iodide positive after 36 h of M. tuberculosis infection. Although, there were striking differences between MON24 and MDM120, there were also some differences in the response to the mycobacterial strains used. Finally, in MDM120 infected with M. tuberculosis H37Rv, a lower percentage of mycobacterial phagosomes accumulated transferrin and a higher percentage co-localized with cathelicidin than in MON24. These results demonstrate that innate responses induced by M. tuberculosis depends upon the stage of differentiation of mononuclear phagocytes and support that terminally differentiated cells are more efficient anti-mycobacterial effectors than the less differentiated ones.

  15. Whole Blood Gene Expression Profiling in Preclinical and Clinical Cattle Infected with Atypical Bovine Spongiform Encephalopathy.

    PubMed

    Xerxa, Elena; Barbisin, Maura; Chieppa, Maria Novella; Krmac, Helena; Vallino Costassa, Elena; Vatta, Paolo; Simmons, Marion; Caramelli, Maria; Casalone, Cristina; Corona, Cristiano; Legname, Giuseppe

    2016-01-01

    Prion diseases, such as bovine spongiform encephalopathies (BSE), are transmissible neurodegenerative disorders affecting humans and a wide variety of mammals. Variant Creutzfeldt-Jakob disease (vCJD), a prion disease in humans, has been linked to exposure to BSE prions. This classical BSE (cBSE) is now rapidly disappearing as a result of appropriate measures to control animal feeding. Besides cBSE, two atypical forms (named H- and L-type BSE) have recently been described in Europe, Japan, and North America. Here we describe the first wide-spectrum microarray analysis in whole blood of atypical BSE-infected cattle. Transcriptome changes in infected animals were analyzed prior to and after the onset of clinical signs. The microarray analysis revealed gene expression changes in blood prior to the appearance of the clinical signs and during the progression of the disease. A set of 32 differentially expressed genes was found to be in common between clinical and preclinical stages and showed a very similar expression pattern in the two phases. A 22-gene signature showed an oscillating pattern of expression, being differentially expressed in the preclinical stage and then going back to control levels in the symptomatic phase. One gene, SEL1L3, was downregulated during the progression of the disease. Most of the studies performed up to date utilized various tissues, which are not suitable for a rapid analysis of infected animals and patients. Our findings suggest the intriguing possibility to take advantage of whole blood RNA transcriptional profiling for the preclinical identification of prion infection. Further, this study highlighted several pathways, such as immune response and metabolism that may play an important role in peripheral prion pathogenesis. Finally, the gene expression changes identified in the present study may be further investigated as a fingerprint for monitoring the progression of disease and for developing targeted therapeutic interventions. PMID

  16. Whole Blood Gene Expression Profiling in Preclinical and Clinical Cattle Infected with Atypical Bovine Spongiform Encephalopathy.

    PubMed

    Xerxa, Elena; Barbisin, Maura; Chieppa, Maria Novella; Krmac, Helena; Vallino Costassa, Elena; Vatta, Paolo; Simmons, Marion; Caramelli, Maria; Casalone, Cristina; Corona, Cristiano; Legname, Giuseppe

    2016-01-01

    Prion diseases, such as bovine spongiform encephalopathies (BSE), are transmissible neurodegenerative disorders affecting humans and a wide variety of mammals. Variant Creutzfeldt-Jakob disease (vCJD), a prion disease in humans, has been linked to exposure to BSE prions. This classical BSE (cBSE) is now rapidly disappearing as a result of appropriate measures to control animal feeding. Besides cBSE, two atypical forms (named H- and L-type BSE) have recently been described in Europe, Japan, and North America. Here we describe the first wide-spectrum microarray analysis in whole blood of atypical BSE-infected cattle. Transcriptome changes in infected animals were analyzed prior to and after the onset of clinical signs. The microarray analysis revealed gene expression changes in blood prior to the appearance of the clinical signs and during the progression of the disease. A set of 32 differentially expressed genes was found to be in common between clinical and preclinical stages and showed a very similar expression pattern in the two phases. A 22-gene signature showed an oscillating pattern of expression, being differentially expressed in the preclinical stage and then going back to control levels in the symptomatic phase. One gene, SEL1L3, was downregulated during the progression of the disease. Most of the studies performed up to date utilized various tissues, which are not suitable for a rapid analysis of infected animals and patients. Our findings suggest the intriguing possibility to take advantage of whole blood RNA transcriptional profiling for the preclinical identification of prion infection. Further, this study highlighted several pathways, such as immune response and metabolism that may play an important role in peripheral prion pathogenesis. Finally, the gene expression changes identified in the present study may be further investigated as a fingerprint for monitoring the progression of disease and for developing targeted therapeutic interventions.

  17. Whole Blood Gene Expression Profiling in Preclinical and Clinical Cattle Infected with Atypical Bovine Spongiform Encephalopathy

    PubMed Central

    Xerxa, Elena; Barbisin, Maura; Chieppa, Maria Novella; Krmac, Helena; Vallino Costassa, Elena; Vatta, Paolo; Simmons, Marion; Caramelli, Maria; Casalone, Cristina; Corona, Cristiano

    2016-01-01

    Prion diseases, such as bovine spongiform encephalopathies (BSE), are transmissible neurodegenerative disorders affecting humans and a wide variety of mammals. Variant Creutzfeldt-Jakob disease (vCJD), a prion disease in humans, has been linked to exposure to BSE prions. This classical BSE (cBSE) is now rapidly disappearing as a result of appropriate measures to control animal feeding. Besides cBSE, two atypical forms (named H- and L-type BSE) have recently been described in Europe, Japan, and North America. Here we describe the first wide-spectrum microarray analysis in whole blood of atypical BSE-infected cattle. Transcriptome changes in infected animals were analyzed prior to and after the onset of clinical signs. The microarray analysis revealed gene expression changes in blood prior to the appearance of the clinical signs and during the progression of the disease. A set of 32 differentially expressed genes was found to be in common between clinical and preclinical stages and showed a very similar expression pattern in the two phases. A 22-gene signature showed an oscillating pattern of expression, being differentially expressed in the preclinical stage and then going back to control levels in the symptomatic phase. One gene, SEL1L3, was downregulated during the progression of the disease. Most of the studies performed up to date utilized various tissues, which are not suitable for a rapid analysis of infected animals and patients. Our findings suggest the intriguing possibility to take advantage of whole blood RNA transcriptional profiling for the preclinical identification of prion infection. Further, this study highlighted several pathways, such as immune response and metabolism that may play an important role in peripheral prion pathogenesis. Finally, the gene expression changes identified in the present study may be further investigated as a fingerprint for monitoring the progression of disease and for developing targeted therapeutic interventions. PMID

  18. A systematic review of four injection therapies for lateral epicondylosis: prolotherapy, polidocanol, whole blood and platelet rich plasma

    PubMed Central

    Best, Thomas M.; Zgierska, Aleksandra E.; Zeisig, Eva; Ryan, Michael; Crane, David

    2009-01-01

    Objective To appraise existing evidence for prolotherapy, polidocanol, autologous whole blood and platelet-rich plasma injection therapies for lateral epicondylosis (LE) Design Systematic Review Data sources Medline, Embase, CINAHL, Cochrane Central Register of Controlled Trials, Allied and Complementary Medicine. Search strategy: names and descriptors of the therapies and LE. Study Selection All human studies assessing the four therapies for LE. Main results Results of five prospective case series and four controlled trials (3 prolotherapy, 2 polidocanol, 3 autologous whole blood and 1 platelet-rich plasma) suggest each of the four therapies is effective for LE. In follow-up periods ranging from 9 to 108 weeks, studies reported sustained, statistically significant(p<0.05) improvement on visual analog scale primary outcome pain score measures and disease specific questionnaires; relative effect sizes ranged from 51% to 94%; Cohen’s d ranged from 0.68 to 6.68. Secondary outcomes also improved, including biomechanical elbow function assessment (polidocanol and prolotherapy), presence of abnormalities and increased vascularity on ultrasound (autologous whole blood and polidocanol). Subjects reported satisfaction with therapies on single-item assessments. All studies were limited by small sample size. Conclusions There is strong pilot-level evidence supporting the use of prolotherapy, polidocanol, autologous whole blood and platelet-rich plasma injections in the treatment of LE. Rigorous studies of sufficient sample size, assessing these injection therapies using validated clinical, radiological and biomechanical measures, and tissue injury/healing-responsive biomarkers, are needed to determine long-term effectiveness and safety, and whether these techniques can play a definitive role in the management of LE and other tendinopathies. PMID:19028733

  19. [Identification of Animal Whole Blood Based on Near Infrared Transmission Spectroscopy].

    PubMed

    Wan, Xiong; Wang, Jian; Liu, Peng-xi; Zhang, Ting-ting

    2016-01-01

    The inspection and classification for blood products are important but complicated in import-export ports or inspection and quarantine departments. For the inspection of whole blood products, open sampling can cause pollution and virulence factors in bloods samples may even endanger inspectors. Thus non-contact classification and identification methods for whole bloods of animals are needed. Spectroscopic techniques adopted in the flowcytometry need sampling blood cells during the detection; therefore they can not meet the demand of non-contact identification and classification for whole bloods of animals. Infrared absorption spectroscopy is a technique that can be used to analyze the molecular structure and chemical bonds of detected samples under the condition of non-contact. To find a feasible spectroscopic approach of non-contact detection for the species variation in whole blood samples, a near infrared transmitted spectra (NITS, 4 497.669 - 7 506.4 cm(-1)) experiment of whole blood samples of three common animals including chickens, dogs and cats has been conducted. During the experiment, the spectroscopic resolution is 5 cm(-1), and each spectrogram is an average of 5 measured spectral data. Experimental results show that all samples have a sharp absorption peak between 5 184 and 5 215 cm(-1), and a gentle absorption peak near 7 000 cm(-1). Besides, the NITS curves of different samples of same animals are similar, and only have slight differences in the whole transmittance. A correlation coefficient (CC) is induced to distinguish the differences of the three animals' whole bloods in NITS curves, and the computed CCs between NITS curves of different samples of the same animals, are greater than 0.99, whereas CCs between NITS curves of the whole bloods of different animals are from 0.509 48 to 0.916 13. Among which CCs between NITS curves of the whole bloods of chickens and cats are from 0.857 23 to 0.912 44, CCs between NITS curves of the whole bloods of

  20. Metabolic effects of microwave radiation and convection heating on human mononuclear leukocytes. Final report, January 1985-May 1986

    SciTech Connect

    Kiel, J.L.; Wong, L.S.; Erwin, D.N.

    1986-01-01

    Investigated here were the effects of microwave (MW) radiation (2450-MHz, continuous-wave, mean specific absorption rate of 103.5 + or - 4.2 W/kg) and convention heating on the nonphosphorylating oxidative metabolism of human peripheral mononuclear leukocytes (96% lymphocytes, 4% monocytes) at 37 C. Metabolic activity, determined by chemiluminescence (CL) of cells challenged with luminol (5-aminO-2, 3-dihydro-1, 4-phthalazinedione) linked to bovine serum albumin, was detected with a brightness photomer. A significant stimulation after after MW exposure (p < 0.005) over total CL of matched 37 C-incubator controls was observed. A similar degree of stimulation, compared to incubator controls, was also detected after sham treatment. No significant difference existed between changes in total CL or stimulation indices of the MW and sham-exposed groups. Exposure to MW radiation, under normothermic (37 + or - 0.03 C) conditions, appears to have no effect on the oxidative metabolic activity of human peripheral mononuclear leukocytes. However, the significant differences between MW or sham-exposed cells and their respective incubator controls occurred because the temperature of the incubator did not exceed 35.9 C, and 39 minutes were required for the temperature to rise from 22 to 35.9 C. Slow heating of incubator controls must be accounted for in thermal and redio-frequency radiation studies in vitro.

  1. Generation of iPS Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors.

    PubMed

    Su, Ruijun Jeanna; Neises, Amanda; Zhang, Xiao-Bing

    2016-01-01

    Peripheral blood is the easy-to-access, minimally invasive, and the most abundant cell source to use for cell reprogramming. The episomal vector is among the best approaches for generating integration-free induced pluripotent stem (iPS) cells due to its simplicity and affordability. Here we describe the detailed protocol for the efficient generation of integration-free iPS cells from peripheral blood mononuclear cells. With this optimized protocol, one can readily generate hundreds of iPS cell colonies from 1 ml of peripheral blood.

  2. Rapid evaluation of fibrinogen levels using the CG02N whole blood coagulation analyzer.

    PubMed

    Hayakawa, Mineji; Gando, Satoshi; Ono, Yuichi; Mizugaki, Asumi; Katabami, Kenichi; Maekawa, Kunihiko; Miyamoto, Daisuke; Wada, Takeshi; Yanagida, Yuichiro; Sawamura, Atsushi

    2015-04-01

    Rapid evaluation of fibrinogen (Fbg) levels is essential for maintaining homeostasis in patients with massive bleeding during severe trauma and major surgery. This study evaluated the accuracy of fibrinogen levels measured by the CG02N whole blood coagulation analyzer (A&T Corporation, Kanagawa, Japan) using heparinized blood drawn for blood gas analysis (whole blood-Fbg). A total of 100 matched pairs of heparinized blood samples and citrated blood samples were simultaneously collected from patients in the intensive care unit. Whole blood-Fbg results were compared with those of citrated plasma (standard-Fbg). The whole blood coagulation analyzer measured fibrinogen levels within 2 minutes. Strong correlations between standard-Fbg and whole blood-Fbg were observed (ρ = 0.91, p < 0.001). Error grid analysis showed that 88% of the values were clinically acceptable, and 12% were in a range with possible effects on clinical decision-making; none were in a clinically dangerous range without appropriate treatment. Using a fibrinogen cutoff value of 1.5 g/L for standard-Fbg, the area under the receiver operating characteristic curve of whole blood-Fbg was 0.980 (95% confidence interval 0.951-1.000, p < 0.001). The whole blood coagulation analyzer can rapidly measure fibrinogen levels in heparinized blood and could be useful in critical care settings where excessive bleeding is a concern.

  3. Survey of the use of whole blood in current blood transfusion practice.

    PubMed

    MacLennan, S; Murphy, M F

    2001-12-01

    Limited use of whole blood has continued despite a general move to blood component therapy in recent years. This paper describes the results of a questionnaire survey which was distributed to haematologists in charge of blood banks in England and North Wales to ascertain how much and for which indications whole blood was being requested. There was a 58% response rate. More than 90% of hospitals that responded had not requested whole blood during the last 12 months. Indications for the use of whole blood were primarily in paediatric practice, mostly for neonatal exchange transfusion or paediatric surgery (cardiac or craniofacial). Infrequent use in adult practice was for "major bleeding" when whole blood was available, and in cardiac surgery, when post-operative bleeding was unresponsive to standard replacement therapy. The evidence for the use of whole blood in preference to component therapy in the massive transfusion setting was reviewed, and no compelling evidence was found for its routine use for this indication. It is worth noting that, as currently supplied in the UK, "whole blood" is not strictly "whole" as the leucocyte-depletion process removes platelets. PMID:11843887

  4. Nanoparticles with Therapeutic Properties Generate Various Response of Human Peripheral Blood Mononuclear Cells.

    PubMed

    Szwed, Marzena; Santos-Oliveira, Ralph

    2016-06-01

    In the present study we report the interactions of four types of different nanoparticles with normal peripheral blood mononuclear cells. To our research we chose four types of nanoparticles which possess therapeutic properties (Trastuzumab, ethylene-diamine-tetra-methylene-phosphonic for breast and bone cancers treatment, respectively) or can be used as the ingredients of sun-protected films (nanoemulsions with or without chitosan). By carrying out XTT survival assay we observed that both types of tested nanoemulsions suppressed the proliferation of normal lymphocytes. However, the survival of peripheral blood mononuclear cells after incubation neither with Trastuzumab nor with ethylene-diamine-tetra-methylene-phosphonic nanoparticles decreased below 80%. If the investigated nanoparticles were analyzed for their effectiveness to the induction of programmed cell death, we proved that only nanoemulsions with or without chitosan provoked an increase of the fraction of apoptotic cells. Moreover we noticed the characteristic, typical for apoptosis changes of cells morphology, which appeared in lymphocytes after all tested nanoparticles treatment. Interestingly, representative for necrosis swollen, enlarged cells were observed after nanoemulsions treatment.

  5. Beryllium alters lipopolysaccharide-mediated intracellular phosphorylation and cytokine release in human peripheral blood mononuclear cells.

    PubMed

    Silva, Shannon; Ganguly, Kumkum; Fresquez, Theresa M; Gupta, Goutam; McCleskey, T Mark; Chaudhary, Anu

    2009-12-01

    Beryllium exposure in susceptible individuals leads to the development of chronic beryllium disease, a lung disorder marked by release of inflammatory cytokine and granuloma formation. We have previously reported that beryllium induces an immune response even in blood mononuclear cells from healthy individuals. In this study, we investigate the effects of beryllium on lipopolysaccharide-mediated cytokine release in blood mononuclear and dendritic cells from healthy individuals. We found that in vitro treatment of beryllium sulfate inhibits the secretion of lipopolysaccharide-mediated interleukin 10, while the release of interleukin 1beta is enhanced. In addition, not all lipopolysaccharide-mediated responses are altered, as interleukin 6 release in unaffected upon beryllium treatment. Beryllium sulfate-treated cells show altered phosphotyrosine levels upon lipopolysaccharide stimulation. Significantly, beryllium inhibits the phosphorylation of signal transducer and activator of transducer 3, induced by lipopolysaccharide. Finally, inhibitors of phosphoinositide-3 kinase mimic the effects of beryllium in inhibition of interleukin 10 release, while they have no effect on interleukin 1beta secretion. This study strongly suggests that prior exposures to beryllium could alter host immune responses to bacterial infections in healthy individuals, by altering intracellular signaling.

  6. Nanoparticles with Therapeutic Properties Generate Various Response of Human Peripheral Blood Mononuclear Cells.

    PubMed

    Szwed, Marzena; Santos-Oliveira, Ralph

    2016-06-01

    In the present study we report the interactions of four types of different nanoparticles with normal peripheral blood mononuclear cells. To our research we chose four types of nanoparticles which possess therapeutic properties (Trastuzumab, ethylene-diamine-tetra-methylene-phosphonic for breast and bone cancers treatment, respectively) or can be used as the ingredients of sun-protected films (nanoemulsions with or without chitosan). By carrying out XTT survival assay we observed that both types of tested nanoemulsions suppressed the proliferation of normal lymphocytes. However, the survival of peripheral blood mononuclear cells after incubation neither with Trastuzumab nor with ethylene-diamine-tetra-methylene-phosphonic nanoparticles decreased below 80%. If the investigated nanoparticles were analyzed for their effectiveness to the induction of programmed cell death, we proved that only nanoemulsions with or without chitosan provoked an increase of the fraction of apoptotic cells. Moreover we noticed the characteristic, typical for apoptosis changes of cells morphology, which appeared in lymphocytes after all tested nanoparticles treatment. Interestingly, representative for necrosis swollen, enlarged cells were observed after nanoemulsions treatment. PMID:27427750

  7. Emergency whole-blood use in the field: a simplified protocol for collection and transfusion.

    PubMed

    Strandenes, Geir; De Pasquale, Marc; Cap, Andrew P; Hervig, Tor A; Kristoffersen, Einar K; Hickey, Matthew; Cordova, Christopher; Berseus, Olle; Eliassen, Håkon S; Fisher, Logan; Williams, Steve; Spinella, Philip C

    2014-05-01

    Military experience and recent in vitro laboratory data provide a biological rationale for whole-blood use in the treatment of exsanguinating hemorrhage and have renewed interest in the reintroduction of fresh whole blood and cold-stored whole blood to patient care in austere environments. There is scant evidence to support, in a field environment, that a whole blood-based resuscitation strategy is superior to a crystalloid/colloid approach even when augmented by a limited number of red blood cell (RBC) and plasma units. Recent retrospective evidence suggests that, in this setting, resuscitation with a full compliment of RBCs, plasma, and platelets may offer an advantage, especially under conditions where evacuation is delayed. No current evacuation system, military or civilian, is capable of providing RBC, plasma, and platelet units in a prehospital environment, especially in austere settings. As a result, for the vast minority of casualties, in austere settings, with life-threatening hemorrhage, it is appropriate to consider a whole blood-based resuscitation approach to provide a balanced response to altered hemostasis and oxygen debt, with the goal of reducing the risk of death from hemorrhagic shock. To optimize the successful use of fresh whole blood/cold-stored whole blood in combat field environments, proper planning and frequent training to maximize efficiency and safety will be required. Combat medics will need proper protocol-based guidance and education if whole-blood collection and transfusion are to be successfully and safely performed in austere environments. In this article, we present the Norwegian Naval Special Operation Commando unit-specific remote damage control resuscitation protocol, which includes field collection and transfusion of whole blood. This protocol can serve as a template for others to use and adjust for their own military or civilian unit-specific needs and capabilities for care in austere environments. PMID:24365879

  8. Leukotriene B/sub 4/ production by stimulated whole blood: comparative studies with isolated polymorphonuclear cells

    SciTech Connect

    Gresele, P.; Arnout, J.; Coene, M.C.; Deckmyn, H.; Vermylen, J.

    1986-05-29

    A new method was developed to study leukotriene B/sub 4/ (LTB/sub 4/) production by stimulated whole blood. The calcium ionophore A23187 and serum-treated zymosan induced LTB/sub 4/ production, measured by radioimmunoassay, in a dose- and time-dependent manner. The pattern of LTB/sub 4/ production by whole blood differed markedly from that observed with isolated, purified polymorphonuclear leukocytes. Higher levels of LTB/sub 4/ were reached and maintained in whole blood. The system allowed to detect drug effects on LTB/sub 4/ takes into account the complex interactions between different cell types which can modulate LTB/sub 4/ metabolism.

  9. Zinc and copper levels in plasma, erythrocytes, and whole blood in cancer patients.

    PubMed

    Aldor, Y; Walach, N; Modai, D; Horn, Y

    1982-04-01

    Zinc and copper levels in erythrocytes, plasma, and whole blood were determined in 35 cancer patients and compared with 24 normal individuals. A decrease in zinc was found in all three blood constituents of the cancer patients. The decrease was significant in plasma and whole blood and nonsignificant in erythrocytes. Copper levels in the cancer group showed a slight and nonsignificant increase in erythrocytes, plasma, and whole blood. The copper to zinc ratio revealed a significant increase only for plasma levels. Further investigations are indicated to determine whether these two elements could serve as indicators for diagnosis or prognosis in cancer patients.

  10. The expression and function of human CD300 receptors on blood circulating mononuclear cells are distinct in neonates and adults

    PubMed Central

    Zenarruzabeitia, Olatz; Vitallé, Joana; García-Obregón, Susana; Astigarraga, Itziar; Eguizabal, Cristina; Santos, Silvia; Simhadri, Venkateswara R.; Borrego, Francisco

    2016-01-01

    Neonates are more susceptible to infections than adults. This susceptibility is thought to reflect neonates’ qualitative and quantitative defects in the adaptive and innate immune responses. Differential expression of cell surface receptors may result in altered thresholds of neonatal immune cell activation. We determined whether the expression and function of the lipid-binding CD300 family of receptors are different on neonatal immune cells compared to adult immune cells. A multiparametric flow cytometry analysis was performed to determine the expression of CD300 receptors on adult peripheral blood mononuclear cells and neonatal cord blood mononuclear cells. The expression of the CD300a inhibitory receptor was significantly reduced on cells from the newborn adaptive immune system, and neonatal antigen presenting cells exhibited a different CD300 receptors expression pattern. We also found differential LPS-mediated regulation of CD300 receptors expression on adult monocytes compared to cord blood monocytes, and that CD300c and CD300e-mediated activation was quantitatively different in neonatal monocytes. This is the first complete study examining the expression of CD300 receptors on human neonatal immune cells compared with adult immune cells. Significant differences in the expression and function of CD300 receptors may help to explain the peculiarities and distinctness of the neonatal immune responses. PMID:27595670

  11. Effect of 900 MHz Electromagnetic Radiation on the Induction of ROS in Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Kazemi, E.; Mortazavi, S. M. J.; Ali-Ghanbari, A.; Sharifzadeh, S.; Ranjbaran, R.; Mostafavi-pour, Z.; Zal, F.; Haghani, M.

    2015-01-01

    Background Despite numerous studies over a decade, it still remains controversial about the biological effects of RF EMF emitted by mobile phone telephony. Objective Here we investigated the effect of 900 MHz GSM on the induction of oxidative stress and the level of intracellular reactive oxygen species (ROS) in human mononuclear cells, monocytes and lymphocytes as defence system cells. Method 6 ml Peripheral Blood samples were obtained from 13 healthy volunteers (21-30 year-old). Each sample was devided into 2 groups: one was exposed RF radiation emitted from a mobile phone simulator for 2 hour and the other used as control group which was not exposed to any fields. After that, mononuclear cells were isolated from peripheral blood by density gradient centrifugation in Ficoll-Paque. The intracellular ROS content in monocytes and lymphocytes was measured by the CM-H2DCFDA fluorescence probe using flowcytometry technique. Results Our results showed significant increase in  ROS production after exposure in population rich in monocytes. This effect was not significant in population rich in lymphocytes in comparison with non exposed cells. Conclusion The results obtained in this study clearly showed the oxidative stress induction capability of RF electromagnetic field in the portion of PBMCs mostly in monocytes, like the case of exposure to micro organisms, although the advantages or disadvantages of this effect should be evaluated. PMID:26396966

  12. The expression and function of human CD300 receptors on blood circulating mononuclear cells are distinct in neonates and adults.

    PubMed

    Zenarruzabeitia, Olatz; Vitallé, Joana; García-Obregón, Susana; Astigarraga, Itziar; Eguizabal, Cristina; Santos, Silvia; Simhadri, Venkateswara R; Borrego, Francisco

    2016-01-01

    Neonates are more susceptible to infections than adults. This susceptibility is thought to reflect neonates' qualitative and quantitative defects in the adaptive and innate immune responses. Differential expression of cell surface receptors may result in altered thresholds of neonatal immune cell activation. We determined whether the expression and function of the lipid-binding CD300 family of receptors are different on neonatal immune cells compared to adult immune cells. A multiparametric flow cytometry analysis was performed to determine the expression of CD300 receptors on adult peripheral blood mononuclear cells and neonatal cord blood mononuclear cells. The expression of the CD300a inhibitory receptor was significantly reduced on cells from the newborn adaptive immune system, and neonatal antigen presenting cells exhibited a different CD300 receptors expression pattern. We also found differential LPS-mediated regulation of CD300 receptors expression on adult monocytes compared to cord blood monocytes, and that CD300c and CD300e-mediated activation was quantitatively different in neonatal monocytes. This is the first complete study examining the expression of CD300 receptors on human neonatal immune cells compared with adult immune cells. Significant differences in the expression and function of CD300 receptors may help to explain the peculiarities and distinctness of the neonatal immune responses. PMID:27595670

  13. Development of a Population Pharmacokinetic Model To Describe Azithromycin Whole-Blood and Plasma Concentrations over Time in Healthy Subjects

    PubMed Central

    Anic-Milic, T.; Oreskovic, K.; Padovan, J.; Brouwer, K. L. R.; Zuo, P.; Schmith, V. D.

    2013-01-01

    Azithromycin (AZI), a broad-spectrum antibiotic, accumulates in polymorphonuclear cells and peripheral blood mononuclear cells. The distribution of AZI in proinflammatory cells may be important to the anti-inflammatory properties. Previous studies have described plasma AZI pharmacokinetics. The objective of this study was to describe the pharmacokinetics of AZI in whole blood (concentration in whole blood [Cb]) and plasma (concentration in plasma [Cp]) of healthy subjects. In this study, 12 subjects received AZI (500 mg once a day for 3 days). AZI Cb and Cp were quantified in serial samples collected up to 3 weeks after the last dose and analyzed using noncompartmental and compartmental methods. After the last dose, Cb was greater than Cp. Importantly, Cb, but not Cp, was quantifiable in all but one subject at 3 weeks. The blood area under the curve during a 24-h dosing interval (AUC24) was ∼2-fold greater than the plasma AUC24, but simulations suggested that Cb was not at steady state by day 3. Upon exploration of numerous models, an empirical 3-compartment model adequately described Cp and Cb, but Cp was somewhat underestimated. Intercompartmental clearance (CL; likely representing cells) was lower than apparent oral CL (18 versus 118 liters/h). Plasma, peripheral, and cell compartmental volumes were 439 liters, 2,980 liters, and 3,084 liters, respectively. Interindividual variability in CL was low (26.2%), while the volume of distribution variability was high (107%). This is the first report to describe AZI Cb in healthy subjects, the distribution parameters between Cp and Cb, and AZI retention in blood for up to 3 weeks following 3 daily doses. The model can be used to predict Cb from Cp for AZI under various dosing regimens. (This study has been registered at ClinicalTrials.gov under registration no. NCT01026064.) PMID:23629714

  14. Thrombin-mediated IL-10 up-regulation involves protease-activated receptor (PAR)-1 expression in human mononuclear leukocytes.

    PubMed

    Naldini, Antonella; Bernini, Claudia; Pucci, Annalisa; Carraro, Fabio

    2005-09-01

    Thrombin, the key enzyme of the coagulation cascade, exerts cellular effects through activation of the protease-activated receptors (PARs). Interleukin (IL)-10, besides its anti-inflammatory properties, is considered a major denominator of the immunosuppressive effect during human endotoxemia. We have recently shown that thrombin inhibits IL-12 production in human mononuclear cells and that such inhibition is accompanied by IL-10 up-regulation. To our knowledge, there are no data available to show that thrombin mediates IL-10 production by its interactions with PAR-1. We here report that human alpha-thrombin enhances IL-10 expression in human peripheral blood mononuclear cells and in established monocytic cell lines and that this up-regulation requires PAR-1 expression. The use of proteolytically inactive thrombin reveals that such enhancement requires thrombin proteolytic activity. Addition of PAR-1 agonist peptides, such as SFLLRN, results in a significant increase of IL-10 production. PAR-1 expression is required for thrombin-induced IL-10 production, as shown by experiments performed with antisense or sense PAR-1 oligonucleotides. Treatment with thrombin or SFLLRN of monocytic cell lines, such as U937 and Mono Mac-6, results in an increased IL-10 production. This suggests that the observed IL-10 up-regulation may be the result of a direct interaction with monocytes. The observation that thrombin-mediated up-regulation of IL-10 may require the expression of the PAR-1 receptor identifies a new, functional link between inflammation and coagulation. Our results may also contribute to better design therapeutic strategies to treat several disorders, characterized by the presence of inflammatory as well as coagulant responses. PMID:15961578

  15. Alteration of the electrophoretic mobility of human peripheral blood mononuclear cells following treatment with dimethyl sulfoxide

    SciTech Connect

    Skrabut, E.M.; Catsimpoolas, N.; Kurtz, S.R.; Griffith, A.L.; Valeri, C.R.

    1983-12-01

    Studies have been conducted to determine the effects of DMSO and freezing on the electrophoretic distribution of peripheral blood mononuclear cells. Sodium (/sup 51/Cr)chromate was used to label the cells, and the distributions of cell number and cell-associated radioactivity were determined. Cells treated with DMSO had a narrower distribution of electrophoretic mobilities when compared with those not treated. DMSO-treated cells also demonstrated a more homogeneous distribution of radioactivity relative to the cell distribution than did the nontreated cells. The freezing of DMSO-treated cells did not result in any additional alteration of electrophoretic pattern compared to DMSO treatment alone. Analysis by linear categorization techniques indicated that the DMSO-treated and nontreated cells were completely distinguished by their electrophoretic behavior.

  16. Intravenous infusion of erythromycin inhibits CXC chemokine production, but augments neutrophil degranulation in whole blood stimulated with Streptococcus pneumoniae.

    PubMed

    Schultz, M J; Speelman, P; Hack, C E; Buurman, W A; van Deventer, S J; van Der Poll, T

    2000-08-01

    Macrolides may influence the inflammatory response to an infection by mechanisms that are unrelated to their antimicrobial effect. Indeed, erythromycin and other macrolides inhibit cytokine production and induce degranulation of neutrophils in vitro. CXC chemokines are small chemotactic cytokines that specifically influence neutrophil functions. To determine the effect of a clinically relevant dose of erythromycin on the production of CXC chemokines and neutrophil degranulation, six healthy humans received a 30 min iv infusion of erythromycin (1000 mg). Whole blood obtained before and at various times after the infusion was stimulated ex vivo with heat-killed Streptococcus pneumoniae. Ex vivo production of the CXC chemokines interleukin 8 (IL-8) and epithelial cell-derived neutrophil attractant 78 (ENA-78), in whole blood obtained after erythromycin infusion, was lower than that in blood drawn before erythromycin infusion (maximum inhibition post-infusion: 32.9 +/- 6.5% and 35.2 +/- 12.6% decrease in production, respectively, expressed as percentage change relative to production before infusion of erythromycin, both P < 0.05). In contrast, infusion of erythromycin was associated with an enhanced capacity of whole blood to release the neutrophil degranulation products bactericidal/permeability increasing protein (BPI), human neutrophil elastase (HNE) and human lactoferrin (HLF) upon stimulation with S. pneumoniae. Effects of erythromycin were greatest 4 h after infusion was stopped, when BPI, HNE and HLF concentrations were increased by +107.6 +/- 33.5%, +134.7 +/- 34.8% and +205.9 +/- 55.9 %, respectively (expressed as percentage change relative to production before infusion of erythromycin) (all P < 0. 05). These results indicate the ability of erythromycin to reduce CXC chemokine production and to enhance neutrophil degranulation in human blood.

  17. Human cord blood mononuclear cells decrease cytokines and inflammatory cells in acute myocardial infarction.

    PubMed

    Henning, Robert J; Shariff, Masood; Eadula, Ujwala; Alvarado, Felipe; Vasko, Mark; Sanberg, Paul R; Sanberg, Cyndy D; Delostia, Vincent

    2008-12-01

    We investigated whether human umbilical cord blood mononuclear cells (HUCBC), which contain hematopoietic and mesenchymal progenitor cells, can limit myocardial cytokine expression and inflammatory cell infiltration in acute myocardial infarction. We permanently ligated the left coronary artery of rats and injected into the myocardium either Isolyte or 4 x 10(6) HUCBC in Isolyte and measured myocardial cytokines with antibody arrays at 2, 6, 12, 24, and 72 hours after infarction. We then measured with flow cytometry myocardial macrophages, neutrophils and lymphocytes at 12, 24, and 72 hours after infarctions in rats treated with either intramyocardial Isolyte or 4 x 10(6) HUCBC. In the Isolyte-treated hearts, between 2 and 12 hours after myocardial infarction, tumor necrosis factor-alpha increased from 6.7 +/- 0.9% to 52.3 +/- 4.7%, monocyte chemoattract protein increased from 9.5 +/- 1.2% to 39.8 +/- 2.1%, fractalkine increased from 11 +/- 1.5% to 28.1 +/- 1.3%, ciliary neurotrophic factor increased from 12.1 +/- 0.02% to 25.9 +/- 1.1%, macrophage inflammatory protein increased from 10.3 +/- 1.5% to 23.9.0 +/- 1.4%, interferon-gamma increased from 8.7 +/- 0.4% to 26.0 +/- 1.6%, interleukin-1beta increased from 6.1 +/- 0.04% to 19.0 +/- 1.2%, and IL-4 increased from 5.9 +/- 0.03% to 15 +/- 1.5% (all p < 0.001 compared with controls). The concentrations of fractalkine remained significantly increased at 72 hours after acute infarction. In contrast, the myocardial concentrations of these cytokines did not significantly change in HUCBC treated hearts at 2, 6, 12, 24, or 72 hours after infarction. The percentage of neutrophils increased from 0.04 +/- 0.2%/50,000 heart cells in the controls to 5.3 +/- 1.2%/50,000 heart cells 12 hours after infarction in Isolyte-treated hearts but averaged only 1.3 +/- 0.7%/50,000 heart cells in HUCBC treated hearts (p < 0.02). Thereafter, the percentages of neutrophils rapidly decreased at 24 and at 72 hours after infarction and

  18. The state of the science of whole blood: lessons learned at Mayo Clinic

    PubMed Central

    Stubbs, James R.; Zielinski, Martin D.; Jenkins, Donald

    2016-01-01

    AABB Standards specify that ABO group-specific whole blood is the only acceptable choice for whole blood transfusions. Although universal donor group O stored whole blood (SWB) was used extensively by the military during the wars of the mid-twentieth century, its use has fallen out of favor and has never been used to great extent in the civilian trauma population. Interest in the use of whole blood has been renewed, particularly in light of its potential value in far-forward military and other austere environments. Evidence of preserved platelet function in SWB has heightened enthusiasm for a “one stop shop” resuscitation product providing volume, oxygen carrying capacity, and hemostatic effects. Experience with universal donor group O SWB is required to ascertain whether its use will be an advance in trauma care. Described here is the process of establishing a universal donor group O SWB at a civilian trauma center in the United States. PMID:27100754

  19. Differences in whole blood serotonin levels based on a typology of parasuicide.

    PubMed

    Rilke, O; Safar, C; Israel, M; Barth, T; Felber, W; Oehler, J

    1998-01-01

    Suicidal behavior has to be considered as a multifactorial phenomenon, which can be analyzed in a classifying-phenomenological manner. We have examined the relation of parasuicide typology to whole blood concentrations of serotonin, HVA, and tryptophan in 58 patients classified into 4 groups of parasuicide typology compared to 22 nonsuicidal depressed patients and 20 healthy subjects. Suicidal patients classified as impetuous, desperate and ambivalent types had significantly reduced whole blood 5-HT levels in comparison with the appealing type as well as nonsuicidal subjects. No differences were detected in the HVA content, but whole blood tryptophan concentrations were significantly reduced in impetuous suicidal patients and depressed patients compared to healthy subjects. This study provides evidence for reduced whole blood serotonin content based on different types of parasuicide.

  20. DIAGNOSING LYMPHOPROLIFERATIVE DISEASE VIRUS IN LIVE WILD TURKEYS (MELEAGRIS GALLOPAVO) USING WHOLE BLOOD.

    PubMed

    Alger, Katrina; Bunting, Elizabeth; Schuler, Krysten; Jagne, Jarra; Whipps, Christopher M

    2015-12-01

    Lymphoproliferative disease virus (LPDV) is a retrovirus that infects wild and domestic turkeys ( Meleagris gallopavo ). The first cases of LPDV in the United States were diagnosed in 2009, and subsequent surveillance has revealed the virus to be widespread in wild turkey populations throughout the eastern half of the country. More research is needed to determine whether LPDV is having a negative effect on turkey populations, but progress has been impeded by the lack of a simple method for diagnosing the virus in living birds. Infected animals may appear asymptomatic, and diagnostics currently rely on tissue or bone marrow, which can be difficult to obtain. This study investigated the reliability of polymerase chain reaction (PCR) to detect LPDV in whole blood, compared with previous methods using buffy coat (concentrated white blood cells) and bone marrow. Paired samples of whole blood and buffy coat were collected from 137 live turkeys and paired samples of whole blood and bone marrow were collected from 32 turkeys postmortem. Compared with buffy coat, whole blood had 97% sensitivity and 100% specificity. When compared with bone marrow, whole blood had 100% sensitivity and 89% specificity. Both comparisons had a high degree of agreement using Cohen's kappa statistic. Based on these results, PCR of whole blood provides detection of LPDV in living birds that is on par with both buffy coat and bone marrow.

  1. Serum/whole blood concentration ratio for ethylglucuronide and ethyl sulfate.

    PubMed

    Høiseth, Gudrun; Morini, Luca; Polettini, Aldo; Christophersen, Asbjørg S; Johnsen, Lene; Karinen, Ritva; Mørland, Jørg

    2009-05-01

    Serum/blood (S/B) concentration ratios for ethyl glucuronide (EtG) and ethyl sulfate (EtS) are missing from the literature, and the aim of this study was to determine these ratios in samples from patients at admission to an alcohol rehabilitation clinic. Two blood samples were collected simultaneously, and EtG and EtS were analyzed in whole blood and serum, respectively, using a liquid chromatography-mass spectrometry method. Separate calibration standards were prepared in both whole blood and serum for the calculation of whole blood and serum concentrations, respectively. Thirteen pairs of serum and whole blood were analyzed. The median S/B value for EtG was 1.69, and the range was 1.33-1.90. For EtS, the median S/B ratio was 1.30, and the range was 1.08-1.47. The S/B ratio was significantly lower for EtS than for EtG (p < 0.001). The higher concentrations of EtG and EtS in serum than in whole blood have to be considered when whole blood results obtained from forensic toxicology are compared to serum or plasma results from clinical laboratories. PMID:19470223

  2. In vitro expansion of Lin{sup +} and Lin{sup −} mononuclear cells from human peripheral blood

    SciTech Connect

    Norhaiza, H. Siti; Zarina, Z. A. Intan; Hisham, Z. A. Shahrul; Rohaya, M. A. W.

    2013-11-27

    Haematopoietic stem cells (HSCs) are used in the therapy of blood disorders due to the ability of these cells to reconstitute haematopoietic lineage cells when transplanted into myeloablative recipients. However, substantial number of cells is required in order for the reconstitution to take place. Since HSCs present in low frequency, larger number of donor is required to accommodate the demand of transplantable HSCs. Therefore, in vitro expansion of HSCs will have profound impact on clinical purposes. The aim of this study was to expand lineage negative (Lin{sup −}) stem cells from human peripheral blood. Total peripheral blood mononuclear cells (PBMNCs) were fractionated from human blood by density gradient centrifugation. Subsequently, PBMNCs were subjected to magnetic assisted cell sorter (MACS) which depletes lineage positive (Lin{sup +}) mononuclear cells expressing lineage positive markers such as CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a to obtained Lin{sup −} cell population. The ability of Lin{sup +} and Lin{sup −} to survive in vitro was explored by culturing both cell populations in complete medium consisting of Alpha-Minimal Essential Medium (AMEM) +10% (v/v) Newborn Calf Serum (NBCS)+ 2% (v/v) pen/strep. In another experiment, Lin{sup +} and Lin{sup −} were cultured with complete medium supplemented with 10ng/mL of the following growth factors: stem cell factor (SCF), interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), 2IU/mL of Erythropoietin (Epo) and 20ng/mL of IL-6. Three samples were monitored in static culture for 22 days. The expansion potential was assessed by the number of total viable cells, counted by trypan blue exclusion assay. It was found that Lin{sup +} mononuclear cells were not able to survive either in normal proliferation medium or proliferation medium supplemented with cytokines. Similarly, Lin{sup −} stem cells were not able to survive in proliferation medium however

  3. Microculture assay for isolation of human immunodeficiency virus type 1 and for titration of infected peripheral blood mononuclear cells.

    PubMed

    Dimitrov, D H; Melnick, J L; Hollinger, F B

    1990-04-01

    To define the optimal conditions for human immunodeficiency virus (HIV) detection in microcultures, experiments were conducted with different ratios of patient and donor peripheral blood mononuclear cells (PBMCs). Donor/patient PBMC ratios ranged from 1:1 to 1:125. Optimal results were obtained when 1,500,000 donor cells were cocultured with equal or smaller quantities of patient PBMCs. Thus, virologic endpoints could be achieved by diluting patient cells. Smaller numbers of donor cells, with or without larger numbers of patients cells, resulted in lower rates of HIV isolation. Similarly, the direct stimulation of patient PBMCs with phytohemagglutinin without the addition of normal donor cells lowered the sensitivity of the assay significantly. We suggest that a microculture procedure using a fixed quantity of donor cells with different dilutions of patient cells may be useful for monitoring changing HIV levels during antiviral therapy.

