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Sample records for isolating cystinotic leukocytes

  1. Tissue Digestion for Stromal Cell and Leukocyte Isolation.

    PubMed

    Nayar, Saba; Campos, Joana; Steinthal, Nathalie; Barone, Francesca

    2017-01-01

    Tissue mechanical disruption is often not sufficient to disrupt cell-to-cell interactions; this is particularly relevant for stromal cells that are embedded within the extracellular matrix. For this reason, different enzyme combinations have been described to enable the isolation of single-cell populations, particularly stromal cells. This chapter aims to describe different methods used for enzymatic digestion of stromal cell and leukocyte populations from secondary and tertiary lymphoid organs. Collagenase D and P and collagenase D and dispase protocols provide a good yield of stromal cells, while a collagenase dispase-only protocol should be used if the main aim of the technique is to retrieve leukocyte populations. However, for isolation of both stroma and leukocyte populations the collagenase D and P protocol would provide the best results. Protocols for these techniques and illustrative results from flow cytometry analysis can be found in this chapter.

  2. Kinetics of reversible-sequestration of leukocytes by the isolated perfused rat lung

    SciTech Connect

    Goliaei, B.

    1980-08-01

    The kinetics and morphology of sequestration and margination of rat leukocytes were studied using an isolated perfused and ventilated rat lung preparation. Whole rat blood, bone marrow suspension, or leukocyte suspensions, were used to perfuse the isolated rat lung. The lung was also perfused with latex particle suspensions and the passage of particles through the lung capillaries was studied. When a leukocyte suspension was perfused through the lung in the single-pass mode, the rate of sequestration decreased as more cells were perfused. In contrast, latex particles of a size comparable to that of leukocytes were totally stopped by the lung. When the leukocyte suspension was recirculated through the lung, cells were rapidly removed from circulation until a steady state was reached, after which no net removal of cells by the lung occurred. These results indicate that leukocytes are reversibly sequestered from circulation. The sequestered cells marginated and attached to the luminal surface of the endothelium of post-capillary venules and veins. A mathematical model was developed based on the assumption that the attachment and detachment of leukocytes to blood vessel walls follows first-order kinetics. The model correctly predicts the following characteristics of the system: (a) the kinetics of the sequestration of leukocytes by the lung; (b) the existence of a steady state when a suspension of leukocytes is recirculated through the lung; and (c) the independence of the fraction of cells remaining in circulation from the starting concentration for all values of starting concentration. (ERB)

  3. Myeloid differentiation architecture of leukocyte transcriptome dynamics in perceived social isolation

    PubMed Central

    Cole, Steven W.; Capitanio, John P.; Chun, Katie; Arevalo, Jesusa M. G.; Ma, Jeffrey; Cacioppo, John T.

    2015-01-01

    To define the cellular mechanisms of up-regulated inflammatory gene expression and down-regulated antiviral response in people experiencing perceived social isolation (loneliness), we conducted integrative analyses of leukocyte gene regulation in humans and rhesus macaques. Five longitudinal leukocyte transcriptome surveys in 141 older adults showed up-regulation of the sympathetic nervous system (SNS), monocyte population expansion, and up-regulation of the leukocyte conserved transcriptional response to adversity (CTRA). Mechanistic analyses in a macaque model of perceived social isolation confirmed CTRA activation and identified selective up-regulation of the CD14++/CD16− classical monocyte transcriptome, functional glucocorticoid desensitization, down-regulation of Type I and II interferons, and impaired response to infection by simian immunodeficiency virus (SIV). These analyses identify neuroendocrine-related alterations in myeloid cell population dynamics as a key mediator of CTRA transcriptome skewing, which may both propagate perceived social isolation and contribute to its associated health risks. PMID:26598672

  4. AN IMPROVED CELL ISOLATION METHOD FOR FLOW CYTOMETRIC AND FUNCTIONAL ANALYSES OF CUTANEOUS WOUND LEUKOCYTES

    PubMed Central

    Brubaker, Aleah L.; Schneider, David F.; Palmer, Jessica L.; Faunce, Douglas E.; Kovacs, Elizabeth J.

    2011-01-01

    Isolation of leukocytes from full-thickness excisional wounds has proven to be a difficult process that results in poor cell yield and holds significant limitations for functional assays. Given the increased interest in the isolation, characterization and functional measurements of wound-derived cell populations, herein we describe a method for preparing wound cell suspensions with an improved yield that enables both phenotypic and functional assessments. PMID:21889511

  5. New lanostanes and naphthoquinones isolated from Antrodia salmonea and their antioxidative burst activity in human leukocytes.

    PubMed

    Shen, Chien-Chang; Shen, Yuh-Chiang; Wang, Yea-Hwey; Lin, Lie-Chwen; Don, Ming-Jaw; Liou, Kuo-Tong; Wang, Wen-Yen; Hou, Yu-Chang; Chang, Tun-Tschu

    2006-02-01

    Four new compounds were isolated from the basidiomata of the fungus Antrodia salmonea, a newly identified species of Antrodia (Aphyllophorales) in Taiwan. These new compounds are named as lanosta-8,24-diene-3beta,15alpha,21-triol (1), 24-methylenelanost-8-ene-3beta,15alpha,21-triol (2), 2,3-dimethoxy-5-(2',5'-dimethoxy-3',4'-methylenedioxyphenyl)-7-methyl-[1,4]-naphthoquinone (3), and 2,3-dimethoxy-6-(2',5'-dimethoxy-3',4'-methylenedioxyphenyl)-7-methyl-[1,4]-naphthoquinone (4), respectively. Their structures were elucidated by spectroscopic methods. An in vitro cellular functional assay was performed to evaluate their anti-oxidative burst activity in human leukocytes. They showed inhibitory effects against phorbol 12-myristate-13-acetate (PMA), a direct protein kinase C activator, induced oxidative burst in neutrophils (PMN) and mononuclear cells (MNC) with 50 % inhibitory concentration (IC(50)) ranging from 3.5 to 25.8 microM. The potency order of these compounds in PMA-activated leukocytes was as 1 > 3 > 4 > 2. They were relatively less effective in formyl-Met-Leu-Phe (fMLP), a G-protein coupled receptor agonist, induced oxidative burst, except for compounds 3 and 4 in fMLP-activated PMN. These results indicated that three (1, 3, and 4) of these four newly identified compounds displayed anti-oxidative effect in human leukocytes with different potency and might confer anti-inflammatory activity to these drugs.

  6. Development of a method for isolating bovine colostrum mononuclear leukocytes for phenotyping and functional studies.

    PubMed

    Meganck, Vanessa; Goddeeris, Bruno M; Stuyven, Edith; Piepers, Sofie; Cox, Eric; Opsomer, Geert

    2014-05-01

    The present study reports a method for isolating bovine colostrum mononuclear cells (CMC) for phenotyping and functional studies. As well as being an important source of immunoglobulins, colostrum also contains leukocytes that may be of greater importance for passive immunity than has previously been thought. Different protocols have been reported for isolating leukocytes from bovine colostrum, although none of these have been validated, and phenotypic analysis of cell populations has not always been performed. In this study, bovine CMC were isolated by density gradient centrifugation. Cell populations were identified by flow cytometry using antibodies against selected bovine cell surface markers and the proliferative capacity of these cells was determined using a (3)H-thymidine proliferation assay. The mean cell count of isolated CMC was 3 × 10(4) and 1 × 10(5) per mL colostrum for the samples used in the flow cytometric assay and the proliferation assay, respectively. A mean of 25.4 ± 17.1% CMC were identified as T lymphocytes, 2.9 ± 3.0% as B lymphocytes and 32.7 ± 13.7% as macrophages. In terms of proliferation, the mean counts per minute were 4.3 × 10(3) and 1.8 × 10(4) for cells cultured in medium only or in the presence of concanavalin A, respectively, showing that CMC are viable and capable of responding to mitogen stimulation. Isolation of CMC and the subsequent phenotypic analysis of the different subpopulations were repeatable, with agreement indices varying between 0.5 and 1.0. Agreement indices for the proliferation assay were estimated at 0.8.

  7. Substrate-Specific Reduction of Tetrazolium Salts by Isolated Mitochondria, Tissues, and Leukocytes.

    PubMed

    Fedotcheva, N I; Litvinova, E G; Zakharchenko, M V; Khunderyakova, N V; Fadeev, R S; Teplova, V V; Fedotcheva, T A; Beloborodova, N V; Kondrashova, M N

    2017-02-01

    Tetrazolium salts are commonly used in cytochemical and biochemical studies as indicators of metabolic activity of cells. Formazans, formed by reduction of tetrazolium salts, behave as pseudo-solutions during initial incubation, which allows monitoring their optical density throughout incubation. The criteria and conditions for measuring oxidative activity of mitochondria and dehydrogenase activity in reduction of nitroblue tetrazolium (NBT) and methyl thiazolyl tetrazolium (MTT) in suspensions of isolated mitochondria, tissue homogenates, and leukocytes were investigated in this work. We found that the reduction of these two acceptors depended on the oxidized substrate - NBT was reduced more readily during succinate oxidation, while MTT - during oxidation of NAD-dependent substrates. Reduction of both acceptors was more sensitive to dehydrogenase inhibitors that to respiratory chain inhibitors. The reduction of NBT in isolated mitochondria, in leukocytes in the presence of digitonin, and in liver and kidney homogenates was completely blocked by succinate dehydrogenase inhibitors - malonate and TTFA. Based on these criteria, activation of succinate oxidation was revealed from the increase in malonate-sensitive fraction of the reduced NBT under physiological stress. The effect of progesterone and its synthetic analogs on oxidation of NAD-dependent substrates by mitochondria was investigated using MTT. Both acceptors are also reduced by superoxide anion; the impact of this reaction is negligible or completely absent under physiological conditions, but can become detectable on generation of superoxide induced by inhibitors of individual enzyme complexes or in the case of mitochondrial dysfunction. The results indicate that the recording of optical density of reduced NBT and MTT is a highly sensitive method for evaluation of metabolic activity of mitochondria applicable for different incubation conditions, it offers certain advantages in comparison with other methods

  8. Cysteamine restores glutathione redox status in cultured cystinotic proximal tubular epithelial cells.

    PubMed

    Wilmer, Martijn J; Kluijtmans, Leo A J; van der Velden, Thea J; Willems, Peter H; Scheffer, Peter G; Masereeuw, Rosalinde; Monnens, Leo A; van den Heuvel, Lambertus P; Levtchenko, Elena N

    2011-06-01

    Recent evidence implies that impaired metabolism of glutathione has a role in the pathogenesis of nephropathic cystinosis. This recessive inherited disorder is characterized by lysosomal cystine accumulation and results in renal Fanconi syndrome progressing to end stage renal disease in the majority of patients. The most common treatment involves intracellular cystine depletion by cysteamine, delaying the development of end stage renal disease by a yet elusive mechanism. However, cystine depletion does not arrest the disease nor cures Fanconi syndrome in patients, indicating involvement of other yet unknown pathologic pathways. Using a newly developed proximal tubular epithelial cell model from cystinotic patients, we investigate the effect of cystine accumulation and cysteamine on both glutathione and ATP metabolism. In addition to the expected increase in cystine and defective sodium-dependent phosphate reabsorption, we observed less negative glutathione redox status and decreased intracellular ATP levels. No differences between control and cystinosis cell lines were observed with respect to protein turnover, albumin uptake, cytosolic and mitochondrial ATP production, total glutathione levels, protein oxidation and lipid peroxidation. Cysteamine treatment increased total glutathione in both control and cystinotic cells and normalized cystine levels and glutathione redox status in cystinotic cells. However, cysteamine did not improve decreased sodium-dependent phosphate uptake. Our data implicate that cysteamine increases total glutathione and restores glutathione redox status in cystinosis, which is a positive side-effect of this agent next to cystine depletion. This beneficial effect points to a potential role of cysteamine as anti-oxidant for other renal disorders associated with enhanced oxidative stress.