  4. Antiepileptic and neuroprotective effects of human umbilical cord blood mononuclear cells in a pilocarpine-induced epilepsy model.

    PubMed

    Costa-Ferro, Zaquer Suzana Munhoz; de Borba Cunha, Fernanda; de Freitas Souza, Bruno Solano; Leal, Marcos Maurício Tosta; da Silva, Adelson Alves; de Bellis Kühn, Telma Ingrid Borges; Forte, Andresa; Sekiya, Eliseo Joji; Soares, Milena Botelho Pereira; Dos Santos, Ricardo Ribeiro

    2014-03-01

    Status epilepticus (SE) is a condition of persistent seizure that leads to brain damage and, frequently, to the establishment of chronic epilepsy. Cord blood is an important source of adult stem cells for the treatment of neurological disorders. The present study aimed to evaluate the effects of human umbilical cord blood mononuclear cells (HUCBC) transplanted into rats after induction of SE by the administration of lithium and pilocarpine chloride. Transplantation of HUCBC into epileptic rats protected against neuronal loss in the hippocampal subfields CA1, CA3 and in the hilus of the dentate gyrus, up to 300 days after SE induction. Moreover, transplanted rats had reduced frequency and duration of spontaneous recurrent seizures (SRS) 15, 120 and 300 days after the SE. Our study shows that HUCBC provide prominent antiepileptic and neuroprotective effects in the experimental model of epilepsy and reinforces that early interventions can protect the brain against the establishment of epilepsy.

  5. Trypanosome-induced Interferon-γ production in whole blood stimulation assays is associated with latent Trypanosoma brucei gambiense infections.

    PubMed

    Ilboudo, Hamidou; Jamonneau, Vincent; Koffi, Mathurin; Kaboré, Jacques; Amoussa, Roukiyath; Holzmuller, Philippe; Garcia, André; Bucheton, Bruno; Courtin, David

    2016-06-01

    Control of human African trypanosomiasis (HAT) is highly dependent on the ability to detect and treat infected individuals. However, a number of individuals exposed to Trypanosoma brucei gambiense are able to control infection to undetectable levels in blood. They are long-term potential reservoirs and thus a threat for control strategies. Cytokine responses in whole blood stimulation assays were quantified in individuals with contrasting HAT status. Trypanosome-induced IFN-γ production was only observed in "trypanotolerant" subjects suspected of harboring latent infections. This result contributes new insights into the immune responses associated with infection control and opens novel diagnosis perspectives regarding HAT elimination. PMID:26993030

  6. Poly(vinyl alcohol)-coated silver nanoparticles: activation of neutrophils and nanotoxicology effects in human hepatocarcinoma and mononuclear cells.

    PubMed

    Paino, Iêda Maria Martinez; Zucolotto, Valtencir

    2015-03-01

    Silver nanoparticles (AgNps) have been described as important for their excellent biocompatibility, biomedical applications. Nevertheless, AgNps can interact with the immune system which is essential to analyze human exposure to assess their potential risk to health and environment. In general, the primary site for accumulation of nanoparticles has been demonstrated to be the liver. Furthermore, the direct activation of neutrophils or oxidative burst by a given nanoparticle is poorly documented. In this paper, we investigated the cell uptake, apoptosis, necrosis, DNA damage in human hepatocarcinoma cells (HepG2), primary normal human peripheral blood mononuclear cells (PBMC) and the direct activation of primary isolated neutrophils through the oxidative burst on exposure to AgNps coated with Polyvinyl-alcohol (PVA). All cell types were incubated in the presence of 1.0 and 50.0 μM of AgNps-PVA for 24h. A significant cyto- and genotoxic-response and the activation of human neutrophils were induced by AgNps-PVA (p<0.05). Our results revealed that AgNps can interact with the normal isolated neutrophils, PBMC and HepG2 cells in vitro, which opens the way for further studies on the toxicological effects of AgNps in the human immune system response and cancer cells. PMID:25681999

  7. Evaluating the role of low-speed centrifugation towards transfecting human peripheral blood mononuclear cell culture.

    PubMed

    Majumdar, M; Ratho, R; Chawla, Y; Singh, M P

    2014-01-01

    The conventional method of transfection of suspension cells by chemical has proven to be very difficult. We present a new transfection protocol, wherein, low-speed centrifugation of cell culture plates immediately after adding the lipid: DNA complex significantly enhances the transfection efficiency. Peripheral blood mononuclear cells (PBMCs) were transfected with BLOCK-iT™ Fluorescent Oligo (scrambled siRNA) and lipofectamine complex using conventional and low-speed centrifugation modified transfection protocols. The efficiency of transfection was determined using flowcytometer and cell viability was checked using MTT assay. Incorporation of low-speed centrifugation significantly enhances the transfection efficiency of BLOCK-iT™ in the suspension culture of PBMCs as compared to conventional transfection method (99.8% vs 28.3%; P < 0.0001), even at a low concentration of 40 picomoles without affecting the cell viability. Centrifugation enhanced transfection (CET) technique is simple, time-saving and novel application without compromising the cell viability in the context of recently popular RNA interference in suspension cultures of PBMCs. This undemanding modification might be applicable to a wide variety of cell lines and solve crucial problem of researchers working with RNA interference in suspension cultures. PMID:24713904

  8. HIV-1 reduces Aβ-degrading enzymatic activities in primary human mononuclear phagocytes1

    PubMed Central

    Lan, Xiqian; Xu, Jiqing; Kiyota, Tomomi; Peng, Hui; Zheng, Jialin C.; Ikezu, Tsuneya

    2011-01-01

    The advent and wide introduction of antiretroviral therapy (ART) has greatly improved the survival and longevity of HIV-infected patients. Unfortunately, despite ART treatment, these patients are still afflicted with many complications including cognitive dysfunction. There is a growing body of reports indicating accelerated deposition of amyloid plaques, which are composed of amyloid-β peptide (Aβ), in HIV-infected brains. Though how HIV viral infection precipitates Aβ accumulation is poorly understood. It is suggested that viral infection leads to increased production and impaired degradation of Aβ. Mononuclear phagocytes (macrophages and microglia) that are productively infected by HIV in brains play a pivotal role in Aβ degradation through the expression and execution of two endopeptidases: neprilysin (NEP) and insulin-degrading enzyme (IDE). Here we report that NEP has the dominant endopeptidase activity towards Aβ in macrophages. Further, we demonstrate that monomeric Aβ degradation by primary cultured macrophages and microglia was significantly impaired by HIV infection. This was accompanied with great reduction of NEP endopeptidase activity, which might be due to the diminished transport of NEP to cell surface and intracellular accumulation at the endoplasmic reticulum and lysosomes. Therefore, these data suggest that malfunction of NEP in infected macrophages may contribute to acceleration of beta amyloidosis in HIV-inflicted brains and modulation of macrophages may be a potential preventative target of Aβ-related cognitive disorders in HIV-affected patients. PMID:21551363

  9. Infection of human peripheral blood mononuclear cells with a temperature-sensitive mutant of measles virus.

    PubMed Central

    Vydelingum, S; Ilonen, J; Salonen, R; Marusyk, R; Salmi, A

    1989-01-01

    A stable temperature-sensitive mutant of measles virus (MV ts38) was used to study the mechanism of virus-mediated immune suppression of peripheral blood mononuclear cells in vitro. Both unstimulated and phytohemagglutinin-stimulated cultures released infectious virus at 32 degrees C, whereas no virus was released at 37 degrees C, although both viral RNA and viral proteins were synthesized. However, the response of the lymphoid cells to phytohemagglutinin, concanavalin A, and herpes simplex virus antigen was decreased in the presence of MV ts38 at 37 degrees C. The viability of infected cells was not diminished, therefore excluding cell death as a reason for immunosuppression. Interleukin 2 did not play a role in the inhibitory effect of MV ts38. Antibodies to alpha interferon partially reversed the inhibitory effect of the virus infection on lymphocyte mitogenesis, thus implying that alpha interferon plays a role in the immunosuppression. Depletion experiments indicated that adherent cells play a greater role in the measles virus-induced immunosuppression than nonadherent cells. However, monocyte maturation to macrophages had no effect on the degree of immunosuppression. Images PMID:2911119

  10. Development of Multiscale Materials in Microfluidic Devices: Case Study for Viral Separation from Whole Blood

    NASA Astrophysics Data System (ADS)

    Surawathanawises, Krissada

    prior to anodization is evaluated to control the growth of AAO. Together with controlled anodizing voltages and electrolytic concentrations, AAO pore and cell sizes are shown to be tunable and controllable with narrow size distributions within submicron range. A high degree of order of AAO pore arrangement is also demonstrated. In addition, overall anodization becomes more time-efficient and stable at high anodizing voltages. Secondly, a three-dimensional (3D) assembly of microbeads is used as a template to fabricate a spherical pore network with small interconnected openings. After depositing and drying a suspension containing both micro- and nanobeads, the microbeads assemble into a 3D close-packed structure while the nanobeads fill the interstitial space. When the nanobeads are melted and microbeads are removed, a spherical pore matrix then form with small interconnected openings. Such the opening size is in submicron range can be adjusted depending on the size of microbead. The advantages of the two macroporous materials are not only controllable and tunable pore size, but also high surface-to-volume ratio due to the nanoscale features. With a ratio on the order of ~1 microm-1, the porous materials provide a significantly large binding surface. Computational and experimental results reveal that porous materials with a pore size matching the nanoparticle size are suitable for their capture. Separation of human immunodeficiency virus (HIV) is used as a model and capture yields of ~99 % and ~80 % are achieved in the nanopost structure and spherical pore network, respectively, after treated with a functional chemistry. Hence, the properties of these two macroporous materials are suitable as a size-exclusion and affinity separation for viral particles. To further explore multiscale separation, i.e. capturing viruses from whole blood, micropatterned arrays of macroporous materials have been designed. In this design, a microscale gap allows the passage of microparticles

  11. Comparison of Human Embryonic Stem Cell-Derived Cardiomyocytes, Cardiovascular Progenitors, and Bone Marrow Mononuclear Cells for Cardiac Repair

    PubMed Central

    Fernandes, Sarah; Chong, James J.H.; Paige, Sharon L.; Iwata, Mineo; Torok-Storb, Beverly; Keller, Gordon; Reinecke, Hans; Murry, Charles E.

    2015-01-01

    Summary Cardiomyocytes derived from human embryonic stem cells (hESC-CMs) can improve the contractility of injured hearts. We hypothesized that mesodermal cardiovascular progenitors (hESC-CVPs), capable of generating vascular cells in addition to cardiomyocytes, would provide superior repair by contributing to multiple components of myocardium. We performed a head-to-head comparison of hESC-CMs and hESC-CVPs and compared these with the most commonly used clinical cell type, human bone marrow mononuclear cells (hBM-MNCs). In a nude rat model of myocardial infarction, hESC-CMs and hESC-CVPs generated comparable grafts. Both similarly improved systolic function and ventricular dilation. Furthermore, only rare human vessels formed from hESC-CVPs. hBM-MNCs attenuated ventricular dilation and enhanced host vascularization without engrafting long-term or improving contractility. Thus, hESC-CMs and CVPs show similar efficacy for cardiac repair, and both are more efficient than hBM-MNCs. However, hESC-CVPs do not form larger grafts or more significant numbers of human vessels in the infarcted heart. PMID:26607951

  12. Impact of Whole-Blood Processing Conditions on Plasma and Serum Concentrations of Cytokines.

    PubMed

    Lee, Jae-Eun; Kim, Jong-Wan; Han, Bok-Ghee; Shin, So-Youn

    2016-02-01

    Pre-analytical variations in plasma and serum samples can occur because of variability in whole-blood processing procedures. The aim of this study was to determine the impact of delayed separation of whole blood on the plasma and serum concentrations of cytokines. The concentrations of 16 cytokines were measured in plasma and serum samples when the centrifugation of whole blood at room temperature was delayed for 4, 6, 24, or 48 h, and the values were compared with those observed after separation within 2 h of whole-blood collection. Receiver operating characteristic (ROC) curve analysis was also performed for cytokines to determine whether cytokine levels in plasma and serum samples can be used to assess delayed separation of whole blood. Plasma concentrations of interleukin (IL)-1β, granulocyte-macrophage colony-stimulating factor (GM-CSF), and soluble CD40 ligand (sCD40L) and serum concentrations of IL-1β, IL-6, IL-8, macrophage inflammatory protein-1α (MIP-1α), and MIP-1β increased significantly (>2-fold) when separation was delayed at room temperature for 24 h. The concentrations of 6 of these cytokines (all except serum IL-1β and IL-6) demonstrated high diagnostic performance (area under the ROC curve >0.8) for delayed separation of whole blood. Furthermore, these cytokine concentrations typically exhibited high sensitivity and specificity at each optimal cutoff point. Conversely, IL-17A was stable in both plasma and serum samples, even when whole-blood centrifugation was delayed at room temperature for 48 h. This study shows that certain cytokines (IL-1β, GM-CSF, sCD40L, IL-8, MIP-1α, and MIP-1β) could be used for assessing the quality of plasma or serum samples. PMID:26808439

  13. Investigation of stillbirths, perinatal mortality and weakness in beef calves with low-selenium whole blood concentrations.

    PubMed

    Davis, Anthony J; Myburgh, Jan G

    2016-07-15

    In this on-farm investigation, we report on stillbirths, weakness and perinatal mortality seen in calves on a commercial beef farm in the Roossenekal area, Mpumalanga province, South Africa. Post-mortem examination of these calves and histopathological examination of organ and tissue samples did not indicate an infectious aetiology. Affected calves had marginal to deficient whole blood selenium concentrations. Whole blood samples collected from adult cattle on this farm and five neighbouring farms were deficient in selenium. The potential contributions of other minerals to the symptoms seen are a subject of ongoing investigation, but selenium deficiency was marked in this herd and required urgent correction. Methods to correct the deficiency included the use of injectable products, and an oral selenium supplement chelated to methionine. Selenium availability to plants is primarily determined by the selenium content of the parent bedrock, the presence of other minerals and the pH of the soil. The apparent sudden onset of this problem implicates a soil factor as being responsible for reducing selenium's bioavailability in this area. Selenium deficiency can have a significant impact on human health. HIV and/or AIDS, various forms of cancer and several specific clinical syndromes are associated with selenium deficiency in humans, and the impact on human health in this area also requires further investigation.

  14. Investigation of stillbirths, perinatal mortality and weakness in beef calves with low-selenium whole blood concentrations.

    PubMed

    Davis, Anthony J; Myburgh, Jan G

    2016-01-01

    In this on-farm investigation, we report on stillbirths, weakness and perinatal mortality seen in calves on a commercial beef farm in the Roossenekal area, Mpumalanga province, South Africa. Post-mortem examination of these calves and histopathological examination of organ and tissue samples did not indicate an infectious aetiology. Affected calves had marginal to deficient whole blood selenium concentrations. Whole blood samples collected from adult cattle on this farm and five neighbouring farms were deficient in selenium. The potential contributions of other minerals to the symptoms seen are a subject of ongoing investigation, but selenium deficiency was marked in this herd and required urgent correction. Methods to correct the deficiency included the use of injectable products, and an oral selenium supplement chelated to methionine. Selenium availability to plants is primarily determined by the selenium content of the parent bedrock, the presence of other minerals and the pH of the soil. The apparent sudden onset of this problem implicates a soil factor as being responsible for reducing selenium's bioavailability in this area. Selenium deficiency can have a significant impact on human health. HIV and/or AIDS, various forms of cancer and several specific clinical syndromes are associated with selenium deficiency in humans, and the impact on human health in this area also requires further investigation. PMID:27543038

  15. On-chip Extraction of Intracellular Molecules in White Blood Cells from Whole Blood

    PubMed Central

    Choi, Jongchan; Hyun, Ji-chul; Yang, Sung

    2015-01-01

    The extraction of virological markers in white blood cells (WBCs) from whole blood—without reagents, electricity, or instruments—is the most important first step for diagnostic testing of infectious diseases in resource-limited settings. Here we develop an integrated microfluidic chip that continuously separates WBCs from whole blood and mechanically ruptures them to extract intracellular proteins and nucleic acids for diagnostic purposes. The integrated chip is assembled with a device that separates WBCs by using differences in blood cell size and a mechanical cell lysis chip with ultra-sharp nanoblade arrays. We demonstrate the performance of the integrated device by quantitatively analyzing the levels of extracted intracellular proteins and genomic DNAs. Our results show that compared with a conventional method, the device yields 120% higher level of total protein amount and similar levels of gDNA (90.3%). To demonstrate its clinical application to human immunodeficiency virus (HIV) diagnostics, the developed chip was used to process blood samples containing HIV-infected cells. Based on PCR results, we demonstrate that the chip can extract HIV proviral DNAs from infected cells with a population as low as 102/μl. These findings suggest that the developed device has potential application in point-of-care testing for infectious diseases in developing countries. PMID:26464211

  16. Lab-on-CD microfluidic platform for rapid separation and mixing of plasma from whole blood.

    PubMed

    Kuo, Ju-Nan; Li, Bo-Shiun

    2014-08-01

    Traditional clinical methods for separating whole blood into blood cells and cell-free plasma are labor intensive and time consuming. Accordingly, the present study proposes a simple compact disk (CD) microfluidic platform for the rapid separation of plasma from whole human blood and the subsequent mixing of the plasma with a suitable reagent. The performance of three CD microfluidic platforms incorporating square-wave mixing channels with different widths is evaluated both numerically and experimentally. The results show that given an appropriate specification of the microchannel geometry and a CD rotation speed of 2000 rpm, a separation efficiency of 95 % can be achieved within 5 ~ 6 s given a diluted blood sample with a hematocrit concentration of 6 %. Moreover, a mixing efficiency of more than 96 % can be obtained within 5 s given a CD rotation speed of 2200 rpm. The practical feasibility of the proposed device is demonstrated by performing a prothrombin time (PT) test. It is shown that while the time required to perform the PT test using a conventional bench top system is around 15 min, the proposed CD microfluidic platform allows the test to be completed within 1 min.

  17. On-chip Extraction of Intracellular Molecules in White Blood Cells from Whole Blood

    NASA Astrophysics Data System (ADS)

    Choi, Jongchan; Hyun, Ji-Chul; Yang, Sung

    2015-10-01

    The extraction of virological markers in white blood cells (WBCs) from whole blood—without reagents, electricity, or instruments—is the most important first step for diagnostic testing of infectious diseases in resource-limited settings. Here we develop an integrated microfluidic chip that continuously separates WBCs from whole blood and mechanically ruptures them to extract intracellular proteins and nucleic acids for diagnostic purposes. The integrated chip is assembled with a device that separates WBCs by using differences in blood cell size and a mechanical cell lysis chip with ultra-sharp nanoblade arrays. We demonstrate the performance of the integrated device by quantitatively analyzing the levels of extracted intracellular proteins and genomic DNAs. Our results show that compared with a conventional method, the device yields 120% higher level of total protein amount and similar levels of gDNA (90.3%). To demonstrate its clinical application to human immunodeficiency virus (HIV) diagnostics, the developed chip was used to process blood samples containing HIV-infected cells. Based on PCR results, we demonstrate that the chip can extract HIV proviral DNAs from infected cells with a population as low as 102/μl. These findings suggest that the developed device has potential application in point-of-care testing for infectious diseases in developing countries.

  18. Optimization of direct whole blood PCR amplification with applications on a static thermostat chip.

    PubMed

    Qu, Bai-Yan; Wu, Zhi-Yong; Tian, Xiao-Xi; Chen, Kun; Fang, Fang

    2007-11-01

    In this paper, direct whole blood PCR amplifications on a static chip thermostat without sample purifications are demonstrated; in these amplifications, problems such as cross-interferences and contaminations could be avoided. The amplification conditions, such as the compositions of reagents and thermal programs, were investigated systematically by a GeneAmp PCR system with a native p53 gene segment (about 543 bp) of human genome and an exterior lambda DNA segment (about 500 bp) as targets. Direct amplifications of p53 and K-ras (about 157 bp) gene segments from 0.5 microL blood samples were successfully demonstrated by a static PCR chip with an indium tin oxide glass substrate. The chip thermostat has a typical size of 25 mm x 25 mm, and a polyethylene tube was used as the PCR vial on the glass surface of the chip. Fuzzy proportional integration-differentiation algorithms were adopted in temperature controls of the chip with an aid of a micro-Pt100 sensor. In the direct PCR with the thermostat chip, the whole process only involves automatic thermal programs. This work demonstrated that a chip PCR for field test without desktop facilities is possible either for a point of care test or for forensic analysis.

  19. Whole Blood Gene Expression and Atrial Fibrillation: The Framingham Heart Study

    PubMed Central

    Lin, Honghuang; Yin, Xiaoyan; Lunetta, Kathryn L.; Dupuis, Josée; McManus, David D.; Lubitz, Steven A.; Magnani, Jared W.; Joehanes, Roby; Munson, Peter J.; Larson, Martin G.; Levy, Daniel; Ellinor, Patrick T.; Benjamin, Emelia J.

    2014-01-01

    Background Atrial fibrillation (AF) involves substantial electrophysiological, structural and contractile remodeling. We hypothesize that characterizing gene expression might uncover important pathways related to AF. Methods and Results We performed genome-wide whole blood transcriptomic profiling (Affymetrix Human Exon 1.0 ST Array) of 2446 participants (mean age 66±9 years, 55% women) from the Offspring cohort of Framingham Heart Study. The study included 177 participants with prevalent AF, 143 with incident AF during up to 7 years follow up, and 2126 participants with no AF. We identified seven genes statistically significantly up-regulated with prevalent AF. The most significant gene, PBX1 (P = 2.8×10−7), plays an important role in cardiovascular development. We integrated differential gene expression with gene-gene interaction information to identify several signaling pathways possibly involved in AF-related transcriptional regulation. We did not detect any statistically significant transcriptomic associations with incident AF. Conclusion We examined associations of gene expression with AF in a large community-based cohort. Our study revealed several genes and signaling pathways that are potentially involved in AF-related transcriptional regulation. PMID:24805109

  20. Whole blood gene expression profiling of neonates with confirmed bacterial sepsis.

    PubMed

    Dickinson, Paul; Smith, Claire L; Forster, Thorsten; Craigon, Marie; Ross, Alan J; Khondoker, Mizan R; Ivens, Alasdair; Lynn, David J; Orme, Judith; Jackson, Allan; Lacaze, Paul; Flanagan, Katie L; Stenson, Benjamin J; Ghazal, Peter

    2015-03-01

    Neonatal infection remains a primary cause of infant morbidity and mortality worldwide and yet our understanding of how human neonates respond to infection remains incomplete. Changes in host gene expression in response to infection may occur in any part of the body, with the continuous interaction between blood and tissues allowing blood cells to act as biosensors for the changes. In this study we have used whole blood transcriptome profiling to systematically identify signatures and the pathway biology underlying the pathogenesis of neonatal infection. Blood samples were collected from neonates at the first clinical signs of suspected sepsis alongside age matched healthy control subjects. Here we report a detailed description of the study design, including clinical data collected, experimental methods used and data analysis workflows and which correspond with data in Gene Expression Omnibus (GEO) data sets (GSE25504). Our data set has allowed identification of a patient invariant 52-gene classifier that predicts bacterial infection with high accuracy and lays the foundation for advancing diagnostic, prognostic and therapeutic strategies for neonatal sepsis.

  1. Whole blood gene expression profiling of neonates with confirmed bacterial sepsis

    PubMed Central

    Dickinson, Paul; Smith, Claire L.; Forster, Thorsten; Craigon, Marie; Ross, Alan J.; Khondoker, Mizan R.; Ivens, Alasdair; Lynn, David J.; Orme, Judith; Jackson, Allan; Lacaze, Paul; Flanagan, Katie L.; Stenson, Benjamin J.; Ghazal, Peter

    2014-01-01

    Neonatal infection remains a primary cause of infant morbidity and mortality worldwide and yet our understanding of how human neonates respond to infection remains incomplete. Changes in host gene expression in response to infection may occur in any part of the body, with the continuous interaction between blood and tissues allowing blood cells to act as biosensors for the changes. In this study we have used whole blood transcriptome profiling to systematically identify signatures and the pathway biology underlying the pathogenesis of neonatal infection. Blood samples were collected from neonates at the first clinical signs of suspected sepsis alongside age matched healthy control subjects. Here we report a detailed description of the study design, including clinical data collected, experimental methods used and data analysis workflows and which correspond with data in Gene Expression Omnibus (GEO) data sets (GSE25504). Our data set has allowed identification of a patient invariant 52-gene classifier that predicts bacterial infection with high accuracy and lays the foundation for advancing diagnostic, prognostic and therapeutic strategies for neonatal sepsis. PMID:26484146

  2. Changes in transcriptional output of human peripheral blood mononuclear cells following resistance exercise.

    PubMed

    Carlson, Lara A; Tighe, S W; Kenefick, R W; Dragon, J; Westcott, N W; Leclair, R J

    2011-12-01

    Various types of exercise alter the population of circulating peripheral blood mononuclear cells (PBMCs) and change their transcriptional output. This work examines changes in PBMC populations and transcription in response to resistance exercise training (RET), and identify key transcriptional changes in PBMCs that may play a role in altering peripheral tissues in response to RET. Ten resistance-trained men (20-24 years), performed an acute bout of RET for ~30 min following a 12 h fast. Venous blood was sampled at rest, immediately following exercise, and at 2 h post-exercise and analyzed for total and differential leukocytes and global gene expression using Affymetrix Genechips. Results showed elevated leukocytes, monocytes, lymphocytes, and lactate values immediately post-exercise (P < 0.05) over baseline. At 2 h post-exercise, leukocytes, and granulocytes remained elevated (P < 0.05), whereas lymphocytes were lower than (P < 0.05) baseline values. Initial microarray results showed the greatest transcriptional changes in pathways related to immune response, inflammation, and cellular communication. The change in PBMC population (2 h time point) correlated with a dramatic decrease in the expression of CD160, and XCL1, markers of lymphocyte populations. At the 2 h recovery time point upregulation of matrix metalloproteinase 9, orosomucoid 1, dishevelled-associated activator of morphogenesis 2, and arginase 1 suggest an induction in muscle damage and repair during this time frame. These results demonstrate that an acute bout of RET disrupts cellular homeostasis, induces a transient redistribution of certain leukocytes, and results in transcriptional changes in PBMCs translating into systemic changes in response to RET. PMID:21437602

  3. Changes in transcriptional output of human peripheral blood mononuclear cells following resistance exercise.

    PubMed

    Carlson, Lara A; Tighe, S W; Kenefick, R W; Dragon, J; Westcott, N W; Leclair, R J

    2011-12-01

    Various types of exercise alter the population of circulating peripheral blood mononuclear cells (PBMCs) and change their transcriptional output. This work examines changes in PBMC populations and transcription in response to resistance exercise training (RET), and identify key transcriptional changes in PBMCs that may play a role in altering peripheral tissues in response to RET. Ten resistance-trained men (20-24 years), performed an acute bout of RET for ~30 min following a 12 h fast. Venous blood was sampled at rest, immediately following exercise, and at 2 h post-exercise and analyzed for total and differential leukocytes and global gene expression using Affymetrix Genechips. Results showed elevated leukocytes, monocytes, lymphocytes, and lactate values immediately post-exercise (P < 0.05) over baseline. At 2 h post-exercise, leukocytes, and granulocytes remained elevated (P < 0.05), whereas lymphocytes were lower than (P < 0.05) baseline values. Initial microarray results showed the greatest transcriptional changes in pathways related to immune response, inflammation, and cellular communication. The change in PBMC population (2 h time point) correlated with a dramatic decrease in the expression of CD160, and XCL1, markers of lymphocyte populations. At the 2 h recovery time point upregulation of matrix metalloproteinase 9, orosomucoid 1, dishevelled-associated activator of morphogenesis 2, and arginase 1 suggest an induction in muscle damage and repair during this time frame. These results demonstrate that an acute bout of RET disrupts cellular homeostasis, induces a transient redistribution of certain leukocytes, and results in transcriptional changes in PBMCs translating into systemic changes in response to RET.

  4. Bisphenol A and its analogs induce morphological and biochemical alterations in human peripheral blood mononuclear cells (in vitro study).

    PubMed

    Michałowicz, Jaromir; Mokra, Katarzyna; Bąk, Agata

    2015-10-01

    Few studies have addressed the cellular effects of bisphenol S (BPS) and bisphenol AF (BPAF) on cells, and no study has been conducted to analyze the mechanism of action of bisphenols in blood cells. In this study, the effect of bisphenol A (BPA), bisphenol F (BPF), BPS and BPAF on human peripheral blood mononuclear cells (PBMCs) was analyzed. It was shown that BPA, BPF and BPAF in particular, decreased cell viability, which was associated with depletion of intracellular ATP level and alterations in PBMCs size and granulation. Bisphenols enhanced ROS (including OH˙) formation, which led to damage to lipids and proteins in PBMCs. The most significant alterations in ROS level were induced by BPF, and particularly BPAF. Moreover, it was shown that BPAF most strongly provoked lipid peroxidation, while BPA and BPS caused the greatest damage to proteins. It may be concluded that BPA and its analogs were capable of inducing oxidative stress and damage in PBMCs in the concentrations ranging from 0.06 to 0.5 μM (0.02-0.1 μg/ml), which may be present in human blood as a result of environmental exposure. Although, most of bisphenols studied decreased cell viability, size and ATP level at higher concentrations, BPAF exhibited its cytotoxic potential at low concentrations ranging from 0.3 to 3 μM (0.1-1.0 μg/ml) that may correspond to concentrations in humans following occupational exposure. PMID:26028149

  5. Bisphenol A and its analogs induce morphological and biochemical alterations in human peripheral blood mononuclear cells (in vitro study).

    PubMed

    Michałowicz, Jaromir; Mokra, Katarzyna; Bąk, Agata

    2015-10-01

    Few studies have addressed the cellular effects of bisphenol S (BPS) and bisphenol AF (BPAF) on cells, and no study has been conducted to analyze the mechanism of action of bisphenols in blood cells. In this study, the effect of bisphenol A (BPA), bisphenol F (BPF), BPS and BPAF on human peripheral blood mononuclear cells (PBMCs) was analyzed. It was shown that BPA, BPF and BPAF in particular, decreased cell viability, which was associated with depletion of intracellular ATP level and alterations in PBMCs size and granulation. Bisphenols enhanced ROS (including OH˙) formation, which led to damage to lipids and proteins in PBMCs. The most significant alterations in ROS level were induced by BPF, and particularly BPAF. Moreover, it was shown that BPAF most strongly provoked lipid peroxidation, while BPA and BPS caused the greatest damage to proteins. It may be concluded that BPA and its analogs were capable of inducing oxidative stress and damage in PBMCs in the concentrations ranging from 0.06 to 0.5 μM (0.02-0.1 μg/ml), which may be present in human blood as a result of environmental exposure. Although, most of bisphenols studied decreased cell viability, size and ATP level at higher concentrations, BPAF exhibited its cytotoxic potential at low concentrations ranging from 0.3 to 3 μM (0.1-1.0 μg/ml) that may correspond to concentrations in humans following occupational exposure.

  6. The immunophenotype of antigen presenting cells of the mononuclear phagocyte system in normal human liver--a systematic review.

    PubMed

    Strauss, Otto; Dunbar, P Rod; Bartlett, Adam; Phillips, Anthony

    2015-02-01

    The mononuclear phagocytic system (MPS), comprised of monocytes, macrophages, and dendritic cells, is essential in tissue homeostasis and in determining the balance of the immune response through its role in antigen presentation. It has been identified as a therapeutic target in infectious disease, cancer, autoimmune disease and transplant rejection. Here, we review the current understanding of the immunophenotype and function of the MPS in normal human liver. Using well-defined selection criteria, a search of MEDLINE and EMBASE databases identified 76 appropriate studies. The majority (n=67) described Kupffer cells (KCs), although the definition of KC differs between sources, and little data were available regarding their function. Only 10 papers looked at liver dendritic cells (DCs), and largely confirmed the presence of the major dendritic cell subsets identified in human blood. Monocytes were thoroughly characterized in four studies that utilized flow cytometry and fluorescent microscopy and highlighted their prominent role in liver homeostasis and displayed subtle differences from circulating monocytes. There was some limited evidence that liver DCs are tolerogenic but neither liver dendritic cell subsets nor macrophages have been thoroughly characterized, using either multi-colour flow cytometry or multi-parameter fluorescence microscopy. The lobular distribution of different subsets of liver MPS cells was also poorly described, and the ability to distinguish between passenger leukocytes and tissue resident cells remains limited. It was apparent that further research, using modern immunological techniques, is now required to accurately characterize the cells of the MPS in human liver.

  7. Effect of human polymorphonuclear and mononuclear leukocytes on chromosomal and plasmid DNA of Escherichia coli. Role of acid DNase

    SciTech Connect

    Rozenberg-Arska, M.; van Strijp, J.A.; Hoekstra, W.P.; Verhoef, J.

    1984-05-01

    Phagocytosis and killing by polymorphonuclear and mononuclear leukocytes are important host resistance factors against invading microorganisms. Evidence showing that killing is rapidly followed by degradation of bacterial components is limited. Therefore, we studied the fate of Escherichia coli DNA following phagocytosis of E. coli by polymorphonuclear and mononuclear leukocytes. (/sup 3/H)Thymidine-labeled, unencapsulated E. coli PC2166 and E. coli 048K1 were incubated in serum, washed, and added to leukocytes. Uptake and killing of the bacteria and degradation of DNA were measured. Although phagocytosis and killing by mononuclear leukocytes was less efficient than that by polymorphonuclear leukocytes, only mononuclear leukocytes were able to degrade E. coli PC2166 DNA. Within 2 h, 60% of the radioactivity added to mononuclear leukocytes was released into the supernate, of which 40% was acid soluble. DNA of E. coli 048K1 was not degraded. To further analyze the capacity of mononuclear leukocytes to degrade E. coli DNA, chromosomal and plasmid DNA was isolated from ingested bacteria and subjected to agarose gel-electrophoresis. Only chromosomal DNA was degraded after phagocytosis. Plasmid DNA of E. coli carrying a gene coding for ampicillin resistance remained intact for a 2-h period after ingestion, and was still able to transform recipient E. coli cells after this period. Although we observed no DNA degradation during phagocytosis by polymorphonuclear leukocytes, lysates of both polymorphonuclear and mononuclear leukocytes contained acid-DNase activity with a pH optimum of 4.9. However, the DNase activity of mononuclear leukocytes was 20 times higher than that of polymorphonuclear leukocytes. No difference was observed between DNase activity from polymorphonuclear and mononuclear leukocytes from a chronic granulomatous disease patient with DNase activity from control polymorphonuclear and mononuclear leukocytes.

  8. Blood Far Forward--a whole blood research and training program for austere environments.

    PubMed

    Strandenes, Geir; Cap, Andrew P; Cacic, Daniel; Lunde, Turid Helen Felli; Eliassen, Håkon Skogrand; Hervig, Tor; Spinella, Philip C

    2013-01-01

    The Blood Far Forward (BFF) research program was established to conduct blood product efficacy and safety studies, donor performance studies, and research on optimal training methods to improve the safety of blood collection and transfusion performed by Norwegian Naval Special Operation Commando soldiers. The use of intravenous fluids for volume replacement during hemorrhagic shock is controversial, but it is currently the standard of care. In the far-forward environment, large volume resuscitation for massive bleeding is a great challenge. Crystalloid and colloid solutions add weight and bulk to the medic's kit, require temperature sensitive storage, and should be warmed before infusion to prevent hypothermia. Excessive use of these solutions causes a dilutional coagulopathy, acidosis, and potentially increased inflammatory injury compared with blood products. Type-specific whole blood from an uninjured combat companion on the other hand is almost always available. It is warm, replaces intravascular volume, and provides oxygen delivery and hemostatic capacity to prevent or treat shock and coagulopathy. Whole blood may be ideal for the resuscitation of combat casualties with hemorrhagic shock. BFF program pilot studies on use of platelet-sparing leukoreduction filters, whole blood transport tolerance, donor performance, and autologous reinfusion of 24-hour ambient temperature stored whole blood have been performed and suggest the feasibility of expanding whole blood use in resuscitation. If successful, the BFF program will change tactics, techniques, and procedures with a new lifesaving capability.

  9. Metal and metalloid multi-elementary ICP-MS validation in whole blood, plasma, urine and hair. Reference values.

    PubMed

    Goullé, Jean-Pierre; Mahieu, Loïc; Castermant, Julien; Neveu, Nicolas; Bonneau, Laurent; Lainé, Gilbert; Bouige, Daniel; Lacroix, Christian

    2005-10-01

    Four multi-elementary metal and metalloid quantification methods using inductively coupled plasma mass spectrometry (ICP-MS) were developed and validated in human whole blood, plasma, urine and hair by means of a single preparation procedure for each sample. The ICP-MS measurements were performed using a Thermo Elemental X7CCT series and PlasmaLab software without a dynamic reaction cell. With this procedure 27-32 elements can be simultaneously quantified in biological matrices: Li, Be, B, Al, V, Cr, Mn, Co, Ni, Cu, Zn, Ga, Ge, As, Se, Rb, Sr, Mo, Pd, Ag, Cd, Sn, Sb, Te, Ba, W, Pt, Hg, Tl, Pb, Bi, U. Whole blood, plasma and urine samples (0.4 ml each) were diluted with purified water, acid, triton X100 and butanol. Rhodium was used as internal standard. The urine sample results were corrected for enzymatic creatinine determination. Twenty-five milligrams hair samples were acid mineralized after a decontamination procedure and diluted as previously described for biological fluids. To be validated, each element had to show linearity with a correlation coefficient higher than 0.99. The intra-assay and inter-assay inaccuracy, measured as the variation coefficient, were below 5 and 10% respectively. Global performance was assessed by a quality control program. Our laboratory is a registered participant of the Institut National de Santé Publique du Québec (Sainte-Foy, Canada) inter-laboratory comparison program for whole blood, urine, and beard hair of non-occupationally exposed individuals spiked with selected elements. In our study multi-element metal and metalloid analysis was assessed for 27 elements in whole blood, 27 elements in plasma, 30 elements in urine and 32 elements in hair, from 0 to 25, or 250 to 1000 ng/ml, depending on the element. Quantification limits ranged from 0.002 ng/ml (U) to 8.1 ng/ml (Al) for whole blood, from 0.002 ng/ml (U) to 7.7 ng/ml (Al) for plasma, from 0.001 ng/ml (U) to 2.2 ng/ml (Se) for urine, and from 0.2 pg/mg (Tl) to 0.5 ng

  10. Schistosome infection is associated with enhanced whole-blood IL-10 secretion in response to cercarial excretory/secretory products.

    PubMed

    Turner, J D; Meurs, L; Dool, P; Bourke, C D; Mbow, M; Dièye, T N; Mboup, S; Polman, K; Mountford, A P

    2013-01-01

    Infection of the human host by schistosome parasites follows exposure of skin to free-swimming cercariae and is aided by the release of excretory/secretory (E/S) material, which is rich in proteases and glycoconjugates. This material provides the initial stimulus to cells of the innate immune system. The study presented here is the first to examine human innate/early immune responsiveness to cercarial E/S in subjects from an area co-endemic for Schistosoma mansoni and S. haematobium. We report that in infected participants, stimulation of whole-blood cultures with cercarial E/S material (termed 0-3 hRP) caused the early (within 24 h) release of greater quantities of regulatory IL-10, compared with uninfected controls. Elevated levels of IL-10 but not pro-inflammatory TNFα or IL-8 were most evident in participants co-infected with S. mansoni and S. haematobium and were accompanied by a higher 0-3 h RP-specific IL-10: TNFα ratio. We also report that glycosylated components within 0-3 h RP appear to be important factors in the stimulation of IL-8, TNFα and IL-10 production by whole-blood cells.

  11. Cluster analysis of host cytokine responses to biodefense pathogens in a whole blood ex vivo exposure model (WEEM)

    PubMed Central

    2012-01-01

    Background Rapid detection and therapeutic intervention for infectious and emerging diseases is a major scientific goal in biodefense and public health. Toward this end, cytokine profiles in human blood were investigated using a human whole blood ex vivo exposure model, called WEEM. Results Samples of whole blood from healthy volunteers were incubated with seven pathogens including Yersinia pseudotuberculosis, Yersinia enterocolitica, Bacillus anthracis, and multiple strains of Yersinia pestis, and multiplexed protein expression profiling was conducted on supernatants of these cultures with an antibody array to detect 30 cytokines simultaneously. Levels of 8 cytokines, IL-1α, IL-1β, IL-6, IL-8, IL-10, IP-10, MCP-1 and TNFα, were significantly up-regulated in plasma after bacterial exposures of 4 hours. Statistical clustering was applied to group the pathogens based on the host response protein expression profiles. The nearest phylogenetic neighbors clustered more closely than the more distant pathogens, and all seven pathogens were clearly differentiated from the unexposed control. In addition, the Y. pestis and Yersinia near neighbors were differentiated from the B. anthracis strains. Conclusions Cluster analysis, based on host response cytokine profiles, indicates that distinct patterns of immunomodulatory proteins are induced by the different pathogen exposures and these patterns may enable further development into biomarkers for diagnosing pathogen exposure. PMID:22607329

  12. Fluorescence detection of the anticancer drug topotecan and related camptothecins in plasma and whole blood by two-photon excitation

    NASA Astrophysics Data System (ADS)

    Burke, Thomas G.; Malak, Henryk M.; Gryczynski, Ignacy; Lakowicz, Joseph R.

    1997-05-01

    Recent FDA-approval of topotecan (9-dimethylaminomethyl-10- hydroxycamptothecin) and camptosar (CPT-11) along with the accelerated clinical development of related camptothecin drugs provides new hope for the successful treatment of human cancer, including neoplasms for which no effective treatments currently exist. Current clinical efforts worldwide are aimed at optimizing the therapeutic efficacies of the camptothecins, with the major focus on the determination of the most effective dosing schedules. To this end, technological advances which provide a direct and rapid means of measuring plasma drug levels (i.e. such that correlations between plasma drug levels and clinical responses can be sought) would be of great utility. Here we report on the direct fluorescence detection of topotecan and SN-38 in human plasma and topotecan in whole blood at micro molar levels using two-photon excitation at 730 or 820 nm. Topotecan was detected at concentrations as low as 0.05 and 1 (mu) M in plasma and whole blood, respectively. Since skin, blood and other tissues are translucent at long wavelengths, our results suggest the attractive possibility of homogeneous or noninvasive clinical sensing of camptothecins in situ using two-photon excitation.

  13. Maternal Gene Expression Profiling During Pregnancy And Preeclampsia In Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Rajakumar, Augustine; Tianjiao, Chu; Handley, Daniel E.; Bunce, Kimberly D.; Burke, Brian; Hubel, Carl A.; Jeyabalan, Arundhathi; Peters, David G.