  9. Handling of L-(/sup 35/S)cystine by cysteamine-pretreated cystinotic and normal fibroblasts

    SciTech Connect

    States, B.; Lee, J.; Segal, S.

    1983-02-01

    In short incubations with 0.1 mM L-(/sup 35/S)cystine in phosphate-buffered saline medium, and long incubations with label in complete minimum Eagle's medium with Earle salts, cystine-depleted cystinotic cells reaccumulate labeled cystine more rapidly than pretreated normal cells. Cysteamine pretreatment of both normal and cystinotic cells resulted in an initial increased conversion of exogenous cystine to intracellular cysteine. In 24-h incubations in complete medium, cysteamine-pretreated cells showed enhanced conversion of 0.1 mM L-(/sup 35/S)cystine to cysteine and reduced glutathione. Addition of cycloheximide to the incubation media decreased the incorporation of /sup 35/S into cellular protein by more than 90% but did not affect the accumulation of intracellular labeled cystine in cystinotic cells. Therefore, the incorporation and release of cystine from protein is not an obligatory source of accumulated cystine and researchers speculate that there may be early extralysosomal entrapment of cystine in cystinotic cells.

  10. 520-d Isolation and confinement simulating a flight to Mars reveals heightened immune responses and alterations of leukocyte phenotype.

    PubMed

    Yi, B; Rykova, M; Feuerecker, M; Jäger, B; Ladinig, C; Basner, M; Hörl, M; Matzel, S; Kaufmann, I; Strewe, C; Nichiporuk, I; Vassilieva, G; Rinas, K; Baatout, S; Schelling, G; Thiel, M; Dinges, D F; Morukov, B; Choukèr, A

    2014-08-01

    During interplanetary exploration, chronic stress caused by long term isolation and confinement in the spacecraft is one of the major concerns of physical and psychological health of space travelers. And for human on Earth, more and more people live in an isolated condition, which has become a common social problem in modern western society. Collective evidences have indicated prolonged chronic stress could bring big influence to human immune function, which may lead to a variety of health problems. However, to what extent long-term isolation can affect the immune system still remains largely unknow. A simulated 520-d Mars mission provided an extraordinary chance to study the effect of prolonged isolation. Six healthy males participated in this mission and their active neuroendocrine and immune conditions were studied with saliva and blood samples from all participants on chosen time points during the isolation period. As a typical neuroendocrine parameter, stress hormone cortisol was measured in the morning saliva samples. Immune phenotype changes were monitored through peripheral leukocyte phenotype analysis. Using an ex vivo viral infection simulation assay we assessed the immune response changes characterized by the ability to produce representative endogenous pro-inflammatory cytokines. The results of this study revealed elevated cortisol levels, increased lymphocyte amount and heightened immune responses, suggesting that prolonged isolation acting as chronic stressors are able to trigger leukocyte phenotype changes and poorly controlled immune responses.

  11. Autochthonous Bacterial Isolates Successfully Stimulate In vitro Peripheral Blood Leukocytes of the European Sea Bass (Dicentrarchus labrax)

    PubMed Central

    Mladineo, Ivona; Bušelić, Ivana; Hrabar, Jerko; Radonić, Ivana; Vrbatović, Anamarija; Jozić, Slaven; Trumbić, Željka

    2016-01-01

    Commercially available probiotics are routinely administered as feed supplements in aquaculture important species. Among them, the European sea bass (Dicentrarchus labrax) is the most widely reared fish in the Mediterranean, whose rearing systems are highly variable between countries, affecting at some level the sustainability of production. After random isolation of autochthonous gut bacteria of the sea bass, their identification and pathogenicity testing, we have selected three potentially probiotic isolates; Pseudoalteromonas sp., Alteromonas sp., and Enterovibrio coralii. Selected isolates were tested and their immunostimulative efficiency was compared with a commercially available Lactobacillus casei isolate, inferring inflammatory, apoptotic and anti-pathogen response of sea bass’ peripheral blood leukocytes. Phagocytic activity, respiratory burst, and expression of lysozyme, Mx protein, caspase 3, TNF-α, IL-10 genes was measured 1, 3, 5, and 12 h post-stimulation by four bacterial isolates to evaluate early kinetics of the responses. Best immunostimulative properties were observed in Pseudoalteromonas-stimulated leukocytes, followed by Alteromonas sp. and L. casei, while Enterovibrio coralii failed to induce significant stimulation. Based on such in vitro assay intestinal autochthonous bacterial isolates showed to have better immunostimulative effect in sea bass compared to aquaculture-widely used L. casei, and further steps need to engage tank and field feeding trials to evaluate long-term prophylactic suitability of the chosen isolates. A panel of biomarkers that represent pro-/anti-inflammatory, pro-/anti-apoptotic, and anti-bacteria/viral responses of the fish should be taken into consideration when evaluating the usefulness of the potential probiotic in aquaculture. PMID:27551281

  12. Pseudomonas aeruginosa variants isolated from patients with cystic fibrosis are killed by a bactericidal protein from human polymorphonuclear leukocytes.

    PubMed Central

    Siefferman, C M; Regelmann, W E; Gray, B H

    1991-01-01

    The susceptibility of paired mucoid and nonmucoid variants of Pseudomonas aeruginosa isolated from 13 patients with cystic fibrosis (CF) to killing by a 55,000-Da bactericidal protein (BP55) from human polymorphonuclear leukocytes was studied. Mucoid and nonmucoid variants were equally sensitive to killing by BP55 at both pH 5.6 and pH 7.2. Eleven of the isolates were resistant to the bactericidal activity of 10% normal human serum but were as sensitive as the serum-sensitive isolates to BP55. Similarly, the 15 isolates with lipopolysaccharides (LPS) containing O-polysaccharide side chains (smooth LPS) were as sensitive to BP55 as those isolates with rough LPS.P. aeruginosa isolates from patients in poor clinical condition were more likely to have LPS of the smooth type and to be resistant to killing by 10% human serum than the isolates from patients in good clinical condition. We have concluded that the susceptibility of the P. aeruginosa isolates from patients with CF to killing by BP55 does not correlate with mucoid or nonmucoid variations, with the presence or absence of smooth LPS, or with the sensitivity or resistance to killing by normal human serum. Images PMID:1903774

  13. Cryopreservation of Human Mucosal Leukocytes

    PubMed Central

    Shu, Zhiquan; Levy, Claire N.; Ferre, April L.; Hartig, Heather; Fang, Cifeng; Lentz, Gretchen; Fialkow, Michael; Kirby, Anna C.; Adams Waldorf, Kristina M.; Veazey, Ronald S.; Germann, Anja; von Briesen, Hagen; McElrath, M. Juliana; Dezzutti, Charlene S.; Sinclair, Elizabeth; Baker, Chris A. R.; Shacklett, Barbara L.; Gao, Dayong; Hladik, Florian

    2016-01-01

    Background Understanding how leukocytes in the cervicovaginal and colorectal mucosae respond to pathogens, and how medical interventions affect these responses, is important for developing better tools to prevent HIV and other sexually transmitted infections. An effective cryopreservation protocol for these cells following their isolation will make studying them more feasible. Methods and Findings To find an optimal cryopreservation protocol for mucosal mononuclear leukocytes, we compared cryopreservation media and procedures using human vaginal leukocytes and confirmed our results with endocervical and colorectal leukocytes. Specifically, we measured the recovery of viable vaginal T cells and macrophages after cryopreservation with different cryopreservation media and handling procedures. We found several cryopreservation media that led to recoveries above 75%. Limiting the number and volume of washes increased the fraction of cells recovered by 10–15%, possibly due to the small cell numbers in mucosal samples. We confirmed that our cryopreservation protocol also works well for both endocervical and colorectal leukocytes. Cryopreserved leukocytes had slightly increased cytokine responses to antigenic stimulation relative to the same cells tested fresh. Additionally, we tested whether it is better to cryopreserve endocervical cells on the cytobrush or in suspension. Conclusions Leukocytes from cervicovaginal and colorectal tissues can be cryopreserved with good recovery of functional, viable cells using several different cryopreservation media. The number and volume of washes has an experimentally meaningful effect on the percentage of cells recovered. We provide a detailed, step-by-step protocol with best practices for cryopreservation of mucosal leukocytes. PMID:27232996

  14. Structural characterization and in vitro inhibitory activities in P-selectin-mediated leukocyte adhesion of polysaccharide fractions isolated from the roots of Physalis alkekengi.

    PubMed

    Tong, Haibin; Wang, Ruifei; Liu, Ximing; Wang, Guiyun; Du, Fengguo; Zeng, Xianlu

    2011-08-01

    Selectin-mediated leukocyte initial attachment and rolling over vessel endothelial surface are crucial steps for inflammatory responses. As P-selectin is a promising target for anti-inflammation therapeutic strategy, recent works have focused on searching for more potent and non-toxic P-selectin antagonists among various natural carbohydrate products. Here, we isolated three water-soluble polysaccharide fractions (PPS-1, PPS-2 and PPS-3) from the roots of Physalis alkekengi by DEAE-cellulose and Sephacryl S-200 chromatography. Their physicochemical and structural characterizations were determined by chemical methods, GC (gas chromatography), HPLC (high performance liquid chromatography), FT-IR (Fourier transform infrared spectrometry), partial acid hydrolysis, methylation and GC-MS (gas chromatography-mass spectrometry) analyses. The inhibitory capacity of the polysaccharide fractions in P-selectin-mediated leukocyte adhesion was evaluated by flow cytometric, static adhesion and laminar flow assays. Results showed that different polysaccharide fractions possess distinct physicochemical and structural properties, including carbohydrate, protein and uronic acid contents, molecular weight, monosaccharide composition and glycosidic linkage type. Among the polysaccharide fractions, PPS-2 could effectively block the interaction between P-selectin and its native ligand.