    2010-01-01

    Preeclampsia is a major obstetrical complication affecting maternal and fetal health. While it is clear that there is a substantial placental contribution to preeclampsia pathogenesis, the maternal contribution is less well characterized. We therefore performed a genome wide transcriptome analysis to explore disease-associated changes in maternal gene expression patterns in peripheral blood mononuclear cells (PBMCs). Methods Preeclampsia was defined as gestational hypertension, proteinuria and hyperurecimia. Total RNA was isolated from PBMCs obtained from women with uncomplicated pregnancies (n=5) and women with preeclamptic pregnancies (n=5). Gene expression analysis was carried out using Agilent oligonucleotide microarrays. Biological pathway analysis was undertaken using Ingenuity Pathway Analysis software. Quantitative real time PCR (QRTPCR) was performed to validate the gene expression changes of selected genes in normotensive and preeclamptic patients (n=12 each). Results We identified a total of 368 genes that were differentially expressed in women with preeclampsia compared to normal controls with false discovery rate (FDR) controlled at 10%. In follow up experiments we further analyzed the expression levels of a number of genes that were identified as altered by the microarray data including survivin (BIRC5), caveolin (CAV1), GATA binding protein-1 (GATA1), signal tranducer and activator of transcription 1 (STAT1), E2F transcription factor-1 (E2F1), fibronectin-1 (FN1), interleukin-4 (IL-4), matrix metalloprotease-9 (MMP-9) and WAP four-disulfide domain protein (WFDC-1) by QRTPCR. Additionally we performed immunoblot analysis and zymography to verify some of these candidate genes at the protein level. Computational analysis of gene function identified an anti-proliferative and altered immune function cellular phenotype in severe preeclamptic samples. Conclusions We have characterized the genome-wide mRNA expression changes associated with preeclampsia

  14. Proteomic methodological recommendations for studies involving human plasma, platelets, and peripheral blood mononuclear cells.

    PubMed

    de Roos, Baukje; Duthie, Susan J; Polley, Abigael C J; Mulholland, Francis; Bouwman, Freek G; Heim, Carolin; Rucklidge, Garry J; Johnson, Ian T; Mariman, Edwin C; Daniel, Hannelore; Elliott, Ruan M

    2008-06-01

    This study was designed to develop, optimize and validate protocols for blood processing prior to proteomic analysis of plasma, platelets and peripheral blood mononuclear cells (PBMC) and to determine analytical variation of a single sample of depleted plasma, platelet and PBMC proteins within and between four laboratories each using their own standard operating protocols for 2D gel electrophoresis. Plasma depleted either using the Beckman Coulter IgY-12 proteome partitioning kit or the Amersham albumin and IgG depletion columns gave good quality gels, but reproducibility appeared better with the single-use immuno-affinity column. The use of the Millipore Filter Device for protein concentration gave a 16% ( p < 0.005) higher recovery of protein in flow-through sample compared with acetone precipitation. The use of OptiPrep gave the lowest level of platelet contamination (1:0.8) during the isolation of PBMC from blood. Several proteins (among which are alpha-tropomyosin, fibrinogen and coagulation factor XIII A) were identified that may be used as biomarkers of platelet contamination in future studies. When identifying preselected spots, at least three out of the four centers found similar identities for 10 out of the 10 plasma proteins, 8 out of the 10 platelet proteins and 8 out of the 10 PBMC proteins. The discrepancy in spot identifications has been described before and may be explained by the mis-selection of spots due to laboratory-to-laboratory variation in gel formats, low scores on the peptide analysis leading to no or only tentative identifications, or incomplete resolution of different proteins in what appears as a single abundant spot. The average within-laboratory coefficient of variation (CV) for each of the matched spots after automatic matching using either PDQuest or ProteomWeaver software ranged between 18 and 69% for depleted plasma proteins, between 21 and 55% for platelet proteins, and between 22 and 38% for PBMC proteins. Subsequent manual

  15. Whole Blood Gene Expression and Interleukin-6 Levels

    PubMed Central

    Lin, Honghuang; Joehanes, Roby; Pilling, Luke C.; Dupuis, Josée; Lunetta, Kathryn L.; Ying, Sai-Xia; Benjamin, Emelia J.; Hernandez, Dena; Singleton, Andrew; Melzer, David; Munson, Peter J.; Levy, Daniel; Ferrucci, Luigi; Murabito, Joanne M.

    2014-01-01

    Background Circulating interleukin-6 levels increase with advancing age and are a risk factor for various diseases and mortality. The characterization of gene expression profiles associated with interleukin-6 levels might suggest important molecular events underlying its regulation. Methods and Results We studied the association of transcriptional profiles with interleukin-6 levels in 2422 participants from Framingham Heart Study Offspring Cohort using Affymetrix Human Exon 1.0 ST Array. We identified 4139 genes that were significantly associated with interleukin-6 levels (FDR<0.05) after adjusting for age, sex and blood cell components. We then replicated 807 genes in the InCHIANTI study with 694 participants. Many of the top genes are involved in inflammation-related pathways or erythrocyte function, including JAK/Stat signaling pathway and interleukin-10 signaling pathway. Conclusion We identified and replicated 807 genes that were associated with circulating interleukin-6 levels. Future characterization of interleukin-6 regulation networks may facilitate the identification of additional potential targets for treating inflammation-related diseases. PMID:25311648

  16. The effects of calpain inhibition upon IL-2 and CD25 expression in human peripheral blood mononuclear cells.

    PubMed

    Schaecher, K E; Goust, J M; Banik, N L

    2001-10-01

    Calcium is an important contributor to T cell activation; it is also the major factor in the activation of the calcium-activated neutral proteinase, calpain. For this reason, we wanted to investigate if calpain has a role in T cell activation and what aspects of this activation calpain affects. As measured by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), calpain inhibition decreased interleukin-2 (IL-2) and CD25 mRNA expression in a dose-dependent manner, at early time points following the initial activation, and over extended periods of time in activated human peripheral blood mononuclear cells (PBMCs). Using an enzyme-linked immuno-sorbent assay (ELISA) specific for human IL-2, we found that calpain inhibition decreased IL-2 secretion in a dose-dependent manner, shortly after activation, and continuously over time. Inhibiting calpain caused a dose-dependent inhibition of CD25 cell surface expression and also inhibited expression shortly after activation and for at least 48 h. This study showed that calpain has an integral role in the synthesis of the two important T cell activation factors, IL-2 and CD25. PMID:11585637

  17. Human Peripheral Blood Mononuclear Cells Cultured in Normal and Hyperglycemic Media in Simulated Microgravity Using NASA Bioreactors

    NASA Technical Reports Server (NTRS)

    Lawless, DeSales

    2003-01-01

    We sought answers to several questions this summer at NASA Johnson Space Center. Initial studies involved the in vitro culture of human peripheral blood mononuclear in cells in different conditioned culture media. Several human cancer clones were similarly studied to determine responses to aberrant glycosylation by the argon laser. The cells were grown at unit gravity in flasks and in simulated microgravity using NASA bioreactors. The cells in each instance were analyzed by flow cytometry. Cell cycle analysis was acquired by staining nuclear DNA with propidium iodide. Responses to the laser stimulation was measured by observing autofluorescence emitted in the green and red spectra after stimulation. Extent of glycosylation correlated with the intensity of the laser stimulated auto-fluorescence. Our particular study was to detect and monitor aberrant glycosylation and its role in etiopathogenesis. Comparisons were made between cells known to be neoplastic and normal cell controls using the same Laser Induced Autofluorescence technique. Studies were begun after extensive literature searches on using the antigen presenting potential of dendritic cells to induce proliferation of antigen specific cytotoxic T-cells. The Sendai virus served as the antigen. Our goal is to generate sufficient numbers of such cells in the simulated microgravity environment for use in autologous transplants of virally infected individuals including those positive for hepatitis and HIV.

  18. Identification of the 11 beta-hydroxysteroid dehydrogenase type 1 mRNA and protein in human mononuclear leukocytes.

    PubMed

    Fiore, C; Nardi, A; Dalla Valle, L; Pellati, D; Krozowski, Z; Colombo, L; Armanini, D

    2009-10-01

    The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the interconversion between inactive 11-ketoglucocorticoids and their active 11beta-hydroxy derivatives, such as cortisol and corticosterone. We have investigated the expression of 11beta-HSD1 in freshly isolated human peripheral mononuclear leukocytes (MNL). The presence of 11beta-HSD1 mRNA was demonstrated in total RNA by RT-PCR using specific primers designed on the 4th and 5th exons of the human 11beta-HSD1 gene. Fragments of the expected size were consistently detected on agarose gels, and sequencing showed complete identity with the corresponding sequence deposited in GenBank. The occurrence of 11beta-HSD1 protein was established by Western immunoblot analysis with a specific polyclonal antibody. Enzyme oxo-reductase activity was investigated by incubating 12 samples of MNL isolated from from 8 subjects with [3H]cortisone and formation of cortisol was established only in 4 subjects (yield range: 0.15-1.3%) after acetylation and TLC, blank subtraction and correction for losses. 18beta-Glycyrrhetinic acid, an inhibitor of 11 beta-HSD1, reduced cortisol production below detection limit. Dehydrogenase activity could not be demonstrated. It is suggested that, although enzyme activity of 11beta-HSD1 in circulating MNL is low, it is apparently ready for enhancement after MNL migration to sites of inflammation. PMID:19235128

  19. Conditioned medium from stimulated mononuclear leukocytes augments human neutrophil-mediated killing of a virulent Acanthamoeba sp.

    PubMed Central

    Ferrante, A; Abell, T J

    1986-01-01

    Human neutrophils in the presence of serum containing anti-amoeba antibody either lacked amoebicidal activity or were poorly amoebicidal for Acanthamoeba culbertsoni. In contrast, neutrophils preexposed for 1 h to supernatants from human peripheral blood mononuclear leukocytes (MNLs) stimulated with phytohemagglutinin demonstrated significant amoeba killing in the presence of serum containing anti-acanthamoeba antibodies. Supernatant from MNL cultured in the absence of phytohemagglutinin were not effective in stimulating significant activity in the neutrophils. Serum containing antibody promoted the adherence of many neutrophils to one amoeba. There was no significant difference between the ability of neutrophils treated with supernatants from stimulated MNLs (stimulated conditioned medium [sCM]) and supernatants from nonstimulated MNLs (nonstimulated conditioned medium [nsCM]) in their binding to acanthamoeba. The effects of sCM on neutrophils was a general phenomenon. For example, the sCM but not the nsCM enhanced the antibody-dependent neutrophil-mediated cytotoxicity against three tumor targets (K562 erythroid myeloid leukemia cell line, B16 melanoma, and P815 (DBA/2 mastocytoma). Furthermore, the sCM but not the nsCM increased the bactericidal (against Staphylococcus aureus and Streptococcus pneumoniae) and fungicidal (against Torulopsis glabrata) activity of the neutrophil. The sCM but not the nsCM contained activities which inhibited neutrophil migration and stimulated a respiratory burst in these leukocytes. These results suggest that the neutrophil antimicrobial power can be increased by exposing the leukocytes to MNL mediators. Images PMID:3943902

  20. Blood leukocyte and spleen lymphocyte immune response of spleen lymphocytes and whole blood leukocytes of hamsters

    SciTech Connect

    Peters, B.A.; Sothmann, M.; Wehrenberg, W.B. )

    1989-01-01

    This study was designed to evaluate the effects of chronic physical activity on the immune response of spleen lymphocytes and whole blood leukocytes of hamsters. Animals were kept sedentary or allowed to exercise spontaneously on running wheels for eight weeks. Physically active animals averaged 12 kilometers per day. The immune response of spleen lymphocytes whole blood leukocytes was evaluated by {sup 3}H-thymidine incorporation in response to Concanavalin A or lipopolysaccharide. There was no treatment effect between physically active and sedentary hamster in response of spleen lymphocytes. The immune response of whole blood leukocytes to these mitogens was significantly greater in physically active vs. sedentary hamsters. These results demonstrate that chronic physical activity has the capacity to modulate immunoresponses.

  1. Cocaine postmortem distribution in three brain structures: a comparison with whole blood and vitreous humour.

    PubMed

    Carvalho, Virginia M; Fukushima, André R; Fontes, Luiz R; Fuzinato, Daniela V; Florio, Jorge C; Chasin, Alice A M

    2013-04-01

    The presence of cocaine (COC) in fluids or tissues does not prove that death was due to drug consumption and the interpretation of postmortem concentrations is more complex than attempts at making such correlations in the living. The purpose of this study was to investigate the distribution of cocaine and its metabolite benzoylecgonine in brain and compare with whole blood and vitreous humour. The distribution in three brain structures (prefrontal cortex, basal ganglia and cerebellum) was homogeneous. There is a strong correlation for cocaine concentrations between vitreous humour and brain, vitreous humour and whole blood, and whole blood and brain in overdose cases. In addition, the comparison of COC/benzoylecgonine (BE) ratios in different experimental specimens proved to be more appropriate for evaluating cocaine-related death than individual drug values. These findings suggest that the comparison of cocaine levels in different compartments is essential to assess the cause of death. PMID:23472791

  2. Serum biochemical changes and chemiluminescent responses of whole blood in Holstein cattle with leukocyte adhesion deficiency.

    PubMed

    Nagahata, H; Tanaka, S; Oba, M; Minami, S; Noda, H

    1994-08-01

    Serum biochemical profile and whole blood chemiluminescent (CL) responses in 8 Holstein cattle affected with leukocyte adhesion deficiency (LAD) were evaluated. Concentrations of sodium, chloride and calcium in serum from cattle affected with LAD were significantly (p < 0.05) decreased as compared with controls. The characteristic changes in serum proteins were hypoalbuminemia and hyperglobulinemia, and the concentrations of albumin and gammaglobulin in serum from normal cattle and cattle affected with LAD were significantly (p < 0.01) different. Significantly (p < 0.01) diminished CL indices and prolonged peak time of CL responses in whole blood were detected in cattle affected with LAD. These findings indicate that the CL response associated with iC3b receptor mediated phagocytic activity is impaired in cattle affected with LAD. The whole blood CL assay appeared to be practical and useful for routine evaluation of blood samples from cattle affected with LAD. PMID:7999886

  3. Development of Multiscale Materials in Microfluidic Devices: Case Study for Viral Separation from Whole Blood

    NASA Astrophysics Data System (ADS)

    Surawathanawises, Krissada

    prior to anodization is evaluated to control the growth of AAO. Together with controlled anodizing voltages and electrolytic concentrations, AAO pore and cell sizes are shown to be tunable and controllable with narrow size distributions within submicron range. A high degree of order of AAO pore arrangement is also demonstrated. In addition, overall anodization becomes more time-efficient and stable at high anodizing voltages. Secondly, a three-dimensional (3D) assembly of microbeads is used as a template to fabricate a spherical pore network with small interconnected openings. After depositing and drying a suspension containing both micro- and nanobeads, the microbeads assemble into a 3D close-packed structure while the nanobeads fill the interstitial space. When the nanobeads are melted and microbeads are removed, a spherical pore matrix then form with small interconnected openings. Such the opening size is in submicron range can be adjusted depending on the size of microbead. The advantages of the two macroporous materials are not only controllable and tunable pore size, but also high surface-to-volume ratio due to the nanoscale features. With a ratio on the order of ~1 microm-1, the porous materials provide a significantly large binding surface. Computational and experimental results reveal that porous materials with a pore size matching the nanoparticle size are suitable for their capture. Separation of human immunodeficiency virus (HIV) is used as a model and capture yields of ~99 % and ~80 % are achieved in the nanopost structure and spherical pore network, respectively, after treated with a functional chemistry. Hence, the properties of these two macroporous materials are suitable as a size-exclusion and affinity separation for viral particles. To further explore multiscale separation, i.e. capturing viruses from whole blood, micropatterned arrays of macroporous materials have been designed. In this design, a microscale gap allows the passage of microparticles

  4. The Levels of Calcium and Magnesium, and of Selected Trace Elements, in Whole Blood and Scalp Hair of Children with Growth Retardation

    PubMed Central

    Ozmen, Habibe; Akarsu, Saadet; Polat, Fatih; Cukurovali, Alaaddin

    2013-01-01

    Objective Metals such as copper (Cu), zinc (Zn), iron (Fe) are essential for human beings. Chronic metabolic disturbances may result from an excess or deficiency of these metals. Ca and Mg are also nutrient elements and play an important role in biological systems. Thus, it is very important to check regularly trace elements concentration in the body. The purpose of this study was to measure the content of Fe, Cu, Zn, Ca and Mg in whole blood and hair of children with growth retardation compared to that of controls. Methods A quantitative elemental analysis of whole blood and scalp hair of children with constitutional growth retardation (n = 27) and matched controls (n = 21) was used to find out correlation and possible changes, between growth retardation and healthy controls. Atomic absorption spectrophotometric (AAS) analysis of quantitative method was used to determine iron, zinc, copper, calcium and magnesium levels of whole blood and scalp hair. Findings The whole blood levels of Fe and Zn were significantly lower in children with growth retardation (P<0.05), but there were no differences in Cu, Ca and Mg concentrations in whole blood between children with growth retardation and healthy controls. The hair levels of Fe, Zn, Ca and Mg were significantly lower in children with growth retardation when compared to that of controls (P<0.05). The Cu concentrations in the hair of children with growth retardation and healthy controls showed no significant differences (P>0.05). Conclusion The usefulness and significance of these elements in growth retardation should be discussed more detailed in the light of the most recent data. PMID:23724170

  5. Development of a Whole Blood Staining Device for use During Space Shuttle Flights

    NASA Technical Reports Server (NTRS)

    Sams, Clarence F.; Crucian, Brian E.; Clift, Vaughan L.; Meinelt, Ellen M.

    1999-01-01

    Exposure to microgravity during space flight results in profound physiologic changes. Numerous studies have shown changes in circulating populations of peripheral blood immune cells immediately after space flight. It is currently unknown if these changes result from exposure to microgravity or are caused by the stress of reentry and readaptation to gravity. We have developed the whole blood staining device as a system for the staining of whole blood collected during space flight for subsequent flow cytometric analysis, This device contains all liquids to address safety issues concerned with space flight and also moves the cells through the staining, lyse/fixation and dilution steps.

  6. The relationship between lead in plasma and whole blood in women.

    PubMed Central

    Smith, Donald; Hernandez-Avila, Mauricio; Téllez-Rojo, Martha Maria; Mercado, Adriana; Hu, Howard

    2002-01-01

    Studies have suggested that plasma lead levels may better reflect the toxicologically labile fraction of circulatory Pb that is more freely available for exchange with target tissues than do Pb levels in whole blood. Studies have also reported an apparent severalfold variation in the relative partitioning of Pb between whole blood and plasma (or serum) for a given whole-blood Pb level. This may reflect inherent differences in the plasma Pb/whole blood Pb partitioning among individuals and/or methodologic challenges associated with the collection and analyses of samples that generally contain < 1-2 ng total Pb. Here, we conducted a longitudinal assessment of the relationship between Pb in whole blood and plasma in environmentally exposed reproductive-age women (n = 63) living in Mexico City, Mexico. We collected whole blood and plasma samples using trace metal clean techniques and analyzed them for Pb using high-resolution inductively coupled plasma mass spectrometry. A subset of subjects provided repeated blood samples weekly for 4 consecutive weeks (n = 17 subjects) or every 1-2 months over a 9-month period (n = 14 subjects). Plasma Pb concentration was significantly positively associated with whole-blood Pb in a curvilinear fashion over the range of blood Pb values observed here (2.13-39.7 microg/dL). This relationship was best described by the function Plasma Pb = e (-2.392 + 0.0898 x blood Pb), where SE(coefficient) = 0.0054, SE(constant) = 0.063 (n = 63 subjects, n = 141 observations). Results from the short- and long-term repeated collection subjects indicated that the within- and between-subject variance components were not significantly different between the two subsets of subjects. The between-subjects component accounts for 78% of the variance in plasma Pb levels, while the residual variance (22%) may be attributed to other unmeasured factors. Collectively, this study demonstrates that plasma Pb measurements may be applied to general clinical settings

  7. Butachlor induced dissipation of mitochondrial membrane potential, oxidative DNA damage and necrosis in human peripheral blood mononuclear cells.

    PubMed

    Dwivedi, Sourabh; Saquib, Quaiser; Al-Khedhairy, Abdulaziz A; Musarrat, Javed

    2012-12-01

    Butachlor is a systemic herbicide widely applied on rice, tea, wheat, beans and other crops; however, it concurrently exerts toxic effects on beneficial organisms like earthworms, aquatic invertebrates and other non-target animals including humans. Owing to the associated risk to humans, this chloroacetanilide class of herbicide was investigated with the aim to assess its potential for the (i) interaction with DNA, (ii) mitochondria membrane damage and DNA strand breaks and (iii) cell cycle arrest and necrosis in butachlor treated human peripheral blood mononuclear (PBMN) cells. Fluorescence quenching data revealed the binding constant (Ka=1.2×10(4)M(-1)) and binding capacity (n=1.02) of butachlor with ctDNA. The oxidative potential of butachlor was ascertained based on its capacity of inducing reactive oxygen species (ROS) and substantial amounts of promutagenic 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) adducts in DNA. Also, the discernible butachlor dose-dependent reduction in fluorescence intensity of a cationic dye rhodamine (Rh-123) and increased fluorescence intensity of 2',7'-dichlorodihydro fluorescein diacetate (DCFH-DA) in treated cells signifies decreased mitochondrial membrane potential (ΔΨm) due to intracellular ROS generation. The comet data revealed significantly greater Olive tail moment (OTM) values in butachlor treated PBMN cells vs untreated and DMSO controls. Treatment of cultured PBMN cells for 24h resulted in significantly increased number of binucleated micronucleated (BNMN) cells with a dose dependent reduction in the nuclear division index (NDI). The flow cytometry analysis of annexin V(-)/7-AAD(+) stained cells demonstrated substantial reduction in live population due to complete loss of cell membrane integrity. Overall the data suggested the formation of butachlor-DNA complex, as an initiating event in butachlor-induced DNA damage. The results elucidated the oxidative role of butachlor in intracellular ROS production, and

  8. Radiofrequency currents exert cytotoxic effects in NB69 human neuroblastoma cells but not in peripheral blood mononuclear cells

    PubMed Central

    HERNÁNDEZ-BULE, MARÍA LUISA; ROLDÁN, ERNESTO; MATILLA, JOAQUÍN; TRILLO, MARÍA ÁNGELES; ÚBEDA, ALEJANDRO

    2012-01-01

    Recently, a number of electric and electrothermal therapies have been applied to the treatment of specific cancer types. However, the cellular and molecular mechanisms involved in the response to such therapies have not been well characterized yet. Capacitive-resistive electric transfer (CRET) therapy uses electric currents at frequencies within the 0.45–0.6 MHz range to induce hyperthermia in target tissues. Preliminary trials in cancer patients have shown consistent signs that CRET could slow down growth of tumor tissues in brain gliomas, without inducing detectable damage in the surrounding healthy tissue. Previous studies by our group have shown that subthermal treatment with 0.57-MHz electric currents can induce a cytostatic, not cytotoxic response in HepG2 human hepatocarcinoma cells; such effect being mediated by cell cycle alterations. In contrast, the study of the response of NB69 human neuroblastoma cells to the same electric treatment revealed consistent indications of cytotoxic effects. The present study extends the knowledge on the response of NB69 cells to the subthermal stimulus, comparing it to that of primary cultures of human peripheral blood mononuclear cells (PBMC) exposed to the same treatment. The results showed no sensitivity of PBMC to the 0.57 MHz subthermal currents and confirmed that the treatment exerts a cytotoxic action in NB69 cells. The data also revealed a previously undetected cytostatic response of the neuroblastoma cell line. CRET currents affected NB69 cell proliferation by significantly reducing the fraction of cells in the phase G2/M of the cell cycle at 12 h of exposure. These data provide new information on the mechanisms of response to CRET therapy, and are consistent with a cytotoxic and/or cytostatic action of the electric treatment, which would affect human cells of tumor origin but not normal cells with a low proliferation rate. PMID:22843038

  9. Determination of cathinones and related ephedrines in forensic whole-blood samples by liquid-chromatography-electrospray tandem mass spectrometry.

    PubMed

    Sørensen, Lambert K

    2011-04-01

    A liquid-chromatography-tandem-mass-spectrometry method using pneumatically assisted electrospray ionisation (LC-ESI-MS/MS) was developed for the simultaneous determination of cathinone, methcathinone, ethcathinone, amfepramone, mephedrone, flephedrone, methedrone, methylone, butylone, cathine, norephedrine, ephedrine, pseudoephedrine, methylephedrine and methylpseudoephedrine in human live and post-mortem whole blood. The blood proteins were precipitated by the addition of methanol, and the extract was purified by ultrafiltration. The separation of diastereomeric ephedrines was achieved on an ethyl-linked phenyl column. Matrix-matched calibrants combined with the isotope dilution of selected substances were used for quantitative analysis. The relative intra-laboratory reproducibility standard deviations were generally better than 7% at concentrations of 20 μg/L, and the mean true recoveries were 87-106% in the concentration range of 10-250 μg/L. The detection limits were in the range of 0.5-3 μg/L. The cathinones were unstable in whole blood and sample extracts under neutral conditions, but the stability could be improved by the acidification of the sample matrix. PMID:21376674

  10. Rapid determination of three anticoagulant rodenticides in whole blood by liquid chromatography coupled with electrospray ionization mass spectrometry.

    PubMed

    Jin, Mi-Cong; Chen, Xiao-Hong

    2006-01-01

    A rapid, sensitive and selective method for the simultaneous determination of bromadiolone, flocoumafen and brodifacoum in whole blood using warfarin as internal standard (IS) by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC/ESI-MS) has been developed and validated. The target compounds were extracted from the whole blood with ethyl acetate and separated on an XDB C18 column (150 mm x 2.1 mm i.d. x 5 microm) by using a mobile phase consisting of 0.2% acetic acid/methanol (12/88, v/v) at a constant flow rate of 0.50 mL/min. The analytes were detected using negative ESI-MS in the selected ion monitoring (SIM) mode. The molecular ions [M-H]- of m/z 527, 541,523 and 307 were selected for the quantification for bromadiolone, flocoumafen, brodifacoum and the IS, respectively. The calibration curves were linear (r2 > 0.995) in the concentration range of 0.50-100.00 ng/mL. The method showed a satisfactory sensitivity (0.05-0.5 ng/mL using 200 microL blood), precision (RSD < 11.9%), accuracy (recovery: 82.0-96.1%) and selectivity. This method was successfully applied to the determination of the analytes for the diagnoses of poisoned human beings and animals. PMID:16921552

  11. Dimethyl fumarate activates the prostaglandin EP2 receptor and stimulates cAMP signaling in human peripheral blood mononuclear cells.

    PubMed

    Fiedler, Sarah E; Kerns, Amelia R; Tsang, Catherine; Tsang, Vivian; Bourdette, Dennis; Salinthone, Sonemany

    2016-06-17

    Dimethyl fumarate (DMF) was recently approved by the FDA for the treatment of relapsing remitting MS. The pathology of MS is a result of both immune dysregulation and oxidative stress induced damage, and DMF is believed to have therapeutic effects on both of these processes. However, the mechanisms of action of DMF are not fully understood. To determine if DMF is able to activate signaling cascades that affect immune dysregulation, we treated human peripheral blood mononuclear cells with DMF. We discovered that DMF stimulates cyclic adenosine monophosphate (cAMP) production after 1 min treatment in vitro. cAMP is a small molecule second messenger that has been shown to modulate immune response. Using pharmacological inhibitors, we determined that adenylyl cyclase mediates DMF induced cAMP production; DMF activated the prostaglandin EP2 receptor to produce cAMP. This response was not due to increased endogenous production of prostaglandin E2 (PGE2), but was enhanced by addition of exogenous PGE2. Furthermore, we determined that the bioactive metabolite of DMF, monomethyl fumarate (MMF), also stimulates cAMP production. These novel findings suggest that DMF may provide protection against MS by inhibiting immune cell function via the cAMP signaling pathway. PMID:27157139

  12. Crotalus durissus collilineatus Venom Induces TNF-α and IL-10 Production in Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Ribeiro, Camila Bastos; dos Santos, Jéssica Cristina; Silva, Jacyelle Medeiros; de Godoi, Pedro Henrique Silva; Magalhães, Marta Regina; Spadafora-Ferreira, Mônica; Fonseca, Simone Gonçalves; Pfrimer, Irmtraut Araci Hoffman

    2014-01-01

    Snake venom has been the subject of numerous studies in an attempt to find properties and biological effects that may be beneficial to man. In this study we evaluated in vitro the effects of Crotalus durissus terrificus (Cdt) and Crotalus durissus collilineatus (Cdc) venom in human peripheral blood mononuclear cells (PBMCs). At 24 h, a significant decrease of viable cells was observed in cells stimulated with the Cdc venom at 0.0005 mg/mL and 0.005 mg/mL compared to the negative control. At 48 h, a significant decrease of viable cells was observed only in cells stimulated with Cdc venom at 0.005 mg/mL. A significant increase of TNF-α and IL-10 was detected 48 hours after culture of PBMC with Cdc, but not with Cdt venom. The expression of CD69 and PD1 (programmed death-1), activation and regulatory cell markers, on CD8+ and CD8− T cells did not change in the presence of Cdt and Cdc venom. Our results suggest the presence of proinflammatory and anti-inflammatory components in the Cdc venom. Further analysis should be done to identify those Cdc venom components as it has been done for the Cdt venom by other authors, indicating that modulatory components are found in the venom of different species of Crotalus snakes. PMID:24563803

  13. Successful simultaneous measurement of cell membrane and cytokine induced phosphorylation pathways [CIPP] in human peripheral blood mononuclear cells.

    PubMed

    Montag, David T; Lotze, Michael T

    2006-06-30

    Phenotyping and simple enumeration of peripheral blood mononuclear cells (PBMC) is of limited value for the assessment of many clinical states. As a preferred alternative, cell surface phenotyping may be combined with functional assays for enhanced assessment of altered cells circulating in patients. One simple, yet informative and rapid approach is to examine signaling within individual cells following brief periods of stimulation via flow cytometry. Although monocytes and lymphoid cells can be distinguished based on size, current permeabilization strategies necessary for identifying intracellular phosphorylated signaling molecules largely compromise the labeling of cell surface proteins used to distinguish individual cellular subsets. We have successfully developed conditions that allow for simultaneous detection of cell surface proteins and intracellular phosphorylated proteins in human PBMC following rapid in vitro cytokine stimulation. We analyzed permeabilized CD4, CD8, CD14, CD19, and CD56 expressing cells together with intracellular pSTAT1, pSTAT3, pSTAT5, pSTAT6, pp38 MAPK, or pERK1/2 within total PBMC. Of the permeabilizing conditions tested, 75% methanol enabled superior simultaneous detection of both cell surface and intracellular epitopes. This method enables the rapid functional analysis of subsets within complex cell mixtures and provides an opportunity for assessing abnormalities arising in the setting of acute or chronic inflammatory states.

  14. Effects of spironolactone on human blood mononuclear cells: mineralocorticoid receptor independent effects on gene expression and late apoptosis induction.

    PubMed

    Sønder, Søren Ulrik Salling; Mikkelsen, Marianne; Rieneck, Klaus; Hedegaard, Chris Juul; Bendtzen, Klaus

    2006-05-01

    1 Spironolactone (SPIR) binds to cytoplasmic mineralocorticoid receptors (MR) and functions as an aldosterone antagonist. Recently, the drug was shown to have an early suppressive effect on several immunoactive and proinflammatory cytokines. 2 To elucidate the mechanism behind this, the four MR-binding steroids SPIR, canrenone, 7alpha-thiomethyl-spironolactone and aldosterone (ALDO) were investigated for effects on lipopolysaccharide- and phytohemagglutinin-A-activated human blood mononuclear cells. Gene expression was examined after 4 h using microarrays, and SPIR affected 1018 transcripts of the (=) 22,000 probed. In contrast, the SPIR-related steroids affected 17 or fewer transcripts. Combining SPIR and ALDO resulted in 940 affected transcripts, indicating that SPIR has an early gene-regulatory effect independent of MR. 3 The affected genes encode a large number of signalling proteins and receptors, including immunoinflammatory response genes and apoptosis and antiapoptosis genes. Apoptosis was evident in CD3-, CD14- and CD19-positive cells, but only after 18 h of exposure to SPIR. 4 The transcriptional network involving the differentially regulated genes was examined and the results indicate that SPIR affects genes controlled by the transcription factors NF-kappaB, CEBPbeta and MYC. 5 These observations provide new insight into the non-MR-mediated effects of SPIR. PMID:16520746

  15. Characterisation of the Immunomodulatory Effects of Meningococcal Opa Proteins on Human Peripheral Blood Mononuclear Cells and CD4+ T Cells

    PubMed Central

    Lewis, Susan; Payne, Isabelle; Saleem, Muhammad; Derrick, Jeremy P.; Pollard, Andrew J.

    2016-01-01

    Opa proteins are major surface-expressed proteins located in the Neisseria meningitidis outer membrane, and are potential meningococcal vaccine candidates. Although Opa proteins elicit high levels of bactericidal antibodies following immunisation in mice, progress towards human clinical trials has been delayed due to previous findings that Opa inhibits T cell proliferation in some in vitro assays. However, results from previous studies are conflicting, with different Opa preparations and culture conditions being used. We investigated the effects of various Opa+ and Opa- antigens from N. meningitidis strain H44/76 in a range of in vitro conditions using peripheral blood mononuclear cells (PBMCs) and purified CD4+ T cells, measuring T cell proliferation by CFSE dilution using flow cytometry. Wild type recombinant and liposomal Opa proteins inhibited CD4+ T cell proliferation after stimulation with IL-2, anti-CD3 and anti-CD28, and these effects were reduced by mutation of the CEACAM1-binding region of Opa. These effects were not observed in culture with ex vivo PBMCs. Opa+ and Opa- OMVs did not consistently exert a stimulatory or inhibitory effect across different culture conditions. These data do not support a hypothesis that Opa proteins would be inhibitory to T cells if given as a vaccine component, and T cell immune responses to OMV vaccines are unlikely to be significantly affected by the presence of Opa proteins. PMID:27111850

  16. Effects of spironolactone on human blood mononuclear cells: mineralocorticoid receptor independent effects on gene expression and late apoptosis induction

    PubMed Central

    Sønder, Søren Ulrik Salling; Mikkelsen, Marianne; Rieneck, Klaus; Hedegaard, Chris Juul; Bendtzen, Klaus

    2006-01-01

    Spironolactone (SPIR) binds to cytoplasmic mineralocorticoid receptors (MR) and functions as an aldosterone antagonist. Recently, the drug was shown to have an early suppressive effect on several immunoactive and proinflammatory cytokines. To elucidate the mechanism behind this, the four MR-binding steroids SPIR, canrenone, 7α-thiomethyl-spironolactone and aldosterone (ALDO) were investigated for effects on lipopolysaccharide- and phytohemagglutinin-A-activated human blood mononuclear cells. Gene expression was examined after 4 h using microarrays, and SPIR affected 1018 transcripts of the (=) 22,000 probed. In contrast, the SPIR-related steroids affected 17 or fewer transcripts. Combining SPIR and ALDO resulted in 940 affected transcripts, indicating that SPIR has an early gene-regulatory effect independent of MR. The affected genes encode a large number of signalling proteins and receptors, including immunoinflammatory response genes and apoptosis and antiapoptosis genes. Apoptosis was evident in CD3-, CD14- and CD19-positive cells, but only after 18 h of exposure to SPIR. The transcriptional network involving the differentially regulated genes was examined and the results indicate that SPIR affects genes controlled by the transcription factors NF-κB, CEBPβ and MYC. These observations provide new insight into the non-MR-mediated effects of SPIR. PMID:16520746

  17. Thrombin inhibits IFN-gamma production in human peripheral blood mononuclear cells by promoting a Th2 profile.

    PubMed

    Naldini, Antonella; Morena, Emilia; Filippi, Irene; Pucci, Annalisa; Bucci, Mariarosaria; Cirino, Giuseppe; Carraro, Fabio

    2006-11-01

    Thrombin, the key enzyme of the coagulation cascade, is involved in inflammation. It was proposed recently that thrombin activity may play an important role in allergic inflammation. Interferon-gamma (IFN-gamma) is a potent Th1-related cytokine secreted by activated T cells and is usually downregulated in allergic inflammation. We recently demonstrated that thrombin enhances interleukin-10 (IL-10) in peripheral blood mononuclear cells (PBMC). Thus, we hypothesized that thrombin may promote a Th2 profile. We here report that human alpha- thrombin downregulates IFN-gamma expression at both protein and mRNA levels in activated PBMCs. The use of proteolytically inactive thrombin and of the specific thrombin receptor agonist peptide, SFLLRN, shows that this downregulation is thrombin specific and requires thrombin proteolytic activity. The addition of an anti- IL-10 monoclonal antibody (mAb) to thrombin-treated PBMCs abolishes IFN-gamma downregulation, suggesting that thrombin exerts its effect through IL-10, a Th2-related cytokine. Furthermore, IFN-gamma reduction was accompanied by increased IL-4 release, as well as by an increase in the proinflammatory cytokine IL-1. In conclusion, the observation that thrombin affects the production of IFN-gamma (Th1 profile) and IL-4 (Th2 profile) provides further evidence for the role played by thrombin in modulating Th1/Th2 cytokine balance, which could be particularly relevant in allergic inflammation. PMID:17115897

  18. Procalcitonin neutralizes bacterial LPS and reduces LPS-induced cytokine release in human peripheral blood mononuclear cells

    PubMed Central

    2012-01-01

    Background Procalcitonin (PCT) is a polypeptide with several cationic aminoacids in its chemical structure and it is a well known marker of sepsis. It is now emerging that PCT might exhibit some anti-inflammatory effects. The present study, based on the evaluation of the in vitro interaction between PCT and bacterial lipopolisaccharide (LPS), reports new data supporting the interesting and potentially useful anti-inflammatory activity of PCT. Results PCT significantly decreased (p < 0.05) the limulus amoebocyte lysate (LAL) assay reactivity of LPS from both Salmonella typhimurium (rough chemotype) and Escherichia coli (smooth chemotype). Subsequently, the in vitro effects of PCT on LPS-induced cytokine release were studied in human peripheral blood mononuclear cells (PBMC). When LPS was pre-incubated for 30 minutes with different concentrations of PCT, the release of interleukin-10 (IL-10) and tumor necrosis factor alpha (TNFα) by PBMC decreased in a concentration-dependent manner after 24 hours for IL-10 and 4 hours for TNFα. The release of monocyte chemotactic protein-1 (MCP-1) exhibited a drastic reduction at 4 hours for all the PCT concentrations assessed, whereas such decrease was concentration-dependent after 24 hours. Conclusions This study provides the first evidence of the capability of PCT to directly neutralize bacterial LPS, thus leading to a reduction of its major inflammatory mediators. PMID:22568957

  19. Brief Report: Whole Blood Serotonin Levels and Gastrointestinal Symptoms in Autism Spectrum Disorder

    ERIC Educational Resources Information Center

    Marler, Sarah; Ferguson, Bradley J.; Lee, Evon Batey; Peters, Brittany; Williams, Kent C.; McDonnell, Erin; Macklin, Eric A.; Levitt, Pat; Gillespie, Catherine Hagan; Anderson, George M.; Margolis, Kara Gross; Beversdorf, David Q.; Veenstra-VanderWeele, Jeremy

    2016-01-01

    Elevated whole blood serotonin levels are observed in more than 25% of children with autism spectrum disorder (ASD). Co-occurring gastrointestinal (GI) symptoms are also common in ASD but have not previously been examined in relationship with hyperserotonemia, despite the synthesis of serotonin in the gut. In 82 children and adolescents with ASD,…

  20. Kit for the selective labeling of red blood cells in whole blood with [sup 99]Tc

    DOEpatents

    Srivastava, S.C.; Babich, J.W.; Straub, R.; Richards, P.

    1992-05-26

    Disclosed herein are a method and kit for the preparation of [sup 99m]Tc labeled red blood cells using whole blood in a closed sterile system containing stannous tin in a form such that it will enter the red blood cells and be available therein for reduction of technetium. No Drawings

  1. THz spectroscopy of whole blood, plasma and cells in mice of SHR line with various pathology

    NASA Astrophysics Data System (ADS)

    Panchenko, A.; Tyndyk, M.; Smolyanskaya, O.; Sulatskiy, M.; Kravtsenyuk, O.; Balbekin, N.; Khodzitsky, M.

    2016-08-01

    This paper is devoted to studying of optical properties of whole blood and blood plasma in SHR mice grafted Ehrlich's carcinoma and mice with chronic inflammation at the terahertz frequency range. Additionally physiological saline solution suspension of ascites Ehrlich's carcinoma cells was explored.

  2. Kit for the selective labeling of red blood cells in whole blood with .sup.9 TC

    DOEpatents

    Srivastava, Suresh C.; Babich, John W.; Straub, Rita; Richards, Powell

    1992-01-01

    Disclosed herein are a method and kit for the preparation of .sup.99m Tc labeled red blood cells using whole blood in a closed sterile system containing stannous tin in a form such that it will enter the red blood cells and be available therein for reduction of technetium.

  3. THE RELATIONSHIP BETWEEN WHOLE BLOOD SEROTONIN AND REPETITIVE BEHAVIORS IN AUTISM

    PubMed Central

    Kolevzon, Alexander; Newcorn, Jeffrey H.; Kryzak, Lauren; Chaplin, William; Watner, Dryden; Hollander, Eric; Smith, Christopher J.; Cook, Edwin H.; Silverman, Jeremy M.

    2009-01-01

    This study was conducted to examine the relationship between whole blood serotonin level and behavioral symptoms in 78 subjects with autism. No significant associations were found between serotonin level and the primary behavioral outcome measures. However, a significant inverse relationship between serotonin level and self-injury was demonstrated. PMID:20044143

  4. New method for the selective labeling of erythrocytes in whole blood with Tc-99m

    DOEpatents

    Srivastava, S.C.; Babich, J.W.; Straub, R.; Richards, P.

    1984-01-27

    Method and kit are described for the preparation of /sup 99m/Tc labeled red blood cells using whole blood in a closed sterile system containing stannous tin in a form such that it will enter the red blood cells and be available therein for the reduction of technetium.

  5. Whole Blood Activation Results in Altered T Cell and Monocyte Cytokine Production Profiles by Flow Cytometry

    NASA Technical Reports Server (NTRS)

    Crucian, Brian E.; Sams, Clarence F.