  15. Inhibition of p38 MAPK during cellular activation modulate gene expression of head kidney leukocytes isolated from Atlantic salmon (Salmo salar) fed soy bean oil or fish oil based diets.

    PubMed

    Holen, E; Winterthun, S; Du, Z-Y; Krøvel, A V

    2011-01-01

    Head kidney leukocytes isolated from Atlantic salmon fed either a diet based on fish oil (FO) or soy bean oil (VO) were used in order to evaluate if different lipid sources could contribute to cellular activation of the salmon innate immune system. A specific inhibitor of p38 MAPK, SB202190, was used to investigate the effect of lipopolysaccharide (LPS) signalling in the head kidney leukocytes. The results show that LPS up regulate IL-1β, TNF-α, Cox2 expression in leukocytes isolated from fish fed either diet. The p38 MAPK inhibitor, SB202190, reduced the LPS induced expression of these genes in both dietary groups. In LPS stimulated leukocytes isolated from VO fed fish, SB202190 showed a clear dose dependent inhibitory effect on IL-1β, TNF-α and Cox2 expression. This effect was also observed for Cox2 in leukocytes isolated from FO fed fish. Furthermore, there was a stronger mean induction of Cox2 in LPS stimulated leucocytes isolated from the VO-group compared to LPS stimulated leukocytes isolated from the FO-group. In both dietary groups, LPS stimulation of salmon head kidney leukocytes increased the induction of CD83, a dendrite cell marker, while the inhibitor reduced CD83 expression in the VO fed fish only. The inhibitor also clearly reduced hsp27 expression in VO fed fish. Indicating a p38 MAPK feedback loop, LPS significantly inhibited the expression of p38MAPK itself in both diets, while SB202190 increased p38MAPK expression especially in the VO diet group. hsp70 expression was not affected by any treatment or feed composition. There were also differences in p38MAPK protein phosphorylation comparing treatment groups but no obvious difference comparing the two dietary groups. The results indicate that dietary fatty acids have the ability to modify signalling through p38 MAPK which may have consequences for the fish's ability to handle infections and stress. Signalling through p38MAPK is ligand dependent and affects gene and protein expression differently.

  16. Specific and azurophilic granules from rabbit polymorphonuclear leukocytes. I. Isolation and characterization of membrane and content subfractions

    PubMed Central

    1983-01-01

    The specific and azurophilic granules of rabbit polymorphonuclear heterophils (PMNs) have been isolated and fractionated into membrane and extractable subfractions. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) revealed several features of the protein composition of the two granules: (a) Whereas each type of granule had 40-60 proteins separable on one-dimensional gradient gels, few of the proteins were common to both granules. (b) The proteins of the extractable fractions (which comprised approximately 98% of the total granule protein) of each granule were distinct from the proteins of the membrane fractions (which comprised approximately 2% of the total granule protein). (c) The extractable proteins co- migrated with those collected from the medium of ionophore-treated, degranulating PMNs and therefore were defined as content proteins. These results were confirmed by radiolabeling studies. Lactoperoxidase- catalyzed iodination of intact granules did not label the content proteins but did label proteins that co-migrated with major granule membrane proteins. Moreover, disruption of the granules before iodination led to labeling of both content and membrane proteins. We conclude that the membranes of specific and azurophilic granules, which arise from different faces of the Golgi complex, are composed of unique sets of membrane proteins some of which are exposed on the cytoplasmic face of the granules. PMID:6833388

  17. cDNA sequence and tissue expression of an antimicrobial peptide, dicentracin; a new component of the moronecidin family isolated from head kidney leukocytes of sea bass, Dicentrarchus labrax.

    PubMed

    Salerno, Giuseppina; Parrinello, Nicolò; Roch, Philippe; Cammarata, Matteo

    2007-04-01

    A 483-bp cDNA was isolated from sea bass (Dicentrarchus labrax) head kidney leukocytes, dicentracin, using PCR primers designed from conserved moronecidin domains. Gene bank analysis revealed that dicentracin cDNA belongs to the moronecidin family. As deduced from alignment with Morone chrysops moronecidin, the precursor of 79 aa appeared to be composed of a signal peptide of 22 aa, followed by the mature AMP (antimicrobial peptide) of 22 aa named dicentracin, and a C-terminal extension of 35 aa. Dicentracin precursor displayed 3 aa substitutions with other moronecidin sequence but none in the mature peptide sequence. Using in situ hybridization assay, dicentracin gene expression was observed in 68-71% of peripheral blood leukocytes, kidney leukocytes or peritoneal cavity leukocytes without significant statistical differences. Dicentracin mRNA was observed in most of the granulocytes, as well as in monocytes from both peripheral blood and head kidney, and in macrophages from peritoneal cavity. No expression was observed in thrombocytes or in lymphocytes.

  18. Cell culture conditions potentiate differences in the response to ionising radiation of peripheral blood leukocytes isolated from breast cancer patients and healthy subjects.

    PubMed

    Adzić, Miroslav; Nićiforović, Ana; Zarić, Bozidarka; Nesković-Konstantinović, Zora; Spasić, Snezana D; Jones, David R; Radojcić, Marija B

    2008-01-01

    To compare the effects of ionising radiation on leukocytes from breast cancer patients and healthy subjects ex vivo, the level of NF-kappaB and the antioxidant enzymes manganese-containing superoxide dismutase (Mn-SOD), copper/zinc-containing superoxide dismutase (CuZn-SOD) and catalase (CAT) in combination with flow cytometric analysis of CD4+ lymphocytes was performed. The level of Mn-SOD protein was significantly increased in the breast cancer study group both before (P < 0.001) and after (P < 0.001) irradiation when compared with healthy subjects. Measurements in parallel indicated that the level of CAT protein was significantly higher in the breast cancer study group after irradiation (2 Gy [P < 0.001] and 9 Gy [P < 0.05]) when compared with healthy subjects. Although the initial number of lymphocytes in the blood of breast cancer patients was not different from healthy subjects, the percentage of apoptotic CD4+ cells was significantly (P < 0.001) lower both before and after irradiation indicating that cell culture conditions induced radioresistance of CD4+ cells in the blood of breast cancer patients. The data presented in this current study indicate that brief ex vivo culture of peripheral blood leukocytes potentiates oxidative stress imposed by a breast cancer tumour.

  19. Towards a computational model of leukocyte adhesion cascade: Leukocyte rolling

    NASA Astrophysics Data System (ADS)

    Khismatullin, Damir

    2005-11-01

    Recruitment of leukocytes into sites of acute and chronic inflammation is a vital component of the innate immune response in humans and plays an important role in cardiovascular diseases, such as ischemia-reperfusion injury and atherosclerosis. Leukocytes extravasate into the inflamed tissue through a multi-step process called "leukocyte adhesion cascade", which involves initial contact of a leukocyte with activated endothelium (tethering), leukocyte rolling, firm adhesion, and transendothelial migration. Recently we developed a fully three-dimensional CFD model of receptor-mediated leukocyte adhesion to endothelium in a parallel-plate flow chamber. The model treats the leukocyte as a viscoelastic cell with the nucleus located in the intracellular space and cylindrical microvilli distributed over the cell membrane. Leukocyte-endothelial adhesion is assumed to be mediated by adhesion molecules expressed on the tips of cell microvilli and on endothelium. We show that the model can predict both shape changes and velocities of rolling leukocytes under physiological flow conditions. Results of this study also indicate that viscosity of the cytoplasm is a critical parameter of leukocyte adhesion, affecting the cell's ability to roll on endothelium. This work is supported by NIH Grant HL- 57446 and NCSA Grant BCS040006 and utilized the NCSA IBM p690.

  20. [Oxygen Leukocyte Larceny].

    PubMed

    Pinto da Costa, Miguel; Pimenta Coelho, Henrique

    2016-05-01

    The authors present a case of a 60-year-old male patient, previously diagnosed with B-cell chronic lymphocytic leukemia, who was admitted to the Emergency Room with dyspnea. The initial evaluation revealed severe anemia (Hgb = 5.0 g/dL) with hyperleukocytosis (800.000/µL), nearly all of the cells being mature lymphocytes, a normal chest X-ray and a low arterial oxygen saturation (89%; pulse oximetry). After red blood cell transfusion, Hgb values rose (9.0 g/dL) and there was a complete reversion of the dyspnea. Yet, subsequent arterial blood gas analysis, without the administration of supplemental oxygen, systematically revealed very low oxygen saturation values (~ 46%), which was inconsistent with the patientâs clinical state and his pulse oximetry values (~ 87%), and these values were not corrected by the administration of oxygen via non-rebreather mask. The investigation performed allowed to establish the diagnosis of oxygen leukocyte larceny, a phenomenon which conceals the true oxygen saturation due to peripheral consumption by leukocytes.

  1. Seminal and colostral protease inhibitors on leukocytes.

    PubMed

    Veselský, L; Cechová, D; Hruban, V; Klaudy, J

    1982-01-01

    For detection of protease inhibitors from cow colostrum (CTI) and bull seminal plasma (BUSI I and BUSI II) on the surface of leukocytes, immunological methods were used. An agglutination and an immunofluorescence test demonstrated components on the surface of bovine, porcine and ovine granulocytes and lymphocytes which were immunologically identical with the protease inhibitors isolated from cow colostrum and bull seminal plasma. When antisera against (CTI, BUSI and BUSI II were absorbed by bovine and porcine liver, kidney and spleen homogenate or by bovine and porcine granulocytes or lymphocytes, the immunological tests were negative.

  2. Photon Counts Statistics in Leukocyte Cell Dynamics

    NASA Astrophysics Data System (ADS)

    van Wijk, Eduard; van der Greef, Jan; van Wijk, Roeland

    2011-12-01

    In the present experiment ultra-weak photon emission/ chemiluminescence from isolated neutrophils was recorded. It is associated with the production of reactive oxygen species (ROS) in the "respiratory burst" process which can be activated by PMA (Phorbol 12-Myristate 13-Acetate). Commonly, the reaction is demonstrated utilizing the enhancer luminol. However, with the use of highly sensitive photomultiplier equipment it is also recorded without enhancer. In that case, it can be hypothesized that photon count statistics may assist in understanding the underlying metabolic activity and cooperation of these cells. To study this hypothesis leukocytes were stimulated with PMA and increased photon signals were recorded in the quasi stable period utilizing Fano factor analysis at different window sizes. The Fano factor is defined by the variance over the mean of the number of photon within the observation time. The analysis demonstrated that the Fano factor of true signal and not of the surrogate signals obtained by random shuffling increases when the window size increased. It is concluded that photon count statistics, in particular Fano factor analysis, provides information regarding leukocyte interactions. It opens the perspective to utilize this analytical procedure in (in vivo) inflammation research. However, this needs further validation.

  3. Selective Harvesting of Marginating-pulmonary Leukocytes.

    PubMed

    Shaashua, Lee; Sorski, Liat; Melamed, Rivka; Ben-Eliyahu, Shamgar

    2016-03-11

    Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.

  4. 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.

  5. p,p'-DDE depresses the immune competence of chinook salmon (Oncorhynchus tshawytscha) leukocytes

    USGS Publications Warehouse

    Misumi, Ichiro; Vella, Anthony T.; Leong, Jo-Ann C.; Nakanishi, Teruyuki; Schreck, Carl B.

    2005-01-01

    p,p′-DDE, the main metabolite of DDT, is still detected in aquatic environments throughout the world. Here, the effects and mechanisms by which p,p′-DDE exposure might affect the immune system of chinook salmon (Oncorhynchus tshawytscha) was studied. Isolated salmon splenic and pronephric leukocytes were incubated with different concentrations of p,p′-DDE, and cell viability, induction of apoptosis, and mitogenic responses were measured by flow cytometry and Alamar Blue assay. p,p′-DDE significantly reduced cell viability and proliferation and increased apoptosis. The effect of p,p′-DDE on pronephric leukocytes was more severe than on splenic leukocytes, likely because pronephric leukocytes had a higher proportion of granulocytes, cells that appear more sensitive to p,p′-DDE. The effect of p,p′-DDE on leukocytes appeared to vary between developmental stages or seasonal differences. The mitogenic response of leukocytes of chinook salmon exposed to p,p′-DDE in vivo exhibited a biphasic dose–response relationship. Only leukocytes isolated from salmon treated with 59 ppm p,p′-DDE had a significantly lower percentage of Ig+ blasting cells than controls, although the response was biphasic. These results support the theory that exposure to chemical contaminants could lead to an increase in disease susceptibility and mortality of fish due to immune suppression.

  6. Analysis of leukocyte binding to depletion filters: role of passive binding, interaction with platelets, and plasma components.

    PubMed

    Henschler, R; Rüster, B; Steimle, A; Hansmann, H L; Walker, W; Montag, T; Seifried, E

    2005-08-01

    Since limited knowledge exists on the mechanisms which regulate cell binding to leukocyte removal filter surfaces, we investigated the binding patterns of leukocytes to individual layers of leukocyte depletion filters. After passage of 1 unit of whole blood, blotting of isolated filter layers on glass slides or elution of cells from filter layers revealed that most leukocytes were located within the first 10 of a total of 28 filter layers, peaking at layers 6 to 8, with granulocytes binding on average to earlier filter layers than lymphocytes. Leukocytes preincubated with inhibitors of actin activation showed unchanged distribution between filter layers, suggesting that cytoskeletal activation does not significantly contribute to their binding. When leukocytes were directly incubated with single filter layers, binding of up to 30% of input cells was recorded in the absence of Ca(2+). Immunohistological analyses showed colocalization of platelets and leukocytes, with co-clustering of platelets and leukocytes. Monocytes and to some degree lymphocytes but not granulocytes competed with platelets for filter binding. Precoating of filter layers with individual plasma components showed that hyaluronic acid, plasma type fibronectin, and fibrinogen all increased the binding of leukocytes compared with albumin coating. In conclusion, leukocytes can bind passively to filters in a process which does not require Ca(2+), which is independent of cytoskeletal activation and which may depend on individual plasma components. These results are of importance when new selective cell enrichment or depletion strategies through specific filters are envisaged.

  7. Mechanisms of Leukocyte Transendothelial Migration

    PubMed Central

    Muller, William A.

    2013-01-01

    Neither the innate nor adaptive immune system “responds” unless leukocytes cross blood vessels. This process occurs through diapedesis, in which the leukocyte moves in an ameboid fashion through tightly apposed endothelial borders and, in some cases, through the endothelial cell itself. This review focuses on the active role of the endothelial cell in diapedesis. Several mechanisms play a critical role in transendothelial migration, including signals derived from clustering of apically disposed intercellular adhesion molecule 1 and vascular cell adhesion molecule 1, disruption or loosening of adherens junctions, and targeted recycling of platelet/endothelial cell adhesion molecule and other molecules from the recently described lateral border recycling compartment. Surprisingly, many of the same molecules and mechanisms that regulate paracellular migration also control transcellular migration. A hypothesis that integrates the various known mechanisms of transmigration is proposed. PMID:21073340

  8. Mechanisms of leukocyte transendothelial migration.

    PubMed

    Muller, William A

    2011-01-01

    Neither the innate nor adaptive immune system "responds" unless leukocytes cross blood vessels. This process occurs through diapedesis, in which the leukocyte moves in an ameboid fashion through tightly apposed endothelial borders and, in some cases, through the endothelial cell itself. This review focuses on the active role of the endothelial cell in diapedesis. Several mechanisms play a critical role in transendothelial migration, including signals derived from clustering of apically disposed intercellular adhesion molecule 1 and vascular cell adhesion molecule 1, disruption or loosening of adherens junctions, and targeted recycling of platelet/endothelial cell adhesion molecule and other molecules from the recently described lateral border recycling compartment. Surprisingly, many of the same molecules and mechanisms that regulate paracellular migration also control transcellular migration. A hypothesis that integrates the various known mechanisms of transmigration is proposed.

  9. Leukocyte integrins: Role in leukocyte recruitment and as therapeutic targets in inflammatory disease

    PubMed Central

    Kourtzelis, Ioannis; Ziogas, Athanassios; Hajishengallis, George; Chavakis, Triantafyllos

    2014-01-01

    Infection or sterile inflammation triggers site-specific attraction of leukocytes. Leukocyte recruitment is a process comprising several steps orchestrated by adhesion molecules, chemokines, cytokines and endogenous regulatory molecules. Distinct adhesive interactions between endothelial cells and leukocytes and signalling mechanisms contribute to the temporal and spatial fine-tuning of the leukocyte adhesion cascade. Central players in the leukocyte adhesion cascade include the leukocyte adhesion receptors of the β2-integrin family, such as the αLβ2 and αMβ2 integrins, or of the β1-integrin family, such as the α4β1- integrin. Given the central involvement of leukocyte recruitment in different inflammatory and autoimmune diseases, the leukocyte adhesion cascade in general, and leukocyte integrins in particular, represent key therapeutic targets. In this context, the present review focuses on the role of leukocyte integrins in the leukocyte adhesion cascade. Experimental evidence that has implicated leukocyte integrins as targets in animal models of inflammatory disorders, such as experimental autoimmune encephalomyelitis, psoriasis, inflammatory bone loss and inflammatory bowel disease as well as preclinical and clinical therapeutic applications of antibodies that target leukocyte integrins in various inflammatory disorders are presented. Finally, we review recent findings on endogenous inhibitors that modify leukocyte integrin function, which could emerge as promising therapeutic targets. PMID:25448040

  10. Dysfunction of polymorphonuclear leukocytes in uremia.

    PubMed

    Haag-Weber, M; Hörl, W H

    1996-05-01

    There is increased incidence of infectious complications in uremic patients, indicating impairment of cellular host defense in these patients. Several reports confirm metabolic and functional abnormalities of polymorphonuclear leukocytes (PMNL) including altered adherence to endothelial cells, altered generation of reactive oxygen species, altered release of microbial enzymes, impaired chemotaxis, phagocytosis, intracellular killing of bacteria, altered carbohydrate metabolism, and/or impaired ATP formation. Several studies report on correlations between PMNL dysfunction, especially phagocytosis and oxidative burst, and ferritin content. Deferoxamine therapy improved PMNL function. Chronic renal failure is a state of increased cytosolic calcium. Increased cytosolic calcium is associated with several alterations of PMNL function and metabolism, which improve by normalization of cytosolic calcium either by calcium channel blockers or by lowering of elevated parathyroid hormone. Each hemodialysis session using bioincompatible membranes triggers neutrophil activation, evidenced by overexpression of adhesion molecules, elevation of cytosolic calcium, release of PMNL granular enzymes, and generation of reactive oxygen species. Several studies claim that this results in chronic downregulation of phagocyte function. Several granulocyte inhibitory compounds have been isolated and characterized from uremic serum. The uremic retention product p-cresol depresses respiratory burst activity. The following granulocyte inhibitory peptides could be isolated from dialysis patients: granulocyte inhibitory protein I and II with homology to light chain proteins and beta 2-microglobulin, degranulation inhibitory protein I and II being identical to angiogenin and complement factor D, and immunoglobulin light chains. These proteins inhibit PMNL function in nanomolar concentrations.

  11. Leukocyte chemoattractant receptors in human disease pathogenesis.

    PubMed

    Zabel, Brian A; Rott, Alena; Butcher, Eugene C

    2015-01-01

    Combinations of leukocyte attractant ligands and cognate heptahelical receptors specify the systemic recruitment of circulating cells by triggering integrin-dependent adhesion to endothelial cells, supporting extravasation, and directing specific intratissue localization via gradient-driven chemotaxis. Chemoattractant receptors also control leukocyte egress from lymphoid organs and peripheral tissues. In this article, we summarize the fundamental mechanics of leukocyte trafficking, from the evolution of multistep models of leukocyte recruitment and navigation to the regulation of chemoattractant availability and function by atypical heptahelical receptors. To provide a more complete picture of the migratory circuits involved in leukocyte trafficking, we integrate a number of nonchemokine chemoattractant receptors into our discussion. Leukocyte chemoattractant receptors play key roles in the pathogenesis of autoimmune diseases, allergy, inflammatory disorders, and cancer. We review recent advances in our understanding of chemoattractant receptors in disease pathogenesis, with a focus on genome-wide association studies in humans and the translational implications of mechanistic studies in animal disease models.

  12. Leukocyte Margination in a Model Microvessel

    NASA Astrophysics Data System (ADS)

    Freund, Jonathan

    2006-11-01

    In the inflammation response, multi-body interactions of blood cells in the microcirculation bring leukocytes (white blood cells) to the vessel walls. We investigated the fluid mechanics of this using numerical simulations of 29 red blood cells and one leukocyte flowing in a two-dimensional microvessel. The cells are modeled as linearly elastic shell membranes. Though obviously simplified, this model reproduced the increasingly blunted velocity profiles and increased leukocyte margination observed at lower shear rates. To study its effect, we varied the relative stiffness of the red cells by over a factor of ten, but the margination was found to be much less correlated with this than to the bluntness of the mean velocity profile. The detailed velocity field around near-wall leukocyte was sensitive to the red cell stiffness, but it changed little for strongly versus weakly marginating cases. In the more strongly marginating cases, however, a red cell is typically leaning on the upstream side of the leukocyte and appears to stabilize it. A well-known feature of the microcirculation is a near-wall cell-free layer. We observed that the leukocyte's most probable position was at the edge of this layer, whose thickness increased following a lubrication scaling. The leukocyte's near-wall position is observed to be less stable with increasing mean stand-off distance, but this distance would have potentially greater effect on adhesion since the range of the molecular binding is so short.