    2001-01-01

    An excellent monitor of the immune balance of peripheral circulating cells is to determine their cytokine production patterns in response to stimuli. Using flow cytometry, a positive identification of cytokine producing cells in a mixed culture may be achieved. Recently, the ability to assess cytokine production following a whole-blood activation culture has been described. In this study, whole blood activation was compared to traditional PBMC activation and the individual cytokine secretion patterns for both T cells, T cell subsets and monocytes was determined by flow cytometry. RESULTS: For T cell cytokine assessment (IFNg/IL-10 and IL-21/L-4) following PMA +ionomycin activation: (1) a Significantly greater percentages of T cells producing IFNgamma and IL-2 were observed following whole-blood culture and (2) altered T cell cytokine production kinetics were observed by varying whole blood culture times. Four-color analysiS was used to allow assessment of cytokine production by specific T cell subsets. It was found that IFNgamma production was significantly elevated in the CD3+/CD8+ T cell population as compared to the CD3+/CD8- population following five hours of whole blood activation. Conversely, IL-2 and IL-10 production were Significantly elevated in the CD3+/CD8- T cell population as compared to the CD3+/CD8+ population. Monocyte cytokine production was assessed in both culture systems following LPS activation for 24 hours. A three-color flow cytometric was used to assess two cytokines (IL-1a/IL-12 and TNFa/IL-10) in conjunction with CD14. Nearly all monocytes were stimulated to produce IL-1a, IL-12 and TNFa. equally well in both culture systems, however monocyte production of IL-10 was significantly elevated in whole blood culture as compared to PBMC culture. IL-12 producing monocytes appeared to be a distinct subpopulation of the IL-1a producing set, whereas IL-10 and TNFa producing monocytes were largely mutually exclusive. IL-10 and TNFa producing

  6. Changes in whole-blood PUFA and their predictors during recovery from severe acute malnutrition.

    PubMed

    Babirekere-Iriso, Esther; Mortensen, Charlotte G; Mupere, Ezekiel; Rytter, Maren J H; Namusoke, Hanifa; Michaelsen, Kim F; Briend, André; Stark, Ken D; Friis, Henrik; Lauritzen, Lotte

    2016-05-28

    Children with severe acute malnutrition (SAM) with complications require in-patient management including therapeutic feeding. Little attention has been given to the effects of these feeds on the essential fatty acid status of children with SAM. The objective of this study was to describe changes in the PUFA composition in whole blood in children with SAM during treatment and to determine predictors of change. This prospective study took place in a paediatric nutrition rehabilitation unit in Kampala, Uganda, and assessed whole-blood fatty acid composition of children with SAM at admission, transition, discharge and follow-up (8 and 16 weeks). ANCOVA was used to identify predictors of change in whole-blood PUFA. The study included 120 children with SAM and twenty-nine healthy control children of similar age and sex. Among the SAM children, 38 % were female and 64 % had oedema. Whole-blood n-6 PUFA proportions increased from admission to follow-up, except for arachidonic acid, which decreased by 0·79 (95 % CI 0·46, 1·12) fatty acid percentage (FA%) from admission to transition and 0·10 (95 % CI 0·23, 0·44) FA% at discharge. n-3 Long-chain (LC) PUFA decreased by 0·21 (95 % CI 0·03, 0·40) FA% at discharge and 0·22 (95 % CI 0·01, 0·42) FA% at 8 weeks of follow-up. This decrease was greater in children from families with recent fish intake and those with nasogastric tube feeding. Current therapeutic feeds do not correct whole-blood levels of LCPUFA, particularly n-3 LCPUFA, in children with SAM. Increased attention is needed to the contents of n-3 LCPUFA in therapeutic feeds. PMID:26996197

  7. Automated processing of whole blood units: operational value and in vitro quality of final blood components

    PubMed Central

    Jurado, Marisa; Algora, Manuel; Garcia-Sanchez, Félix; Vico, Santiago; Rodriguez, Eva; Perez, Sonia; Barbolla, Luz

    2012-01-01

    Background The Community Transfusion Centre in Madrid currently processes whole blood using a conventional procedure (Compomat, Fresenius) followed by automated processing of buffy coats with the OrbiSac system (CaridianBCT). The Atreus 3C system (CaridianBCT) automates the production of red blood cells, plasma and an interim platelet unit from a whole blood unit. Interim platelet unit are pooled to produce a transfusable platelet unit. In this study the Atreus 3C system was evaluated and compared to the routine method with regards to product quality and operational value. Materials and methods Over a 5-week period 810 whole blood units were processed using the Atreus 3C system. The attributes of the automated process were compared to those of the routine method by assessing productivity, space, equipment and staffing requirements. The data obtained were evaluated in order to estimate the impact of implementing the Atreus 3C system in the routine setting of the blood centre. Yield and in vitro quality of the final blood components processed with the two systems were evaluated and compared. Results The Atreus 3C system enabled higher throughput while requiring less space and employee time by decreasing the amount of equipment and processing time per unit of whole blood processed. Whole blood units processed on the Atreus 3C system gave a higher platelet yield, a similar amount of red blood cells and a smaller volume of plasma. Discussion These results support the conclusion that the Atreus 3C system produces blood components meeting quality requirements while providing a high operational efficiency. Implementation of the Atreus 3C system could result in a large organisational improvement. PMID:22044958

  8. Evaluation of whole blood zinc and copper levels in children with autism spectrum disorder.

    PubMed

    Crăciun, Elena Cristina; Bjørklund, Geir; Tinkov, Alexey A; Urbina, Mauricio A; Skalny, Anatoly V; Rad, Florina; Dronca, Eleonora

    2016-08-01

    Zinc (Zn) and copper (Cu) are important trace elements for cognitive development and normal neurological functioning. Autism spectrum disorder (ASD) is a common neurological disorder, which has previously been associated with the levels of some trace elements in the blood. However, clinical data regarding the potential implication of Zn and Cu in patients with ASD are still insufficient. Therefore, the aim of the present study was to investigate the whole blood levels of Zn and Cu in a cohort of 28 children with ASD and 28 age- and gender-matched healthy controls. Whole blood Zn and Cu levels were assessed using inductively-coupled plasma-sector field mass spectrometry. Both in the control and in the ASD group, the values of whole blood Cu and Zn were characterized by a Gaussian distribution. The results indicate that the ASD children were characterized by ~10 % (p = 0.005) and ~12 % (p = 0.015) lower levels of whole blood Zn and Zn/Cu ratio, respectively, in comparison to controls. No significant difference in whole blood Cu was observed. However, Cu/Zn ratio was ~15 % (p = 0.008) higher in ASD children than that in the control ones. The results of the present study may be indicative of Zn deficiency in ASD children. Taking into account Zn-mediated up-regulation of metallothionein (MT) gene expression, these findings suggest a possible alteration in the functioning of the neuroprotective MT system. However, further investigations are required to test this hypothesis.

  9. Changes in whole-blood PUFA and their predictors during recovery from severe acute malnutrition.

    PubMed

    Babirekere-Iriso, Esther; Mortensen, Charlotte G; Mupere, Ezekiel; Rytter, Maren J H; Namusoke, Hanifa; Michaelsen, Kim F; Briend, André; Stark, Ken D; Friis, Henrik; Lauritzen, Lotte

    2016-05-28

    Children with severe acute malnutrition (SAM) with complications require in-patient management including therapeutic feeding. Little attention has been given to the effects of these feeds on the essential fatty acid status of children with SAM. The objective of this study was to describe changes in the PUFA composition in whole blood in children with SAM during treatment and to determine predictors of change. This prospective study took place in a paediatric nutrition rehabilitation unit in Kampala, Uganda, and assessed whole-blood fatty acid composition of children with SAM at admission, transition, discharge and follow-up (8 and 16 weeks). ANCOVA was used to identify predictors of change in whole-blood PUFA. The study included 120 children with SAM and twenty-nine healthy control children of similar age and sex. Among the SAM children, 38 % were female and 64 % had oedema. Whole-blood n-6 PUFA proportions increased from admission to follow-up, except for arachidonic acid, which decreased by 0·79 (95 % CI 0·46, 1·12) fatty acid percentage (FA%) from admission to transition and 0·10 (95 % CI 0·23, 0·44) FA% at discharge. n-3 Long-chain (LC) PUFA decreased by 0·21 (95 % CI 0·03, 0·40) FA% at discharge and 0·22 (95 % CI 0·01, 0·42) FA% at 8 weeks of follow-up. This decrease was greater in children from families with recent fish intake and those with nasogastric tube feeding. Current therapeutic feeds do not correct whole-blood levels of LCPUFA, particularly n-3 LCPUFA, in children with SAM. Increased attention is needed to the contents of n-3 LCPUFA in therapeutic feeds.

  10. Long-term three-dimensional perfusion culture of human adult bone marrow mononuclear cells in bioreactors.

    PubMed

    Schmelzer, Eva; Finoli, Anthony; Nettleship, Ian; Gerlach, Jörg C

    2015-04-01

    The construction and long-term maintenance of three-dimensional in vitro bone marrow models is of great interest but still quite challenging. Here we describe the use of a multi-compartment hollow-fiber membrane based three-dimensional perfusion bioreactor for long-term culture of whole human bone marrow mononuclear cells. We also investigated bioreactors with incorporated open-porous foamed hydroxyapatite scaffolds, mimicking the in vivo bone matrix. Cells in bioreactors with and without scaffolds were cultured to 6 weeks and compared to Petri dish controls. Cells were analyzed for gene expression, surface markers by flow cytometry, metabolic activity, hematopoietic potential, viability, and attachment by immunocytochemistry. Cells in bioreactors were metabolic active during long-term culture. The percentages of hematopoietic stem cell and mature endothelial cell fractions were maintained in bioreactors. The expression of most of the analyzed genes stabilized and increased after long-term culture of 6 weeks. Compared to Petri dish culture controls, bioreactor perfusion culture improved in both the short and long-term, the colony formation unit capacity of hematopoietic progenitors. Cells attached to the ample surface area provided by hydroxyapatite scaffolds. The implementation of a hydroxyapatite scaffold did not influence colony formation capacity, percentages of cell type specific fractions, gene expression, cell viability or metabolic turnover when compared to control cells cultured in bioreactors without scaffolds. In conclusion, three-dimensional perfusion bioreactor culture enables long-term maintenance of primary human bone marrow cells, with hydroxyapatite scaffolds providing an in vivo-like scaffold for three-dimensional culture.

  11. Mononuclear phagocyte intercellular crosstalk facilitates transmission of cell-targeted nanoformulated antiretroviral drugs to human brain endothelial cells.

    PubMed

    Kanmogne, Georgette D; Singh, Sangya; Roy, Upal; Liu, Xinming; McMillan, Joellyn; Gorantla, Santhi; Balkundi, Shantanu; Smith, Nathan; Alnouti, Yazen; Gautam, Nagsen; Zhou, You; Poluektova, Larisa; Kabanov, Alexander; Bronich, Tatiana; Gendelman, Howard E

    2012-01-01

    Despite the successes of antiretroviral therapy (ART), HIV-associated neurocognitive disorders remain prevalent in infected people. This is due, in part, to incomplete ART penetration across the blood-brain barrier (BBB) and lymph nodes and to the establishment of viral sanctuaries within the central nervous system. In efforts to improve ART delivery, our laboratories developed a macrophage-carriage system for nanoformulated crystalline ART (nanoART) (atazanavir, ritonavir, indinavir, and efavirenz). We demonstrate that nanoART transfer from mononuclear phagocytes (MP) to human brain microvascular endothelial cells (HBMEC) can be realized through cell-to-cell contacts, which can facilitate drug passage across the BBB. Coculturing of donor MP containing nanoART with recipient HBMEC facilitates intercellular particle transfer. NanoART uptake was observed in up to 52% of HBMEC with limited cytotoxicity. Folate coating of nanoART increased MP to HBMEC particle transfer by up to 77%. To translate the cell assays into relevant animal models of disease, ritonavir and atazanavir nanoformulations were injected into HIV-1-infected NOD/scid-γ(c)(null) mice reconstituted with human peripheral blood lymphocytes. Atazanavir and ritonavir levels in brains of mice treated with folate-coated nanoART were three- to four-fold higher than in mice treated with noncoated particles. This was associated with decreased viral load in the spleen and brain, and diminished brain CD11b-associated glial activation. We postulate that monocyte-macrophage transfer of nanoART to brain endothelial cells could facilitate drug entry into the brain.

  12. Revisiting the IFN-γ release assay: Whole blood or PBMC cultures? - And other factors of influence.

    PubMed

    Hartmann, Sofie Bruun; Emnéus, Jenny; Wolff, Anders; Jungersen, Gregers

    2016-07-01

    The interferon-γ release assay (IGRA) is a widely used test for the presence of a cell-mediated immune (CMI) response in vitro. This measure is used to test for infection with intracellular pathogens or for validating vaccine efficacy, and it is a widely used test for both human as well as cattle. However, there is no consensus whether to use whole blood cultures or purified PBMCs for the assay, and both cell populations are being used and results compared. Therefore the aim of this study was to compare different culture settings using immune cells from previously vaccinated calves, and to shed light on external factors that could influence the read out in terms of IFN-γ levels. It was found that optimal culture conditions varied between individual animals; when polyclonal activated, cells from whole blood cultures were most responsive, but when activated specifically, the optimal cell concentration/population varied with whole blood, 10×10(6)cells/ml PBMC and 5×10(6)cells/ml PBMC being the highest performing conditions. A further investigation of the distribution of cell populations in PBMCs compared to whole blood was conducted, and a significant (p<0.001) decrease in the percentage of CD3(+) T lymphocytes within the PBMCs was found. More specifically, this reduction was due to a significant (p<0.01) decrease in the percentage of γδ(+) T lymphocytes. Thus measuring immune responses on purified PBMCs might not give a physiologically relevant output. Additionally, it was tested if the choice of incubation plate would interfere with the level of secreted IFN-γ in whole blood cultures from five calves. Six plates (a-f) were tested and no significant difference in absolute levels of IFN-γ was detected in the six plates when cells were polyclonal and specifically activated. However, we observed a significant (p<0.05) higher background level in a flat-bottom plate from Corning® (cat# 3595) (plate d) compared to two different flat-bottom plates from Corning

  13. Revisiting the IFN-γ release assay: Whole blood or PBMC cultures? - And other factors of influence.

    PubMed

    Hartmann, Sofie Bruun; Emnéus, Jenny; Wolff, Anders; Jungersen, Gregers

    2016-07-01

    The interferon-γ release assay (IGRA) is a widely used test for the presence of a cell-mediated immune (CMI) response in vitro. This measure is used to test for infection with intracellular pathogens or for validating vaccine efficacy, and it is a widely used test for both human as well as cattle. However, there is no consensus whether to use whole blood cultures or purified PBMCs for the assay, and both cell populations are being used and results compared. Therefore the aim of this study was to compare different culture settings using immune cells from previously vaccinated calves, and to shed light on external factors that could influence the read out in terms of IFN-γ levels. It was found that optimal culture conditions varied between individual animals; when polyclonal activated, cells from whole blood cultures were most responsive, but when activated specifically, the optimal cell concentration/population varied with whole blood, 10×10(6)cells/ml PBMC and 5×10(6)cells/ml PBMC being the highest performing conditions. A further investigation of the distribution of cell populations in PBMCs compared to whole blood was conducted, and a significant (p<0.001) decrease in the percentage of CD3(+) T lymphocytes within the PBMCs was found. More specifically, this reduction was due to a significant (p<0.01) decrease in the percentage of γδ(+) T lymphocytes. Thus measuring immune responses on purified PBMCs might not give a physiologically relevant output. Additionally, it was tested if the choice of incubation plate would interfere with the level of secreted IFN-γ in whole blood cultures from five calves. Six plates (a-f) were tested and no significant difference in absolute levels of IFN-γ was detected in the six plates when cells were polyclonal and specifically activated. However, we observed a significant (p<0.05) higher background level in a flat-bottom plate from Corning® (cat# 3595) (plate d) compared to two different flat-bottom plates from Corning

  14. Quantification and characterization of ACTH-related peptides produced by human peripheral blood mononuclear cells

    SciTech Connect

    Pepper, G.M.; Maimon, J.; Schneider, B.S. )

    1991-04-01

    The authors have used a sensitive radioimmunoassay to quantify and characterize PBMC-associated immunoreactive ACTH (ACTH-IR). Mean ACTH content of freshly isolated human PBMCs was 3.8 {plus minus} 0.72 pg (SEM) per 10(6) cells. During 3 days of incubation ACTH-IR in conditioned media of control PBMCs increased significantly, p less than 0.02. Gel filtration chromatography revealed a minor peak of ACTH-IR coeluting with ACTH (1-39) and a major peak coeluting with ACTH (11-24). Treatment with 15 nM CRH did not alter the amount of ACTH-IR secreted or its gel pattern. Synthetic ACTH (11-24), was radioiodinated and was used for binding experiments that demonstrated specific high- and low-affinity binding sites for ACTH (11-24) on a human T cell line. These results add support for a role of ACTH and related peptides in immune regulatory systems and suggest that cell-specific post-translational processing of PBMC may generate an expanding number of biologically active moieties.

  15. Human Peripheral Blood Mononuclear Cell Function and Dendritic Cell Differentiation Are Affected by Bisphenol-A Exposure.

    PubMed

    Camarca, Alessandra; Gianfrani, Carmen; Ariemma, Fabiana; Cimmino, Ilaria; Bruzzese, Dario; Scerbo, Roberta; Picascia, Stefania; D'Esposito, Vittoria; Beguinot, Francesco; Formisano, Pietro; Valentino, Rossella

    2016-01-01

    Environmental pollutants, including endocrine disruptor chemicals (EDCs), interfere on human health, leading to hormonal, immune and metabolic perturbations. Bisphenol-A (BPA), a main component of polycarbonate plastics, has been receiving increased attention due to its worldwide distribution with a large exposure. In humans, BPA, for its estrogenic activity, may have a role in autoimmunity, inflammatory and allergic diseases. To this aim, we assessed the effect of low BPA doses on functionality of human peripheral blood mononuclear cells (PBMCs), and on in vitro differentiation of dendritic cells from monocytes (mDCs). Fresh peripheral blood samples were obtained from 12 healthy adult volunteers. PBMCs were left unstimulated or were activated with the mitogen phytohemagglutinin (PHA) or the anti-CD3 and anti-CD28 antibodies and incubated in presence or absence of BPA at 0.1 and 1nM concentrations. The immune-modulatory effect of BPA was assessed by evaluating the cell proliferation and the levels of interferon-γ (IFN-γ), interleukin-4 (IL-4), interleukin-10 (IL-10) and interleukin-13 (IL-13) secreted by PBMCs. mDCs were differentiated with IL-4 and GC-CSF with or without BPA and the expression of differentiation/maturation markers (CD11c, CD1a, CD86, HLA-DR) was evaluated by flow cytometry; furthermore, a panel of 27 different cytokines, growth factors and chemokines were assayed in the mDC culture supernatants. PBMCs proliferation significantly increased upon BPA exposure compared to BPA untreated cells. In addition, a significant decrease in IL-10 secretion was observed in PBMCs incubated with BPA, either in unstimulated or mitogen-stimulated cells, and at both 0.1 and 1nM BPA concentrations. Similarly, IL-13 was reduced, mainly in cells activated by antiCD3/CD28. By contrast, no significant changes in IFN-γ and IL-4 production were found in any condition assayed. Finally, BPA at 1nM increased the density of dendritic cells expressing CD1a and concomitantly

  16. Human Peripheral Blood Mononuclear Cell Function and Dendritic Cell Differentiation Are Affected by Bisphenol-A Exposure

    PubMed Central

    Ariemma, Fabiana; Cimmino, Ilaria; Bruzzese, Dario; Scerbo, Roberta; Picascia, Stefania; D’Esposito, Vittoria; Beguinot, Francesco; Formisano, Pietro

    2016-01-01

    Environmental pollutants, including endocrine disruptor chemicals (EDCs), interfere on human health, leading to hormonal, immune and metabolic perturbations. Bisphenol-A (BPA), a main component of polycarbonate plastics, has been receiving increased attention due to its worldwide distribution with a large exposure. In humans, BPA, for its estrogenic activity, may have a role in autoimmunity, inflammatory and allergic diseases. To this aim, we assessed the effect of low BPA doses on functionality of human peripheral blood mononuclear cells (PBMCs), and on in vitro differentiation of dendritic cells from monocytes (mDCs). Fresh peripheral blood samples were obtained from 12 healthy adult volunteers. PBMCs were left unstimulated or were activated with the mitogen phytohemagglutinin (PHA) or the anti-CD3 and anti-CD28 antibodies and incubated in presence or absence of BPA at 0.1 and 1nM concentrations. The immune-modulatory effect of BPA was assessed by evaluating the cell proliferation and the levels of interferon-γ (IFN-γ), interleukin-4 (IL-4), interleukin-10 (IL-10) and interleukin-13 (IL-13) secreted by PBMCs. mDCs were differentiated with IL-4 and GC-CSF with or without BPA and the expression of differentiation/maturation markers (CD11c, CD1a, CD86, HLA-DR) was evaluated by flow cytometry; furthermore, a panel of 27 different cytokines, growth factors and chemokines were assayed in the mDC culture supernatants. PBMCs proliferation significantly increased upon BPA exposure compared to BPA untreated cells. In addition, a significant decrease in IL-10 secretion was observed in PBMCs incubated with BPA, either in unstimulated or mitogen-stimulated cells, and at both 0.1 and 1nM BPA concentrations. Similarly, IL-13 was reduced, mainly in cells activated by antiCD3/CD28. By contrast, no significant changes in IFN-γ and IL-4 production were found in any condition assayed. Finally, BPA at 1nM increased the density of dendritic cells expressing CD1a and concomitantly

  17. Barcoding of live human peripheral blood mononuclear cells for multiplexed mass cytometry.

    PubMed

    Mei, Henrik E; Leipold, Michael D; Schulz, Axel Ronald; Chester, Cariad; Maecker, Holden T

    2015-02-15

    Mass cytometry is developing as a means of multiparametric single-cell analysis. In this study, we present an approach to barcoding separate live human PBMC samples for combined preparation and acquisition on a cytometry by time of flight instrument. Using six different anti-CD45 Ab conjugates labeled with Pd104, Pd106, Pd108, Pd110, In113, and In115, respectively, we barcoded up to 20 samples with unique combinations of exactly three different CD45 Ab tags. Cell events carrying more than or less than three different tags were excluded from analyses during Boolean data deconvolution, allowing for precise sample assignment and the electronic removal of cell aggregates. Data from barcoded samples matched data from corresponding individually stained and acquired samples, at cell event recoveries similar to individual sample analyses. The approach greatly reduced technical noise and minimizes unwanted cell doublet events in mass cytometry data, and it reduces wet work and Ab consumption. It also eliminates sample-to-sample carryover and the requirement of instrument cleaning between samples, thereby effectively reducing overall instrument runtime. Hence, CD45 barcoding facilitates accuracy of mass cytometric immunophenotyping studies, thus supporting biomarker discovery efforts, and it should be applicable to fluorescence flow cytometry as well.

  18. The expression of pluripotency genes and neuronal markers after neurodifferentiation in fibroblasts co-cultured with human umbilical cord blood mononuclear cells.

    PubMed

    Marinowic, D R; Domingues, M F; Machado, D C; DaCosta, J C

    2015-01-01

    Human umbilical cord blood is an attractive source of stem cells; however, it has a heterogeneous cell population with few mesenchymal stem cells. Cell reprogramming induced by different methodologies can confer pluripotency to differentiated adult cells. The objective of this study was to evaluate the reprogramming of fibroblasts and their subsequent neural differentiation after co-culture with umbilical cord blood mononuclear cells. Cells were obtained from four human umbilical cords. The mononuclear cells were cultured for 7 d and subsequently co-cultured with mouse fibroblast NIH-3T3 cells for 6 d. The pluripotency of the cells was evaluated by RT-PCR using primers specific for pluripotency marker genes. The pluripotency was also confirmed by adipogenic and osteogenic differentiation. Neural differentiation of the reprogrammed cells was evaluated by immunofluorescence. All co-cultured cells showed adipogenic and osteogenic differentiation capacity. After co-cultivation, cells expressed the pluripotency gene KLF4. Statistically significant differences in cell area, diameter, optical density, and fractal dimension were observed by confocal microscopy in the neurally differentiated cells. Contact in the form of co-cultivation of fibroblasts with umbilical cord blood mononuclear fraction for 6 d promoted the reprogramming of these cells, allowing the later induction of neural differentiation. PMID:25134818

  19. [Determination of Al, Be, Cd, Co, Cr, Mn, Ni, Pb, Se and Tl in whole blood by atomic absorption spectrometry without preliminary sample digestion].

    PubMed

    Ivanenko, N B; Ivanenko, A A; Solov'ev, N D; Navolotskiĭ, D V; Pavlova, O V; Ganeev, A A

    2014-01-01

    Methods of whole blood trace element determination by Graphite furnace atomic absorption spectrometry (in the variant of Zeeman's modulation polarization spectrometry) have been proposed. They do not require preliminary sample digestion. Furnace programs, modifiers and blood dilution factors were optimized. Seronorm™ human whole blood reference materials were used for validation. Dynamic ranges (for undiluted blood samples) were: Al 8 ¸ 210 мg/L; Be 0.3 ¸ 50 мg/L; Cd 0.2 ¸ 75 мg/L; Сo 5 ¸ 350 мg/L; Cr 10 ¸ 100 мg/L; Mn 6 ¸ 250 мg/L; Ni 10 ¸ 350 мg/L; Pb 3 ¸ 240 мg/L; Se 10 ¸ 500 мg/L; Tl 2 ¸ 600 мg/L. Precision (RSD) for the middle of dynamic range ranged from 5% for Mn to 11 for Se.

  20. Biotin uptake into human peripheral blood mononuclear cells increases early in the cell cycle, increasing carboxylase activities.

    PubMed

    Stanley, J Steven; Mock, Donald M; Griffin, Jacob B; Zempleni, Janos

    2002-07-01

    Cells respond to proliferation with increased accumulation of biotin, suggesting that proliferation enhances biotin demand. Here we determined whether peripheral blood mononuclear cells (PBMC) increase biotin uptake at specific phases of the cell cycle, and whether biotin is utilized to increase biotinylation of carboxylases. Biotin uptake was quantified in human PBMC that were arrested chemically at specific phases of the cell cycle, i.e., biotin uptake increased in the G1 phase of the cycle [658 +/- 574 amol biotin/(10(6) cells x 30 min)] and remained increased during phases S, G2, and M compared with quiescent controls [200 +/- 62 amol biotin/(10(6) cells x 30 min)]. The abundance of the sodium-dependent multivitamin transporter (SMVT, which transports biotin) was similar at all phases of the cell cycle, suggesting that transporters other than SMVT or splicing variants of SMVT may account for the increased biotin uptake observed in proliferating cells. Activities of biotin-dependent 3-methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase were up to two times greater in proliferating PBMC compared with controls. The abundance of mRNA encoding 3-methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase paralleled carboxylase activities, suggesting that PBMC respond to proliferation with increased expression of genes encoding carboxylases. Similarly, expression of the gene encoding holocarboxylase synthetase (which catalyzes binding of biotin to carboxylases) increased in response to proliferation, suggesting that cellular capacity to biotinylate carboxylases was increased. In summary, these findings suggest that PBMC respond to proliferation with increased biotin uptake early in the cell cycle, and that biotin is utilized to increase activities of two of the four biotin-requiring carboxylases.

  1. Effect of asbestos exposure on differentiation of cytotoxic T lymphocytes in mixed lymphocyte reaction of human peripheral blood mononuclear cells.

    PubMed

    Kumagai-Takei, Naoko; Nishimura, Yasumitsu; Maeda, Megumi; Hayashi, Hiroaki; Matsuzaki, Hidenori; Lee, Suni; Hiratsuka, Junichi; Otsuki, Takemi

    2013-07-01

    Asbestos fibers are associated with tumorigenicity, and are thought to cause mesothelioma. However, their effect on immune response remains unclear. We examined the effect of asbestos exposure on differentiation of cytotoxic T lymphocytes (CTLs) in mixed lymphocyte reactions (MLR) of human peripheral blood mononuclear cells (PBMCs) upon exposure to chrysotile B (CB) or crocidolite (CR) asbestos at 5 μg/ml for 7 days. Exposure to CB during MLR suppressed increases in the percentage and number of CD8⁺ T cells in response to allogenic cells. The cytotoxicity for allogenic targets decreased in PBMCs exposed to CB, but not CR, when compared with PBMCs without any exposure during MLR. Exposure to CB during MLR resulted in suppression of increases in granzyme B⁺ cells and IFN-γ⁺ cells. CB exposure also resulted in suppression of increases in CD45RO⁺ effector/memory cells and CD25⁺-activated cells in CD8⁺ lymphocytes, and a decrease in CD45RA⁺ cells. CB exposure suppressed the proliferation of CD8⁺ lymphocytes without an increase in annexin V⁺ apoptotic cells in CD8⁺ lymphocytes. Moreover, the production of IL-10, IFN-γ, and TNF-α, but not IL-2, decreased in the presence of CB. These results suggest that exposure to asbestos potentially suppresses the differentiation of cytotoxic T lymphocyte, accompanied by decreases in IFN-γ and TNF-α.

  2. Members of the Candida parapsilosis Complex and Candida albicans are Differentially Recognized by Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Estrada-Mata, Eine; Navarro-Arias, María J.; Pérez-García, Luis A.; Mellado-Mojica, Erika; López, Mercedes G.; Csonka, Katalin; Gacser, Attila; Mora-Montes, Héctor M.

    2016-01-01

    The systemic infections caused by members of the Candida parapsilosis complex are currently associated to high morbility and mortality rates, and are considered as relevant as those caused by Candida albicans. Since the fungal cell wall is the first point of contact with the host cells, here we performed a comparison of this organelle in members of the C. parapsilosis complex, and its relevance during interaction with human peripheral blood mononuclear cells (PBMCs). We found that the wall of the C. parapsilosis complex members is similar in composition, but differs to that from C. albicans, with less mannan content and more β-glucan and porosity levels. Furthermore, lectin-based analysis showed increased chitin and β1,3-glucan exposure at the surface of C. parapsilosis sensu lato when compared to C. albicans. Yeast cells of members of the C. parapsilosis complex stimulated more cytokine production by human PBMCs than C. albicans cells; and this significantly changed upon removal of O-linked mannans, indicating this wall component plays a significant role in cytokine stimulation by C. parapsilosis sensu lato. When inner wall components were exposed on the wall surface, C. parapsilosis sensu stricto and C. metapsilosis, but not C. orthopsilosis, stimulated higher cytokine production. Moreover, we found a strong dependency on β1,3-glucan recognition for the members of the C. parapsilosis complex, but not for live C. albicans cells; whereas TLR4 was required for TNFα production by the three members of the complex, and stimulation of IL-6 by C. orthopsilosis. Mannose receptor had a significant role during TNFα and IL-1β stimulation by members of the complex. Finally, we demonstrated that purified N- and O-mannans from either C. parapsilosis sensu lato or C. albicans are capable to block the recognition of these pathogens by human PBMCs. Together; our results suggest that the innate immune recognition of the members of the C. parapsilosis complex is differential

  3. A smart pipette for equipment-free separation and delivery of plasma for on-site whole blood analysis.

    PubMed

    Im, Sung B; Kim, Sang C; Shim, Joon S

    2016-02-01

    A novel device of smart pipette has been suggested to extract and deliver plasma from whole blood in a disposable format. By operating an on-chip disposable micropump, approximately 30 μL of plasma was obtained from 100 μL of whole blood within 5 min without any external equipment for point-of-care blood analysis. PMID:26718913

  4. Brief Report: Whole Blood Serotonin Levels and Gastrointestinal Symptoms in Autism Spectrum Disorder

    PubMed Central

    Marler, Sarah; Ferguson, Bradley J.; Lee, Evon Batey; Peters, Brittany; Williams, Kent C.; McDonnell, Erin; Macklin, Eric A.; Levitt, Pat; Gillespie, Catherine Hagan; Anderson, George M.; Margolis, Kara Gross; Beversdorf, David Q.; Veenstra-VanderWeele, Jeremy

    2016-01-01

    Elevated whole blood serotonin levels are observed in more than 25 % of children with autism spectrum disorder (ASD). Co-occurring gastrointestinal (GI) symptoms are also common in ASD but have not previously been examined in relationship with hyperserotonemia, despite the synthesis of serotonin in the gut. In 82 children and adolescents with ASD, we observed a correlation between a quantitative measure of lower GI symptoms and whole blood serotonin levels. No significant association was seen between functional constipation diagnosis and serotonin levels in the hyperserotonemia range, suggesting that this correlation is not driven by a single subgroup. More specific assessment of gut function, including the microbiome, will be necessary to evaluate the contribution of gut physiology to serotonin levels in ASD. PMID:26527110

  5. Brief Report: Whole Blood Serotonin Levels and Gastrointestinal Symptoms in Autism Spectrum Disorder.

    PubMed

    Marler, Sarah; Ferguson, Bradley J; Lee, Evon Batey; Peters, Brittany; Williams, Kent C; McDonnell, Erin; Macklin, Eric A; Levitt, Pat; Gillespie, Catherine Hagan; Anderson, George M; Margolis, Kara Gross; Beversdorf, David Q; Veenstra-VanderWeele, Jeremy

    2016-03-01

    Elevated whole blood serotonin levels are observed in more than 25% of children with autism spectrum disorder (ASD). Co-occurring gastrointestinal (GI) symptoms are also common in ASD but have not previously been examined in relationship with hyperserotonemia, despite the synthesis of serotonin in the gut. In 82 children and adolescents with ASD, we observed a correlation between a quantitative measure of lower GI symptoms and whole blood serotonin levels. No significant association was seen between functional constipation diagnosis and serotonin levels in the hyperserotonemia range, suggesting that this correlation is not driven by a single subgroup. More specific assessment of gut function, including the microbiome, will be necessary to evaluate the contribution of gut physiology to serotonin levels in ASD.

  6. Device and method for automated separation of a sample of whole blood into aliquots

    DOEpatents

    Burtis, Carl A.; Johnson, Wayne F.

    1989-01-01

    A device and a method for automated processing and separation of an unmeasured sample of whole blood into multiple aliquots of plasma. Capillaries are radially oriented on a rotor, with the rotor defining a sample chamber, transfer channels, overflow chamber, overflow channel, vent channel, cell chambers, and processing chambers. A sample of whole blood is placed in the sample chamber, and when the rotor is rotated, the blood moves outward through the transfer channels to the processing chambers where the blood is centrifugally separated into a solid cellular component and a liquid plasma component. When the rotor speed is decreased, the plasma component backfills the capillaries resulting in uniform aliquots of plasma which may be used for subsequent analytical procedures.

  7. Hydrodynamic and label-free sorting of circulating tumor cells from whole blood

    NASA Astrophysics Data System (ADS)

    Geislinger, Thomas M.; Stamp, Melanie E. M.; Wixforth, Achim; Franke, Thomas

    2015-11-01

    We demonstrate continuous, passive, and label-free sorting of different in vitro cancer cell lines (MV3, MCF7, and HEPG2) as model systems for circulating tumor cells (CTCs) from undiluted whole blood employing the non-inertial lift effect as driving force. This purely viscous, repulsive cell-wall interaction is sensitive to cell size and deformability differences and yields highly efficient cell separation and high enrichment factors. We show that the performance of the device is robust over a large range of blood cell concentrations and flow rates as well as for the different cell lines. The collected samples usually contain more than 90% of the initially injected CTCs and exhibit average enrichment factors of more than 20 for sorting from whole blood samples.

  8. Acoustofluidics and whole-blood manipulation in surface acoustic wave counterflow devices.

    PubMed

    Travagliati, Marco; Shilton, Richie J; Pagliazzi, Marco; Tonazzini, Ilaria; Beltram, Fabio; Cecchini, Marco

    2014-11-01

    On-chip functional blocks for sample preprocessing are necessary elements for the implementation of fully portable micrototal analysis systems (μTAS). We demonstrate and characterize the microparticle and whole-blood manipulation capabilities of surface acoustic wave (SAW) driven counterflow micropumps. The motion of suspended cells in this system is governed by the two dominant acoustic forces associated with the scattered SAW (of wavelength λf): acoustic-radiation force and acoustic-streaming Stokesian drag force. We show that by reducing the microchannel height (h) beyond a threshold value the balance of these forces is shifted toward the acoustic-radiation force and that this yields control of two different regimes of microparticle dynamics. In the regime dominated by the acoustic radiation force (h ≲ λf), microparticles are collected in the seminodes of the partial standing sound-wave arising from reflections off microchannel walls. This enables the complete separation of plasma and corpuscular components of whole blood in periodical predetermined positions without any prior sample dilution. Conversely, in the regime dominated by acoustic streaming (h ≫ λf), the microbeads follow vortical streamlines in a pattern characterized by three different phases during microchannel filling. This makes it possible to generate a cell-concentration gradient within whole-blood samples, a behavior not previously reported in any acoustic-streaming device. By careful device design, a new class of SAW pumping devices is presented that allows the manipulation and pretreatment of whole-blood samples for portable and integrable biological chips and is compatible with hand-held battery-operated devices.

  9. Whole blood mRNA in prostate cancer reveals a four-gene androgen regulated panel.

    PubMed

    Thomas, Benjamin C; Kay, Jonathan D; Menon, Suraj; Vowler, Sarah L; Dawson, Sarah N; Bucklow, Laura J; Luxton, Hayley J; Johnston, Thomas; Massie, Charlie E; Pugh, Michelle; Warren, Anne Y; Barker, Peter; Burling, Keith; Lynch, Andy G; George, Anne; Burge, Johanna; Corcoran, Marie; Stearn, Sara; Lamb, Alastair D; Sharma, Naomi L; Shaw, Greg L; Neal, David E; Whitaker, Hayley C

    2016-10-01

    Due to increased sensitivity, the expression of circulating nucleotides is rapidly gaining popularity in cancer diagnosis. Whole blood mRNA has been used in studies on a number of cancers, most notably two separate studies that used whole blood mRNA to define non-overlapping signatures of prostate cancer that has become castration independent. Prostate cancer is known to rely on androgens for initial growth, and there is increasing evidence on the importance of the androgen axis in advanced disease. Using whole blood mRNA samples from patients with prostate cancer, we have identified the four-gene panel of FAM129A, MME, KRT7 and SOD2 in circulating mRNA that are differentially expressed in a discovery cohort of metastatic samples. Validation of these genes at the mRNA and protein level was undertaken in additional cohorts defined by risk of relapse following surgery and hormone status. All the four genes were downregulated at the mRNA level in the circulation and in primary tissue, but this was not always reflected in tissue protein expression. MME demonstrated significant differences in the hormone cohorts, whereas FAM129A is downregulated at the mRNA level but is raised at the protein level in tumours. Using published ChIP-seq data, we have demonstrated that this may be due to AR binding at the FAM129A and MME loci in multiple cell lines. These data suggest that whole blood mRNA of androgen-regulated genes has the potential to be used for diagnosis and monitoring of prostate cancer. PMID:27578825

  10. Multiple electrode aggregometry: a new device to measure platelet aggregation in whole blood.

    PubMed

    Tóth, Orsolya; Calatzis, Andreas; Penz, Sandra; Losonczy, Hajna; Siess, Wolfgang

    2006-12-01

    Several methods are used to analyse platelet function in whole blood. A new device to measure whole blood platelet aggregation has been developed, called multiple electrode platelet aggregometry (MEA). Our aim was to evaluate MEA in comparison with the single platelet counting (SPC) method for the measurement of platelet aggregation and platelet inhibition by aspirin or apyrase in diluted whole blood. Platelet aggregation induced by different concentrations of ADP, collagen and TRAP-6 and platelet inhibition by apyrase or aspirin were determined in citrateor hirudin-anticoagulated blood by MEA and SPC. MEA indicated that spontaneous platelet aggregation was lower, and stimulated platelet aggregation was higher in hirudin- than citrate-anticoagulated blood. In hirudin-anticoagulated, but not citrate-anticoagulated blood, spontaneous platelet aggregation measured by MEA was inhibited by apyrase. For MEA compared with SPC the dose response-curves of agonist-induced platelet aggregation in citrate- and hirudin-blood showed similar EC50 values for TRAP, and higher EC50 values for ADP (non-significant) and collagen (p < 0.05). MEA and the SPC method gave similar results concerning platelet-inhibition by apyrase and aspirin. MEA was more sensitive than SPC to the inhibitory effect of aspirin in collagen-induced aggregation. In conclusion, MEA is an easy, reproducible and sensitive method for measuring spontaneous and stimulated platelet aggregation, and evaluating antiplatelet drugs in diluted whole blood. The use of hirudin as an anticoagulant is preferable to the use of citrate. MEA is a promising technique for experimental and clinical applications. PMID:17139373

  11. Selected epidemiological aspects of fresh whole blood application in the Polish Field Hospital in Afghanistan.

    PubMed

    Olszewski, Adam; Korzeniewski, Krzysztof; Lass, Anna

    2014-01-01

    Minimisation of blood transmitted diseases is a basic element of all blood transfusion strategies. Civilian health service standards used in peacetime may be difficult to implement in a battlefield. The risk of blood-borne diseases depends on the applied donor qualification procedures and the epidemiological situation in the areas of military operations. The authors discuss various epidemiological aspects considered when selecting potential donors of fresh whole blood for a Walking Blood Bank at the Polish Field Hospital in Afghanistan.

  12. Integrated separation of blood plasma from whole blood for microfluidic paper-based analytical devices.

    PubMed

    Yang, Xiaoxi; Forouzan, Omid; Brown, Theodore P; Shevkoplyas, Sergey S

    2012-01-21

    Many diagnostic tests in a conventional clinical laboratory are performed on blood plasma because changes in its composition often reflect the current status of pathological processes throughout the body. Recently, a significant research effort has been invested into the development of microfluidic paper-based analytical devices (μPADs) implementing these conventional laboratory tests for point-of-care diagnostics in resource-limited settings. This paper describes the use of red blood cell (RBC) agglutination for separating plasma from finger-prick volumes of whole blood directly in paper, and demonstrates the utility of this approach by integrating plasma separation and a colorimetric assay in a single μPAD. The μPAD was fabricated by printing its pattern onto chromatography paper with a solid ink (wax) printer and melting the ink to create hydrophobic barriers spanning through the entire thickness of the paper substrate. The μPAD was functionalized by spotting agglutinating antibodies onto the plasma separation zone in the center and the reagents of the colorimetric assay onto the test readout zones on the periphery of the device. To operate the μPAD, a drop of whole blood was placed directly onto the plasma separation zone of the device. RBCs in the whole blood sample agglutinated and remained in the central zone, while separated plasma wicked through the paper substrate into the test readout zones where analyte in plasma reacted with the reagents of the colorimetric assay to produce a visible color change. The color change was digitized with a portable scanner and converted to concentration values using a calibration curve. The purity and yield of separated plasma was sufficient for successful operation of the μPAD. This approach to plasma separation based on RBC agglutination will be particularly useful for designing fully integrated μPADs operating directly on small samples of whole blood.

  13. Damage Associated Molecular Pattern Molecule-Induced microRNAs (DAMPmiRs) in Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Unlu, Sebnem; Tang, Siuwah; Wang, E. na; Martinez, Ivan; Tang, Daolin; Bianchi, Marco E.; Zeh, Herbert J.; Lotze, Michael T.

    2012-01-01

    Endogenous damage associated molecular pattern molecules (DAMPs) released from necrotic, damaged or stressed cells are associated with an inflammatory response. Whether the microRNA (miR) expression signature of this response is different from that of a pathogen associated molecular pattern (PAMP)-stimulated inflammatory response is unknown. We report here that miR-34c and miR-214 are significantly expressed in fresh human peripheral blood mononuclear cells (PBMCs) exposed to DAMP-containing freeze-thaw lysates, or to conditioned media from serum-starved and glucose-deprived cells (p<6×10−4 and p<3.7×10−3), respectively. Interestingly, only miR-34c expression was differentially expressed in PBMCs exposed to freeze-thaw lysates or conditioned media from wildtype High Mobility Group B1 (HMGB1+/+) mouse embryonic fibroblast (MEF) cells, when compared to cultures exposed to lysates or conditioned media from HMGB1−/− MEFs. miR-155 expression in these cultures was negligible, but was significantly expressed in PBMCs stimulated with Lipopolysaccahride (LPS) or most other Toll-like receptor (TLR) ligands, making it the prototypic “PAMPmiR”. Exposure to a damaged human colorectal carcinoma cell line lysate (HCT116) similarly resulted in increased miR-34c and miR-214 levels. When PBMCs were pre-transfected with anti-miR-34c and then exposed to lysate, expression levels of IKKγ mRNA, a putative target of miR-34c, increased, while protein levels of IKKγ in cultures transfected with a pre-miR-34c were abrogated. Levels of miR-34c expression (as well as pro-inflammatory cytokines, IL-1β and TNFα) decreased when PBMC cultures were briefly pre-incubated with the K+ channel (inflammasome) inhibitor, glybenclamide, suggesting that inflammasome activation is upstream of miR-34c expression in response to DAMPs. Our findings demonstrate that a specific microRNA expression signature is associated with the inflammatory response to damaged/injured cells and carries

  14. Development of a simple device for processing whole blood samples into measured aliquots of plasma

    SciTech Connect

    Burtis, C.A.; Johnson, W.F.; Walker, W.A.

    1986-01-01

    A capillary processor and aliquoter (CPA) has been designed and fabricated that is capable of accepting aliquots of whole blood and automatically processing them into discrete aliquots of plasma. The device consists of two disks, each of which contains 16 individual capillaries and a processing rotor. One of the disks accepts larger capillaries, each of which will hold approx. 100 ..mu..L of whole blood. The second disk, which accepts 2.54-cm-long precision capillaries of varying internal diameter, provides for exact sample volumes ranging from 1 to 10 ..mu..L. The processing rotor consists of 16 individual compartments and chambers to accept both disks. Gravimetric and photometric evaluation of the CPA indicates that it is capable of entraining and delivering microliter volumes of liquids with a degree of precision and accuracy (1 to 2%) approaching that of a state-of-the-art mechanical pipette. In addition, we have demonstrated that aliquots of whole blood can be transferred into the chambers of the processing unit and separated into their cellular and plasma fractions, which can then be analyzed with an acceptable degree of precision (i.e., C.V.s of approx. +-3% for serum enzyme measurements). 15 refs., 6 figs., 4 tbls.

  15. [Determination of strontium content in whole blood and urine by icp-ms].

    PubMed

    Ulanova, T S; Gileva, O V; Stenno, E V; Veikhman, G A; Nedochitova, A V

    2015-01-01

    Parameters of strontium determination in the whole blood and urine of children living near ore deposits containing up to 20% strontium sulfate have been determined. The average strontium content in the whole blood of two children groups of 109.52 ± 11.07 mg/L and 131.62 ± 12.95 mg/L, significantly exceeded the level in the comparison group 44.2 ± 4.24 mg/L. The average strontium contents of two groups of children in urine were 1252.3 ± 332.2 mg/L and 1341.5 ± 241.8 mg/L, these values were 4.2 and 4.5 times higher than in the comparison group 296.4 ± 61.5 mg/L. The conditions for blood and urine sample preparation were optimized to reduce measure errors and to determine strontium at the reference concentration level. The accuracy of the results has been confirmed by analysis of the standard samples Seronorm™ Whole Blood L1, L2, L3 and Seronorm™ Urine.

  16. Tacrolimus whole blood concentrations correlate closely to side-effects in renal transplant recipients

    PubMed Central

    Böttiger, Y; Brattström, C; Tydén, G; Säwe, J; Groth, C-G

    1999-01-01

    Aims To evaluate the relationship between tacrolimus whole blood concentrations and side-effects and rejections in 14 renal transplant recipients. Methods Tacrolimus was measured by MEIA in whole blood in samples collected repeatedly during the first year after transplantation. Retrospectively, tacrolimus trough concentrations on the days with adverse events (n = 172) or rejection (n = 28) were related to the total distribution of the concentration values (n = 656). Results Side-effects (one or more) were noted in connection with 76% of tacrolimus concentrations above 30 ng ml−1, with 41% of concentrations within the interval of 20–30 ng ml−1, with 26% of the concentrations within the interval of 10–20 ng ml−1 and with only 5.3% on the concentrations lower than 10 ng ml−1. No relation to the tacrolimus concentration was seen for rejection episodes. Conclusions We conclude that therapeutic drug monitoring may be helpful in the management of tacrolimus therapy and that tacrolimus whole blood trough concentrations (MEIA) should preferably be kept below 20 ng ml−1 to avoid side-effects, such as nephro-and neurotoxicity and infections. The lower limit of the therapeutic range has yet to be defined. PMID:10510159

  17. The effect of ex vivo anticoagulants on whole blood platelet aggregation.

    PubMed

    Kalb, Madeleine L; Potura, Lukasz; Scharbert, Gisela; Kozek-Langenecker, Sibylle A

    2009-02-01

    Pre- and intraoperative platelet function monitoring is increasingly recommended in order to detect risk factors for bleeding and to target coagulation management. The ideal anticoagulant for accurate platelet aggregometry remains controversial. The aim of this experimental trial was to compare platelet aggregability in whole blood stored in citrate, heparin and direct thrombin inhibitors. Whole blood was drawn from 11 healthy adult volunteers who had not taken any medication in the previous 14 days. Blood was stored in trisodium citrate, unfractionated heparin, melagatran, lepirudin and argatroban. Platelet aggregation was performed using the impedance aggregometer Multiplate (Dynabyte, Munich, Germany) with adenosine diphosphate (ADP), thrombin receptor activating peptide (TRAP), collagen, arachidonic acid and ristocetin as agonists. Samples were analysed immediately after blood sampling (baseline), as well as 30 and 120 min afterwards. At baseline there were no significant differences in aggregability between samples containing direct thrombin inhibitors and heparin. In contrast, aggregation in response to all agonists except for ristocetin was significantly impaired in citrated blood. During storage the response to arachidonic acid and collagen was maintained by direct thrombin inhibitors and heparin, whereas ADP-, TRAP- and ristocetin-induced aggregation varied considerably over time in all ex vivo anticoagulants tested. Pre-analytical procedures should be standardized because storage duration and anticoagulants significantly affect platelet aggregability in whole blood. For point-of-care monitoring with immediate analysis after blood withdrawal all tested direct thrombin inhibitors as well as unfractionated heparin can be used as anticoagulants whereas citrate is not recommended. PMID:19172515

  18. [Determination of strontium content in whole blood and urine by icp-ms].

    PubMed

    Ulanova, T S; Gileva, O V; Stenno, E V; Veikhman, G A; Nedochitova, A V

    2015-01-01

    Parameters of strontium determination in the whole blood and urine of children living near ore deposits containing up to 20% strontium sulfate have been determined. The average strontium content in the whole blood of two children groups of 109.52 ± 11.07 mg/L and 131.62 ± 12.95 mg/L, significantly exceeded the level in the comparison group 44.2 ± 4.24 mg/L. The average strontium contents of two groups of children in urine were 1252.3 ± 332.2 mg/L and 1341.5 ± 241.8 mg/L, these values were 4.2 and 4.5 times higher than in the comparison group 296.4 ± 61.5 mg/L. The conditions for blood and urine sample preparation were optimized to reduce measure errors and to determine strontium at the reference concentration level. The accuracy of the results has been confirmed by analysis of the standard samples Seronorm™ Whole Blood L1, L2, L3 and Seronorm™ Urine. PMID:26539868

  19. Release of adenosine triphosphate by adenosine diphosphate in whole blood and in erythrocyte suspensions.

    PubMed

    Knöfler, R; Weissbach, G; Kuhlisch, E

    1997-12-01

    In whole blood samples from thrombocytopenic patients, large amounts of ATP were released by ADP, exceeding the level obtained with samples from normal persons by far. Because we suspected that the high potential of ATP in erythrocytes would be the main source for this phenomenon, the release of ATP by ADP was measured in whole blood samples from normal, thrombocytopenic, and leukocytopenic persons and in suspensions of washed erythrocytes. The release was recorded by a Whole Blood Lumi-Aggregometer type 500 VS (Chrono-Log Corporation, Havertown, PA) using the luciferin-luciferase system. Not only in samples from thrombocytopenic persons but also with normal platelet count, increasing amounts of ATP were released with increasing ADP concentrations, finally exceeding the ATP releasable from thrombocytes by thrombin. The amounts of ADP required to match the ATP release of thrombin were closely correlated with the platelet counts in the samples. With lower platelet counts, the release mechanism from erythrocytes could be stimulated more easily by low concentrations of ADP. The binding of ADP to platelets occurred with ostensibly higher affinity. The phenomenon of overshooting ATP release was also observed in samples from extremely leukocytopenic patients. A very large release of ATP was also achieved in suspensions of washed erythrocytes. In this way our hypothesis of ATP release from erythrocytes by ADP was confirmed again. The mechanism of the release from erythrocytes remains unclear. We speculate that its purpose is to regulate extracellular nucleotides in the circulating blood.

  20. Association between DNA Methylation in Whole Blood and Measures of Glucose Metabolism: KORA F4 Study.

    PubMed

    Kriebel, Jennifer; Herder, Christian; Rathmann, Wolfgang; Wahl, Simone; Kunze, Sonja; Molnos, Sophie; Volkova, Nadezda; Schramm, Katharina; Carstensen-Kirberg, Maren; Waldenberger, Melanie; Gieger, Christian; Peters, Annette; Illig, Thomas; Prokisch, Holger; Roden, Michael; Grallert, Harald

    2016-01-01

    Epigenetic regulation has been postulated to affect glucose metabolism, insulin sensitivity and the risk of type 2 diabetes. Therefore, we performed an epigenome-wide association study for measures of glucose metabolism in whole blood samples of the population-based Cooperative Health Research in the Region of Augsburg F4 study using the Illumina HumanMethylation 450 BeadChip. We identified a total of 31 CpG sites where methylation level was associated with measures of glucose metabolism after adjustment for age, sex, smoking, and estimated white blood cell proportions and correction for multiple testing using the Benjamini-Hochberg (B-H) method (four for fasting glucose, seven for fasting insulin, 25 for homeostasis model assessment-insulin resistance [HOMA-IR]; B-H-adjusted p-values between 9.2x10(-5) and 0.047). In addition, DNA methylation at cg06500161 (annotated to ABCG1) was associated with all the aforementioned phenotypes and 2-hour glucose (B-H-adjusted p-values between 9.2x10(-5) and 3.0x10(-3)). Methylation status of additional three CpG sites showed an association with fasting insulin only after additional adjustment for body mass index (BMI) (B-H-adjusted p-values = 0.047). Overall, effect strengths were reduced by around 30% after additional adjustment for BMI, suggesting that this variable has an influence on the investigated phenotypes. Furthermore, we found significant associations between methylation status of 21 of the aforementioned CpG sites and 2-hour insulin in a subset of samples with seven significant associations persisting after additional adjustment for BMI. In a subset of 533 participants, methylation of the CpG site cg06500161 (ABCG1) was inversely associated with ABCG1 gene expression (B-H-adjusted p-value = 1.5x10(-9)). Additionally, we observed an enrichment of the top 1,000 CpG sites for diabetes-related canonical pathways using Ingenuity Pathway Analysis. In conclusion, our study indicates that DNA methylation and diabetes

  1. Association between DNA Methylation in Whole Blood and Measures of Glucose Metabolism: KORA F4 Study

    PubMed Central

    Wahl, Simone; Kunze, Sonja; Molnos, Sophie; Volkova, Nadezda; Schramm, Katharina; Carstensen-Kirberg, Maren; Waldenberger, Melanie; Gieger, Christian; Peters, Annette; Illig, Thomas; Prokisch, Holger; Roden, Michael; Grallert, Harald

    2016-01-01

    Epigenetic regulation has been postulated to affect glucose metabolism, insulin sensitivity and the risk of type 2 diabetes. Therefore, we performed an epigenome-wide association study for measures of glucose metabolism in whole blood samples of the population-based Cooperative Health Research in the Region of Augsburg F4 study using the Illumina HumanMethylation 450 BeadChip. We identified a total of 31 CpG sites where methylation level was associated with measures of glucose metabolism after adjustment for age, sex, smoking, and estimated white blood cell proportions and correction for multiple testing using the Benjamini-Hochberg (B-H) method (four for fasting glucose, seven for fasting insulin, 25 for homeostasis model assessment-insulin resistance [HOMA-IR]; B-H-adjusted p-values between 9.2x10-5 and 0.047). In addition, DNA methylation at cg06500161 (annotated to ABCG1) was associated with all the aforementioned phenotypes and 2-hour glucose (B-H-adjusted p-values between 9.2x10-5 and 3.0x10-3). Methylation status of additional three CpG sites showed an association with fasting insulin only after additional adjustment for body mass index (BMI) (B-H-adjusted p-values = 0.047). Overall, effect strengths were reduced by around 30% after additional adjustment for BMI, suggesting that this variable has an influence on the investigated phenotypes. Furthermore, we found significant associations between methylation status of 21 of the aforementioned CpG sites and 2-hour insulin in a subset of samples with seven significant associations persisting after additional adjustment for BMI. In a subset of 533 participants, methylation of the CpG site cg06500161 (ABCG1) was inversely associated with ABCG1 gene expression (B-H-adjusted p-value = 1.5x10-9). Additionally, we observed an enrichment of the top 1,000 CpG sites for diabetes-related canonical pathways using Ingenuity Pathway Analysis. In conclusion, our study indicates that DNA methylation and diabetes-related traits

  2. Inhibition of platelet function with clopidogrel, as measured with a novel whole blood impedance aggregometer in horses.

    PubMed

    Roscher, Katja A; Failing, Klaus; Moritz, Andreas

    2015-03-01

    This study aimed to validate a loading and maintenance clopidogrel dosing scheme for the inhibition of platelet function, measured by whole blood impedance aggregometry in healthy adult horses. Ten Warmblood horses received oral clopidogrel once daily. Doses were based on 50 kg weight categories and resulted in one loading dose of 6-6.5 mg/kg bodyweight and maintenance doses of 1.2-1.4 mg/kg over the next 4 days. Platelet function was measured via whole blood multiple electrode impedance aggregometry prior to (T0) and at 6, 12, 24, 48, 72, 96, 144, 192 and 240 h following the loading dose. Aggregometries for collagen (COLtest), arachidonic acid (ASPItest), adenosine diphosphate (ADPtest) and ADP with prostaglandin E1 (ADPtestHS) were performed. Statistical analyses included one way repeated measures ANOVAs and subsequent Dunnett's tests. Platelet aggregation induced by collagen remained unchanged. There were significant inhibitions in the ASPItest (P <0.01 at 192 h, and P <0.05 at 240 h) and the ADPtest and ADPtestHS (P < 0.01, with the exception of 240 h). The loading dose of clopidogrel induced rapid inhibition of platelet function within hours, and the low dose was suitable for maintaining the inhibition over the 4 days of therapy. Recovery of platelet function was restored 6 days after the cessation of medication, determined with the ADPtest and ADPtestHS, but remained inhibited with the ASPItest. The prolonged effect of clopidogrel may indicate differences in the activation of platelets between horses and humans that were previously unknown.

  3. Quantification of 33 antidepressants by LC-MS/MS--comparative validation in whole blood, plasma, and serum.

    PubMed

    Montenarh, Deborah; Wernet, Mathias P; Hopf, Markus; Maurer, Hans H; Schmidt, Peter H; Ewald, Andreas H

    2014-09-01

    In the present study, a liquid chromatography-mass spectrometry (LC-MS/MS) multi-analyte approach based on a simple liquid-liquid extraction was developed for fast target screening and quantification of 33 antidepressants in whole blood, plasma, and serum. The method was validated with respect to selectivity, matrix effects, recovery, process efficiency, accuracy and precision, stabilities, and limits. In addition, cross-calibration between the three biosamples was done to assess the impact of the different matrices on the calibration. Whole blood, plasma, and serum (500 μL each) were extracted twice at pH 7.4 and at pH 10 with ether-ethyl acetate (1:1). Separation, detection, and quantification were performed using LC-MS/MS with electrospray ionization in positive mode. For accuracy and precision, full calibration was performed with ranges from subtherapeutic to toxic concentrations. The approach was sensitive and selective for 33 analytes in whole blood and 31 analytes in plasma and serum and accurate and precise for 30 of the 33 tested drugs in whole blood, 31 in plasma, and 28 in serum. Cross-calibration was successful only for 13 analytes in whole blood and 16 analytes in serum calculated over a calibration curve made in plasma, 12 analytes in whole blood and 15 analytes in plasma calculated over a calibration curve made in serum, and 10 analytes in plasma and 15 analytes in serum calculated over a calibration curve made in whole blood.

  4. The Impact of Glyphosate, Its Metabolites and Impurities on Viability, ATP Level and Morphological changes in Human Peripheral Blood Mononuclear Cells.

    PubMed

    Kwiatkowska, Marta; Jarosiewicz, Paweł; Michałowicz, Jaromir; Koter-Michalak, Maria; Huras, Bogumiła; Bukowska, Bożena

    2016-01-01

    The toxicity of herbicides to animals and human is an issue of worldwide concern. The present study has been undertaken to assess toxic effect of widely used pesticide-glyphosate, its metabolites: aminomethylphosphonic acid (AMPA) and methylphosphonic acid and its impurities: N-(phosphonomethyl)iminodiacetic acid (PMIDA), N-methylglyphosate, hydroxymethylphosphonic acid and bis-(phosphonomethyl)amine on human peripheral blood mononuclear cells (PBMCs). We have evaluated the effect of those compounds on viability, ATP level, size (FSC-A parameter) and granulation (SSC-A parameter) of the cells studied. Human peripheral blood mononuclear cells were exposed to different concentrations of glyphosate, its metabolites and impurities (0.01-10 mM) for 4 and 24 h. It was found that investigated compounds caused statistically significant decrease in viability and ATP level of PBMCs. The strongest changes in cell viability and ATP level were observed after 24 h incubation of PBMCs with bis-(phosphonomethyl)amine, and particularly PMIDA. Moreover, all studied compounds changed cell granularity, while PMIDA and bis-(phosphonomethyl)amine altered PBMCs size. It may be concluded that bis-(phosphonomethyl)amine, and PMIDA caused a slightly stronger damage to PBMCs than did glyphosate. Changes in the parameters studied in PBMCs were observed only at high concentrations of the compounds examined, which clearly shows that they may occur in this cell type only as a result of acute poisoning of human organism with these substances. PMID:27280764

  5. The Impact of Glyphosate, Its Metabolites and Impurities on Viability, ATP Level and Morphological changes in Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Kwiatkowska, Marta; Jarosiewicz, Paweł; Michałowicz, Jaromir; Koter-Michalak, Maria; Huras, Bogumiła; Bukowska, Bożena

    2016-01-01

    The toxicity of herbicides to animals and human is an issue of worldwide concern. The present study has been undertaken to assess toxic effect of widely used pesticide—glyphosate, its metabolites: aminomethylphosphonic acid (AMPA) and methylphosphonic acid and its impurities: N-(phosphonomethyl)iminodiacetic acid (PMIDA), N-methylglyphosate, hydroxymethylphosphonic acid and bis-(phosphonomethyl)amine on human peripheral blood mononuclear cells (PBMCs). We have evaluated the effect of those compounds on viability, ATP level, size (FSC-A parameter) and granulation (SSC-A parameter) of the cells studied. Human peripheral blood mononuclear cells were exposed to different concentrations of glyphosate, its metabolites and impurities (0.01–10 mM) for 4 and 24 h. It was found that investigated compounds caused statistically significant decrease in viability and ATP level of PBMCs. The strongest changes in cell viability and ATP level were observed after 24 h incubation of PBMCs with bis-(phosphonomethyl)amine, and particularly PMIDA. Moreover, all studied compounds changed cell granularity, while PMIDA and bis-(phosphonomethyl)amine altered PBMCs size. It may be concluded that bis-(phosphonomethyl)amine, and PMIDA caused a slightly stronger damage to PBMCs than did glyphosate. Changes in the parameters studied in PBMCs were observed only at high concentrations of the compounds examined, which clearly shows that they may occur in this cell type only as a result of acute poisoning of human organism with these substances. PMID:27280764

  6. Ellagic Acid, a Dietary Polyphenol, Inhibits Tautomerase Activity of Human Macrophage Migration Inhibitory Factor and Its Pro-inflammatory Responses in Human Peripheral Blood Mononuclear Cells.

    PubMed

    Sarkar, Souvik; Siddiqui, Asim A; Mazumder, Somnath; De, Rudranil; Saha, Shubhra J; Banerjee, Chinmoy; Iqbal, Mohd S; Adhikari, Susanta; Alam, Athar; Roy, Siddhartha; Bandyopadhyay, Uday

    2015-05-27

    Ellagic acid (EA), a phenolic lactone, inhibited tautomerase activity of human macrophage migration inhibitory factor (MIF) noncompetitively (Ki = 1.97 ± 0.7 μM). The binding of EA to MIF was determined by following the quenching of tryptophan fluorescence. We synthesized several EA derivatives, and their structure-activity relationship studies indicated that the planar conjugated lactone moiety of EA was essential for MIF inhibition. MIF induces nuclear translocation of NF-κB and chemotaxis of peripheral blood mononuclear cells (PBMCs) to promote inflammation. We were interested in evaluating the effect of EA on nuclear translocation of NF-κB and chemotactic activity in human PBMCs in the presence of MIF. The results showed that EA inhibited MIF-induced NF-κB nuclear translocation in PBMCs, as evident from confocal immunofluorescence microscopic data. EA also inhibited MIF-mediated chemotaxis of PBMCs. Thus, we report MIF-inhibitory activity of EA and inhibition of MIF-mediated proinflammatory responses in PBMCs by EA.

  7. Rapid, sensitive, and specific lateral-flow immunochromatographic point-of-care device for detection of herpes simplex virus type 2-specific immunoglobulin G antibodies in serum and whole blood.

    PubMed

    Laderman, Elisabeth I; Whitworth, Emma; Dumaual, Erickson; Jones, Mark; Hudak, Andrew; Hogrefe, Wayne; Carney, Jim; Groen, Jan

    2008-01-01

    Herpes simplex virus type 2 (HSV-2) is a common human pathogen that can cause a variety of clinical manifestations in humans. In order to provide near-patient results to allow for faster counseling and treatment, a rapid point-of-care test that is accurate and simple to use is desirable. Here, we describe the development and evaluation of an HSV-2 immunoglobulin G (IgG)-specific antibody lateral-flow immunochromatographic assay (LFIA) based on colloidal gold nanoparticles. A total of 359 serum samples and 100 whole-blood samples were tested in the newly developed HSV-2 LFIA. Serum results were compared to those from the HerpeSelect HSV-2 enzyme-linked immunosorbent assay (ELISA), and whole-blood sample results were compared to those of both ELISA and HerpeSelect HSV-1 and -2 immunoblotting (IB). The sensitivity of the HSV-2 LFIA compared to that of the HerpeSelect ELISA was 100% (89/89), and the specificity was 97.3% (257/264). Cross-reactivity with HSV-1 IgG-positive serum samples was observed in 2.6% (5/196) of samples, 2.9% (1/34) for rubella virus, and 6.2% (1/16) for Epstein-Barr virus. No cross-reactivity in varicella-zoster virus or cytomegalovirus IgG-positive serum samples was observed. No interference was observed from bilirubin-, triglyceride-, albumin-, or hemoglobin-spiked samples. The concordance of the LFIA results between capillary whole blood, EDTA-treated venous whole blood, heparin-treated venous whole blood, and serum was 99% (99/100). In conclusion, the LFIA for HSV-2 IgG-specific antibodies demonstrated excellent sensitivity, specificity, and concordance for both serum and whole-blood samples compared to the sensitivity, specificity, and concordance of both HSV-2 ELISA and IB.

  8. Thrombocytopenia model with minimal manipulation of blood cells allowing whole blood assessment of platelet function.

    PubMed

    Tiedemann Skipper, Mette; Rubak, Peter; Halfdan Larsen, Ole; Hvas, Anne-Mette

    2016-06-01

    In vitro models of thrombocytopenia are useful research tools. Previously published models have shortcomings altering properties of platelets and other blood components. The aim of the present study was to develop a whole blood method to induce thrombocytopenia with minimal manipulation, and to describe platelet function in induced thrombocytopenia in individuals with healthy platelets. Hirudin anticoagulated blood was obtained from 20 healthy volunteers. One part of the blood was gently centrifuged at 130g for 15 minutes. The platelet-rich plasma was replaced with phosphate-buffered saline to establish thrombocytopenia. Various levels of thrombocytopenia were achieved by combining different volumes of baseline whole blood and thrombocytopenic blood. Platelet counts were measured by flow cytometry (Navios, Beckman Coulter) and routine haematological analyser (Sysmex XE-5000). Platelet function was analysed by impedance aggregometry (Multiplate® Analyzer, Roche) and by flow cytometry (Navios, Beckman Coulter) using collagen, adenosine diphosphate, thrombin receptor activating peptide-6 and ristocetin as agonists. Median baseline platelet count was 227×10(9)/l. The in vitro model yielded median platelet counts at 51×10(9)/l (range 26-93×10(9)/l). We observed minor, yet significant, changes in platelet size and maturity from baseline to modelled thrombocytopenia. In the thrombocytopenic samples, significant and positive linear associations were found between platelet count and platelet aggregation across all agonists (all p-values<0.001). Platelet function assessed by flow cytometry showed minimal alterations in the thrombocytopenic samples. A new whole blood-based model of thrombocytopenia was established and validated. This new model serves as a useful future tool, particularly to explore platelet function in patients with thrombocytopenia.

  9. LC-MS-MS analysis of buprenorphine and norbuprenorphine in whole blood from suspected drug users.

    PubMed

    Seldén, Tor; Roman, Markus; Druid, Henrik; Kronstrand, Robert

    2011-06-15

    A liquid chromatography tandem mass spectrometry method is described for the analysis of buprenorphine and norbuprenorphine in whole blood. Linearity was achieved between 0.2-5 ng/g for buprenorphine and 0.5-5 ng/g for norbuprenorphine. Stability studies on spiked whole blood and an authentic sample showed no degradation of buprenorphine- and norbuprenorphine-glucuronide to their respective aglycones. Buprenorphine and norbuprenorphine showed some degradation when stored at 4°C for three weeks, but was stable when stored at -20°C for 4 weeks. The method was applied to forensic cases of driving under the influence of drugs (DUID) and petty drug offences (PDO) during 2007-2009. Out of 2459 cases analyzed, 322 were positive for both buprenorphine and norbuprenorphine (13%), 219 for buprenorphine only (9%), and 12 for norbuprenorphine only (0.5%). The mean and median concentrations (N=322) were 1.7 and 1.0 ng/g, respectively, for buprenorphine and norbuprenorphine. The mean and median norbuprenorphine/buprenorphine ratios were 1.5 and 1.1, respectively. There was no significant difference in concentration ratios for DUID and PDO cases (p>0.05). We conclude that the described method for analysis of buprenorphine and norbuprenorphine in whole blood could be used to investigate use or misuse of buprenorphine but that many of the cases presented with very low concentrations of buprenorphine. We also conclude that analysis should be performed within two weeks unless samples are stored frozen prior to analysis. PMID:21306845

  10. Automated high-throughput purification of genomic DNA from whole blood using Promega's MagneSilTM paramagnetic particles with either the Max Yield or MagneSilTM ONE normalized purification methods

    NASA Astrophysics Data System (ADS)

    Bitner, Rex M.; Koller, Susan C.; Sankbeil, Jacqui

    2003-07-01

    Two different methods of automated high throughput purification of genomic DNA from human whole blood in 96 well plates are described. One method uses MagneSilTM paramagnetic particles to purify a maximal amount of the DNA present in the sample. Another method, the MagnesilTM ONE system, allows for the purification of a predetermined amount of DNA from human whole blood. Protocols for the purification of 100 ng or, alternatively 1 ug, of human genomic DNA from whole blood using MagneSilTM paramagnetic particles and a Beckman BioMekTM FX robot are described. With the maximal yield purification system, typical DNA yields fall in the range of 4-9 ug of DNA from 200ul of human whole blood, depending upon the white cell content of donor sample. For situations where DNA achiving is desired, or when the number of downstream sample applications is not clearly defined (e.g. multiple SNP analyses) the maximal yield method is usually preferred. However, in situations with a defined downstream application (e.g. criminal databasing or use of a defined set of amplifications) where purifying DNA in a narrow concentrate range streamlines the high throughput purification and analysis process, the automated MagneSilTM ONE purification system is the method of choice. DNA from either method is suitable for applications such as PCR, STR, READITTM SNP analysis, and multiplexed PCR systems such as Promega's Y-chromosome deletion detection system.

  11. Lead levels in whole blood of sheep from different areas of the Nile delta.

    PubMed

    Takla, P G; Mohamed, H A; Wright, J; Fahmy, F

    1989-03-25

    The concentration of lead has been determined in samples of whole blood from Osimi and Rahmani sheep grazing near main highways in three areas of the Nile delta region of Egypt. Between 117 and 200 blood samples were collected from each area. The mean values for the blood lead content were 0.062, 0.067 and 0.083 micrograms/ml, and were low in all the locations. The value of 0.083 micrograms/ml obtained for the samples taken from the partly industralised Enshass region was significantly higher than the mean values from the other sites. PMID:2728270

  12. Centrifugal extraction of plasma from whole blood on a rotating disk.

    PubMed

    Haeberle, Stefan; Brenner, Thilo; Zengerle, Roland; Ducrée, Jens

    2006-06-01

    We present a centrifugal process for the extraction of plasma from sediment by a decanting structure, terminating with metered plasma which is readily available for subsequent on-disk processing. Our technique supplies 2 microl plasma from 5 microl of whole blood at moderate spinning frequencies of 40 Hz within 20 s, only. The residual cell concentration in the purified plasma amounts to less than 0.11%, independent of the frequency of rotation. A capillary duct connects the extracted plasma to subsequent on-disk processing units.

  13. Homologous whole blood transfusion during treatment of severe anemia in a chimpanzee (Pan troglodytes).

    PubMed

    Debenham, John James; Atencia, Rebeca

    2014-09-01

    A 12-yr-old female chimpanzee (Pan troglodytes) was presented as severely emaciated and with generalized muscle weakness. Hematology and biochemistry revealed severe anemia and hypokalemia. The chimpanzee was treated supportively and symptomatically; although initially stable, the animal deteriorated rapidly on day 5, becoming depressed and jaundiced with further deterioration of anemia. To address the decline, a prompt transfusion of compatible and cross-matched fresh whole blood from a healthy adult male chimpanzee was administered over 120 min. During transfusion, an immediate reduction in the recipient's tachycardia was noted and substantial clinical improvement continued over 24 hr posttransfusion; no adverse transfusion reactions were observed.

  14. Analysis of serum and whole blood values in relation to helminth and ectoparasite infections of feral pigs in Texas.

    PubMed

    Shender, Lisa A; Botzler, Richard G; George, T Luke

    2002-04-01

    In the summers of 1996 and 1997, 60 wild pigs (Sus scrofa) were necropsied from three sites in south Texas (USA) to test the hypothesis that serum and whole blood parameters vary significantly (P < or = 0.05) with the prevalence and intensity of parasites infecting wild pigs. We found ten parasite species: five nematodes (Metastrongylus salmi, Metastrongylus pudentotectus, Stephanurus dentatus, Oesophagostomum dentatum, and Physocephalus sexalatus); four ixodid ticks (Amblyomma cajennense, Amblyomma maculatum, Amblyomma americanum, and Dermacentor variabilis); and one trematode (Fascioloides magna). Among juvenile pigs, the intensity of the four species of ticks, collectively, was negatively correlated (P < or = 0.05) with whole blood principal component number one (PC-1); this factor was positively associated with lymphocytes and eosinophils. Lungworm intensity (Metastrongylus spp.) among adult pigs was negatively correlated (P < or = 0.05) with whole blood PC-2; this factor was negatively associated with segmented neutrophils and monocytes. There were no significant correlations found between parasite prevalences and either serum or whole blood principal component factors. The correlations observed between parasite intensities and serum and whole blood parameters generally were weak. Thus, we found no strong evidence that serum and whole blood parameters provided good predictive information on parasite infections in wild pigs for most practical management decisions.

  15. Detection of Zika virus RNA in whole blood of imported Zika virus disease cases up to 2 months after symptom onset, Israel, December 2015 to April 2016.

    PubMed

    Lustig, Yaniv; Mendelson, Ella; Paran, Nir; Melamed, Sharon; Schwartz, Eli

    2016-06-30

    Zika virus RNA presence in serum, whole-blood and urine samples from six Israeli travellers symptomatic for Zika virus disease was examined. Whole-blood samples were positive for as late as 2 months (58 days) post-symptom onset, longer than for urine (26 days) and serum (3 days). These findings suggest the utility of whole blood in Zika infection diagnosis.

  16. Antidepressants detection and quantification in whole blood samples by GC-MS/MS, for forensic purposes.

    PubMed

    Truta, Liliana; Castro, André L; Tarelho, Sónia; Costa, Pedro; Sales, M Goreti F; Teixeira, Helena M

    2016-09-01

    Depression is among the most prevalent psychiatric disorders of our society, leading to an increase in antidepressant drug consumption that needs to be accurately determined in whole blood samples in Forensic Toxicology Laboratories. For this purpose, this work presents a new gas chromatography tandem mass spectrometry (GC-MS/MS) method targeting the simultaneous and rapid determination of 14 common Antidepressants in whole blood: 13 Antidepressants (amitriptyline, citalopram, clomipramine, dothiepin, fluoxetine, imipramine, mianserin, mirtazapine, nortryptiline, paroxetine, sertraline, trimipramine and venlafaxine) and 1 Metabolite (N-desmethylclomipramine). Solid-phase extraction was used prior to chromatographic separation. Chromatographic and MS/MS parameters were selected to improve sensitivity, peak resolution and unequivocal identification of the eluted analyte. The detection was performed on a triple quadrupole tandem MS in selected ion monitoring (SIM) mode in tandem, using electronic impact ionization. Clomipramine-D3 and trimipramine-D3 were used as deutered internal standards. The validation parameters included linearity, limits of detection, lower limit of quantification, selectivity/specificity, extraction efficiency, carry-over, precision and robustness, and followed internationally accepted guidelines. Limits of quantification and detection were lower than therapeutic and sub-therapeutic concentration ranges. Overall, the method offered good selectivity, robustness and quick response (<16min) for typical concentration ranges, both for therapeutic and lethal levels. PMID:27376459

  17. Simultaneous measurement of red blood cell aggregation and whole blood coagulation using high-frequency ultrasound.

    PubMed

    Nam, Kweon-Ho; Yeom, Eunseop; Ha, Hojin; Lee, Sang Joon

    2012-03-01

    This study aims to investigate the feasibility of using high-frequency ultrasound (HFUS) for simultaneous monitoring of blood coagulation and red blood cell (RBC) aggregation. Using a 35-MHz ultrasound scanner, ultrasound speckle data were acquired from whole blood samples of three experimental groups of rats, including 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS)-treated, noncoagulation and normal control groups. The variations of blood echogenicity, the shape parameters of probability distribution of speckle intensity (skewness and kurtosis) and the correlation coefficient between two consecutive speckle data were calculated as a function of time starting from immediately after taking blood. The blood echogenicity increases rapidly to plateaus at the early stage of measurement for all the experimental groups caused by the formation of RBC aggregates. The DIDS-treated group exhibits the lowest echogenicity level due to the inhibitory effect of DIDS on RBC aggregation. The correlation analysis between consecutive speckle patterns seems to be useful to examine the variation of blood fluidity and the progress of clot formation. Whole blood coagulation is observed to be accelerated by DIDS treatment. In addition, the results of skewness and kurtosis analysis indicated that RBC aggregates may be disrupted during blood coagulation. The present study suggests that HFUS has good potential for simultaneous monitoring of RBC aggregation and blood coagulation to examine the relationship between them.

  18. A validated method for quantifying hypoglycin A in whole blood by UHPLC-HRMS/MS.

    PubMed

    Carlier, Jérémy; Guitton, Jérôme; Moreau, Cécile; Boyer, Baptiste; Bévalot, Fabien; Fanton, Laurent; Habyarimana, Jean; Gault, Gilbert; Gaillard, Yvan

    2015-01-26

    Hypoglycin A (HGA) is the toxic principle in ackee (Blighia sapida Koenig), a nutritious and readily available fruit which is a staple of the Jamaican working-class and rural population. The aril of the unripe fruit has high concentrations of HGA, the cause of Jamaican vomiting sickness, which is very often fatal. HGA is also present in the samara of several species of maple (Acer spp.) which are suspected to cause seasonal pasture myopathy in North America and equine atypical myopathy in Europe, often fatal for horses. The aim of this study was to develop a method for quantifying HGA in blood that would be sensitive enough to provide toxicological evidence of ackee or maple poisoning. Analysis was carried out using solid-phase extraction (HILIC cartridges), dansyl derivatization and UHPLC-HRMS/MS detection. The method was validated in whole blood with a detection limit of 0.35 μg/L (range: 0.8-500 μg/L). This is the first method applicable in forensic toxicology for quantifying HGA in whole blood. HGA was quantified in two serum samples from horses suffering from atypical myopathy. The concentrations were 446.9 and 87.8 μg/L. HGA was also quantified in dried arils of unripe ackee fruit (Suriname) and seeds of sycamore maple (Acer pseudoplatanus L.) (France). The concentrations were 7.2 and 0.74 mg/g respectively.

  19. Comparison of Proteins in Whole Blood and Dried Blood Spot Samples by LC/MS/MS

    NASA Astrophysics Data System (ADS)

    Chambers, Andrew G.; Percy, Andrew J.; Hardie, Darryl B.; Borchers, Christoph H.

    2013-09-01

    Dried blood spot (DBS) sampling methods are desirable for population-wide biomarker screening programs because of their ease of collection, transportation, and storage. Immunoassays are traditionally used to quantify endogenous proteins in these samples but require a separate assay for each protein. Recently, targeted mass spectrometry (MS) has been proposed for generating highly-multiplexed assays for biomarker proteins in DBS samples. In this work, we report the first comparison of proteins in whole blood and DBS samples using an untargeted MS approach. The average number of proteins identified in undepleted whole blood and DBS samples by liquid chromatography (LC)/MS/MS was 223 and 253, respectively. Protein identification repeatability was between 77 %-92 % within replicates and the majority of these repeated proteins (70 %) were observed in both sample formats. Proteins exclusively identified in the liquid or dried fluid spot format were unbiased based on their molecular weight, isoelectric point, aliphatic index, and grand average hydrophobicity. In addition, we extended this comparison to include proteins in matching plasma and serum samples with their dried fluid spot equivalents, dried plasma spot (DPS), and dried serum spot (DSS). This work begins to define the accessibility of endogenous proteins in dried fluid spot samples for analysis by MS and is useful in evaluating the scope of this new approach.

  20. Counting leukocytes from whole blood using a lab-on-a-chip Coulter counter.

    PubMed

    Mei, Zhe; Cho, Sung Hwan; Zhang, Arthur; Dai, Jie; Wu, Tsung-Feng; Lo, Yu-Hwa

    2012-01-01

    A microfluidic lab-on-a-chip Coulter counter was demonstrated to count micro particles and leukocytes from whole blood. Instead of electroplated or deposited metal electrodes, off-the-shelf gold pins were used as electrodes to simplify fabrication process, reduce cost, enhance device durability, and above all, achieve superior uniformity in E-field distribution for improved signal quality. A custom-designed, low-cost demodulation circuit was developed to detect the AC impedance signals of the particles and cells passing the detection area defined by the microfluidic channels. A mixture of polystyrene beads with three different sizes was used to characterize the device. The results showed high throughput at 2000 particles/s and clear separation among different sizes of beads with coefficients of variation (CV) of 13.53%, 10.35% and 5.67% for 7.66 µm, 10.5 µm and 14.7 µmbeads, respectively. To demonstrate the potential for a point-of-care or self-administered device for cancer patients going through chemotherapy, we have used the lab-on-a-chip device to count leukocytes from whole blood, generating encouraging preliminary results comparable to the results from a commercial flow cytometer.

  1. The in vitro effect of aspirin on increased whole blood platelet aggregation in oral contraceptive users.

    PubMed

    Norris, L A; Bonnar, J

    1994-05-01

    The effects of triphasic oral contraceptives on whole blood platelet aggregation in 36 Italian women are reported here. Aspirin's effects on platelet aggregation were also studied. 18 women took a triphasic oral contraceptive; 10 women took Trinordiol, while 8 took Trinovum for at least 90 days. The remaining 18 women took nothing and served as controls. The study was aligned with each woman's birth control pill cycle. Blood was taken daily on days 15-21 of their cycle. Either saline solution or acetylsalicylic acid was added to the blood samples and compared. All data was statistically analyzed using unpaired student's t-test. Effects of 3 aggregating agents, ADP, PAF, and EDTA, on platelet aggregation were studied. Arachidonic acid and adrenalin bitartrate were also studied in this manner. An increase in platelet aggregation was observed in women taking oral contraceptives. No difference was found between patients taking Trinordiol and those taking Trinovum. The results of this study indicate an increase in whole blood platelet sensitivity to collagen, adrenalin, and arachidonic acid when using oral contraceptives. Aspirin, at low doses, may have a role in preventing early thrombus formation in women taking oral contraceptives. PMID:8042198

  2. Prospective Study of Metal Fume-Induced Responses of Global Gene Expression Profiling in Whole Blood

    PubMed Central

    Wang, Zhaoxi; Neuberg, Donna; Su, Li; Kim, Jee Young; Chen, Jiu-Chiuan; Christiani, David C.

    2008-01-01

    Metal particulate inhalation causes pulmonary and cardiovascular diseases. Our previous results showed that systemic responses to short-term occupational welding-fume exposure could be assessed by microarray analyses in whole-blood total RNA sampled before and after exposure. To expand our understanding of the duration of particulate-induced gene expression changes, we conducted a study using a similar population 1 yr after the original study and extended our observations in the postexposure period. We recruited 15 individuals with welding fume exposure and 7 nonexposed individuals. Thirteen of the 22 individuals (9 in exposed group and 4 in nonexposed group) had been monitored in the previous study. Whole-blood total RNA was analyzed at 3 time points, including baseline, immediately following exposure (approximately 5 h after baseline), and 24 h after baseline, using cDNA microarray technology. We replicated the patterns of Gene Ontology (GO) terms associated with response to stimulus, cell death, phosphorus metabolism, localization, and regulation of biological processes significantly enriched with altered genes in the nonsmoking exposed group. Most of the identified genes had opposite expression changes between the exposure and postexposure periods in nonsmoking welders. In addition, we found dose-dependent patterns that were affected by smoking status. In conclusion, short-term occupational exposure to metal particulates causes systemic responses in the peripheral blood. Furthermore, the acute particulate-induced effects on gene expression profiling were transient in nonsmoking welders, with most effects diminishing within 19 h following exposure. PMID:18951227

  3. Keishibukuryogan, a Traditional Japanese Medicine, Inhibits Platelet Aggregation in Guinea Pig Whole Blood

    PubMed Central

    Terawaki, Kiyoshi; Noguchi, Masamichi; Yuzurihara, Mitsutoshi; Omiya, Yuji; Ikarashi, Yasushi; Kase, Yoshio

    2015-01-01

    Effects of keishibukuryogan (KBG) on platelet aggregation were investigated. To ensure the specificity of KBG, tokishakuyakusan (TSS) and kamisyoyosan (KSS), which are known to have platelet aggregation-inhibiting effects, and rikkunshito (RKT) and shakuyakukanzoto (SKT), which are considered to be devoid of such effects, were used for comparison. The platelet aggregation of each test drug was measured by the screen filtration pressure method using whole blood of guinea pigs and expressed as a collagen-induced pressure rate (%) or a collagen concentration required for 50% increase in the pressure rate (PATI value). KBG suppressed the collagen-induced whole blood pressure rate increase and increased the PATI value, like TSS and KSS. Neither RKT nor SKT showed these effects. The Moutan cortex and Cinnamomi cortex, the constituent crude drugs of KBG, showed KBG-like pressure rate suppression and PATI-increasing effects. Furthermore, paeonol, a representative component of Moutan cortex, and aspirin which is known to have platelet aggregation-inhibiting activity (COX-1 inhibitor) also showed similar effects. These results suggest that the platelet aggregation-inhibiting activity of the constituent crude drugs Moutan cortex and Cinnamomi cortex is involved in the improving effects of KBG on impaired microcirculation and that paeonol plays a role in these effects. PMID:26379740

  4. [Comparison of rheologic properties between Ca-alginate hydrogel microspheres suspension and whole blood].