  13. Adhesion of human leukocytes to biomaterials: an in vitro study using alkanethiolate monolayers with different chemically functionalized surfaces.

    PubMed

    Barbosa, Judite N; Barbosa, Mário A; Aguas, Artur P

    2003-06-15

    The adhesion of human leukocytes to self-assembled monolayers of well-defined surface chemistry was investigated in vitro. Polymorphonuclear (PMN) and mononuclear leukocytes were isolated from human blood by centrifugation techniques. The effect on adhesion of cell activation produced by pre-incubation of leukocytes with phytohemagglutinin (PHA) and phorbol 12-myristate 13-acetate (PMA) was also studied. Gold substrates were modified by treatment with alkanethiols with three different terminal chemical groups: COOH, OH, and CH(3). After incubation with the two subpopulations of leukocytes, the monolayers were washed, treated with fixative, stained with a Giemsa method, and observed by light microscopy to quantify the number of attached leukocytes. Comparative quantification of the density of leukocyte adhesion to the three types of self-assembled monolayers was determined. The hydrophobic surface expressing CH(3) was found to be the one that induced the highest adhesion density of leukocytes, both of PMN and mononuclear cells. In vitro activation of both mononuclear and PMN leukocytes further increased cell adhesion to the chemically defined monolayers that were used. This enhancement was higher for PHA-activated than for PMA-stimulated mononuclear cells, whereas PMA treatment of neutrophils resulted in a higher rate of adhesion of these cells than PHA stimulation.

  14. Nanowire array chips for molecular typing of rare trafficking leukocytes with application to neurodegenerative pathology

    NASA Astrophysics Data System (ADS)

    Kwak, Minsuk; Kim, Dong-Joo; Lee, Mi-Ri; Wu, Yu; Han, Lin; Lee, Sang-Kwon; Fan, Rong

    2014-05-01

    Despite the presence of the blood-brain barrier (BBB) that restricts the entry of immune cells and mediators into the central nervous system (CNS), a small number of peripheral leukocytes can traverse the BBB and infiltrate into the CNS. The cerebrospinal fluid (CSF) is one of the major routes through which trafficking leukocytes migrate into the CNS. Therefore, the number of leukocytes and their phenotypic compositions in the CSF may represent important sources to investigate immune-to-brain interactions or diagnose and monitor neurodegenerative diseases. Due to the paucity of trafficking leucocytes in the CSF, a technology capable of efficient isolation, enumeration, and molecular typing of these cells in the clinical settings has not been achieved. In this study, we report on a biofunctionalized silicon nanowire array chip for highly efficient capture and multiplexed phenotyping of rare trafficking leukocytes in small quantities (50 microliters) of clinical CSF specimens collected from neurodegenerative disease patients. The antibody coated 3D nanostructured materials exhibited vastly improved rare cell capture efficiency due to high-affinity binding and enhanced cell-substrate interactions. Moreover, our platform creates multiple cell capture interfaces, each of which can selectively isolate specific leukocyte phenotypes. A comparison with the traditional immunophenotyping using flow cytometry demonstrated that our novel silicon nanowire-based rare cell analysis platform can perform rapid detection and simultaneous molecular characterization of heterogeneous immune cells. Multiplexed molecular typing of rare leukocytes in CSF samples collected from Alzheimer's disease patients revealed the elevation of white blood cell counts and significant alterations in the distribution of major leukocyte phenotypes. Our technology represents a practical tool for potentially diagnosing and monitoring the pathogenesis of neurodegenerative diseases by allowing an effective

  15. Defect of In Vitro Digestive Ability of Polymorphonuclear Leukocytes in Paracoccidioidomycosis

    PubMed Central

    Goihman-Yahr, Mauricio; Essenfeld-Yahr, Ervin; De Albornoz, María C.; Yarzábal, Luis; De Gómez, MaríA H.; Martín, Blanca San; Ocanto, Ana; Gil, Francisco; Convit, Jacinto

    1980-01-01

    Selected functions of polymorphonuclear leukocytes were studied in patients with paracoccidioidomycosis (South American blastomycosis), in healthy control individuals, and in patients with diseases unrelated to paracoccidioidomycosis. Patients with paracoccidioidomycosis were also evaluated by standard immunological techniques. Phagocytosis and digestion of Paracoccidioides brasiliensis yeastlike cells in vitro was estimated by an original method. It was based on the appearance of phagocytosed P. brasiliensis in preparations stained by a modification of the Papanicolaou method and examined with phase-contrast optics. Interpretation of such findings was confirmed by electron microscopy. Two strains of P. brasiliensis were used. Strain 8506 was freshly isolated from a patient. Strain Pb9 was known to be nonpathogenic and to have a peculiar cell wall composition. Yeastlike cells of the Pb9 strain were digested significantly better than those of strain 8506. A higher number of leukocytes per fungus cells led to a higher proportion of digested P. brasiliensis. Leukocytes from patients with paracoccidioidomycosis phagocytosed the fungus in a normal way, but had a significant lower ability to digest it in vitro. When individual cases were analyzed, there was an excellent correlation between clinical evolution and digestive ability of polymorphonuclear leukocytes. There was good correlation between both of these and immunological parameters. Leukocytes from all groups behaved comparably in tests of general leukocyte function and in their abilities to kill and digest Candida albicans. Our results indicate that, as a group, polymorphonuclear leukocytes from patients with paracoccidioidomycosis had a significant, rather specific, defect in their in vitro digestive capacity against phagocytosed P. brasiliensis. There was also an inverse correlation between strain pathogenicity and its susceptibility to in vitro digestion by polymorphonuclear leukocytes. Our findings are

  16. Nanowire array chips for molecular typing of rare trafficking leukocytes with application to neurodegenerative pathology.

    PubMed

    Kwak, Minsuk; Kim, Dong-Joo; Lee, Mi-Ri; Wu, Yu; Han, Lin; Lee, Sang-Kwon; Fan, Rong

    2014-06-21

    Despite the presence of the blood-brain barrier (BBB) that restricts the entry of immune cells and mediators into the central nervous system (CNS), a small number of peripheral leukocytes can traverse the BBB and infiltrate into the CNS. The cerebrospinal fluid (CSF) is one of the major routes through which trafficking leukocytes migrate into the CNS. Therefore, the number of leukocytes and their phenotypic compositions in the CSF may represent important sources to investigate immune-to-brain interactions or diagnose and monitor neurodegenerative diseases. Due to the paucity of trafficking leucocytes in the CSF, a technology capable of efficient isolation, enumeration, and molecular typing of these cells in the clinical settings has not been achieved. In this study, we report on a biofunctionalized silicon nanowire array chip for highly efficient capture and multiplexed phenotyping of rare trafficking leukocytes in small quantities (50 microliters) of clinical CSF specimens collected from neurodegenerative disease patients. The antibody coated 3D nanostructured materials exhibited vastly improved rare cell capture efficiency due to high-affinity binding and enhanced cell-substrate interactions. Moreover, our platform creates multiple cell capture interfaces, each of which can selectively isolate specific leukocyte phenotypes. A comparison with the traditional immunophenotyping using flow cytometry demonstrated that our novel silicon nanowire-based rare cell analysis platform can perform rapid detection and simultaneous molecular characterization of heterogeneous immune cells. Multiplexed molecular typing of rare leukocytes in CSF samples collected from Alzheimer's disease patients revealed the elevation of white blood cell counts and significant alterations in the distribution of major leukocyte phenotypes. Our technology represents a practical tool for potentially diagnosing and monitoring the pathogenesis of neurodegenerative diseases by allowing an effective

  17. Leukocyte filtration in lung transplantation.

    PubMed

    Kurusz, Mark; Roach, John D; Vertrees, Roger A; Girouard, Mark K; Lick, Scott D

    2002-05-01

    Controlled reperfusion of the transplanted lung has been used in nine consecutive patients to decrease manifestations of lung reperfusion injury. An extracorporeal circuit containing a roller pump, heat exchanger and leukodepleting filter is primed with substrate-enhanced reperfusion solution mixed with approximately 2000 ml of the patient's blood. This solution is slowly recirculated to remove leukocytes prior to reperfusion. When the pulmonary anastomoses are completed, the pulmonary artery is cannulated through the untied anastomosis using a catheter containing a pressure lumen for measurement of infusion pressure. An atrial clamp is left in place on the patient's native atrial cuff to decrease the risk of systemic air embolism during the brief period of reperfusion from the extracorporeal reservoir. During reperfusion, the water bath to the heat exchanger is kept at 35 degrees C and the flow rate for reperfusion solution is between 150 and 200 m/min, keeping the pulmonary artery pressure <14 mmHg. Eight of nine patients were ventilated on 40% inspired oxygen within a few hours of operation and 7/9 were extubated on or before postoperative day 1. Six of nine patients are long-term survivors.

  18. Estrogen binding by leukocytes during phagocytosis,

    PubMed Central

    1977-01-01

    Estradiol binds covalently to normal leukocytes during phagocytosis. The binding involves three cell types, neutrophils, eosinophils, and monocytes and at least two reaction mechanisms, one involving the peroxidase of neutrophils and monocytes (myeloperoxidase [MPO]) and possibly the eosinophil peroxidase, and the second involving catalase. Binding is markedly reduced when leukocytes from patients with chronic granulomatous disease (CGD), severe leukocytic glucose 6-phosphate dehydrogenase deficiency, and familial lipochrome histiocytosis are employed and two populations of neutrophils, one which binds estradiol and one which does not, can be demonstrated in the blood of a CGD carrier. Leukocytes from patients with hereditary MPO deficiency also bind estradiol poorly although the defect is not as severe as in CGD. These findings are discussed in relation to the inactivation of estrogens during infection and the possible role of estrogens in neutrophil function. PMID:858996

  19. Leukocytes in Mammary Development and Cancer

    PubMed Central

    Coussens, Lisa M.; Pollard, Jeffrey W.

    2011-01-01

    Leukocytes, of both the innate and adaptive lineages, are normal cellular components of all tissues. These important cells not only are critical for regulating normal tissue homeostasis, but also are significant paracrine regulators of all physiologic and pathologic tissue repair processes. This article summarizes recent insights regarding the trophic roles of leukocytes at each stage of mammary gland development and during cancer development, with a focus on Murids and humans. PMID:21123394

  20. Leukocyte margination in a model microvessel

    NASA Astrophysics Data System (ADS)

    Freund, Jonathan B.