    PubMed

    Xu, Pei; Wang, Xiang; Li, Yaojin; Wang, Feifei; Duan, Ming; Yang, Li

    2013-02-01

    Starting from the form of red blood cells and the hematocrit (Hct, about 45 vol% of whole blood), we tried to prepare a kind of microspheres suspension to imitate non-Newtonian fluid property of whole blood, exploring its potentiality to be applied in blood viscosity quality control substance. In our study, we produced Ca-alginate hydrogel microspheres using emulsion polymerization, then we suspended the microspheres in 0.9 wt% NaCl solution to obtain a kind of liquid sample with the microspheres taking 45% volume. Then we used two types of viscometers to measure and analyse the changes of sample viscosity at different shear rate. We observed the forms of Ca-alginate hydrogel microspheres with microscope, and found them to be relatively complete, and their diameters to be normally distributed. Diameters of about 90% of the microspheres were distributed in a range from 6 to 22 micron. The samples were examined with viscometer FASCO-3010 and LG-R-80c respectively, both of which have shown a shear-thinning effect. After 5-week stability test, the CV of viscosity results corresponding to the two instruments were 7.3% to 13.8% and 8.9% to 14.2%, respectively. Although some differences existed among the results under the same shear rate, the general variation trends of the corresponding results were consistent, so the sample had the potentiality to be widely used in calibrating a different type of blood viscometer. PMID:23488147

  5. Effect of delay and storage on whole-blood clotting analysis as determined by thrombelastography.

    PubMed

    Orlikowski, C E; Murray, W B; Rocke, D A

    1993-01-01

    The thrombelastogram (TEG) measures the viscoelastic properties of clotting blood, displaying a visual trace of all phases of coagulation and fibrinolysis. Thrombelastography can be performed on whole blood (WBTEG) or on citrated blood or plasma, citrated samples facilitating delayed analysis but requiring recalcification of the sample. The aim of this study was to investigate the effect of delay and storage method on WBTEG measurement. Thrombelastographic analysis of coagulation in whole blood was investigated after delays of 3 and 6 minutes in polystyrene syringes (PS3 and PS6) and 3 minutes in silicone-coated glass tubes (SG3). Thrombelastograms of the delayed samples were compared with those measured immediately. Silicone-coated glass tubes activated coagulation, as seen by shorter r times (p < 0.01), shorter r + k times (p < 0.01), and larger maximum amplitude (ma) values (p < 0.01) compared with TEG values determined immediately after sampling. In the SG3 group, 20% of samples had clotted by 3 minutes, and the use of SG tubes for this purpose cannot be recommended. A delay of 6 minutes in PS had less effect on the activation of clotting in the earlier stages in that the r time was prolonged (p < 0.01). However, there appeared to be some activation later in that k time was shorter (p < 0.01) and ma was wider (p < 0.05). Overall, a 3-minute delay in PS produced the best values.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Metallic Profile of Whole Blood and Plasma in a Series of 99 Healthy Children.

    PubMed

    Goullé, Jean-Pierre; Le Roux, Pascal; Castanet, Mireille; Mahieu, Loïc; Guyet-Job, Stéphanie; Guerbet, Michel

    2015-01-01

    In recent years, special emphasis has been put on heavy metals. Children are very sensitive to accumulation of metals. Furthermore, as regards elements, the reference values in children are scarce in the literature as it is difficult to obtain the large quantity of blood necessary to analyze many metals by the conventional atomic absorption spectrometry technique. An inductively coupled plasma mass spectrometry (ICP-MS) procedure that uses a reduced sample of 0.3 mL whole blood or plasma is adapted to multielemental determinations. We applied a previously validated technique for adults that simultaneously quantifies 25 elements by ICP-MS in whole blood and 23 in plasma in a series of 99 healthy children ranging from under 5 years to <18 years, without exposure to metal or drug-containing metals. The aims of the study were to compare metallic concentrations according to the age among children and metallic concentration differences between children and adults. The blood and plasma pediatric metallic profile is a practical useful tool for many purposes in clinical toxicology, forensic toxicology and any cases of metal environmental exposure.

  7. Pattern recognition of neuron specific enolase and carcinoembryonic antigen in whole blood samples.

    PubMed

    Stefan-van Staden, Raluca-Ioana; Comnea-Stancu, Ionela Raluca; Surdu-Bob, Carmen Cristina; Stanciu-Gavan, Camelia

    2015-02-01

    New tools and methods for pattern recognition of neuron specific enolase (NSE) and carcinoembryonic antigen (CEA) were proposed for the screening of whole blood samples. The new tools were based on stochastic sensors designed using nanoporous gold microspheres, graphite, graphene, diamond paste as well as α-CDs, and 5,10,15,20-tetraphenyl-21H,23H-porphyrin. The best sensor for the assay of CEA was the one based on P/graphite (the limit of determination was 16 fg/ml and sensitivity was 2.32 × 10(7)  s mg(-1)  ml), while for the assay of NSE the, best sensor was the one based on P/graphene (the limit of determination was 7.45 pg/ml and sensitivity was 2.49 × 10(8)  s mg(-1)  ml). The sensor of choice for simultaneous detection of NSE and CEA is the one based on P/graphene because we need high sensitivity and low limit of determination for NSE. To our knowledge, this is the only one screening test for early detection of lung cancer, by identification of NSE and CEA in whole blood samples. PMID:25604868

  8. Metallic Profile of Whole Blood and Plasma in a Series of 99 Healthy Children.

    PubMed

    Goullé, Jean-Pierre; Le Roux, Pascal; Castanet, Mireille; Mahieu, Loïc; Guyet-Job, Stéphanie; Guerbet, Michel

    2015-01-01

    In recent years, special emphasis has been put on heavy metals. Children are very sensitive to accumulation of metals. Furthermore, as regards elements, the reference values in children are scarce in the literature as it is difficult to obtain the large quantity of blood necessary to analyze many metals by the conventional atomic absorption spectrometry technique. An inductively coupled plasma mass spectrometry (ICP-MS) procedure that uses a reduced sample of 0.3 mL whole blood or plasma is adapted to multielemental determinations. We applied a previously validated technique for adults that simultaneously quantifies 25 elements by ICP-MS in whole blood and 23 in plasma in a series of 99 healthy children ranging from under 5 years to <18 years, without exposure to metal or drug-containing metals. The aims of the study were to compare metallic concentrations according to the age among children and metallic concentration differences between children and adults. The blood and plasma pediatric metallic profile is a practical useful tool for many purposes in clinical toxicology, forensic toxicology and any cases of metal environmental exposure. PMID:26265286

  9. The clinical significance of whole blood viscosity in (cardio)vascular medicine

    PubMed Central

    Pop, G.A.M.; Duncker, D.J.; Gardien, M.; Vranckx, P.; Versluis, S.; Hasan, D.; Slager, C.J.

    2002-01-01

    Whole blood is a non-Newtonian fluid, which means that its viscosity depends on shear rate. At low shear, blood cells aggregate, which induces a sharp increase in viscosity, whereas at higher shear blood cells disaggregate, deform and align in the direction of flow. Other important determinants of blood viscosity are the haematocrit, the presence of macro-molecules in the medium, temperature and, especially at high shear, the deformability of red blood cells. At the sites of severe atherosclerotic obstructions or at vasospastic locations, when change of vessel diameter is limited, blood viscosity contributes to stenotic resistance thereby jeopardising tissue perfusion. However, blood viscosity plays its most important role in the microcirculation where it contributes significantly to peripheral resistance and may cause sludging in the postcapillary venules. Apart from the direct haemodynamic significance, an increase in blood viscosity at low shear by red blood cell aggregation is also associated with increased thrombotic risk, as has been demonstrated in atrial fibrillation. Furthermore, as increased red blood cell aggregation is a reflection of inflammation, hyperviscosity has been shown to be a marker of inflammatory activity. Thus, because of its potential role in haemodynamics, thrombosis and inflammation, determination of whole blood viscosity could provide useful information for diagnostics and therapy of (cardio)vascular disease. ImagesFigure 3 PMID:25696056

  10. Determination of buprenorphine, fentanyl and LSD in whole blood by UPLC-MS-MS.

    PubMed

    Berg, Thomas; Jørgenrud, Benedicte; Strand, Dag Helge

    2013-04-01

    A sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method has been developed and validated for the quantification of buprenorphine, fentanyl and lysergic acid diethylamide (LSD) in whole blood. Sample preparation was performed by liquid-liquid extraction (LLE) with methyl tert-butyl ether. UPLC-MS-MS analysis was performed with a mobile phase consisting of ammonium formate (pH 10.2) and methanol. Positive electrospray ionization MS-MS detection was performed with two multiple reaction monitoring transitions for each of the analytes and the deuterium labeled internal standards. Limit of detection values of buprenorphine, fentanyl and LSD were 0.28, 0.044 and 0.0097 ng/mL and limit of quantification values were 0.94, 0.14 and 0.036 ng/mL, respectively. Most phospholipids were removed during LLE. No or only minor matrix effects were observed. The method has been routinely used at the Norwegian Institute of Public Health since September 2011 for qualitative and quantitative detections of buprenorphine, fentanyl and/or LSD in more than 400 whole blood samples with two replicates per sample.

  11. Ochratoxin A inhibits the production of tissue factor and plasminogen activator inhibitor-2 by human blood mononuclear cells: Another potential mechanism of immune-suppression

    SciTech Connect

    Rossiello, Maria R.; Rotunno, Crescenzia; Coluccia, Addolorata; Carratu, Maria R.; Di Santo, Angelomaria; Evangelista, Virgilio; Semeraro, Nicola; Colucci, Mario

    2008-06-01

    The mycotoxin ochratoxin A (OTA), an ubiquitous contaminant of food products endowed with a wide spectrum of toxicity, affects several functions of mononuclear leukocytes. Monocytes/macrophages play a major role in fibrin accumulation associated with immune-inflammatory processes through the production of tissue factor (TF) and plasminogen activator inhibitor 2 (PAI-2). We studied the effect of OTA on TF and PAI-2 production by human blood mononuclear cells (MNC). The cells were incubated for 3 or 18 h at 37 deg. C with non toxic OTA concentrations in the absence and in the presence of lipopolysaccharide (LPS) or other inflammatory agents. TF activity was measured by a one-stage clotting test. Antigen assays were performed by specific ELISAs in cell extracts or conditioned media and specific mRNAs were assessed by RT-PCR. OTA had no direct effect on TF and PAI-2 production by MNC. However, OTA caused a dose-dependent reduction in LPS-induced TF (activity, antigen and mRNA) and PAI-2 (antigen and mRNA) production with > 85% inhibition at 1 {mu}g/ml. Similar results were obtained when monocyte-enriched preparations were used instead of MNC. TF production was also impaired by OTA (1 {mu}g/ml) when MNC were stimulated with phorbol myristate acetate (98% inhibition), IL-1{beta} (83%) or TNF-{alpha} (62%). The inhibition of TF and PAI-2 induction might represent a hitherto unrecognized mechanism whereby OTA exerts immunosuppressant activity.

  12. Transcriptome analysis of the human T lymphocyte cell line Jurkat and human peripheral blood mononuclear cells exposed to deoxynivalenol (DON): New mechanistic insights

    SciTech Connect

    Katika, Madhumohan R.; Hendriksen, Peter J.M.; Shao, Jia; Loveren, Henk van; Peijnenburg, Ad

    2012-10-01

    Deoxynivalenol (DON) or vomitoxin is a commonly encountered type-B trichothecene mycotoxin, produced by Fusarium species predominantly found in cereals and grains. DON is known to exert toxic effects on the gastrointestinal, reproductive and neuroendocrine systems, and particularly on the immune system. Depending on dose and exposure time, it can either stimulate or suppress immune function. The main objective of this study was to obtain a deeper insight into DON-induced effects on lymphoid cells. For this, we exposed the human T-lymphocyte cell line Jurkat and human peripheral blood mononuclear cells (PBMCs) to various concentrations of DON for various times and examined gene expression changes by DNA microarray analysis. Jurkat cells were exposed to 0.25 and 0.5 μM DON for 3, 6 and 24 h. Biological interpretation of the microarray data indicated that DON affects various processes in these cells: It upregulates genes involved in ribosome structure and function, RNA/protein synthesis and processing, endoplasmic reticulum (ER) stress, calcium-mediated signaling, mitochondrial function, oxidative stress, the NFAT and NF-κB/TNF-α pathways, T cell activation and apoptosis. The effects of DON on the expression of genes involved in ER stress, NFAT activation and apoptosis were confirmed by qRT-PCR. Other biochemical experiments confirmed that DON activates calcium-dependent proteins such as calcineurin and M-calpain that are known to be involved in T cell activation and apoptosis. Induction of T cell activation was also confirmed by demonstrating that DON activates NFATC1 and induces its translocation from the cytoplasm to the nucleus. For the gene expression profiling of PBMCs, cells were exposed to 2 and 4 μM DON for 6 and 24 h. Comparison of the Jurkat microarray data with those obtained with PBMCs showed that most of the processes affected by DON in the Jurkat cell line were also affected in the PBMCs. -- Highlights: ► The human T cell line Jurkat and human

  13. Validation of the QIAsymphony RGQ system for DNA quantitation of different BK virus genotypes in whole blood samples.

    PubMed

    Burrel, Sonia; Brunet, Christel; Hamm, Nathalie; Gits-Muselli, Maud; Hermet, Laurence; Aimé, Catherine; Agut, Henri; Boutolleau, David

    2014-02-01

    Human polyomavirus BK (BKV) is increasingly recognized as an opportunistic pathogen in transplant recipients. The aim of this work was to evaluate the artus(®) BK Virus QS-RGQ assay on the QIAsymphony RGQ system in whole blood (WB) samples (tests performed in an off-label capacity) according to different BKV genotypes by comparison with a laboratory-developed assay. BKV loads were measured in 111 WB samples and BKV genotype was determined by sequencing the full-length VP1 gene. The artus(®) assay exhibited a limit of detection of 77copies/mL, a linearity range from 3.0 to 6.0log10copies/mL, intra-assay and inter-assay coefficients of variation ranging from 0.65% to 5.18%. Regarding BKV quantitation, artus(®) and laboratory-developed assays were highly correlated (Spearman correlation coefficient Rho=0.79; P<0.0001) with an excellent overall agreement (96.4%) and no significant quantitative difference according to Bland-Altman analysis (mean difference: -0.34log10copies/mL). The results did not show any influence of BKV genotype on BKV quantitation by the artus(®) assay, except a potential underquantitation of BKV subtype Ia which deserves further confirmation. In conclusion, the QIAsymphony RGQ system appears to be appropriate for the quantitation of BKV load in WB samples.

  14. The in vitro effects of artificial and natural sweeteners on the immune system using whole blood culture assays.

    PubMed

    Rahiman, F; Pool, E J

    2014-01-01

    This article investigates the effects of commercially available artificial (aspartame, saccharin, sucralose) and natural sweeteners (brown sugar, white sugar, molasses) on the immune system. Human whole blood cultures were incubated with various sweeteners and stimulated in vitro with either phytohemagglutinin or endotoxin. Harvested supernatants were screened for cytotoxicity and cytokine release. Results showed that none of the artificial or natural sweeteners proved to be cytotoxic, indicating that no cell death was induced in vitro. The natural sweetener, sugar cane molasses (10 ug/mL), enhanced levels of the inflammatory biomarker IL-6 while all artificial sweeteners (10 ug/mL) revealed a suppressive effect on IL-6 secretion (P < 0.001). Exposure of blood cells to sucralose-containing sweeteners under stimulatory conditions reduced levels of the biomarker of humoral immunity, Interleukin-10 (P < 0.001). The cumulative suppression of Interleukin-6 and Interleukin-10 levels induced by sucralose may contribute to the inability in mounting an effective humoral response when posed with an exogenous threat. PMID:24063614

  15. The in vitro effects of artificial and natural sweeteners on the immune system using whole blood culture assays.

    PubMed

    Rahiman, F; Pool, E J

    2014-01-01

    This article investigates the effects of commercially available artificial (aspartame, saccharin, sucralose) and natural sweeteners (brown sugar, white sugar, molasses) on the immune system. Human whole blood cultures were incubated with various sweeteners and stimulated in vitro with either phytohemagglutinin or endotoxin. Harvested supernatants were screened for cytotoxicity and cytokine release. Results showed that none of the artificial or natural sweeteners proved to be cytotoxic, indicating that no cell death was induced in vitro. The natural sweetener, sugar cane molasses (10 ug/mL), enhanced levels of the inflammatory biomarker IL-6 while all artificial sweeteners (10 ug/mL) revealed a suppressive effect on IL-6 secretion (P < 0.001). Exposure of blood cells to sucralose-containing sweeteners under stimulatory conditions reduced levels of the biomarker of humoral immunity, Interleukin-10 (P < 0.001). The cumulative suppression of Interleukin-6 and Interleukin-10 levels induced by sucralose may contribute to the inability in mounting an effective humoral response when posed with an exogenous threat.

  16. Bleeding management in remote environment: the use of fresh whole blood transfusion and lyophilised plasma.

    PubMed

    Sicard, Bruno; Marouzé, Frédéric; Roche, Céline; Carron, Mathieu; Ausset, Sylvain; Sailliol, Anne

    2016-01-01

    To mitigate medical risks in remote environments, the authors have implemented an innovative integrated medical support solution for bleeding management on board ships since 2013. Fresh whole blood transfusion (FWBT) and lyophilised plasma were put in place to address life threatening haemorrhages in maritime operations in the Arctic and Antarctica. The authors are illustrating the bleeding risks with an actual case occurring in Antarctica prior to the implementation of these procedures. They are presenting the different steps involved in the complex process of FWBT, from blood donors' qualifications to actual transfusions. The pros and cons of blood transfusion in extreme remote environment are discussed, including the training of health care professionals, equipment requirements, legal and ethical issues, decision making in complex blood group matching, medical benefits and risks. PMID:27364172

  17. A validated GC/MS method for the determination of amisulpride in whole blood.

    PubMed

    Papoutsis, Ioannis; Rizopoulou, Anna; Nikolaou, Panagiota; Pistos, Constantinos; Spiliopoulou, Chara; Athanaselis, Sotiris

    2014-02-01

    A sensitive GC/MS method for the determination of amisulpride in whole blood was developed, optimized and validated. Sample preparation included solid-phase extraction using HF Bond Elut C18 cartridges and further derivatization with heptafluorobutyric anhydride (HFBA). The limits of detection and quantification were 3.00 and 10.0 μg/L, respectively. The calibration curves were linear up to 1000 μg/L (R(2)≥0.991). Absolute recovery ranged from 94.2 to 101%. Accuracy was found to be between -8.7 and 1.9% and imprecision was less than 10.0%. The developed method covers the generally accepted therapeutic range but it can also cover levels above them. This makes our method suitable for the determination of amisulpride not only for clinical purposes on psychiatric patients, but also during the investigation of forensic cases where amisulpride is involved.

  18. Standardized Whole-Blood Transcriptional Profiling Enables the Deconvolution of Complex Induced Immune Responses.

    PubMed

    Urrutia, Alejandra; Duffy, Darragh; Rouilly, Vincent; Posseme, Céline; Djebali, Raouf; Illanes, Gabriel; Libri, Valentina; Albaud, Benoit; Gentien, David; Piasecka, Barbara; Hasan, Milena; Fontes, Magnus; Quintana-Murci, Lluis; Albert, Matthew L

    2016-09-01

    Systems approaches for the study of immune signaling pathways have been traditionally based on purified cells or cultured lines. However, in vivo responses involve the coordinated action of multiple cell types, which interact to establish an inflammatory microenvironment. We employed standardized whole-blood stimulation systems to test the hypothesis that responses to Toll-like receptor ligands or whole microbes can be defined by the transcriptional signatures of key cytokines. We found 44 genes, identified using Support Vector Machine learning, that captured the diversity of complex innate immune responses with improved segregation between distinct stimuli. Furthermore, we used donor variability to identify shared inter-cellular pathways and trace cytokine loops involved in gene expression. This provides strategies for dimension reduction of large datasets and deconvolution of innate immune responses applicable for characterizing immunomodulatory molecules. Moreover, we provide an interactive R-Shiny application with healthy donor reference values for induced inflammatory genes. PMID:27568558

  19. Psychoneuroimmunology and natural killer cells: the chromium release whole blood assay.

    PubMed

    Fletcher, Mary Ann; Barnes, Zachary; Broderick, Gordon; Klimas, Nancy G

    2012-01-01

    Natural killer (NK) cells are an essential component of innate immunity. These lymphocytes are also sensitive barometers of the effects of endogenous and exogenous stressors on the immune system. This chapter will describe a chromium ((51)Cr) release bioassay designed to measure the target cell killing capacity of NK cells (NKCC). Key features of the cytotoxicity assay are that it is done with whole blood and that numbers of effector cells are determined for each sample by flow cytometry and lymphocyte count. Effector cells are defined as CD3-CD56+ lymphocytes. Target cells are the K562 eyrthroleukemia cell line. Killing capacity is defined as number of target cells killed per effector cell, at an effector cell/target cell ratio of 1:1 during a 4 h in vitro assay.

  20. Storage of Heparinised Canine Whole Blood for the Measurement of Glutathione Peroxidase Activity.

    PubMed

    van Zelst, Mariëlle; Hesta, Myriam; Gray, Kerry; Janssens, Geert P J

    2016-08-01

    Glutathione peroxidase activity is used as a biomarker of selenium status in dogs. Freshly collected blood samples are usually measured, due to the lack of knowledge on the effect of storing the samples. This study investigated if the analysis of glutathione peroxidase activity in whole blood collected from dogs was affected by storage of between 5 and 164 days. Results indicated that glutathione peroxidase activity was more variable in the freshly analysed samples compared to the stored samples. Although the mean differences between fresh and stored samples were not always equal to zero, this is thought to be caused by the variability of reagent preparation rather than by storage, as no consistent increase or decrease in glutathione peroxidase activity was found. Therefore, it can be concluded that heparinised dog blood samples can be successfully stored up to 164 days before analysis of glutathione peroxidase activity. PMID:26701335

  1. MicroRNA Profiling from RSV-Infected Biofluids, Whole Blood, and Tissue Samples.

    PubMed

    Anderson, Lydia; Jorquera, Patricia A; Tripp, Ralph A

    2016-01-01

    Several studies have shown that respiratory syncytial virus (RSV) can modulate the host innate immune response by dysregulation of host microRNAs (miRNAs) related to the antiviral response, a feature that also affects the memory immune response to RSV (Thornburg et al. MBio 3(6), 2012). miRNAs are small, endogenous, noncoding RNAs that function in posttranscriptional gene regulation. Here, we explain a compilation of methods for the purification, quantification, and characterization of miRNA expression profiles in biofluids, whole blood samples, and tissue samples obtained from in vivo studies. In addition, this chapter describes methods for the isolation of exosomal miRNA populations. Understanding alterations in miRNA expression profiles and identifying miRNA targets genes, and their contribution to the pathogenesis of RSV, may help elucidate novel mechanism of host-virus interaction (Rossi et al., Pediatr Pulmonol, 2015). PMID:27464696

  2. Micro-elastometry on whole blood clots using actuated surface-attached posts (ASAPs)

    PubMed Central

    Judith, Robert M.; Fisher, Jay K.; Spero, Richard Chasen; Fiser, Briana L.; Turner, Adam; Oberhardt, Bruce; Taylor, R.M.; Falvo, Michael R.; Superfine, Richard

    2015-01-01

    We present a novel technology for microfluidic elastometry and demonstrate its ability to measure stiffness of blood clots as they form. A disposable micro-capillary strip draws small volumes (20 μL) of whole blood into a chamber containing a surface-mounted micropost array. The posts are magnetically actuated, thereby applying a shear stress to the blood clot. The posts’ response to magnetic field changes as the blood clot forms; this response is measured by optical transmission. We show that a quasi-static model correctly predicts the torque applied to the microposts. We experimentally validate the ability of the system to measure clot stiffness by correlating our system with a commercial thromboelastograph. We conclude that actuated surface-attached post (ASAP) technology addresses a clinical need for point-of-care and small-volume elastic haemostatic assays. PMID:25592158

  3. Cytokine release by lipopolysaccharide-stimulated whole blood from patients with typhoid fever.

    PubMed

    House, Deborah; Chinh, Nguyen T; Hien, Tran T; Parry, Christopher P; Ly, Nguyen T; Diep, To S; Wain, John; Dunstan, Sarah; White, Nicholas J; Dougan, Gordon; Farrar, Jeremy J

    2002-07-15

    The ex vivo cytokine response to lipopolysaccharide (LPS) of whole blood from patients with typhoid fever was investigated. Tumor necrosis factor (TNF)-alpha release by LPS-stimulated blood was found to be lower during acute typhoid fever than after a course of antimicrobial therapy (P

  4. Psychoneuroimmunology and natural killer cells: the chromium release whole blood assay.

    PubMed

    Fletcher, Mary Ann; Barnes, Zachary; Broderick, Gordon; Klimas, Nancy G

    2012-01-01

    Natural killer (NK) cells are an essential component of innate immunity. These lymphocytes are also sensitive barometers of the effects of endogenous and exogenous stressors on the immune system. This chapter will describe a chromium ((51)Cr) release bioassay designed to measure the target cell killing capacity of NK cells (NKCC). Key features of the cytotoxicity assay are that it is done with whole blood and that numbers of effector cells are determined for each sample by flow cytometry and lymphocyte count. Effector cells are defined as CD3-CD56+ lymphocytes. Target cells are the K562 eyrthroleukemia cell line. Killing capacity is defined as number of target cells killed per effector cell, at an effector cell/target cell ratio of 1:1 during a 4 h in vitro assay. PMID:22933153

  5. Rapid, Sensitive, and Specific Lateral-Flow Immunochromatographic Device To Measure Anti-Anthrax Protective Antigen Immunoglobulin G in Serum and Whole Blood

    PubMed Central

    Biagini, Raymond E.; Sammons, Deborah L.; Smith, Jerome P.; MacKenzie, Barbara A.; Striley, Cynthia A. F.; Snawder, John E.; Robertson, Shirley A.; Quinn, Conrad P.

    2006-01-01

    Evidence from animals suggests that anti-anthrax protective antigen (PA) immunoglobulin G (IgG) from vaccination with anthrax vaccine adsorbed (AVA) is protective against Bacillus anthracis infection. Measurement of anti-PA IgG in human sera can be performed using either enzyme-linked immunosorbent assay or fluorescent covalent microsphere immunoassay (ELISA) (R. E. Biagini, D. L. Sammons, J. P. Smith, B. A. MacKenzie, C. A. Striley, V. Semenova, E. Steward-Clark, K. Stamey, A. E. Freeman, C. P. Quinn, and J. E. Snawder, Clin. Diagn. Lab. Immunol. 11:50-55, 2004). Both these methods are laboratory based. We describe the development of a rapid lateral-flow immunochromatographic assay (LFIA) test kit for the measurement of anti-PA IgG in serum or whole-blood samples (30-μl samples) using colloidal gold nanoparticles as the detection reagent and an internal control. Using sera from 19 anthrax AVA vaccinees (anti-PA IgG range, 2.4 to 340 μg/ml) and 10 controls and PA-supplemented whole-blood samples, we demonstrated that the LFIA had a sensitivity of approximately 3 μg/ml anti-PA IgG in serum and ∼14 μg/ml anti-PA IgG in whole blood. Preabsorption of sera with PA yielded negative anti-PA LFIAs. The diagnostic sensitivity and specificity of the assay were 100% using ELISA-measured anti-PA IgG as the standard. This kit has utility in determining anti-PA antibody reactivity in the sera of individuals vaccinated with AVA or individuals with clinical anthrax. PMID:16682473

  6. Maternal exposure to perfluoroalkyl acids measured in whole blood and birth outcomes in offspring.

    PubMed

    Callan, A C; Rotander, A; Thompson, K; Heyworth, J; Mueller, J F; Odland, J Ø; Hinwood, A L

    2016-11-01

    Perfluoralkyl and polyfluoralkyl substances have been measured in plasma and serum of pregnant women as a measure of prenatal exposure. Increased concentrations of individual perfluoroalkyl acids (PFAAs), (typically perfluorooctanoic acid (PFOA) and perfluoroctane sulfonate (PFOS) have been reported to be associated with reductions in birth weight and other birth outcomes. We undertook a study of 14 PFAAs in whole blood (including PFOS, PFHxS, PFHpA, PFOA, PFNA, PFDA and PFUnDA) from 98 pregnant women in Western Australia from 2008 to 2011. Median concentrations (in μg/L) were: PFOS 1.99; PFHxS 0.33; PFOA 0.86; PFNA 0.30; PFDA 0.12 and PFUnDA 0.08. Infants born to women with the highest tertile of PFHxS exposure had an increased odds of being <95% of their optimal birth weight (OR 3.5, 95% CI 1.1-11.5). Conversely, maternal blood concentrations of PFUnDA were associated with non-significant increases in average birth weight (+102g, 95% CI -41, 245) and significant increases in proportion of optimal birth weight (+4.7%, 95% CI 0.7, 8.8) per ln-unit change. This study has reported a range of PFAAs in the whole blood of pregnant women and suggests that PFHxS and PFUnDA may influence foetal growth and warrant further attention. Additional studies are required to identify the sources of PFAA exposure with a view to prevention, in addition to further studies investigating the long term health effects of these ubiquitous chemicals. PMID:27387804

  7. DNA-AP sites generation by Etoposide in whole blood cells

    PubMed Central

    2009-01-01

    Background Etoposide is currently one of the most commonly used antitumor drugs. The mechanisms of action proposed for its antitumor activity are based mainly on its interaction with topoisomerase II. Etoposide effects in transformed cells have been described previously. The aim of the present study was to evaluate the genotoxic effects of this drug in non-transformed whole blood cells, such as occurs as collateral damage induced by some chemotherapies. Methods To determine etoposide genotoxicity, we employed Comet assay in two alkaline versions. To evaluate single strand breaks and delay repair sites we use pH 12.3 conditions and pH >13 to evidence alkali labile sites. With the purpose to quantified apurinic or apyrimidine (AP) sites we employed a specific restriction enzyme. Etoposide effects were determined on whole blood cells cultured in absence or presence of phytohemagglutinin (PHA) treated during 2 and 24 hours of cultured. Results Alkaline (pH > 13) single cell gel electrophoresis (SCGE) assay experiments revealed etoposide-induced increases in DNA damage in phytohemaglutinine (PHA)-stimulated blood and non-stimulated blood cells. When the assay was performed at a less alkaline pH, 12.3, we observed DNA damage in PHA-stimulated blood cells consistent with the existence of alkali labile sites (ALSs). In an effort to elucidate the molecular events underlying this result, we applied exonuclease III (Exo III) in conjunction with a SCGE assay, enabling detection of DNA-AP sites along the genome. More DNA AP-sites were revealed by Exo III and ALSs were recognized by the SCGE assay only in the non-stimulated blood cells treated with etoposide. Conclusion Our results indicate that etoposide induces DNA damage specifically at DNA-AP sites in quiescent blood cells. This effect could be involved in the development of secondary malignancies associated with etoposide chemotherapy. PMID:19917085

  8. Profiling gene expression in whole blood samples following an in-vitro challenge.

    PubMed

    Spijker, Sabine; van de Leemput, Joyce C H; Hoekstra, Chantal; Boomsma, Dorret I; Smit, August B

    2004-12-01

    Genomics tools (gene- and protein-expression studies) can be used to find possible target genes involved in a quantifiable trait or disease state. However in many instances, cells and tissues directly involved in the trait's expression, for example, brain tissue, are not amenable for gene expression analysis. Whole blood cells share a molecular make-up for cellular communication and gene regulation systems with many other cell types, for example, neuronal cells, and have the advantage of being very accessible for gene profiling. We investigated the feasibility of nationwide blood sample collection for lymphocyte RNA isolation and real-time PCR analysis to quantify genomic responses. We tested several designs for blood collection and storage: blood sampling in PAXgene blood collection tubes and storage at -20 degrees C, blood sampling in heparin tubes and decanting the samples (with or without in-vitro stimulus) into either PAXgene blood collection tubes and storage at -20 degrees C, or polypropylene tubes followed by snap-freezing and storage at -80 degrees C. The latter procedure is the best cost-wise when only small amounts of total RNA are needed for downstream applications. Lymphocyte gene expression studies are most likely hampered by the quality of isolated RNA rather than the sampling method. We show that large-scale nationwide sample collections did not alter RNA quality or gene expression levels when compared to sampling and processing in a more controlled way. To this end, we present an optimized protocol for easy and standardized isolation of high quality RNA using the PAXgene isolation kit. Based on these results, we suggest that whole blood genomic data can be used as a genomic probe in experimental and clinical research.

  9. Rapid Detection of Thrombin and Other Protease Activity Directly in Whole Blood

    NASA Astrophysics Data System (ADS)

    Yu, Johnson Chung Sing

    Thrombin is a serine protease that plays a key role in the clotting cascade to promote hemostasis following injury to the endothelium. From a clinical diagnostic perspective, in-vivo thrombin activity is linked to various blood clotting disorders, as well as cardiovascular disease (DVT, arteriosclerosis, etc). Thus, the ability to rapidly measure protease activity directly in whole blood will provide important new diagnostics, and clinical researchers with a powerful tool to further elucidate the relationship between circulating protease levels and disease. The ultimate goal is to design novel point of care (POC) diagnostic devices that are capable of monitoring protease activities directly in whole blood and biological sample. A charge-changing substrate specific to the thrombin enzyme was engineered and its functionality was confirmed by a series of experiments. This led to the preliminary design, construction, and testing of two device platforms deemed fully functional for the electrophoretic separation and focusing of charged peptide fragments. The concept of using the existing charge-changing substrate platform for bacterial protease detection was also investigated. Certain strains of E coli are associated with severe symptoms such as abdominal cramps, bloody diarrhea, and vomiting. The OmpT protease is expressed on the outer membrane of E coli and plays a role in the cleavage of antimicrobial peptides, the degradation of recombinant heterologous proteins, and the activation of plasminogen in the host. Thus, a synthetic peptide substrate specific to the OmpT protease was designed and modeled for the purpose of detecting E coli in biological sample.

  10. Whole Blood Gene Expression Profile Associated with Spontaneous Preterm Birth in Women with Threatened Preterm Labor

    PubMed Central

    Heng, Yujing Jan; Pennell, Craig Edward; Chua, Hon Nian; Perkins, Jonathan Edward; Lye, Stephen James

    2014-01-01

    Threatened preterm labor (TPTL) is defined as persistent premature uterine contractions between 20 and 37 weeks of gestation and is the most common condition that requires hospitalization during pregnancy. Most of these TPTL women continue their pregnancies to term while only an estimated 5% will deliver a premature baby within ten days. The aim of this work was to study differential whole blood gene expression associated with spontaneous preterm birth (sPTB) within 48 hours of hospital admission. Peripheral blood was collected at point of hospital admission from 154 women with TPTL before any medical treatment. Microarrays were utilized to investigate differential whole blood gene expression between TPTL women who did (n = 48) or did not have a sPTB (n = 106) within 48 hours of admission. Total leukocyte and neutrophil counts were significantly higher (35% and 41% respectively) in women who had sPTB than women who did not deliver within 48 hours (p<0.001). Fetal fibronectin (fFN) test was performed on 62 women. There was no difference in the urine, vaginal and placental microbiology and histopathology reports between the two groups of women. There were 469 significant differentially expressed genes (FDR<0.05); 28 differentially expressed genes were chosen for microarray validation using qRT-PCR and 20 out of 28 genes were successfully validated (p<0.05). An optimal random forest classifier model to predict sPTB was achieved using the top nine differentially expressed genes coupled with peripheral clinical blood data (sensitivity 70.8%, specificity 75.5%). These differentially expressed genes may further elucidate the underlying mechanisms of sPTB and pave the way for future systems biology studies to predict sPTB. PMID:24828675

  11. Detection of reticulated platelets in whole blood of rats using flow cytometry.

    PubMed

    Pankraz, Alexander; Ledieu, David; Pralet, Dominique; Provencher-Bolliger, Anne

    2008-09-01

    As opposed to erythropoiesis, which is regularly assessed in the peripheral blood of animals by reticulocyte count, thrombopoiesis is rarely assessed in assays that detect immature platelets in the peripheral blood. An assessment of recent thrombopoiesis is feasible with the analysis of reticulated platelets in the peripheral blood via flow cytometry, but rarely performed. The aim of this study was to establish an assay for the detection of reticulated platelets in whole blood of rats via flow cytometry, using a two-color staining method with a platelet-specific antibody (CD61-PE) and thiazole orange to detect RNA-containing platelets. Platelets were detected in K3EDTA-anticoagulated, paraformaldehyde-fixed samples, using a CD61-PE antibody as well as a gate specific for the light scatter properties of platelets. The intra-assay coefficient of variation varied between 3.6% and 8.3% (n=6 animals). The stability of the assay was determined by storing blood prior to staining, storing stained samples for up to 2h at room temperature, and by diluting the blood prior to analysis with autologous plasma to create samples with artificial anemia and thrombocytopenia. Only samples stored at room temperature prior to analysis showed a significantly lower percentage of reticulated platelets. Percentage of reticulated platelets in the reference population (n=41 rats) was 10.0+/-1.3% reticulated platelets (mean+/-SD; min=6.2%; max=12.5%). These data show that the detection of reticulated platelets in whole blood of rats using a platelet-specific antibody is feasible. This test presents a minimal-invasive method to assess thrombopoiesis in rats that can be used for example in preclinical toxicological studies.

  12. A nonlethal sampling method to obtain, generate and assemble whole blood transcriptomes from small, wild mammals.

    PubMed

    Huang, Zixia; Gallot, Aurore; Lao, Nga T; Puechmaille, Sébastien J; Foley, Nicole M; Jebb, David; Bekaert, Michaël; Teeling, Emma C

    2016-01-01

    The acquisition of tissue samples from wild populations is a constant challenge in conservation biology, especially for endangered species and protected species where nonlethal sampling is the only option. Whole blood has been suggested as a nonlethal sample type that contains a high percentage of bodywide and genomewide transcripts and therefore can be used to assess the transcriptional status of an individual, and to infer a high percentage of the genome. However, only limited quantities of blood can be nonlethally sampled from small species and it is not known if enough genetic material is contained in only a few drops of blood, which represents the upper limit of sample collection for some small species. In this study, we developed a nonlethal sampling method, the laboratory protocols and a bioinformatic pipeline to sequence and assemble the whole blood transcriptome, using Illumina RNA-Seq, from wild greater mouse-eared bats (Myotis myotis). For optimal results, both ribosomal and globin RNAs must be removed before library construction. Treatment of DNase is recommended but not required enabling the use of smaller amounts of starting RNA. A large proportion of protein-coding genes (61%) in the genome were expressed in the blood transcriptome, comparable to brain (65%), kidney (63%) and liver (58%) transcriptomes, and up to 99% of the mitogenome (excluding D-loop) was recovered in the RNA-Seq data. In conclusion, this nonlethal blood sampling method provides an opportunity for a genomewide transcriptomic study of small, endangered or critically protected species, without sacrificing any individuals. PMID:26186236

  13. Whole blood gene expression profile associated with spontaneous preterm birth in women with threatened preterm labor.

    PubMed

    Heng, Yujing Jan; Pennell, Craig Edward; Chua, Hon Nian; Perkins, Jonathan Edward; Lye, Stephen James

    2014-01-01

    Threatened preterm labor (TPTL) is defined as persistent premature uterine contractions between 20 and 37 weeks of gestation and is the most common condition that requires hospitalization during pregnancy. Most of these TPTL women continue their pregnancies to term while only an estimated 5% will deliver a premature baby within ten days. The aim of this work was to study differential whole blood gene expression associated with spontaneous preterm birth (sPTB) within 48 hours of hospital admission. Peripheral blood was collected at point of hospital admission from 154 women with TPTL before any medical treatment. Microarrays were utilized to investigate differential whole blood gene expression between TPTL women who did (n = 48) or did not have a sPTB (n = 106) within 48 hours of admission. Total leukocyte and neutrophil counts were significantly higher (35% and 41% respectively) in women who had sPTB than women who did not deliver within 48 hours (p<0.001). Fetal fibronectin (fFN) test was performed on 62 women. There was no difference in the urine, vaginal and placental microbiology and histopathology reports between the two groups of women. There were 469 significant differentially expressed genes (FDR<0.05); 28 differentially expressed genes were chosen for microarray validation using qRT-PCR and 20 out of 28 genes were successfully validated (p<0.05). An optimal random forest classifier model to predict sPTB was achieved using the top nine differentially expressed genes coupled with peripheral clinical blood data (sensitivity 70.8%, specificity 75.5%). These differentially expressed genes may further elucidate the underlying mechanisms of sPTB and pave the way for future systems biology studies to predict sPTB. PMID:24828675

  14. Cross-talk between human neural stem/progenitor cells and peripheral blood mononuclear cells in an allogeneic co-culture model.

    PubMed

    Zhang, Hongxia; Shao, Bei; Zhuge, Qichuan; Wang, Peng; Zheng, Chengcai; Huang, Weilong; Yang, Chenqi; Wang, Brian; Su, Dong-Ming; Jin, Kunlin

    2015-01-01

    Transplantation of human neural stem/progenitor cells (hNSCs) as a regenerative cell replacement therapy holds great promise. However, the underlying mechanisms remain unclear. We, here, focused on the interaction between hNSCs and allogeneic peripheral blood mononuclear cells (PBMCs) in a co-culture model. We found that hNSCs significantly decrease the CD3+ and CD8+ T cells, reduce the gamma delta T cells and increase the regulatory T cells, along with reduced pro-inflammatory cytokines and increased anti-inflammatory cytokines after co-culture. We also found that PBMCs, in turn, significantly promote the proliferation and differentiation of hNSCs. Our data suggest that hNSCs cross-talk with immune cells.

  15. An isomeric mixture of novel cerebrosides isolated from Impatiens pritzellii reduces lipopolysaccharide-induced release of IL-18 from human peripheral blood mononuclear cells.

    PubMed

    Zhou, Xuefeng; Tang, Lan; Liu, Yonghong

    2009-08-01

    An isomeric mixture of two cerebrosides, soya-cerebrosides I and II, was isolated from an ethno drug, the rhizomes of Impatiens pritzellii Hook. f. var. hupehensis Hook. f., and their structures were identified by spectroscopic (NMR, MS) analysis. In order to determine the immunomodulatory activities of soya-cerebrosides I and II, the effects of the mixture of cerebrosides (MC) on cytotoxicity of human peripheral blood mononuclear cells (PBMC) and the inhibitory activities to lipopolysaccharide (LPS)-induced interleukin (IL)-18 in PBMC were studied. The MC at concentrations of 10 and 1 microM, without toxicity to PBMC in 24 h, showed obvious inhibitory activity on IL-18 secretion. Because of this effect of modulating the cellular immune response, soya-cerebrosides I and II were considered to be the active substances of this ethno drug.

  16. Woodchuck dendritic cells generated from peripheral blood mononuclear cells and transduced with recombinant human adenovirus serotype 5 induce antigen-specific cellular immune responses.

    PubMed

    Ochoa-Callejero, Laura; Berraondo, Pedro; Crettaz, Julien; Olagüe, Cristina; Vales, Africa; Ruiz, Juan; Prieto, Jesús; Tennant, Bud C; Menne, Stephan; González-Aseguinolaza, Gloria

    2007-05-01

    Woodchucks infected with the woodchuck hepatitis virus (WHV) is the best available animal model for testing the immunotherapeutic effects of dendritic cells (DCs) in the setting of a chronic infection, as woodchucks develop a persistent infection resembling that seen in humans infected with the hepatitis B virus. In the present study, DCs were generated from woodchuck peripheral blood mononuclear cells (wDCs) in the presence of human granulocyte macrophage colony-stimulating factor (hGM-CSF) and human interleukin 4 (hIL-4). After 7 days of culture, cells with morphology similar to DCs were stained positively with a cross-reactive anti-human CD86 antibody. Functional analysis showed that uptake of FITC-dextran by wDCs was very efficient and was partially inhibited after LPS-induced maturation. Furthermore, wDCs stimulated allogenic lymphocytes and induced proliferation. Moreover, wDCs were transduced efficiently with a human adenovirus serotype 5 for the expression of beta-galactosidase. Following transduction and in vivo administration of such DCs into woodchucks, an antigen-specific cellular immune response was induced. These results demonstrate that wDCs can be generated from the peripheral blood. Following transfection with a recombinant adenovirus wDCs can be used as a feasible and effective tool for eliciting WHV-specific T-cell responses indicating their potential to serve as prophylactic and therapeutic vaccines. PMID:17385694

  17. Microfluidic Chip-Based Detection and Intraspecies Strain Discrimination of Salmonella Serovars Derived from Whole Blood of Septic Mice

    PubMed Central

    Patterson, Adriana S.; Heithoff, Douglas M.; Ferguson, Brian S.; Soh, H. Tom; Mahan, Michael J.