    2007-02-01

    The physiological inflammation response depends upon the multibody interactions of blood cells in the microcirculation that bring leukocytes (white blood cells) to the vessel walls. We investigate the fluid mechanics of this using numerical simulations of 29 red blood cells and one leukocyte flowing in a two-dimensional microvessel, with the cells modeled as linearly elastic shell membranes. Despite its obvious simplifications, this model successfully reproduces the increasingly blunted velocity profiles and increased leukocyte margination observed at lower shear rates in actual microvessels. Red cell aggregation is shown to be unnecessary for margination. The relative stiffness of the red cells in our simulations is varied by over a factor of 10, but the margination is found to be much less correlated with this than it is to changes associated with the blunting of the mean velocity profile at lower shear rates. While velocity around the leukocyte when it is near the wall depends upon the red cell properties, it changes little for strongly versus weakly marginating cases. In the more strongly marginating cases, however, a red cell is frequently observed to be leaning on the upstream side of the leukocyte and appears to stabilize it, preventing other red cells from coming between it and the wall. A well-known feature of the microcirculation is a near-wall cell-free layer. In our simulations, it is observed that the leukocyte's most probable position is at the edge of this layer. This wall stand-off distance increases with velocity following a scaling that would be expected for a lubrication mechanism, assuming that there were a nearly constant force pushing the cells toward the wall. The leukocyte's near-wall position is observed to be less stable with increasing mean stand-off distance, but this distance would have potentially greater effect on adhesion since the range of the molecular binding is so short.

  1. Moojenactivase, a novel pro-coagulant PIIId metalloprotease isolated from Bothrops moojeni snake venom, activates coagulation factors II and X and induces tissue factor up-regulation in leukocytes.

    PubMed

    Sartim, Marco A; Costa, Tassia R; Laure, Helen J; Espíndola, Milena S; Frantz, Fabiani G; Sorgi, Carlos A; Cintra, Adélia C O; Arantes, Eliane C; Faccioli, Lucia H; Rosa, José C; Sampaio, Suely V

    2016-05-01

    Coagulopathies following snakebite are triggered by pro-coagulant venom toxins, in which metalloproteases play a major role in envenomation-induced coagulation disorders by acting on coagulation cascade, platelet function and fibrinolysis. Considering this relevance, here we describe the isolation and biochemical characterization of moojenactivase (MooA), a metalloprotease from Bothrops moojeni snake venom, and investigate its involvement in hemostasis in vitro. MooA is a glycoprotein of 85,746.22 Da, member of the PIIId group of snake venom metalloproteases, composed of three linked disulfide-bonded chains: an N-glycosylated heavy chain, and two light chains. The venom protease induced human plasma clotting in vitro by activating on both blood coagulation factors II (prothrombin) and X, which in turn generated α-thrombin and factor Xa, respectively. Additionally, MooA induced expression of tissue factor (TF) on the membrane surface of peripheral blood mononuclear cells (PBMC), which led these cells to adopt pro-coagulant characteristics. MooA was also shown to be involved with production of the inflammatory mediators TNF-α, IL-8 and MCP-1, suggesting an association between MooA pro-inflammatory stimulation of PBMC and TF up-regulation. We also observed aggregation of washed platelets when in presence of MooA; however, the protease had no effect on fibrinolysis. Our findings show that MooA is a novel hemostatically active metalloprotease, which may lead to the development of coagulopathies during B. moojeni envenomation. Moreover, the metalloprotease may contribute to the development of new diagnostic tools and pharmacological approaches applied to hemostatic disorders.

  2. Lactobacillus casei prevents the upregulation of ICAM-1 expression and leukocyte recruitment in experimental colitis.

    PubMed

    Angulo, Sandra; Llopis, Marta; Antolín, María; Gironella, Meritxell; Sans, Miquel; Malagelada, Juan Ramon; Piqué, Josep Maria; Guarner, Francisco; Panés, Julián

    2006-12-01

    Lactobacillus casei has been shown to attenuate the severity of experimental colitis. The objective of the present study was to determine whether the effects of L. casei on colitis are related to modulation of leukocyte recruitment into the inflamed intestine. Rats with a colonic segment excluded from fecal transit were surgically prepared. The segment was decontaminated with antibiotics and recolonized with normal flora isolated from the inflamed rat colon, associated or not to L. casei. Control and colitic [2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced] animals were studied. Leukocyte-endothelial cell interactions were characterized in the colonic microcirculation by intravital microscopy, and ICAM-1 and VCAM-1 expression was measured by the radiolabeled antibody technique. Compared with the noninflamed colonic segment, induction of colitis by TNBS provoked a marked increase in the number of leukocytes firmly adherent to the venular wall (0.5 +/- 0.1 vs. 2.1 +/- 0.6 leukocytes/100 mum, P < 0.01). Colonization with L. casei significantly reduced the number of adherent leukocytes (1.3 +/- 0.4 leukocytes/100 mum; P < 0.05) but did not affect the increased rolling interactions associated with the induction of colitis. Compared with the noncolitic group, induction of colitis was associated with a marked increase in ICAM-1 expression (117 +/- 4 vs. 180 +/- 3 ng antibody/g tissue) that was abrogated when the colitic segment was colonized by L. casei (117 +/- 3 ng antibody/g tissue, P < 0.05). However, L. casei administration did not modify VCAM-1 upregulation in colitic animals. L. casei attenuates leukocyte recruitment observed in experimental colitis induced by TNBS. This effect is possibly related to abrogation of ICAM-1 upregulation.

  3. Cigarette smoking and leukocyte subpopulations in men.

    PubMed

    Freedman, D S; Flanders, W D; Barboriak, J J; Malarcher, A M; Gates, L

    1996-07-01

    Because of previously reported associations among the total leukocyte count, cigarette smoking, and risk of cardiovascular disease, we examined the relation of cigarette smoking to various leukocyte subpopulations among 3467 men aged 31 to 45 years. The median total leukocyte count was 36% higher (7840 vs. 5760 cells/mL) among current cigarette smokers than among men who had never smoked, and both stratification and regression analyses were used to examine independent associations with leukocyte subpopulations. At equivalent counts of other subpopulations, CD4+ lymphocytes and neutrophils were the cell types most strongly associated with cigarette smoking; each standard deviation change in counts of these subpopulations increased the odds of current (vs. never) smoking by approximately threefold. Furthermore, whereas 15% of the 238 men with relatively low (< 25 percentile) counts of both neutrophils and CD4+ lymphocytes were cigarette smokers, 96% of the 249 men with relatively high counts of both subpopulations were current smokers. Counts of T lymphocytes also tended to be higher among the 32 men with self-reported ischemic heart disease than among other men. These results, along with previous reports of immunologically active T lymphocytes in atherosclerotic plaques, suggest that this subpopulation may be of particular interest in studies examining the relation of leukocytes to cardiovascular disease.

  4. Imaging Leukocyte Responses in the Kidney.

    PubMed

    Finsterbusch, Michaela; Kitching, A Richard; Hickey, Michael J

    2017-03-01

    The kidney can be negatively affected by a range of innate and adaptive immune responses, resulting in alterations in the functions of the kidney and, in some cases, progression to renal failure. In many of these responses, infiltration of blood-borne leukocytes into the kidney is central to the response. In addition, a large population of mononuclear phagocytes resident in the kidney can modulate these responses. A great deal of research has investigated both the mechanisms of leukocyte recruitment to the kidney and the actions of immune cells resident within the kidney. Because of the dynamic nature of the processes whereby leukocytes enter sites of inflammation, in vivo imaging has been one of the key approaches used for understanding leukocyte recruitment as it occurs throughout the body, and this is also true for kidney. However, imaging this organ and its complicated microvasculature during different forms of renal pathology presents a unique set of challenges. In this review, we examine the approaches used for intravital imaging of the kidney and summarize the insights gained from these studies regarding the mechanisms of leukocyte entry into the kidney during inflammation and the actions of immune cells within this organ.

  5. Halloysite nanotube coatings suppress leukocyte spreading

    PubMed Central

    Hughes, Andrew D.; Marsh, Graham; Waugh, Richard E.; Foster, David G.; King, Michael R.

    2016-01-01

    The nanoscale topography of adhesive surfaces is known to be an important factor governing cellular behavior. Previous work has shown that surface coatings composed of halloysite nanotubes enhances the adhesion, and therefore capture, of rare target cells such as circulating tumor cells. Here, we demonstrate a unique feature of these coatings in its ability to reduce the adhesion of leukocytes and prevent leukocyte spreading. Surfaces were prepared with coatings of halloysite nanotubes and functionalized for leukocyte adhesion with E-selectin, and the dilution of nanotube concentration revealed a threshold concentration below which cell spreading became comparable with smooth surfaces. Evaluation of surface roughness characteristics determined that the average distance between discrete surface features correlated with adhesion metrics, with a separation distance of approximately 2 μm identified as the critical threshold. Computational modeling of the interaction of leukocytes with halloysite nanotube coated surfaces of varying concentrations demonstrates that the geometry of the cell surface and adhesive counter-surface produce a significantly diminished effective contact area compared to a leukocyte interacting with a smooth surface. PMID:26605493

  6. Characterization of porcine peripheral blood leukocytes by light-scattering flow cytometry.

    PubMed Central

    Wang, F I; Williams, T J; el-Awar, F Y; Pang, V F; Hahn, E C

    1987-01-01

    As a basis for other experiments using flow cytometry of porcine peripheral blood leukocytes, cell fractions were isolated by various methods and analyzed by forward angle light scatter and 90 degree light scatter. Cytospin smears of cell samples were also studied by leukocyte differential counts and nonspecific esterase staining. Three main populations of peripheral blood leukocytes [lymphocytes, monocytes, and granulocytes (primarily neutrophils)], were defined in the log 90 degree light scatter by forward angle light scatter histogram. Partial overlap was observed between lymphocyte and monocyte, and between monocyte and granulocyte domains. Correlation between leukocyte differential counts and flow cytometric quantification based on bitmap statistics of appropriate domains was between r = 0.872-0.892 for lymphocyte and granulocyte. Percoll density gradients were used for subfractionation of leukocyte populations, especially for the enrichment of granulocytes. The specific densities were calculated for lymphocytes (1.0585-1.0819 g/cc), monocytes (1.0585-1.0702 g/cc), granulocyte (1.0819-1.0936 g/cc), and erythrocytes (greater than 1.0952 g/cc). We suggest that light scatter characterization is a basis for future studies of porcine blood by flow cytometry. PMID:3453262

  7. Lamellar leukocyte infiltration and involvement of IL-6 during oligofructose-induced equine laminitis development.

    PubMed

    Visser, Michelle B; Pollitt, Christopher C

    2011-11-15

    Laminitis is known to involve deregulation of proteases and destruction of the lamellar basement membrane with the host inflammatory response also playing a role. Leukocyte infiltration has been well characterized in the black walnut model of laminitis induction, but not in carbohydrate induced models. Increased gene expression of multiple cytokines, including IL-6, has also been implicated in laminitis development. Using real time PCR, immunohistochemistry and zymography methods, we characterize leukocyte infiltration and IL-6 gene expression in oligofructose (OF) induced laminitis. As well, we use two in vitro models to investigate a role for IL-6 in protease regulation. Laminitis was induced in normal standardbred horses (n=5) by alimentary OF dosing and lamellar biopsies were obtained throughout the 48 h experimental period. Lamellar explants and keratinocytes were also isolated from clinically normal horses for in vitro experiments. We found infiltration of calprotectin-positive leukocytes (monocytes and neutrophils) at 18-24h post oligofructose dosing, while IL-6 gene expression was increased as early as 12h post dosing. Additionally, while we found that IL-6 did not cause significant BM damage in vitro, it did result in increased secreted proMMP-9 levels from lamellar explants. Thus, we find that leukocyte infiltration does occur during oligofructose-induced laminitis development, however, IL-6 gene expression in the lamellae may precede leukocyte infiltration. Additionally, we show IL-6 plays a role in increasing the level of proMMP-9 in vivo in a manner that does not involve keratinocytes.