    2013-01-01

    Salmonella is a zoonotic pathogen that poses a considerable public health and economic burden in the United States and worldwide. Resultant human diseases range from enterocolitis to bacteremia to sepsis and are acutely dependent on the particular serovar of Salmonella enterica subsp. enterica, which comprises over 99% of human-pathogenic S. enterica isolates. Point-of-care methods for detection and strain discrimination of Salmonella serovars would thus have considerable benefit to medical, veterinary, and field applications that safeguard public health and reduce industry-associated losses. Here we describe a single, disposable microfluidic chip that supports isothermal amplification and sequence-specific detection and discrimination of Salmonella serovars derived from whole blood of septic mice. The integrated microfluidic electrochemical DNA (IMED) chip consists of an amplification chamber that supports loop-mediated isothermal amplification (LAMP), a rapid, single-temperature amplification method as an alternative to PCR that offers advantages in terms of sensitivity, reaction speed, and amplicon yield. The amplification chamber is connected via a microchannel to a detection chamber containing a reagentless, multiplexed (here biplex) sensing array for sequence-specific electrochemical DNA (E-DNA) detection of the LAMP products. Validation of the IMED device was assessed by the detection and discrimination of S. enterica subsp. enterica serovars Typhimurium and Choleraesuis, the causative agents of enterocolitis and sepsis in humans, respectively. IMED chips conferred rapid (under 2 h) detection and discrimination of these strains at clinically relevant levels (<1,000 CFU/ml) from whole, unprocessed blood collected from septic animals. The IMED-based chip assay shows considerable promise as a rapid, inexpensive, and portable point-of-care diagnostic platform for the detection and strain-specific discrimination of microbial pathogens. PMID:23354710

  18. The Interchangeability of Plasma and Whole Blood Metal Ion Measurement in the Monitoring of Metal on Metal Hips

    PubMed Central

    Malek, Ibrahim A.; Rogers, Joanne; King, Amanda Christina; Clutton, Juliet; Winson, Daniel; John, Alun

    2015-01-01

    One hundred and twenty six paired samples of plasma and whole blood were measured with inductively coupled plasma mass spectrometry technique for metal ions analysis to determine a relationship between them. There was a significant difference between the mean plasma and whole blood concentrations of both cobalt (Co) and chromium (Cr) (p < 0.0001 for both Co and Cr). The mean ratio between plasma and whole blood Cr and Co was 1.56 (range: 0.39–3.85) and 1.54 (range: 0.64–18.26), respectively, but Bland and Altman analysis illustrated that this relationship was not universal throughout the range of concentrations. There was higher variability at high concentrations for both ions. We conclude that both these concentrations should not be used interchangeably and conversion factors are unreliable due to concentration dependent variability. PMID:26798516

  19. Human mononuclear cell function after 4 degrees C storage during 1-G and microgravity conditions of spaceflight

    NASA Technical Reports Server (NTRS)

    Meehan, R.; Taylor, G.; Lionetti, F.; Neale, L.; Curren, T.

    1989-01-01

    Future space missions of long duration may require that autologous leukocytes be stored in flight for infusion to restore normal immune competence in crewmembers. Peripheral blood mononuclear cells (PBMNCs), as leukocyte concentrates in autologous plasma and 2% dextrose, were stored in the microgravity conditions provided by the U.S. Space Shuttle Columbia (Mission 61-C). Activity of PBMNC after space flight was compared with that from a series of preflight ground control experiments, which demonstrated in culture a progressive daily loss in mitogen-stimulated protein synthesis at 24 h and thymidine uptake at 72 h after storage for 7 d at 4 degrees C. Post-storage viabilities were at least 90% as determined by trypan dye exclusion. A progressive reduction in the percentage of PBMNC expressing cell-surface phenotype markers, which was similar for monocytes, B cells, and T-cell subsets, also occurred after storage. The ability of PBMNC, stored for 8 d in Columbia's middeck, to become activated and proliferate in vitro was similar to that of cells that remained in identical flight lockers on the ground as 1-G controls, thus indicating that PBMNCs were not adversely affected by storage under microgravity conditions.

  20. Flow cytometry analysis of hormone receptors on human peripheral blood mononuclear cells to identify stress-induced neuroendocrine effects

    NASA Technical Reports Server (NTRS)

    Meehan, R. T.

    1986-01-01

    Understanding the role of circulating peptide hormones in the pathogenesis of space-flight induced disorders would be greatly facilitated by a method which monitors chronic levels of hormones and their effects upon in vivo cell physiology. Single and simultaneous multiparameter flow cytometry analysis was employed to identify subpopulations of mononuclear cells bearing receptors for ACTH, Endorphin, and Somatomedin-C using monoclonal antibodies and monospecific antisera with indirect immunofluorescence. Blood samples were obtained from normal donors and subjects participating in decompression chamber studies (acute stress), medical student academic examination (chronic stress), and a drug study (Dexamethasone). Preliminary results indicate most ACTH and Endorphin receptor positive cells are monocytes and B-cells, exhibit little diurnal variation but the relative percentages of receptor positive cells are influenced by exposure to various stressors and ACTH inhibition. This study demonstrates the capability of flow cytometry analysis to study cell surface hormone receptor regulation which should allow insight into neuroendocrine modulation of the immune and other cellular systems during exposure to stress or microgravity.

  1. Expression levels of seven candidate genes in human peripheral blood mononuclear cells and their association with preeclampsia

    PubMed Central

    Garza-Veloz, I.; Carrillo-Sanchez, K.; Martinez-Gaytan, V.; Cortes-Flores, R.; Ochoa-Torres, M. A.; Guerrero, G. G.; Rodriguez-Sanchez, I. P.; Cancela-Murrieta, C. O.; Zamudio-Osuna, M.; Badillo-Almaraz, J. I.; Castruita-De la Rosa, C.

    2014-01-01

    Objective To evaluate the peripheral blood mononuclear cell (PBMC) expression levels of hemeoxygenase 1 (HMOX-1), superoxide dismutase 1 (SOD-1), vascular endothelial growth factor A (VEGF-A), transforming growth factor beta 1 (TGF-β1), interleukin (IL)-6, IL-15 and AdipoQ genes to study their association with preeclampsia (PE). Methods A total of 177 pregnant women were recruited: 108 cases and 69 controls. Quantification of gene expression was measured by quantitative real-time polymerase chain reaction (PCR) using TaqMan probes. Results Underexpression of VEGF-A and TGF-β1 was a constant in most of the cases (80.91% and 76.36%, respectively) and their expression was associated with onset and/or severity of disease (p values < 0.05). IL-6, IL-15 and AdipoQ, showed low or no expression in PBMC samples evaluated. Conclusion PBMC underexpression of VEGF-A and TGF-β1 is a hallmark of PE in the study population. PMID:24295154

  2. Mononuclear cells in dementia.

    PubMed

    Mandas, Antonella; Dessì, Sandra

    2014-04-20

    According to the World Health Organization statistics, dementias are the largest contributors to disease burden in advanced market economies, and the leading cause of disability and dependence among older people worldwide. So far, several techniques have been developed to identify dementias with reasonable accuracy while the patient is still alive, however, no single of them has proven to be ideal, especially if you need to have a satisfactory early diagnosis. Studies of early onset dementia are largely limited by the inaccessibility to direct examination of the living human brain: it appears therefore that for a correct biochemical and molecular characterization of dementias, potential surrogate tissues must be identified. In this context, peripheral blood mononuclear cells (PBMCs) appear particularly attractive because they can be obtained in a minimally invasive manner and can be easily analyzed. This review focuses on the most representative methodologies and strategies in detecting and quantifying fluctuation in dementia that are currently being developed. In addition it provides a comprehensive evaluation of the diagnostic sensitivity of PBMCs in patients with dementia. Finally, it discusses the data supporting the use of the determination of neutral lipids (NLs) in PBMCs by Oil Red O (ORO) staining, which is a minimally invasive, cheap, easy and fast procedure, as the promising method for early detection of dementia and to search for new effective treatments.

  3. Whole Blood Activation Results in Enhanced Detection of T Cell and Monocyte Cytokine Production by Flow Cytometry

    NASA Technical Reports Server (NTRS)

    Sams, Clarence F.; Crucian, Brian E.

    2001-01-01

    An excellent monitor of the immune balance of peripheral circulating cells is to determine their cytokine production patterns in response to stimuli. Using flow cytometry a positive identification of cytokine producing cells in a mixed culture may be achieved. Recently, the ability to assess cytokine production following a wholeblood activation culture has been described. We compared whole blood culture to standard PBMC culture and determined the individual cytokine secretion patterns for both T cells and monocytes via flow cytometry. For T cells cytokine assessment following PMA +ionomycin activation: (1) a significantly greater percentages of T cells producing IFNgamma and IL-2 were observed following whole-blood culture; (2) altered T cell cytokine production kinetics were observed by varying whole blood culture times. In addition, a four-color cytometric analysis was used to allow accurate phenotyping and quantitation of cytokine producing lymphocyte populations. Using this technique we found IFNgamma production to be significantly elevated in the CD3+/CD8+ T cell population as compared to the CD3+/CD8- population following five hours of whole blood activation. Conversely, IL-2 and IL-10 production were significantly elevated in the CD3+/CD8- T cell population as compared to the CD3+/CD8+ population. Monocyte cytokine production was assessed in both culture systems following LPS activation for 24 hours. A three-color flow cytometric was used to assess two cytokines in conjunction with CD 14. The cytokine pairs used for analysis were IL-1a/IL-12, and IL-10ITNFa. Nearly all monocytes were stimulated to produce IL-1a, IL-12 and TNFalpha equally well in both culture systems. Monocyte production of IL-10 was significantly elevated following whole blood culture as compared to PBMC culture. IL-12 producing monocytes appeared to be a distinct subpopulation of the IL-1a producing set, whereas IL-10 and TNFa producing monocytes were largely mutually exclusive. IL-10 and

  4. Selenium concentration levels in whole blood of Belgian blood bank donors, as determined by direct graphite furnace atomic absorption spectrometry.

    PubMed

    Van Cauwenbergh, R; Robberecht, H; Deelstra, H

    1990-12-01

    Direct graphite furnace atomic absorption spectrometry after appropriate sample dilution is applied to whole blood of Belgian blood bank donors from different geographical areas. Highest values were obtained for the northern part of the country (Flanders). Different consumption habits may explain the significant difference in selenium level found for the two cultural communities (Flemish and Walloon). No sex difference was obtained in both parts of the country and no influence of age was observed for the two communities. Values obtained are compared with literature data on whole blood selenium content for other European countries.

  5. A novel role of h2-calponin in regulating whole blood thrombosis and platelet adhesion during physiologic flow.

    PubMed

    Hines, Patrick C; Gao, Xiufeng; White, Jennell C; D'Agostino, Ashley; Jin, Jian-Ping

    2014-12-01

    Calponin is an actin filament-associated protein reported in platelets, although the specific isoform expressed and functional role were not identified. The h2-calponin isoform is expressed in myeloid-derived peripheral blood monocytes, where it regulates adhesion. Our objective was to characterize the presence and function of the h2 isoform of calponin in platelets. H2-calponin was detected in human and mouse platelets via Western blotting. Immunofluorescent staining demonstrated h2-calponin and actin colocalized in both human and wild-type mouse platelets at rest and following collagen activation. The kinetics of platelet adhesion and whole blood thrombosis during physiologic flow was evaluated in a microfluidic flow-based thrombosis assay. The time to initiation of rapid platelet/thrombus accumulation (lag time) was significantly longer in h2-calponin knockout versus wild-type mouse blood (130.02 ± 3.74 sec and 72.95 ± 16.23 sec, respectively, P < 0.05). There was no significant difference in the rate of platelet/thrombus accumulation during the rapid phase or the maximum platelet/thrombus accumulation. H2-calponin knockout mice also had prolonged bleeding time and blood loss. H2-calponin in platelets facilitates early interactions between platelets and collagen during physiologic flow, but does not significantly affect the rate or magnitude of platelet/thrombus accumulation. H2-calponin knockout mice take 2.3 times longer to achieve hemostasis compared to wild-type controls in a tail bleeding model. The ability to delay platelet accumulation without inhibiting downstream thrombotic potential would be of significant therapeutic value, thus h2-calponin may be a novel target for therapeutic platelet inhibition. PMID:25472609

  6. A novel role of h2‐calponin in regulating whole blood thrombosis and platelet adhesion during physiologic flow

    PubMed Central

    Hines, Patrick C.; Gao, Xiufeng; White, Jennell C.; D'Agostino, Ashley; Jin, Jian‐Ping

    2014-01-01

    Abstract Calponin is an actin filament‐associated protein reported in platelets, although the specific isoform expressed and functional role were not identified. The h2‐calponin isoform is expressed in myeloid‐derived peripheral blood monocytes, where it regulates adhesion. Our objective was to characterize the presence and function of the h2 isoform of calponin in platelets. H2‐calponin was detected in human and mouse platelets via Western blotting. Immunofluorescent staining demonstrated h2‐calponin and actin colocalized in both human and wild‐type mouse platelets at rest and following collagen activation. The kinetics of platelet adhesion and whole blood thrombosis during physiologic flow was evaluated in a microfluidic flow‐based thrombosis assay. The time to initiation of rapid platelet/thrombus accumulation (lag time) was significantly longer in h2‐calponin knockout versus wild‐type mouse blood (130.02 ± 3.74 sec and 72.95 ± 16.23 sec, respectively, P < 0.05). There was no significant difference in the rate of platelet/thrombus accumulation during the rapid phase or the maximum platelet/thrombus accumulation. H2‐calponin knockout mice also had prolonged bleeding time and blood loss. H2‐calponin in platelets facilitates early interactions between platelets and collagen during physiologic flow, but does not significantly affect the rate or magnitude of platelet/thrombus accumulation. H2‐calponin knockout mice take 2.3 times longer to achieve hemostasis compared to wild‐type controls in a tail bleeding model. The ability to delay platelet accumulation without inhibiting downstream thrombotic potential would be of significant therapeutic value, thus h2‐calponin may be a novel target for therapeutic platelet inhibition. PMID:25472609

  7. A novel μ-fluidic whole blood coagulation assay based on Rayleigh surface-acoustic waves as a point-of-care method to detect anticoagulants

    PubMed Central

    Meyer dos Santos, Sascha; Zorn, Anita; Guttenberg, Zeno; Picard-Willems, Bettina; Kläffling, Christina; Nelson, Karen; Klinkhardt, Ute; Harder, Sebastian

    2013-01-01

    A universal coagulation test that reliably detects prolonged coagulation time in patients, irrespective of the anticoagulant administered, has not been available to date. An easily miniaturised, novel μ-fluidic universal coagulation test employing surface acoustic waves (SAW) is presented here. SAW was employed to instantly mix and recalcify 6 μl citrated whole blood and image correlation analysis was used to quantify clot formation kinetics. The detection of clinically relevant anticoagulant dosing with old anticoagulants (unfractionated heparin, argatroban) and new anticoagulants (dabigatran, rivaroxaban) has been tested and compared to standard plasma coagulation assays. The applicability of this novel method has been confirmed in a small patient population. Coagulation was dose-proportionally prolonged with heparin, argatroban, dabigatran, and rivaroxaban, comparable to standard tests. Aspirin and clopidogrel did not interfere with the SAW-induced clotting time (SAW-CT), whereas the strong GPIIb/IIIa-inhibitor abciximab did interfere. Preliminary clinical data prove the suitability of the SAW-CT in patients being treated with warfarin, rivaroxaban, or dabigatran. The system principally allows assessment of whole blood coagulation in humans in a point-of-care setting. This method could be used in stroke units, emergency vehicles, general and intensive care wards, as well as for laboratory and home testing of coagulation. PMID:24404078

  8. Preparation of Hemocompatible Poly(lactic-co-glycolic acid)-F127 Nanospheres and Their Application to Biosensor for Analysis of Whole Blood.

    PubMed

    Sun, Chong; Niu, Yanlian; Yang, Xujie; Liu, Min; Yang, Xiaodi; Huang, Xiaohua; Zhao, Wenbo

    2015-01-01

    In this paper, the novel poly(lactic-co-glycolic acid)-F127 nanospheres (PLGA-F127 NSs) were synthesized and used to establish an amperometric glucose biosensor that can be applied in whole blood directly. This property of glucose biosensor was based on the antibiofouling property of PLGA-F127 NSs. More details of preparing PLGA-F127 NSs and immobilizing glucose oxidase (GOx) on (PLGA-F127)/glass carbon electrode (GCE) were presented. Then, the electrochemical behaviors of the biosensor in whole blood were studied. The cyclic voltammetric results indicated that GOx immobilized on PLGA-F127 NSs exhibited direct electron transfer reaction, which led to stable amperometric biosensing for glucose with a detection limit of 5.57 x 10(-6) M (S/N = 3). The glucose biosensor did not respond to ascorbic acid (AA) and uric.acid (UA) at their concentration normally encountered in blood. The development of materials science will bring significant input to high-performance biosensors relevant to diagnostics and therapy of interest for human health. PMID:26328311

  9. Is platelet function as measured by Thrombelastograph monitoring in whole blood affected by platelet inhibitors?

    PubMed

    Bailey, Lori A; Sistino, Joseph J; Uber, Walter E

    2005-03-01

    Platelet inhibitors, especially the glycoprotein (GP) IIb/IIIa receptor antagonists, have demonstrated their effectiveness in reducing the acute ischemic complications of percutaneous coronary intervention (PCI) and in improving clinical outcomes in patients with acute coronary crisis. Three common platelet inhibitors observed in emergent cardiopulmonary bypass (CPB) for failed PCI are abciximab, eptifibatide, and tirofiban. An in vitro model was constructed in two parts to determine whether platelet aggregation inhibition induced by platelet inhibitors would be demonstrated by the Thrombelastograph (TEG) monitor when compared with baseline samples with no platelet inhibitor. In part A, 20 mL of fresh whole blood was divided into four groups: group I = baseline, group A = abcix-imab microg/mL, group E = eptifibatide ng/mL, and group T = tirofiban ng/mL. Platelet inhibitor concentrations in whole blood were derived starting with reported serum concentrations with escalation to achieve 80% platelet inhibition using the Medtronic hemoSTATUS and/or Lumi-aggregometer. A concentration range determined by our in vitro tests were chosen for each drug using concentrations achieving less than, equal to, or greater than 80% platelet inhibition. In part B, TEG analysis was then performed using baseline and concentrations for each drug derived in part A. Parameters measured were clot formation reaction time (R), coagulation time (K), maximum amplitude (MA) and alpha angle (A). Groups E1000 and E2000 extended R over control by 37% and 23%, respectively (p = 0.01 and 0.03). Groups E1000 and E2000 increased K times by 45% and 58% (p = .02 and .04). T160 samples prolonged K by 20% (p = 0.01). The angle or clot strength (A) was decreased in groups T160 and E1000 by 23% (+ 7.06 SD) and 18% (+ 11.23 SD), respectively (p = 0.001 and 0.01). The MA decrease was statistically significant in the T160, E1000 and E2000 by 9%, 6% and 13% respectively (p = 0.01). Samples treated with abciximab

  10. Whole Blood Gene Expression Differentiates between Atrial Fibrillation and Sinus Rhythm after Cardioversion

    PubMed Central

    Raman, Kripa; Aeschbacher, Stefanie; Bossard, Matthias; Hochgruber, Thomas; Zimmermann, Andreas J.; Kaufmann, Beat A.; Pumpol, Katrin; Rickenbacker, Peter

    2016-01-01

    Background Treatment to restore sinus rhythm among patients with atrial fibrillation (AF) has limited long-term success rates. Gene expression profiling may provide new insights into AF pathophysiology. Objective To identify biomarkers and improve our understanding of AF pathophysiology by comparing whole blood gene expression before and after electrical cardioversion (ECV). Methods In 46 patients with persistent AF that underwent ECV, whole blood samples were collected 1–2 hours before and 4 to 6 weeks after successful cardioversion. The paired samples were sent for microarray and plasma biomarker comparison. Results Of 13,942 genes tested, expression of SLC25A20 and PDK4 had the strongest associations with AF. Post-cardioversion, SLC25A20 and PDK4 expression decreased by 0.8 (CI 0.7–0.8, p = 2.0x10-6) and 0.7 (CI 0.6–0.8, p = 3.0x10-5) fold respectively. Median N-terminal pro B-type natriuretic peptide (NT-proBNP) concentrations decreased from 127.7 pg/mL to 44.9 pg/mL (p = 2.3x10-13) after cardioversion. AF discrimination models combining NT-proBNP and gene expression (NT-proBNP + SLC25A20 area under the curve = 0.88, NT-proBNP + PDK4 AUC = 0.86) had greater discriminative capacity as compared with NT-proBNP alone (AUC = 0.82). Moreover, a model including NT-proBNP, SLC25A20 and PDK4 significantly improved AF discrimination as compared with other models (AUC = 0.87, Net Reclassification Index >0.56, p<5.8x10-3). We validated the association between SLC25A20 and PDK4 with AF in an independent sample of 17 patients. Conclusion This study demonstrates that SLC25A20, PDK4, and NT-proBNP have incremental utility as biomarkers discriminating AF from sinus rhythm. Elevated SLC25A20 and PDK4 expression during AF indicates an important role for energy metabolism in AF. PMID:27332823

  11. Measurement of pH in whole blood by near-infrared spectroscopy

    SciTech Connect

    Alam, M. Kathleen; Maynard, John D.; Robinson, M. Ries Niemczyk, Thomas M. Rohrscheib, Mark R.

    1999-03-01

    Whole blood pH has been determined {ital in vitro} by using near-infrared spectroscopy over the wavelength range of 1500 to 1785 nm with multivariate calibration modeling of the spectral data obtained from two different sample sets. In the first sample set, the pH of whole blood was varied without controlling cell size and oxygen saturation (O{sub 2} Sat) variation. The result was that the red blood cell (RBC) size and O{sub 2} Sat correlated with pH. Although the partial least-squares (PLS) multivariate calibration of these data produced a good pH prediction cross-validation standard error of prediction (CVSEP)=0.046, R{sup 2}=0.982, the spectral data were dominated by scattering changes due to changing RBC size that correlated with the pH changes. A second experiment was carried out where the RBC size and O{sub 2} Sat were varied orthogonally to the pH variation. A PLS calibration of the spectral data obtained from these samples produced a pH prediction with an R{sup 2} of 0.954 and a cross-validated standard error of prediction of 0.064 pH units. The robustness of the PLS calibration models was tested by predicting the data obtained from the other sets. The predicted pH values obtained from both data sets yielded R{sup 2} values greater than 0.9 once the data were corrected for differences in hemoglobin concentration. For example, with the use of the calibration produced from the second sample set, the pH values from the first sample set were predicted with an R{sup 2} of 0.92 after the predictions were corrected for bias and slope. It is shown that spectral information specific to pH-induced chemical changes in the hemoglobin molecule is contained within the PLS loading vectors developed for both the first and second data sets. It is this pH specific information that allows the spectra dominated by pH-correlated scattering changes to provide robust pH predictive ability in the uncorrelated data, and visa versa. {copyright} {ital 1999} {ital Society for Applied

  12. Comparative genomics analysis of mononuclear phagocyte subsets confirms homology between lymphoid tissue-resident and dermal XCR1(+) DCs in mouse and human and distinguishes them from Langerhans cells.

    PubMed

    Carpentier, Sabrina; Vu Manh, Thien-Phong; Chelbi, Rabie; Henri, Sandrine; Malissen, Bernard; Haniffa, Muzlifah; Ginhoux, Florent; Dalod, Marc

    2016-05-01

    Dendritic cells (DC) are mononuclear phagocytes which exhibit a branching (dendritic) morphology and excel at naïve T cell activation. DC encompass several subsets initially identified by their expression of cell surface molecules and later shown to possess distinct functions. DC subset differentiation is orchestrated by transcription factors, growth factors and cytokines. Identifying DC subsets is challenging as very few cell surface molecules are uniquely expressed on any one of these cell populations. There is no standard consensus to identify mononuclear phagocyte subsets; varying antigens are employed depending on the tissue and animal species studied and between laboratories. This has led to confusion in how to accurately define and classify DCs across tissues and between species. Here we report a comparative genomics strategy that enables universal definition of DC and other mononuclear phagocyte subsets across species. We performed a meta-analysis of several public datasets of human and mouse mononuclear phagocyte subsets isolated from blood, spleen, skin or cutaneous lymph nodes, including by using a novel and user friendly software, BubbleGUM, which generates and integrates gene signatures for high throughput gene set enrichment analysis. This analysis demonstrates the equivalence between human and mouse skin XCR1(+) DCs, and between mouse and human Langerhans cells.

  13. Comparative genomics analysis of mononuclear phagocyte subsets confirms homology between lymphoid tissue-resident and dermal XCR1(+) DCs in mouse and human and distinguishes them from Langerhans cells.

    PubMed

    Carpentier, Sabrina; Vu Manh, Thien-Phong; Chelbi, Rabie; Henri, Sandrine; Malissen, Bernard; Haniffa, Muzlifah; Ginhoux, Florent; Dalod, Marc

    2016-05-01

    Dendritic cells (DC) are mononuclear phagocytes which exhibit a branching (dendritic) morphology and excel at naïve T cell activation. DC encompass several subsets initially identified by their expression of cell surface molecules and later shown to possess distinct functions. DC subset differentiation is orchestrated by transcription factors, growth factors and cytokines. Identifying DC subsets is challenging as very few cell surface molecules are uniquely expressed on any one of these cell populations. There is no standard consensus to identify mononuclear phagocyte subsets; varying antigens are employed depending on the tissue and animal species studied and between laboratories. This has led to confusion in how to accurately define and classify DCs across tissues and between species. Here we report a comparative genomics strategy that enables universal definition of DC and other mononuclear phagocyte subsets across species. We performed a meta-analysis of several public datasets of human and mouse mononuclear phagocyte subsets isolated from blood, spleen, skin or cutaneous lymph nodes, including by using a novel and user friendly software, BubbleGUM, which generates and integrates gene signatures for high throughput gene set enrichment analysis. This analysis demonstrates the equivalence between human and mouse skin XCR1(+) DCs, and between mouse and human Langerhans cells. PMID:26966045

  14. Comparative genomics analysis of mononuclear phagocyte subsets confirms homology between lymphoid tissue-resident and dermal XCR1+ DCs in mouse and human and distinguishes them from Langerhans cells

    PubMed Central

    Carpentier, Sabrina; Vu Manh, Thien-Phong; Chelbi, Rabie; Henri, Sandrine; Malissen, Bernard; Haniffa, Muzlifah; Ginhoux, Florent; Dalod, Marc

    2016-01-01

    Dendritic cells (DC) are mononuclear phagocytes which exhibit a branching (dendritic) morphology and excel at naïve T cell activation. DC encompass several subsets initially identified by their expression of cell surface molecules and later shown to possess distinct functions. DC subset differentiation is orchestrated by transcription factors, growth factors and cytokines. Identifying DC subsets is challenging as very few cell surface molecules are uniquely expressed on any one of these cell populations. There is no standard consensus to identify mononuclear phagocyte subsets; varying antigens are employed depending on the tissue and animal species studied and between laboratories. This has led to confusion in how to accurately define and classify DCs across tissues and between species. Here we report a comparative genomics strategy that enables universal definition of DC and other mononuclear phagocyte subsets across species. We performed a meta-analysis of several public datasets of human and mouse mononuclear phagocyte subsets isolated from blood, spleen, skin or cutaneous lymph nodes, including by using a novel and user friendly software, BubbleGUM, which generates and integrates gene signatures for high throughput gene set enrichment analysis. This analysis demonstrates the equivalence between human and mouse skin XCR1+ DCs, and between mouse and human Langerhans cells. PMID:26966045

  15. In vitro synthesis of primary specific anti-breast cancer antibodies by normal human peripheral blood mononuclear cells

    PubMed Central

    Norkina, Oxana; Lum, Lawrence G.

    2013-01-01

    In this study, we developed a unique in vitro model to mimic the endogenous tumor microenvironment to understand the effect of immunotherapy with activated T-cells (ATC) armed with anti-CD3 × anti-Her2 bispecific antibody (aATC) on antibody response by naive immune cells. This model contained a co-culture of naïve peripheral blood mononuclear cells (PBMC), breast cancer cells (SK-BR-3), ATC or aATC and CpG ODNs. Culture supernatants were tested at various time points for anti-SK-BR-3 antibodies by ELISA, Western blot and flow cytometry. PBMC cocultured with non-irradiated aATC or irradiated (*) aATC showed significant increases in anti-tumor antibody production at day 14 (P < 0.0001) in the presence of CpG-ODN compared to unstimulated PBMC cultures (n = 9). Antibody specificity was confirmed by ELISA, Western blot and flow cytometry. Co-cultures containing *aATC and CpG showed significantly enhanced levels of IgG2 (P < 0.001) and cytokines that promote IgG2 synthesis including IL-13 (P < 0.02), IFNγ (P < 0.01) and GM-CSF (P < 0.05) compared to unstimulated PBMC control (n = 3). We show that aATC targeting and lysis of tumor cells induces an anti-tumor antibody response in our in vitro model. This model provides a unique opportunity to evaluate the interactions of T-cells, B-cells, and antigen-presenting cells leading to specific anti-tumor antibody responses. PMID:21713642

  16. Pulsed photoacoustic flow imaging of whole blood with low frequency detection

    NASA Astrophysics Data System (ADS)

    van den Berg, Pim J.; Daoudi, Khalid; Steenbergen, Wiendelt

    2016-03-01

    Ultrasound flow imaging is widely used for quantification of blood flow in vivo, but estimating low flow velocities remains challenging. Pulsed photoacoustic flowmetry could be an alternative - but has not been shown capable of deep in vivo imaging so far. A new photoacoustic system is proposed, that has more potential for deep in-vivo applications. In this work the system is tested in vitro. In contrast to earlier research, 1064 nm NIR laser irradiation is used, that would allow deeper in-vivo light penetration. For detection, a 15 MHz transducer with lower in-vivo tissue ultrasound attenuation is used. Both changes are not trivial, as they reduce the overall visibility of the photoacoustic signals from red blood cells. This work shows the flow estimation performance of this system in vitro, and aims to serve as a point of reference when moving to an in-vivo application. Using this 15 MHz transducer, 1D flow profiles of flowing blood were extracted. The jitter (standard error) in the velocity estimation over the profiles was 10% when estimating flow of particles and 20% with whole blood; the bias in flow estimation was roughly 30%.

  17. Stability study of propoxur (Baygon) in whole blood and urine stored at varying temperature conditions.

    PubMed

    Ramagiri, Suma; Kosanam, Hari; Sai Prakash, P K

    2006-06-01

    A stability study has been initiated for propoxur (Baygon) in whole blood and urine samples stored over a period of 60 days at four different temperature conditions (room temperature, 4 degrees C, -20 degrees C, and -80 degrees C). Stability data was established on day 0, 1, 7, 14, 28, 42, and 60. Sample purification was done by solid-phase extraction using a weak cation exchange cartridge (Isolute CBA), and quantitation was carried out by a validated high-performance liquid chromatographic method with a photodiode-array UV detector. Propoxur was spiked at two different concentration levels in both blood and urine samples [low concentration (10 microg/L) and high concentration (100 microg/L)]. Isopropoxy phenol was observed as the major degradation product in blood and urine samples and confirmed by liquid chromatography-electrospray ionization-mass spectrometry. At room temperature, a substantial decrease in concentration of about 95% was observed at the end of the stability study in both blood and urine samples. However, at 4 degrees C, the concentration of propoxur observed after 60 days was around 60% in both samples. A decrease in temperature reduced the degradation, and finally propoxur was found to be stable at -80 degrees C and -20 degrees C for the whole observation period (60 days). The data collected suggests that knowledge about time-dependent decrease of propoxur in urine and blood samples is of considerable significance in forensic toxicology, and, therefore, forensic cases should be interpreted with caution.

  18. Influence of Relative Humidity on the Spreading Dynamics of a Drying Drop of Whole Blood

    NASA Astrophysics Data System (ADS)

    Bou Zeid, Wassim; Brutin, David

    2013-11-01

    Newtonian and non-Newtonian fluids start spreading after coming into contact with a solid substrate till the anchoring of the triple line. Our experimental work aims to study the effect of the relative humidity on the spreading dynamics of drops of whole blood. Drops of blood of same volume (+/-4.8%) are injected using a digital micropipette and gently deposited onto microscope ultraclean glass substrates. Experiments are conducted in a glove box at ambient temperature and a range of investigated relative humidities between 13.0% and 80.0%. The recorded images are post-processed using ImageJ in which the position of the contact line is measured every 20 ms. We show that the spreading dynamics is, in a first time, governed by relative humidity and later no more influence by relative humidity. Two spreading regimes are observed and analyzed compared to classical viscous drops. In previous work, we show that relative humidity influences the contact angle and the initial wetting radius. In the first regime, we find a spreading power law exponent that decreases for an increasing relative humidity. In the second regime, all the data collapse on each other and the evolution of the dimensionless radius no more depend on relative humidity.

  19. On-chip recalcification of citrated whole blood using a microfluidic herringbone mixer.

    PubMed

    Lehmann, Marcus; Wallbank, Alison M; Dennis, Kimberly A; Wufsus, Adam R; Davis, Kara M; Rana, Kuldeepsinh; Neeves, Keith B

    2015-11-01

    In vitro assays of platelet function and coagulation are typically performed in the presence of an anticoagulant. The divalent cation chelator sodium citrate is among the most common because its effect on coagulation is reversible upon reintroduction of divalent cations. Adding divalent cations into citrated blood by batch mixing leads to platelet activation and initiation of coagulation after several minutes, thus limiting the time blood can be used before spontaneously clotting. In this work, we describe a herringbone microfluidic mixer to continuously introduce divalent cations into citrated blood. The mixing ratio, defined as the ratio of the volumetric flow rates of citrated blood and recalcification buffer, can be adjusted by changing the relative inlet pressures of these two solutions. This feature is useful in whole blood assays in order to account for differences in hematocrit, and thus viscosity. The recalcification process in the herringbone mixer does not activate platelets. The advantage of this continuous mixing approach is demonstrated in microfluidic vascular injury model in which platelets and fibrin accumulate on a collagen-tissue factor surface under flow. Continuous recalcification with the herringbone mixer allowed for flow assay times of up to 30 min, more than three times longer than the time achieved by batch recalcification. This continuous mixer allows for measurements of thrombus formation, remodeling, and fibrinolysis in vitro over time scales that are relevant to these physiological processes. PMID:26634014

  20. Investigation of electrolyte measurement in diluted whole blood using spectroscopic and chemometric methods

    NASA Technical Reports Server (NTRS)

    Soller, Babs R.; Favreau, Janice; Idwasi, Patrick O.

    2003-01-01

    The feasibility of using near-infrared (NIR) spectroscopy in combination with partial least-squares (PLS) regression was explored to measure electrolyte concentration in whole blood samples. Spectra were collected from diluted blood samples containing randomized, clinically relevant concentrations of Na+, K+, and Ca2+. Sodium was also studied in lysed blood. Reference measurements were made from the same samples using a standard clinical chemistry instrument. Partial least squares (PLS) was used to develop calibration models for each ion with acceptable results (Na+, R2 = 0.86, CVSEP = 9.5 mmol/L; K+, R2 = 0.54, CVSEP = 1.4 mmol/L; Ca2+, R2 = 0.56, CVSEP = 0.18 mmol/L). Slightly improved results were obtained using a narrower wavelength region (470-925 nm) where hemoglobin, but not water, absorbed indicating that ionic interaction with hemoglobin is as effective as water in causing measurable spectral variation. Good models were also achieved for sodium in lysed blood, illustrating that cell swelling, which is correlated with sodium concentration, is not required for calibration model development.

  1. A new specific method to detect cyanide in body fluids, especially whole blood, by fluorimetry.

    PubMed

    Felscher, D; Wulfmeyer, M

    1998-09-01

    This study shows a simple, rapid, and specific method for the quantitative determination of cyanide ion in body fluids, especially blood, by fluorimetry. It is based upon the transformation of cyanide ion into hydrocyanic acid, which then reacts with 2,3-naphthalenedialdehyde and taurine in a self-contained system. The 1-cyano-2-benzoisoindole derivate thus formed is suitable for fluorimetric measurement (lambdaEX = 418 nm; lambdaEM = 460 nm). The fluorescence intensity can be determined by spectrophotometry or by high-performance liquid chromatography (HPLC) with fluorescence detection. The detection limit is 0.002 microg/mL. Linearity was excellent from 0.002 to 1 microg/mL for spectrophotometry and from 0.002 to 5 microg/mL for HPLC with fluorescence detection. The coefficient of variation for repeatability was 8% or less. Thiocyanate and sulfide did not interfere, even at high concentrations (200 microg/mL). The method was applicable to whole blood, so it should be suitable for both clinical and forensic purposes. PMID:9737330

  2. Magnetic fingerprints of rolling cells for quantitative flow cytometry in whole blood

    PubMed Central

    Reisbeck, Mathias; Helou, Michael Johannes; Richter, Lukas; Kappes, Barbara; Friedrich, Oliver; Hayden, Oliver

    2016-01-01

    Over the past 50 years, flow cytometry has had a profound impact on preclinical and clinical applications requiring single cell function information for counting, sub-typing and quantification of epitope expression. At the same time, the workflow complexity and high costs of such optical systems still limit flow cytometry applications to specialized laboratories. Here, we present a quantitative magnetic flow cytometer that incorporates in situ magnetophoretic cell focusing for highly accurate and reproducible rolling of the cellular targets over giant magnetoresistance sensing elements. Time-of-flight analysis is used to unveil quantitative single cell information contained in its magnetic fingerprint. Furthermore, we used erythrocytes as a biological model to validate our methodology with respect to precise analysis of the hydrodynamic cell diameter, quantification of binding capacity of immunomagnetic labels, and discrimination of cell morphology. The extracted time-of-flight information should enable point-of-care quantitative flow cytometry in whole blood for clinical applications, such as immunology and primary hemostasis. PMID:27596736

  3. Longitudinal analysis of whole blood transcriptomes to explore molecular signatures associated with acute renal allograft rejection.

    PubMed

    Shin, Heesun; Günther, Oliver; Hollander, Zsuzsanna; Wilson-McManus, Janet E; Ng, Raymond T; Balshaw, Robert; Keown, Paul A; McMaster, Robert; McManus, Bruce M; Isbel, Nicole M; Knoll, Greg; Tebbutt, Scott J

    2014-01-01

    In this study, we explored a time course of peripheral whole blood transcriptomes from kidney transplantation patients who either experienced an acute rejection episode or did not in order to better delineate the immunological and biological processes measureable in blood leukocytes that are associated with acute renal allograft rejection. Using microarrays, we generated gene expression data from 24 acute rejectors and 24 nonrejectors. We filtered the data to obtain the most unambiguous and robustly expressing probe sets and selected a subset of patients with the clearest phenotype. We then performed a data-driven exploratory analysis using data reduction and differential gene expression analysis tools in order to reveal gene expression signatures associated with acute allograft rejection. Using a template-matching algorithm, we then expanded our analysis to include time course data, identifying genes whose expression is modulated leading up to acute rejection. We have identified molecular phenotypes associated with acute renal allograft rejection, including a significantly upregulated signature of neutrophil activation and accumulation following transplant surgery that is common to both acute rejectors and nonrejectors. Our analysis shows that this expression signature appears to stabilize over time in nonrejectors but persists in patients who go on to reject the transplanted organ. In addition, we describe an expression signature characteristic of lymphocyte activity and proliferation. This lymphocyte signature is significantly downregulated in both acute rejectors and nonrejectors following surgery; however, patients who go on to reject the organ show a persistent downregulation of this signature relative to the neutrophil signature.

  4. Magnetic fingerprints of rolling cells for quantitative flow cytometry in whole blood.

    PubMed

    Reisbeck, Mathias; Helou, Michael Johannes; Richter, Lukas; Kappes, Barbara; Friedrich, Oliver; Hayden, Oliver

    2016-01-01

    Over the past 50 years, flow cytometry has had a profound impact on preclinical and clinical applications requiring single cell function information for counting, sub-typing and quantification of epitope expression. At the same time, the workflow complexity and high costs of such optical systems still limit flow cytometry applications to specialized laboratories. Here, we present a quantitative magnetic flow cytometer that incorporates in situ magnetophoretic cell focusing for highly accurate and reproducible rolling of the cellular targets over giant magnetoresistance sensing elements. Time-of-flight analysis is used to unveil quantitative single cell information contained in its magnetic fingerprint. Furthermore, we used erythrocytes as a biological model to validate our methodology with respect to precise analysis of the hydrodynamic cell diameter, quantification of binding capacity of immunomagnetic labels, and discrimination of cell morphology. The extracted time-of-flight information should enable point-of-care quantitative flow cytometry in whole blood for clinical applications, such as immunology and primary hemostasis. PMID:27596736

  5. Plasma contributes to the antimicrobial activity of whole blood against Mycobacterium tuberculosis.

    PubMed

    López-Medrano, Ramiro; Guerra-Laso, José Manuel; López-Fidalgo, Eduardo; Diez-Tascón, Cristina; García-García, Silvia; Blanco-Conde, Sara; Rivero-Lezcano, Octavio Miguel

    2016-10-01

    The whole blood model for infection has proven useful to analyze the immunological response to Mycobacterium tuberculosis, because it exerts a significant antimicrobial activity. Although this activity has been generally assumed to be cellular, we have found that the leukocyte fraction of blood from healthy volunteers did not kill the bacilli. We have discovered that plasma was responsible for a large proportion, but not all, of the antimicrobial activity. Furthermore, infected monocytes controlled the mycobacterial multiplication when cultivated in the presence of plasma. Intriguingly, serum from the same donors did not share this activity, although it was able to eliminate the non-pathogenic Mycobacterium gordonae To identify the remaining components that participate in the antimycobacterial activity we fractionated blood in leukocytes, plasma, erythrocytes and platelets, and analyzed the bactericidal power of each fraction and their combinations using a factorial design. We found that erythrocytes, but not platelets, participated and showed by flow cytometry that mycobacteria physically associated with erythrocytes. We propose that in exposed healthy individuals that show 'early clearance' of the mycobacteria, the innate response is predominantly humoral, probably through the effect of antimicrobial peptides and proteins. PMID:27554054

  6. Rapid detection of cancer related DNA nanoparticulate biomarkers and nanoparticles in whole blood

    NASA Astrophysics Data System (ADS)

    Heller, Michael J.; Krishnan, Raj; Sonnenberg, Avery

    2010-08-01

    The ability to rapidly detect cell free circulating (cfc) DNA, cfc-RNA, exosomes and other nanoparticulate disease biomarkers as well as drug delivery nanoparticles directly in blood is a major challenge for nanomedicine. We now show that microarray and new high voltage dielectrophoretic (DEP) devices can be used to rapidly isolate and detect cfc-DNA nanoparticulates and nanoparticles directly from whole blood and other high conductance samples (plasma, serum, urine, etc.). At DEP frequencies of 5kHz-10kHz both fluorescent-stained high molecular weight (hmw) DNA, cfc-DNA and fluorescent nanoparticles separate from the blood and become highly concentrated at specific DEP highfield regions over the microelectrodes, while blood cells move to the DEP low field-regions. The blood cells can then be removed by a simple fluidic wash while the DNA and nanoparticles remain highly concentrated. The hmw-DNA could be detected at a level of <260ng/ml and the nanoparticles at <9.5 x 109 particles/ml, detection levels that are well within the range for viable clinical diagnostics and drug nanoparticle monitoring. Disease specific cfc-DNA materials could also be detected directly in blood from patients with Chronic Lymphocytic Leukemia (CLL) and confirmed by PCR genotyping analysis.