  8. Urotensin II upregulates migration and cytokine gene expression in leukocytes of the African clawed frog, Xenopus laevis.

    PubMed

    Tomiyama, Shiori; Nakamachi, Tomoya; Uchiyama, Minoru; Matsuda, Kouhei; Konno, Norifumi

    2015-05-15

    Urotensin II (UII) exhibits diverse physiological actions including vasoconstriction, locomotor activity, osmoregulation, and immune response via the UII receptor (UTR) in mammals. However, in amphibians the function of the UII-UTR system remains unknown. In the present study, we investigated the potential immune function of UII using leukocytes isolated from the African clawed frog, Xenopus laevis. Stimulation of male frogs with lipopolysaccharide increased mRNA expression of UII and UTR in leukocytes, suggesting that inflammatory stimuli induce activation of the UII-UTR system. Migration assays showed that both UII and UII-related peptide enhanced migration of leukocytes in a dose-dependent manner, and that UII effect was inhibited by the UTR antagonist urantide. Inhibition of Rho kinase with Y-27632 abolished UII-induced migration, suggesting that it depends on the activation of RhoA/Rho kinase. Treatment of isolated leukocytes with UII increased the expression of several cytokine genes including tumor necrosis factor-α, interleukin-1β, and macrophage migration inhibitory factor, and the effects were abolished by urantide. These results suggest that in amphibian leukocytes the UII-UTR system is involved in the activation of leukocyte migration and cytokine gene expression in response to inflammatory stimuli.

  9. Leukocytes Crossing the Endothelium: A Matter of Communication.

    PubMed

    Timmerman, Ilse; Daniel, Anna E; Kroon, Jeffrey; van Buul, Jaap D

    2016-01-01

    Leukocytes cross the endothelial vessel wall in a process called transendothelial migration (TEM). The purpose of leukocyte TEM is to clear the causing agents of inflammation in underlying tissues, for example, bacteria and viruses. During TEM, endothelial cells initiate signals that attract and guide leukocytes to sites of tissue damage. Leukocytes react by attaching to these sites and signal their readiness to move back to endothelial cells. Endothelial cells in turn respond by facilitating the passage of leukocytes while retaining overall integrity. In this review, we present recent findings in the field and we have endeavored to synthesize a coherent picture of the intricate interplay between endothelial cells and leukocytes during TEM.

  10. Reactive oxygen species in phagocytic leukocytes

    PubMed Central

    2008-01-01

    Phagocytic leukocytes consume oxygen and generate reactive oxygen species in response to appropriate stimuli. The phagocyte NADPH oxidase, a multiprotein complex, existing in the dissociated state in resting cells becomes assembled into the functional oxidase complex upon stimulation and then generates superoxide anions. Biochemical aspects of the NADPH oxidase are briefly discussed in this review; however, the major focus relates to the contributions of various modes of microscopy to our understanding of the NADPH oxidase and the cell biology of phagocytic leukocytes. PMID:18597105

  11. A simple skin blister technique for the study of in vivo transmigration of human leukocytes.

    PubMed

    Davidsson, Lisa; Björkman, Lena; Christenson, Karin; Alsterholm, Mikael; Movitz, Charlotta; Thorén, Fredrik B; Karlsson, Anna; Welin, Amanda; Bylund, Johan

    2013-07-31

    The study of human leukocytes is almost exclusively conducted using cells isolated from peripheral blood. This is especially true for neutrophils, despite the fact that these cells are of main (pathological) importance in extravascular tissues upon e.g., infection and/or tissue damage. The journey from circulation to tissue is typically associated with a number of cellular changes, making the cells primed, or hyper-responsive, and in many aspects distinct from the cells present in circulation. Models to obtain in vivo transmigrated leukocytes from human tissue are available, but not widely used. We describe here an easy-to-use model for the study of local inflammation, stemming from limited tissue damage, which can be used to isolate viable and functional leukocytes. The model is based on the generation of aseptic skin blisters, formed by the application of negative pressure, and allows for investigations of the cellular infiltrate as well as of soluble mediators present in the exudate. We believe that this method, combined with modern analysis equipment suitable for small volumes and cell numbers, could be of great use for increasing our understanding of the nature and function of leukocytes that have left circulation and transmigrated to inflamed tissues.

  12. A Semianalytic Model of Leukocyte Rolling

    PubMed Central

    Krasik, Ellen F.; Hammer, Daniel A.

    2004-01-01

    Rolling allows leukocytes to maintain adhesion to vascular endothelium and to molecularly coated surfaces in flow chambers. Using insights from adhesive dynamics, a computational method for simulating leukocyte rolling and firm adhesion, we have developed a semianalytic model for the steady-state rolling of a leukocyte. After formation in a force-free region of the contact zone, receptor-ligand bonds are transported into the trailing edge of the contact zone. Rolling velocity results from a balance of the convective flux of bonds and the rate of dissociation at the back edge of the contact zone. We compare the model's results to that of adhesive dynamics and to experimental data on the rolling of leukocytes, with good agreement. We calculate the dependence of rolling velocity on shear rate, intrinsic forward and reverse reaction rates, bond stiffness, and reactive compliance, and use the model to calculate a state diagram relating molecular parameters and the dynamic state of adhesion. A dimensionless form of the analytic model permits exploration of the parameters that control rolling. The chemical affinity of a receptor-ligand pair does not uniquely determine rolling velocity. We elucidate a fundamental relationship between off-rate, ligand density, and reactive compliance at the transition between firm and rolling adhesion. The model provides a rapid method for screening system parameters for the potential to mediate rolling. PMID:15315955

  13. Fröhlich electromagnetic radiation from human leukocytes: implications for leukocyte adherence inhibition test.

    PubMed

    Pokorný, J; Jandová, A; Kobilková, J; Heyberger, K; Hraba, T

    1983-05-21

    The Fröhlich coherent vibrations may be a source of an electromagnetic field generated by living cells in the frequency range from 0.1 to 10 THz. The electromagnetic field may cause the time dependent orientation (i.e. rotation or rocking) of the polar molecules of the ambient liquid medium and may attract them. The attracted molecules move together with the cell and the friction coefficient of the cellular motion, therefore, may depend on the field. The cell-generated electromagnetic field may interact with the surface charge of various solid-state materials causing attractive forces. These interaction attractive forces may be significant in the process of the leukocyte adherence to the surfaces of various materials. The hypothesis presented in this paper assumes that the exposition of leukocytes from immune individuals to antigen causes changes of the Fröhlich coherent vibrations resulting in decrease of the leukocyte adherence observed in the leukocyte adherence inhibition test.

  14. Diterpenoids from Tetraclinis articulata that inhibit various human leukocyte functions.

    PubMed

    Barrero, Alejandro F; Quílez del Moral, José F; Lucas, Rut; Payá, Miguel; Akssira, Mohamed; Akaad, Said; Mellouki, Fouad

    2003-06-01

    Ten new compounds, eight of them pimarane derivatives (1-8), together with a menthane dimer (9) and a totarane diterpenoid (10), were isolated from the leaves and wood of Tetraclinis articulata. The structures of 1-10 were established by using spectroscopic techniques, including 2D NMR spectra. Pimaranes 1-5 were found to possess an unusual cis interannular union of the B and C rings, which, from a biogenetic perspective, could be derived from the hydration of a carbocation at C-8. Compounds 4-6 and a mixture of 7 and 11 modulated different human leukocyte functions at a concentration of 10 microM, mainly the degranulation process measured as myeloperoxidase release and, to a lesser extent, the superoxide production measured by chemiluminescence.

  15. Gestational diabetes mellitus is associated with increased leukocyte peroxisome proliferator-activated receptor γ expression

    PubMed Central

    Mac-Marcjanek, Katarzyna; Nadel, Iwona; Woźniak, Lucyna; Cypryk, Katarzyna

    2015-01-01

    Introduction Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated transcription factor of the nuclear receptor superfamily that is involved in lipid and carbohydrate metabolism as well as inflammation; thereby it participates in metabolic diseases including diabetes. Although PPARγ expression has been observed in different tissues of diabetic patients, its level in leukocytes from subjects affected by gestational diabetes mellitus (GDM) has not yet been reported. This study aimed to investigate leukocyte PPARG expression in GDM patients at 24–33 weeks of gestation and, in turn, to correlate these alterations with anthropometric and metabolic parameters of patients. Material and methods Leukocytes were isolated from the blood of normal glucose tolerant (NGT; n = 34) and GDM (n = 77) pregnant women between 24 and 33 weeks of gestation. Leukocyte PPARG mRNA expression was determined by semi-quantitative polymerase chain reaction. Univariate correlation analysis was performed to investigate associations between PPARG expression and clinical characteristics of patients. Results Leukocyte PPARG mRNA level was significantly higher in GDM than NGT women (p < 0.05). In the whole study group, PPARG expression positively correlated with plasma glucose concentrations at 1 h (r = 0.222, p = 0.049) and 2 h (r = 0.315, p = 0.020) of 75 g oral glucose tolerance test (OGTT), and negatively correlated with plasma HDL cholesterol concentration (r = -0.351, p = 0.010). Conclusions The correlation between leukocyte PPARG overexpression and hyperglycaemia suggests that PPARG mRNA expression in these cells might be up-regulated in high-glucose conditions in GDM patients at 24–33 weeks of gestation. PMID:26322090

  16. Nicotinamide phosphoribosyltransferase leukocyte overexpression in Graves' opthalmopathy.

    PubMed

    Sawicka-Gutaj, Nadia; Budny, Bartłomiej; Zybek-Kocik, Ariadna; Sowiński, Jerzy; Ziemnicka, Katarzyna; Waligórska-Stachura, Joanna; Ruchała, Marek