  7. Selective suppression of cytokine secretion in whole blood cell cultures of patients with colorectal cancer.

    PubMed Central

    Lahm, H.; Schindel, M.; Frikart, L.; Cerottini, J. P.; Yilmaz, A.; Givel, J. C.; Fischer, J. R.

    1998-01-01

    We have investigated the secretion of interferon alpha (IFN-alpha), IFN-gamma, interleukin-1alpha (IL-1alpha), IL-1beta, IL-2 and tumour necrosis factor alpha (TNF-alpha) in whole blood cell cultures (WBCCs) of colorectal cancer patients upon mitogen stimulation. Whereas the values for IL-1beta and TNF-alpha remained virtually unchanged in comparison with healthy control subjects, WBCCs of colorectal cancer patients secreted significantly lower amounts of IFN-alpha (P < 0.005), IFN-gamma (P < 0.0001), IL-1alpha (P < 0.0001) and IL-2 (P < 0.05). This reduction correlated with the progression of the disease. The total leucocyte and monocyte population were almost identical in both groups. In contrast, a dramatic depletion of lymphocytes was observed in colorectal cancer patients, which affected both lymphocyte counts (P < 0.0005) and their distribution (P < 0.0001). Our results suggest a selective suppression of cytokines in colorectal cancer patients that is related to tumour burden. Several mechanisms might account for this phenomenon, one of which might be lymphocyte depletion. PMID:9792144

  8. Magnetic fingerprints of rolling cells for quantitative flow cytometry in whole blood

    NASA Astrophysics Data System (ADS)

    Reisbeck, Mathias; Helou, Michael Johannes; Richter, Lukas; Kappes, Barbara; Friedrich, Oliver; Hayden, Oliver

    2016-09-01

    Over the past 50 years, flow cytometry has had a profound impact on preclinical and clinical applications requiring single cell function information for counting, sub-typing and quantification of epitope expression. At the same time, the workflow complexity and high costs of such optical systems still limit flow cytometry applications to specialized laboratories. Here, we present a quantitative magnetic flow cytometer that incorporates in situ magnetophoretic cell focusing for highly accurate and reproducible rolling of the cellular targets over giant magnetoresistance sensing elements. Time-of-flight analysis is used to unveil quantitative single cell information contained in its magnetic fingerprint. Furthermore, we used erythrocytes as a biological model to validate our methodology with respect to precise analysis of the hydrodynamic cell diameter, quantification of binding capacity of immunomagnetic labels, and discrimination of cell morphology. The extracted time-of-flight information should enable point-of-care quantitative flow cytometry in whole blood for clinical applications, such as immunology and primary hemostasis.

  9. Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood.

    PubMed

    Park, Yang-Seok; Sunkara, Vijaya; Kim, Yubin; Lee, Won Seok; Han, Ja-Ryoung; Cho, Yoon-Kyoung

    2016-01-01

    Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins. PMID:27167836

  10. The effects of selective serotonin reuptake inhibitors on platelet function in whole blood and platelet concentrates.

    PubMed

    Reikvam, Anne-Grete; Hustad, Steinar; Reikvam, Håkon; Apelseth, Torunn Oveland; Nepstad, Ina; Hervig, Tor Audun

    2012-01-01

    Several studies report that patients who are treated with selective serotonin reuptake inhibitors (SSRIs) for depression may have increased risk of bleeding, particularly from the gastrointestinal tract. This may be related to low intraplatelet serotonin concentrations. Several blood banks do not store platelets from donors using SSRIs for transfusion, although the possible effects of SSRIs on platelet storage are not documented. We conducted a case-control pilot study of apheresis platelet concentrates prepared from donors using SSRIs (n=8) and from donors without medication (n=10). The platelet concentrates were stored for 5 days. Light transmission aggregometry (LTA), thrombelastography (TEG), and flow cytometric analyses were preformed for in vitro measurements of platelet function. Platelet function and platelet serotonin content were investigated in whole blood and in platelet concentrates stored for up to 5 days. LTA, TEG, and flow cytometric analysis of glycoprotein expression did not reveal any significant differences between the two groups. All 18 platelet concentrates performed well according to the standards set for platelet quality in relation to transfusion. Blood donors using SSRIs had significantly lower platelet serotonin compared to blood donors without medication. The results from our pilot study indicate that platelets from donors using SSRIs may be suitable for transfusion after storage for 5 days, but further laboratory and clinical studies are necessary to confirm this.

  11. Measurement of glycated haemoglobin in whole blood by a novel fluorescence quenching assay.

    PubMed

    Blincko, S; Anzetse, J; Edwards, R

    2000-07-01

    We describe a method for the specific measurement of glycated Hb (GHb) by fluorescence quenching. Whole blood is added to lysing solution, then the lysate is mixed with eosin-boronic acid solution and reacted for at least 5 min at room temperature. The quenching of the fluorescence of the eosin-boronic acid solution is proportional to the concentration of GHb present. Total Hb concentration was measured by absorbance and the GHb expressed as a percentage of the total Hb. Comparison with a commercial high-performance liquid chromatography (HPLC) system for HbA1c showed: %GHb=1.30 (SD 0.04) %HbA1c + 1.36 (SD 0.30), S(y/x) 0.803, n=95, r=0.965 (SD=standard deviation). Intra-assay coefficients of variation were <2.5% (for GHb concentrations in the range 6-20%) and inter-assay coefficients of variation were <4.1% (10 assays on six samples with GHb concentrations in the range 6-20%). Linearity of response was demonstrated by dilution. The effect of adding exogenous glucose, bilirubin and triglycerides was tested on samples with low, medium and high GHb concentrations. No significant interference was found. Variation of haematocrit over the range 0.4-0.6 also had no significant effect on percentage GHb. Preliminary results with samples containing variant Hb (HbAS and HbAC) indicated good agreement with HPLC for these samples also. PMID:10902866

  12. An economic evaluation of plasma production via erythroplasmapheresis and whole blood collection.

    PubMed

    Connelly, Luke B; Pink, Anne

    2002-10-01

    This paper presents cost-effectiveness analyses (CEAs) of plasma collection via two alternative methods: whole blood collection (WBC) and erythroplasmapheresis collection (EPC). The objective of the study is to provide an answer to the question 'What is the least-cost method of plasma production'. This question is answered, both from the viewpoint of the blood collection agency (using financial CEA) and from that of 'society' as a whole (using economic CEA). We employ detailed financial data and economic survey data for collections made by a blood collection agency and to WBC and EPC donors in Brisbane, Australia. The results indicate that, despite the superior yield provided by EPC, WBC is actually more cost-effective. This result is robust to thorough sensitivity analysis and arises regardless of whether an economic or financial perspective is taken. We conclude that, ceteris paribus, the cost of recruiting new plasma donors would need to be quite substantial for marginal investments in EPC to be considered cost-effective. PMID:12350045

  13. Determination of buprenorphine and norbuprenorphine in whole blood by liquid chromatography-mass spectrometry.

    PubMed

    Hoja, H; Marquet, P; Verneuil, B; Lotfi, H; Dupuy, J L; Lachâtre, G

    1997-01-01

    A sensitive and highly specific method for the determination of buprenorphine and norbuprenorphine in postmortem and hemolyzed whole blood using combined liquid chromatography and mass spectrometry (LC-MS) with electrospray ionization was developed. After enzymatic hydrolysis and deproteinization with acetonitrile, Extrelut-3 cartridges were used for a preliminary basic sample cleanup. Elution by toluene-ether (50:50, v/v) was followed by an acid wash with 0.05M H3PO4 and a basic re-extraction into ether. A Nucleosil C18 (150 x 1-mm i.d.) reversed-phase column was used for the chromatographic separation together with a mixture of 2mM ammonium formate buffer (pH 3) and acetonitrile (55:45, v/v) as the mobile phase. Recoveries were between 56 and 60%, and detection limits were 0.05 ng/mL for both analytes. The coefficient of variation for repeatability was lower than 4%. Limits of quantitation were 0.1 ng/mL for buprenorphine and norbuprenorphine. Reproducibility was good, and linearity was excellent in the range of 0.1 to 100 ng/mL (r > 0.9999, n = 7). PMID:9083835

  14. Smart point-of-care systems for molecular diagnostics based on nanotechnology: whole blood glucose analysis

    NASA Astrophysics Data System (ADS)

    Devadhasan, Jasmine P.; Kim, Sanghyo

    2015-07-01

    Complementary metal oxide semiconductor (CMOS) image sensors are received great attention for their high efficiency in biological applications. The present work describes a CMOS image sensor-based whole blood glucose monitoring system through a point-of-care (POC) approach. A simple poly-ethylene terephthalate (PET) film chip was developed to carry out the enzyme kinetic reaction at various concentrations of blood glucose. In this technique, assay reagent was adsorbed onto amine functionalized silica (AFSiO2) nanoparticles in order to achieve glucose oxidation on the PET film chip. The AFSiO2 nanoparticles can immobilize the assay reagent with an electrostatic attraction and eased to develop the opaque platform which was technically suitable chip to analyze by the camera module. The oxidized glucose then produces a green color according to the glucose concentration and is analyzed by the camera module as a photon detection technique. The photon number decreases with increasing glucose concentration. The simple sensing approach, utilizing enzyme immobilized AFSiO2 nanoparticle chip and assay detection method was developed for quantitative glucose measurement.

  15. Optimization of a Whole-Blood Gamma Interferon Assay for Detection of Mycobacterium bovis-Infected Cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Antigens of Mycobacterium bovis elicit a cell-mediated immune response upon intradermalinjection in cattle. In vitro, such antigens stimulate the production of interferon (IFN)-gamma by bovine T-cells in whole blood culture (IFN-gamma assay). We have analyzed various parameters of the in vitro IFN-g...

  16. A novel hydrophilic polymer-brush pattern for site-specific capture of blood cells from whole blood.

    PubMed

    Hou, Jianwen; Shi, Qiang; Ye, Wei; Fan, Qunfu; Shi, Hengchong; Wong, Shing-Chung; Xu, Xiaodong; Yin, Jinghua

    2015-03-11

    A novel hydrophilic PAMPS-PAAm brush pattern is fabricated to selectively capture blood cells from whole blood. PAMPS brushes provide antifouling surfaces to resist protein and cell adhesion while PAAm brushes effectively entrap targeted proteins for site-specific and cell-type dependent capture of blood cells. PMID:25469596

  17. Method and kit for the selective labeling of red blood cells in whole blood with Tc-99m

    DOEpatents

    Srivastava, S.C.; Babich, J.W.; Straub, R.; Richards, P.

    1988-07-05

    Disclosed herein are a method and kit for the preparation of [sup 99m]Tc labeled red blood cells using whole blood in a closed sterile system containing stannous tin in a form such that it will enter the red blood cells and be available for the reduction of technetium. No Drawings

  18. Method and kit for the selective labeling of red blood cells in whole blood with TC-99M

    DOEpatents

    Srivastava, Suresh C.; Babich, John W.; Straub, Rita; Richards, Powell

    1988-01-01

    Disclosed herein are a method and kit for the preparation of .sup.99m Tc labeled red blood cells using whole blood in a closed sterile system containing stannous tin in a form such that it will enter the red blood cells and be available therein for the reduction of technetium.

  19. Brief Report: Are Autistic-Behaviors in Children Related to Prenatal Vitamin Use and Maternal Whole Blood Folate Concentrations?

    ERIC Educational Resources Information Center

    Braun, Joseph M.; Froehlich, Tanya; Kalkbrenner, Amy; Pfeiffer, Christine M.; Fazili, Zia; Yolton, Kimberly; Lanphear, Bruce P.

    2014-01-01

    Prenatal multivitamin/folic acid supplement use may reduce the risk of autism spectrum disorders. We investigated whether 2nd trimester prenatal vitamin use and maternal whole blood folate (WBF) concentrations were associated with Social Responsiveness Scale (SRS) scores at 4-5 years of age in a prospective cohort of 209 mother-child pairs. After…

  20. Rapid on-chip recalcification and drug dosing of citrated whole blood using microfluidic buffer sheath flow.

    PubMed

    Muthard, Ryan W; Diamond, Scott L

    2014-01-01

    Millions of clotting tests each year require recalcification of blood treated with sodium citrate, a calcium chelator that prevents prothrombinase assembly. We validated a converging trifurcated microfluidic device to measure platelet and fibrin accumulation following on-chip recalcification of citrated whole blood. Recalcification was accomplished by sheathing the blood with Ca2+ buffer. Fluorescein rapidly diffused across the buffer-blood interface (achieving 62.5% of maximum centerline concentration within ~4 cm of flow), while albumin remained relatively unchanged in blood due to its lower diffusivity (<20% decrease). Since Ca2+ diffuses faster than fluorescein, full recalcification of whole blood was achieved within ~1 cm of flow prior to encountering a collagen/tissue surface. Platelet and fibrin were reduced by 87.3% and 99.0%, respectively, when the sheath buffer was Ca2+-free. A 30-min preincubation of citrated whole blood prior to on-chip recalcification increased platelet (159%) and fibrin (86.6%) deposition, compared to 5-min preincubation, likely due to factor XIIa generation in citrated blood. The P2Y1 inhibitor, MRS-2179, was delivered by diffusion into flowing blood and inhibited platelet deposition on collagen with a calculated IC50 of 0.155 μM. On-chip recalcification and drug dosing of citrated blood allows for assays of platelet function in a whole blood milieu under flow.

  1. 76 FR 5386 - Draft Guidance for Industry: Pre-Storage Leukocyte Reduction of Whole Blood and Blood Components...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-31

    ... same title dated January 2001 (January 31, 2001, 66 FR 8410). The draft guidance, when finalized, will... of Whole Blood and Blood Components Intended for Transfusion; Availability AGENCY: Food and Drug... Blood and Blood Components Intended for Transfusion'' dated January 2011. The draft guidance...

  2. REACTIVE OXYGEN SPECIES IN WHOLE BLOOD, BLOOD PLASMA AND BREAST MILK: VALIDATION OF A POTENTIAL MARKER OF EXPOSURE AND EFFECT

    EPA Science Inventory

    Reactive oxygen species (ROS) are recognized to contribute to the pathobiology of many diseases. We have applied a simple chemiluminescent (CL) probe to detect ROS in various biological fluids (plasma, whole blood, urine and breast milk) in an environmental arsenic drinking wate...

  3. Effects of argon laser on in vitro aggregation of platelets in platelet rich plasma and whole blood

    SciTech Connect

    Doerger, P.T.; Glueck, H.I.; McGill, M.

    1988-06-01

    The effects of an Argon laser on platelet aggregation were studied, since platelets may be exposed to laser energy when used intravascularly. Various preparations of platelets in platelet rich plasma (PRP) and whole blood, with or without aspirin, were tested with the aggregating agents ADP, collagen, thrombin, and epinephrine. Simultaneous release of ATP was also measured in PRP. At relatively low levels of irradiation, platelet aggregation was potentiated. Enhancement was evidenced by an increase in percent aggregation, earlier onset of the reaction, and reduction in the amount of aggregating agent required. In PRP, the mechanism of laser potentiation appeared to be the release of endogenous ATP from platelets. At relatively high levels of irradiation, platelets were destroyed and aggregation abolished. In whole blood, the mechanism was somewhat more complicated since release of ATP occurred from RBCs as well as platelets. Spontaneous aggregation following laser treatment occurred in isolated instances in PRP and in every trial in whole blood preparations. Aspirin ingestion inhibited the laser's effects in PRP but not in whole blood. These results may have important clinical implications for laser angioplasty, and the potentiated aggregation response may prove useful in laboratory studies of platelet function.

  4. A lateral flow immunosensor for direct, sensitive, and highly selective detection of hemoglobin A1c in whole blood.

    PubMed

    Ang, Shu Hwang; Thevarajah, T Malathi; Woi, Pei Meng; Alias, Yatimah binti; Khor, Sook Mei

    2016-03-15

    An immunosensor that operates based on the principles of lateral flow was developed for direct detection of hemoglobin A1c (HbA1c) in whole blood. We utilized colloidal gold-functionalized antibodies to transduce the specific signal generated when sandwich immuno-complexes were formed on the strip in the presence of HbA1c. The number and intensity of the test lines on the strips indicate normal, under control, and elevated levels of HbA1c. In addition, a linear relationship between HbA1c levels and immunosensor signal intensity was confirmed, with a dynamic range of 4-14% (20-130 mmol mol(-1)) HbA1c. Using this linear relationship, we determined the HbA1c levels in blood as a function of the signal intensity on the strips. Measurements were validated using the Bio-Rad Variant II HPLC and DCA Vantage tests. Moreover, the immunosensor was verified to be highly selective for detection of HbA1c against HbA0, glycated species of HbA0, and HbA2. The limit of detection was found to be 42.5 μg mL(-1) (1.35 mmol mol(-1)) HbA1c, which is reasonably sensitive compared to the values reported for microarray immunoassays. The shelf life of the immunosensor was estimated to be 1.4 months when stored at ambient temperature, indicating that the immunoassay is stable. Thus, the lateral flow immunosensor developed here was shown to be capable of performing selective, accurate, rapid, and stable detection of HbA1c in human blood samples. PMID:26927875

  5. Evaluation of apoptosis induction in human peripheral blood mononuclear cells and synovial cells in patients with rheumatoid arthritis.

    PubMed

    Demian, Soheir R; Abo-Shousha, Seham A; Sultan, Hussein E; Zarka, Wael El

    2005-01-01

    Rheumatoid arthritis (RA) is a chronic inflammatory destructive disease involving the joint and characterized by T-lymphocyte accumulation within the synovial compartment. It is dominated by the presence of macrophages, plasma cells and synovial fibroblasts which are the main pathogenic factors leading to the destruction of bone and cartilage. The survival of these cells may be promoted by inadequate apoptosis leading to synovial hyperplasia. So, the aim of the present study was to evaluate the apoptosis levels before and after induction of apoptosis using anti-Fas mAb, both in peripheral blood (PB) and synovial fluid (SF) infiltrating mononuclear cells (MCs) of patients with RA. CD4+ T cell subsets and cell survival assays were also done to investigate correlations between these parameters. The study was conducted on 15 patients with RA, 10 individual volunteers as a control group and 10 patients with osteoarthritis (OA) as a control group for SF evaluations (have defective Fas expression on their cells). Results of this work revealed that in vitro induction of apoptosis by anti-Fas mAb resulted in increase of: percent (%) reduction of cell viability in PBMCs and SFMCs, % reduction of CD4+ T cell subsets and apoptotic cell % in all studied groups than before induction. The increase in the three parameters is only significant in SF of RA group compared to PB while it is non significant in OA group due to the defective Fas expression on OA cells. Our results also showed a significant positive correlation between CD4+ T cell and viability percentages before induction of apoptosis in SF of RA and between apoptosis levels and CD4+ T cell percentage after induction of apoptosis in the SF of RA group. In conclusion, activated T cells infiltrating SF of RA patients have functional Fas antigen which enable them to undergo in vitro apoptosis using anti-Fas mAb. The cytotoxicity of which is more specific to local lesion such as SF of RA patients suggesting that local

  6. Evaluation of apoptosis induction in human peripheral blood mononuclear cells and synovial cells in patients with rheumatoid arthritis.

    PubMed

    Demian, Soheir R; Abo-Shousha, Seham A; Sultan, Hussein E; Zarka, Wael El

    2005-01-01

    Rheumatoid arthritis (RA) is a chronic inflammatory destructive disease involving the joint and characterized by T-lymphocyte accumulation within the synovial compartment. It is dominated by the presence of macrophages, plasma cells and synovial fibroblasts which are the main pathogenic factors leading to the destruction of bone and cartilage. The survival of these cells may be promoted by inadequate apoptosis leading to synovial hyperplasia. So, the aim of the present study was to evaluate the apoptosis levels before and after induction of apoptosis using anti-Fas mAb, both in peripheral blood (PB) and synovial fluid (SF) infiltrating mononuclear cells (MCs) of patients with RA. CD4+ T cell subsets and cell survival assays were also done to investigate correlations between these parameters. The study was conducted on 15 patients with RA, 10 individual volunteers as a control group and 10 patients with osteoarthritis (OA) as a control group for SF evaluations (have defective Fas expression on their cells). Results of this work revealed that in vitro induction of apoptosis by anti-Fas mAb resulted in increase of: percent (%) reduction of cell viability in PBMCs and SFMCs, % reduction of CD4+ T cell subsets and apoptotic cell % in all studied groups than before induction. The increase in the three parameters is only significant in SF of RA group compared to PB while it is non significant in OA group due to the defective Fas expression on OA cells. Our results also showed a significant positive correlation between CD4+ T cell and viability percentages before induction of apoptosis in SF of RA and between apoptosis levels and CD4+ T cell percentage after induction of apoptosis in the SF of RA group. In conclusion, activated T cells infiltrating SF of RA patients have functional Fas antigen which enable them to undergo in vitro apoptosis using anti-Fas mAb. The cytotoxicity of which is more specific to local lesion such as SF of RA patients suggesting that local

  7. Leukotriene C/sub 4/ (LTC/sub 4/) produced by a human T-T hybridoma suppresses pokeweed mitogen (PWM) induced immunoglobulin (Ig) production by human mononuclear cells

    SciTech Connect

    Jurgensen, C.H.; Ambrus, J.L. Jr.; Witzel, N.; Lewis, R.A.; Butler, J.L.; Fauci, A.S.

    1986-03-05

    Human T lymphocytes are capable of secreting many different lymphokines which regulate Ig production. The authors produced a human T-T fusion between T cells from a normal donor and the T lymphoma line CEM-6. From this fusion 3 of 180 clones produced a factor which suppressed polyclonal Ig production by PWM-stimulated mononuclear cells. All 3 clones expressed both T4 and T8 surface markers; CEM-6 is T4/sup +//T8/sup +/. Suppression of Ig production did not involve cytotoxicity and was most effective when the hybridoma supernatant was added during the first 3 d of culture. Production of the suppressive factor could be inhibited by corticosteroids but not indomethacin. Analysis of the hybridoma supernatant by reverse phase chromatography revealed a peak of suppressive activity in the fraction where purified LTC/sub 4/ could be eluted. Crude hybridoma supernatant contained 10-50 ng/ml of LTC/sub 4/, but no LTB/sub 4/ by radioimmunoassay (RIA). In addition, LTC/sub 4/ produced by the hybridomas co-eluted with purified LTC/sub 4/ as assessed by RIA. No LTC/sub 4/ was found in nonsuppressive hybridoma supernatants. Furthermore, antibody to LTC/sub 4/ could absorb suppressive activity from hybridoma supernatants. Thus, human T lymphocytes produce LTC/sub 4/ which suppresses polyclonal Ig production by human mononuclear cells.

  8. Variation in RNA-Seq Transcriptome Profiles of Peripheral Whole Blood from Healthy Individuals with and without Globin Depletion

    PubMed Central

    Fishbane, Nick; Ruan, Jian; Zhou, Mi; Balshaw, Robert; Wilson-McManus, Janet E.; Ng, Raymond T.; McManus, Bruce M.; Tebbutt, Scott J.

    2014-01-01

    Background The molecular profile of circulating blood can reflect physiological and pathological events occurring in other tissues and organs of the body and delivers a comprehensive view of the status of the immune system. Blood has been useful in studying the pathobiology of many diseases. It is accessible and easily collected making it ideally suited to the development of diagnostic biomarker tests. The blood transcriptome has a high complement of globin RNA that could potentially saturate next-generation sequencing platforms, masking lower abundance transcripts. Methods to deplete globin mRNA are available, but their effect has not been comprehensively studied in peripheral whole blood RNA-Seq data. In this study we aimed to assess technical variability associated with globin depletion in addition to assessing general technical variability in RNA-Seq from whole blood derived samples. Results We compared technical and biological replicates having undergone globin depletion or not and found that the experimental globin depletion protocol employed removed approximately 80% of globin transcripts, improved the correlation of technical replicates, allowed for reliable detection of thousands of additional transcripts and generally increased transcript abundance measures. Differential expression analysis revealed thousands of genes significantly up-regulated as a result of globin depletion. In addition, globin depletion resulted in the down-regulation of genes involved in both iron and zinc metal ion bonding. Conclusions Globin depletion appears to meaningfully improve the quality of peripheral whole blood RNA-Seq data, and may improve our ability to detect true biological variation. Some concerns remain, however. Key amongst them the significant reduction in RNA yields following globin depletion. More generally, our investigation of technical and biological variation with and without globin depletion finds that high-throughput sequencing by RNA-Seq is highly

  9. Simple decomposition procedure for determination of selenium in whole blood, serum and urine by hydride generation atomic absorption spectroscopy.

    PubMed

    Tiran, B; Tiran, A; Rossipal, E; Lorenz, O

    1993-12-01

    A digestion procedure for selenium determination by hydride generation atomic absorption spectroscopy (AAS) in whole blood, serum and urine is described, it employs sulfuric acid, hydrogen peroxide and vanadium (V) sulfuric acid reagent solution. The method is rapid, uses no explosive reagents and can be performed at a constant temperature of 100 degrees C. Therefore, it is easily applicable in a routine clinical laboratory for a large amount of samples. The coefficient of intra-assay variation was 4.3-5.6%, the coefficient for inter-assay variation was 5-5.9% in the medium and high concentration range, and 5.8-8.6% in the low range. In analyzing several commercial reference materials our results showed good agreement with the target values. Analytical recovery by addition of sodium selenite and seleno-DL-methionine to samples ranged between 97 and 104%. The correlation between the described digestion procedure and the nitric, sulfuric and perchloric acid digestion procedure recommended by the International Union of Pure and Applied Chemistry showed good agreement for whole blood, serum and for urine. We determined selenium in serum (n = 58) and whole blood (n = 50) in a collective of healthy children from 1 to 5 years living in Styria, Austria. The low values in serum (35 +/- 11 micrograms/L) and whole blood (42 +/- 6 micrograms/L) at one year of life increased significantly to 48 +/- 13 mu/L (p = 0.033) and 55 +/- 6 micrograms/L (p = 0.004) at three years of life in serum and whole blood, respectively. The selenium concentration showed no further increase up to five years of age.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Ultra-sensitive method for determination of ethanol in whole blood by headspace capillary gas chromatography with cryogenic oven trapping.

    PubMed

    Watanabe-Suzuki, K; Seno, H; Ishii, A; Kumazawa, T; Suzuki, O

    1999-04-30

    We have established an ultra-sensitive method for determination of ethanol in whole blood by headspace capillary gas chromatography (GC) with cryogenic oven trapping. After heating a blood sample containing ethanol and isobutyl alcohol (internal standard, IS) in a 7.0-ml vial at 55 degrees C for 15 min, 5 ml of the headspace vapor was drawn into a glass syringe and injected into a GC port. All vapor was introduced into an Rtx-BAC2 wide-bore capillary column in the splitless mode at -60 degrees C oven temperature to trap entire analytes, and then the oven temperature was programmed up to 240 degrees C for GC measurements with flame ionization detection. The present method gave sharp peaks of ethanol and IS, and low background noise for whole blood samples. The mean partition into the gaseous phase for ethanol and IS was 3.06+/-0.733 and 8.33+/-2.19%, respectively. The calibration curves showed linearity in the range 0.02-5.0 microg/ml whole blood. The detection limit was estimated to be 0.01 microg/ml. The coefficients of intra-day and inter-day variation for spiked ethanol were 8.72 and 9.47%, respectively. Because of the extremely high sensitivity, we could measure low levels of endogenous ethanol in whole blood of subjects without drinking. The concentration of endogenous ethanol measured for 10 subjects under uncontrolled conditions varied from 0 to 0.377 microg/ml (mean, 0.180 microg/ml). Data on the diurnal changes of endogenous ethanol in whole blood of five subjects under strict food control are also presented; they are in accordance with the idea that endogenous blood ethanol is of enteric bacterial origin.

  11. SMN Protein Can Be Reliably Measured in Whole Blood with an Electrochemiluminescence (ECL) Immunoassay: Implications for Clinical Trials

    PubMed Central

    Taylor, Shannon; Sunshine, Sara S.; McCarthy, Kathleen; Risher, Nicole; Newcomb, Tara; Weetall, Marla; Prior, Thomas W.; Swoboda, Kathryn J.; Chen, Karen S.; Paushkin, Sergey

    2016-01-01

    Spinal muscular atrophy (SMA) is caused by defects in the survival motor neuron 1 (SMN1) gene that encodes survival motor neuron (SMN) protein. The majority of therapeutic approaches currently in clinical development for SMA aim to increase SMN protein expression and there is a need for sensitive methods able to quantify increases in SMN protein levels in accessible tissues. We have developed a sensitive electrochemiluminescence (ECL)-based immunoassay for measuring SMN protein in whole blood with a minimum volume requirement of 5μL. The SMN-ECL immunoassay enables accurate measurement of SMN in whole blood and other tissues. Using the assay, we measured SMN protein in whole blood from SMA patients and healthy controls and found that SMN protein levels were associated with SMN2 copy number and were greater in SMA patients with 4 copies, relative to those with 2 and 3 copies. SMN protein levels did not vary significantly in healthy individuals over a four-week period and were not affected by circadian rhythms. Almost half of the SMN protein was found in platelets. We show that SMN protein levels in C/C-allele mice, which model a mild form of SMA, were high in neonatal stage, decreased in the first few weeks after birth, and then remained stable throughout the adult stage. Importantly, SMN protein levels in the CNS correlated with SMN levels measured in whole blood of the C/C-allele mice. These findings have implications for the measurement of SMN protein induction in whole blood in response to SMN-upregulating therapy. PMID:26953792

  12. Levels of arsenic, mercury, cadmium, copper, lead, zinc and manganese in serum and whole blood of resident adults from mining and non-mining communities in Ghana.

    PubMed

    Obiri, Samuel; Yeboah, Philip O; Osae, Shiloh; Adu-Kumi, Sam

    2016-08-01

    Human beings working or living near an industrial site where toxic chemicals such as As, Hg, Cd, Cu, Mn, Pb, Zn and or their compounds are used or indiscriminately discharged into the environment, are constantly exposed to such chemicals via ingestion (drinking or eating), dermal contact or inhalation (breathing). However, in developing countries such as Ghana, limited data on levels of the aforementioned chemicals in whole blood and serum of human beings as a result of exposure to the aforementioned chemicals from mining communities and non-mining communities is preventing effective policy formulation to protect human health. Hence, this study was undertaken to measure the levels of the aforementioned toxic chemicals in whole blood and serum of 300 resident adults from mining (Tarkwa Nsuaem Municipality Assembly (TNMA) and Prestea Huni Valley District (PHVD)) and non-mining (Cape Coast Metropolis) communities in Ghana, using neutron activation analysis (NAA). Blood samples were taken from 200 resident adults (105 males and 95 females) from mining and 100 resident adults (60 males and 40 males) from non-mining communities in the study area following the completion of an informed consent and the issuance of ethical clearance by the Ghana Health Service Ethical Committee. The mean concentrations for As, Hg, Cd, Cu, Mn, Pb and Zn in whole blood of residents from mining communities were as follows: 38 ± 320 μg/L, 63 ± 0.23 μg/L, 303 ± 117 μg/L, 3300 ± 953, 195 ± 90 μg/L, 28 ± 14 μg/L and 1405 ± 458 μg/L, respectively; while the levels of measured toxic chemicals in the serum of resident adults from mining communities were as follows: 65 ± 14 μg/L, 358 ± 22 μg/l, 134 ± 12 μg/L, 3590 ± 254 μg/L, 401 ± 113 μg/L, 58 ± 5.8 μg/L and 49 ± 31 μg/L, respectively, for As, Hg, Cd, Cu, Mn, Pb and Zn and were found to have exceeded the permissible WHO guideline values. PMID:27178291

  13. Levels of arsenic, mercury, cadmium, copper, lead, zinc and manganese in serum and whole blood of resident adults from mining and non-mining communities in Ghana.

    PubMed

    Obiri, Samuel; Yeboah, Philip O; Osae, Shiloh; Adu-Kumi, Sam

    2016-08-01

    Human beings working or living near an industrial site where toxic chemicals such as As, Hg, Cd, Cu, Mn, Pb, Zn and or their compounds are used or indiscriminately discharged into the environment, are constantly exposed to such chemicals via ingestion (drinking or eating), dermal contact or inhalation (breathing). However, in developing countries such as Ghana, limited data on levels of the aforementioned chemicals in whole blood and serum of human beings as a result of exposure to the aforementioned chemicals from mining communities and non-mining communities is preventing effective policy formulation to protect human health. Hence, this study was undertaken to measure the levels of the aforementioned toxic chemicals in whole blood and serum of 300 resident adults from mining (Tarkwa Nsuaem Municipality Assembly (TNMA) and Prestea Huni Valley District (PHVD)) and non-mining (Cape Coast Metropolis) communities in Ghana, using neutron activation analysis (NAA). Blood samples were taken from 200 resident adults (105 males and 95 females) from mining and 100 resident adults (60 males and 40 males) from non-mining communities in the study area following the completion of an informed consent and the issuance of ethical clearance by the Ghana Health Service Ethical Committee. The mean concentrations for As, Hg, Cd, Cu, Mn, Pb and Zn in whole blood of residents from mining communities were as follows: 38 ± 320 μg/L, 63 ± 0.23 μg/L, 303 ± 117 μg/L, 3300 ± 953, 195 ± 90 μg/L, 28 ± 14 μg/L and 1405 ± 458 μg/L, respectively; while the levels of measured toxic chemicals in the serum of resident adults from mining communities were as follows: 65 ± 14 μg/L, 358 ± 22 μg/l, 134 ± 12 μg/L, 3590 ± 254 μg/L, 401 ± 113 μg/L, 58 ± 5.8 μg/L and 49 ± 31 μg/L, respectively, for As, Hg, Cd, Cu, Mn, Pb and Zn and were found to have exceeded the permissible WHO guideline values.

  14. Vibration Induces BAFF Overexpression and Aberrant O-Glycosylation of IgA1 in Cultured Human Tonsillar Mononuclear Cells in IgA Nephropathy

    PubMed Central

    Ye, Muyao; Liu, Chan; Yan, Wenzhe; Peng, Xiaofei; He, Liyu; Liu, Hong; Liu, Fuyou

    2016-01-01

    Objective. To investigate the influence of in vitro vibratory stimulation of human tonsillar mononuclear cells (TMCs). Methods. Fourteen IgA nephropathy (IgAN) patients with chronic tonsillitis (CT) and 12 CT patients with no renal pathology were enrolled. Group A TMCs were collected after 24 hours of culture and used to determine baseline levels. TMCs in groups B, C, D, E, and F were exposed to vibratory stimulation (60 Hz) for 0 (as the control group), 1, 3, 5, and 10 minutes, respectively. Results. Baseline concentrations of B-cell-activation factor (BAFF) and IgA1, BAFF mRNA expression, and aberrant O-glycosylation IgA1 level were higher in the IgAN group as compared to that in the CT group, and all increased after vibratory stimulation. Baseline mRNA expressions of core β1,3-galactosyltransferase (C1GALT1) and core β1,3GalT-specific molecular chaperone (Cosmc) were lower in the IgAN group; the levels decreased further after vibratory stimulation. Conclusion. In patients with IgAN, vibratory stimulation of TMCs appears to induce IgA1 secretion through activation of BAFF release and to aberrant O-glycosylation IgA1 by suppressing C1GALT1 and Cosmc expression. In vitro vibratory stimulation of human TMCs mimics the vibratory simulation of palatine tonsils produced by vocal cords during phonation.

  15. Stable Delivery of CCR5-Directed shRNA into Human Primary Peripheral Blood Mononuclear Cells and Hematopoietic Stem/Progenitor Cells via a Lentiviral Vector

    PubMed Central

    Shimizu, Saki; Yadav, Swati Seth; An, Dong Sung

    2016-01-01

    RNAi is a powerful tool to achieve suppression of a specific gene expression and therefore it has tremendous potential for gene therapy applications. A number of vector systems have been developed to express short-hairpin RNAs (shRNAs) to produce siRNAs within mammalian T-cells, primary hematopoietic stem/progenitor cells (HSPC), human peripheral blood mononuclear cells, and in animal model systems. Among these, vectors based on lentivirus backbones have significantly transformed our ability to transfer shRNAs into nondividing cells, such as HSPC, resulting in high transduction efficiencies. However, delivery and long-term expression of shRNAs should be carefully optimized for efficient knock down of target gene without causing cytotoxicity in mammalian cells. Here, we describe our protocols for the development of shRNA against a major HIV co-receptor/chemokine receptor CCR5 and the use of lentiviral vectors for stable shRNA delivery and expression in primary human PBMC and HSPC. PMID:26472455

  16. Proliferation and TH1/TH2 Cytokine Production in Human Peripheral Blood Mononuclear Cells after Treatment with Cypermethrin and Mancozeb In Vitro

    PubMed Central

    Mandarapu, Rajesh; Ajumeera, Rajanna; Venkatesan, Vijayalakshmi; Prakhya, Balakrishna Murthy

    2014-01-01

    In recent times, human cell-based assays are gaining attention in assessments of immunomodulatory effects of chemicals. In the study here, the possible effects of cypermethrin and mancozeb on lymphocyte proliferation and proinflammatory (tumor necrosis factor (TNF-) α) and immunoregulatory cytokine (interferon- (IFN-) γ, interleukins (IL) 2, 4, 6, and 10) formation in vitro were investigated. Human peripheral blood mononuclear cells (PBMC) were isolated and exposed for 6 hr to noncytotoxic doses (0.45–30 µM) of cypermethrin or mancozeb in the presence of activating rat S9 fraction. Cultures were then further incubated for 48 or 72 hr in fresh medium containing phytohemagglutinin (10 µg/mL) to assess, respectively, effects on cell proliferation (BrdU-ELISA method) and cytokine formation (flow cytometric bead immunoassays). Mancozeb induced dose-dependent increases in lymphocyte proliferation, inhibition of production of TNFα and the TH2 cytokines IL-6 and IL-10, and an increase in IFNγ (TH1 cytokine) production (at least 2-fold compared to control); mancozeb also induced inhibition of IL-4 (TH2) and stimulated IL-2 (TH1) production, albeit only in dose-related manners for each. In contrast, cypermethrin exposure did not cause significant effects on proliferation or cytokine profiles. Further studies are needed to better understand the functional significance of our in vitro findings. PMID:25328518

  17. Proliferation and TH1/TH2 cytokine production in human peripheral blood mononuclear cells after treatment with cypermethrin and mancozeb in vitro.

    PubMed

    Mandarapu, Rajesh; Ajumeera, Rajanna; Venkatesan, Vijayalakshmi; Prakhya, Balakrishna Murthy

    2014-01-01

    In recent times, human cell-based assays are gaining attention in assessments of immunomodulatory effects of chemicals. In the study here, the possible effects of cypermethrin and mancozeb on lymphocyte proliferation and proinflammatory (tumor necrosis factor (TNF-) α) and immunoregulatory cytokine (interferon- (IFN-) γ, interleukins (IL) 2, 4, 6, and 10) formation in vitro were investigated. Human peripheral blood mononuclear cells (PBMC) were isolated and exposed for 6 hr to noncytotoxic doses (0.45-30 µM) of cypermethrin or mancozeb in the presence of activating rat S9 fraction. Cultures were then further incubated for 48 or 72 hr in fresh medium containing phytohemagglutinin (10 µg/mL) to assess, respectively, effects on cell proliferation (BrdU-ELISA method) and cytokine formation (flow cytometric bead immunoassays). Mancozeb induced dose-dependent increases in lymphocyte proliferation, inhibition of production of TNFα and the TH2 cytokines IL-6 and IL-10, and an increase in IFNγ (TH1 cytokine) production (at least 2-fold compared to control); mancozeb also induced inhibition of IL-4 (TH2) and stimulated IL-2 (TH1) production, albeit only in dose-related manners for each. In contrast, cypermethrin exposure did not cause significant effects on proliferation or cytokine profiles. Further studies are needed to better understand the functional significance of our in vitro findings. PMID:25328518

  18. Investigating the specific core genetic-and-epigenetic networks of cellular mechanisms involved in human aging in peripheral blood mononuclear cells

    PubMed Central

    Li, Cheng-Wei; Wang, Wen-Hsin; Chen, Bor-Sen

    2016-01-01

    Aging is an inevitable part of life for humans, and slowing down the aging process has become a main focus of human endeavor. Here, we applied a systems biology approach to construct protein-protein interaction networks, gene regulatory networks, and epigenetic networks, i.e. genetic and epigenetic networks (GENs), of elderly individuals and young controls. We then compared these GENs to extract aging mechanisms using microarray data in peripheral blood mononuclear cells, microRNA (miRNA) data, and database mining. The core GENs of elderly individuals and young controls were obtained by applying principal network projection to GENs based on Principal Component Analysis. By comparing the core networks, we identified that to overcome the accumulated mutation of genes in the aging process the transcription factor JUN can be activated by stress signals, including the MAPK signaling, T-cell receptor signaling, and neurotrophin signaling pathways through DNA methylation of BTG3, G0S2, and AP2B1 and the regulations of mir-223 let-7d, and mir-130a. We also address the aging mechanisms in old men and women. Furthermore, we proposed that drugs designed to target these DNA methylated genes or miRNAs may delay aging. A multiple drug combination comprising phenylalanine, cholesterol, and palbociclib was finally designed for delaying the aging process. PMID:26895224

  19. Vibration Induces BAFF Overexpression and Aberrant O-Glycosylation of IgA1 in Cultured Human Tonsillar Mononuclear Cells in IgA Nephropathy

    PubMed Central

    Ye, Muyao; Liu, Chan; Yan, Wenzhe; Peng, Xiaofei; He, Liyu; Liu, Hong; Liu, Fuyou

    2016-01-01

    Objective. To investigate the influence of in vitro vibratory stimulation of human tonsillar mononuclear cells (TMCs). Methods. Fourteen IgA nephropathy (IgAN) patients with chronic tonsillitis (CT) and 12 CT patients with no renal pathology were enrolled. Group A TMCs were collected after 24 hours of culture and used to determine baseline levels. TMCs in groups B, C, D, E, and F were exposed to vibratory stimulation (60 Hz) for 0 (as the control group), 1, 3, 5, and 10 minutes, respectively. Results. Baseline concentrations of B-cell-activation factor (BAFF) and IgA1, BAFF mRNA expression, and aberrant O-glycosylation IgA1 level were higher in the IgAN group as compared to that in the CT group, and all increased after vibratory stimulation. Baseline mRNA expressions of core β1,3-galactosyltransferase (C1GALT1) and core β1,3GalT-specific molecular chaperone (Cosmc) were lower in the IgAN group; the levels decreased further after vibratory stimulation. Conclusion. In patients with IgAN, vibratory stimulation of TMCs appears to induce IgA1 secretion through activation of BAFF release and to aberrant O-glycosylation IgA1 by suppressing C1GALT1 and Cosmc expression. In vitro vibratory stimulation of human TMCs mimics the vibratory simulation of palatine tonsils produced by vocal cords during phonation. PMID:27672662