    2016-08-01

    To investigate the role of NAMPT/visfatin in euthyroid patients with Graves' disease without (GD) and with Graves' ophthalmopathy (GO), we analyzed NAMPT leukocyte expression and its serum concentration. This was a single-center, cross-sectional study with consecutive enrollment. In total, 149 patients diagnosed with Graves' disease were enrolled in the study. We excluded subjects with hyper- or hypothyroidism, diabetes mellitus, other autoimmune disorders, active neoplastic disease, and infection. The control group was recruited among healthy volunteers adjusted for age, sex, and BMI with normal thyroid function and negative thyroid antibodies. Serum levels of visfatin, TSH, FT4, FT3, antibodies against TSH receptor (TRAb), antithyroperoxidase antibodies, antithyroglobulin antibodies, fasting glucose, and insulin were measured. NAMPT mRNA leukocyte expression was assessed using RT-qPCR. NAMPT/visfatin serum concentration was higher in GD (n = 44) and GO (n = 49) patients than in the control group (n = 40) (p = 0.0275). NAMPT leukocyte expression was higher in patients with GO (n = 30) than in GD patients (n = 27) and the control group (n = 29) (p < 0.0001). Simple linear regression analysis revealed that NAMPT/visfatin serum concentration was significantly associated with GD (β = 1.5723; p = 0.021). When NAMPT leukocyte expression was used as a dependent variable, simple regression analysis found association with TRAb, fasting insulin level, HOMA-IR, GD, and GO. In the stepwise multiple regression analysis, we confirmed the association between higher serum NAMPT/visfatin level and GD (coefficient = 1.5723; p = 0.0212), and between NAMPT leukocyte expression and GO (coefficient = 2.4619; p = 0.0001) and TRAb (coefficient = 0.08742; p = 0.006). Increased NAMPT leukocyte expression in patients with GO might suggest a presently undefined role in the pathogenesis of GO.

  17. Endothelial activation drives lateral migration and diapedesis of leukocytes.

    PubMed

    Stock, Christian; Riethmuller, Christoph

    2011-01-01

    To invade a tissue, leukocytes have to overcome the endothelial barrier. Prior to trans-endothelial migration, leukocytes move laterally on the endothelial surface-searching for an emigration site. It is still unclear, how the actual diapedesis step is initiated and whether the endothelium has a decisive role. Here, video-microscopy was employed to investigate, whether lateral migration of leukocytes is correlated to their diapedesis rate. To address the contribution of each cell type, selective stimulation of either leukocytes or endothelial cells with TNFα was performed. Stimulation of endothelial cells alone was sufficient for maximal effects, thereby underlining their decisive role for leukocyte diapedesis. Concomitant to the TNFα-enhanced diapedesis rate, leukocyte adhesion was intensified and, unexpectedly, the lateral leukocyte migration was accelerated.

  18. Endothelial cell regulation of leukocyte infiltration in inflammatory tissues

    PubMed Central

    Mantovani, A.; Introna, M.; Dejana, E.

    1995-01-01

    Endothelial cells play an important, active role in the onset and regulation of inflammatory and immune reactions. Through the production of chemokines they attract leukocytes and activate their adhesive receptors. This leads to the anchorage of leukocytes to the adhesive molecules expressed on the endothelial surface. Leukocyte adhesion to endothelial cells is frequently followed by their extravasation. The mechanisms which regulate the passage of leukocytes through endothelial clefts remain to be clarified. Many indirect data suggest that leukocytes might transfer signals to endothelial cells both through the release of active agents and adhesion to the endothelial cell surface. Adhesive molecules (such as PECAM) on the endothelial cell surface might also ‘direct’ leukocytes through the intercellular junction by haptotaxis. The information available on the molecular structure and functional properties of endothelial chemokines, adhesive molecules or junction organization is still fragmentary. Further work is needed to clarify how they interplay in regulating leukocyte infiltration into tissues. PMID:18475659

  19. Indium-111 leukocyte scanning and fracture healing

    SciTech Connect

    Mead, L.P.; Scott, A.C.; Bondurant, F.J.; Browner, B.D. )

    1990-01-01

    This study was undertaken to determine the specificity of indium-111 leukocyte scans for osteomyelitis when fractures are present. Midshaft tibial osteotomies were performed in 14 New Zealand white rabbits, seven of which were infected postoperatively with Staphylococcus aureus per Norden's protocol. All 14 rabbits were scanned following injection with 75 microCi of indium 111 at 72 h after osteotomy and at weekly intervals for 4 weeks. Before the rabbits were killed, the fracture sites were cultured to document the presence or absence of infection. The results of all infected osteotomy sites were positive, whereas no positive scans were found in the noninfected osteotomies. We concluded from this study that uncomplicated fracture healing does not result in a positive indium-111 leukocyte scan.

  20. Passive deformation analysis of human leukocytes.

    PubMed

    Dong, C; Skalak, R; Sung, K L; Schmid-Schönbein, G W; Chien, S

    1988-02-01

    The following analysis presents an experimental and theoretical study of the passive viscoelastic behavior of human leukocytes. Individual neutrophils in EDTA were observed both during their partial aspiration into a small micropipette and after expulsion from a large micropipette where the cell had been totally aspirated and deformed into a sausage shape. To analyze the data, a passive model of leukocyte rheology has been developed consisting of a cortical shell containing a Maxwell fluid which describes the average properties of the cell cytoplasm. The cortical shell represents a crosslinked actin layer near the surface of the cell and is assumed to be under pre-stressed tension. This model can reproduce the results of experiments using micropipette for both short-time small deformation and slow recovery data after large deformation. In addition, a finite element scheme has been established for the same model which shows close agreement with the analytical solution.

  1. Blood spotlight on leukocytes and obesity

    PubMed Central

    Carvalheira, Jose Barreto Campello; Qiu, Yifu

    2013-01-01

    The rise of obesity and its attendant pathological sequelae, including type 2 diabetes and coronary artery disease, constitute an ongoing public health catastrophe in both the developed and, more recently, the developing world. Although the underlying pathophysiology is complex, chronic low-grade inflammation has emerged as a central driver of both primary metabolic dysfunction and subsequent tissue failure. Importantly, this inflammation has been shown to arise as a consequence of both the disruption of homeostatic tissue resident leukocytes and the recruitment of antagonistic effector cells from the circulation. In this review, we discuss the roles of visceral adipose tissue’s salient leukocyte lineages in the transition to obesity and highlight key points at which this emerging immune axis may be manipulated for therapeutic effect. PMID:24065242

  2. Getting Leukocytes to the Site of Inflammation

    PubMed Central

    Muller, W. A.

    2013-01-01

    There is no “response” in either the innate or adaptive immune response unless leukocytes cross blood vessels. They do this through the process of diapedesis, in which the leukocyte moves in ameboid fashion through tightly apposed endothelial borders (paracellular transmigration) and in some cases through the endothelial cell itself (transcellular migration). This review summarizes the steps leading up to diapedesis, then focuses on the molecules and mechanisms responsible for transendothelial migration. Surprisingly, many of the same molecules and mechanisms that regulate paracellular migration also control transcellular migration, including a major role for membrane from the recently described lateral border recycling compartment. A hypothesis that integrates the various known mechanisms of transmigration is proposed. PMID:23345459

  3. Oxidative stress and reduced responsiveness of challenged circulating leukocytes following pulmonary instillation of metal-rich particulate matter in rats

    PubMed Central

    2014-01-01

    Welding fume is an exposure that consists of a mixture of metal-rich particulate matter with gases (ozone, carbon monoxide) and/or vapors (VOCs). Data suggests that welders are immune compromised. Given the inability of pulmonary leukocytes to properly respond to a secondary infection in animal models, the question arose whether the dysfunction persisted systemically. Our aim was to evaluate the circulating leukocyte population in terms of cellular activation, presence of oxidative stress, and functionality after a secondary challenge, following welding fume exposure. Rats were intratracheally instilled (ITI) with PBS or 2 mg of welding fume collected from a stainless steel weld. Rats were sacrificed 4 and 24 h post-exposure and whole blood was collected. Whole blood was used for cellular differential counts, RNA isolation with subsequent microarray and Ingenuity Pathway Analysis, and secondary stimulation with LPS utilizing TruCulture technology. In addition, mononuclear cells were isolated 24 h post-exposure to measure oxidative stress by flow cytometry and confocal microscopy. Welding fume exposure had rapid effects on the circulating leukocyte population as identified by relative mRNA expression changes. Instillation of welding fume reduced inflammatory protein production of circulating leukocytes when challenged with the secondary stimulus LPS. The effects were not related to transcription, but were observed in conjunction with oxidative stress. These findings support previous studies of an inadequate pulmonary immune response following a metal-rich exposure and extend those findings showing leukocyte dysfunction occurs systemically. PMID:25123171

  4. Age- and disease-related innate immunity of human leukocytes ex vivo.

    PubMed

    Jatczak, Bogna; Leszek, Jerzy; Siemieniec, Iwona; Sochocka, Marta; Wiśniewska, Agnieszka; Tarkowski, Radosław; Bębenek, Marek; Błach-Olszewska, Zofia

    2012-01-01

    Two mechanisms of innate immunity, i.e. resistance to viral infection and the production of cytokines by leukocytes, were compared in blood isolated from four groups of donors: healthy young (19-35 years old), healthy elderly (over 60), elderly Alzheimer's disease (AD) patients, and elderly patients with alimentary tract cancer (CA). Peripheral blood leukocytes (PBLs) were isolated by gradient centrifugation in Gradisol G. The degree of resistance was calculated from the kinetics of vesicular stomatitis virus (VSV) replication in the PBLs. Cytokine (TNFα, IFNα, IFNγ, IL-12, and IL-10) levels were determined by ELISA. The antiviral resistance of the PBLs varied, but a difference was observed only between the young and elderly groups and not between the healthy elderly controls and those with AD or cancer. Differences observed in all the groups concerned the ability and intensity of cytokine production. The most impressive results were obtained for spontaneous TNF and IFNα release. While TNF was released spontaneously by the PBLs of the elderly CA patients and the young healthy group, it was usually undetected in the AD and only sometimes in the healthy elderly group. Leukocytes isolated from the elderly groups responded to VSV infection with more intense IFNα and IFNγ production than the younger group.

  5. Bovine leukocyte adhesion deficiency (BLAD): a review.

    PubMed

    Nagahata, Hajime

    2004-12-01

    Bovine leukocyte adhesion deficiency (BLAD) in Holstein cattle is an autosomal recessive congenital disease characterized by recurrent bacterial infections, delayed wound healing and stunted growth, and is also associated with persistent marked neutrophilia. The molecular basis of BLAD is a single point mutation (adenine to guanine) at position 383 of the CD18 gene, which caused an aspartic acid to glycine substitution at amino acid 128 (D128G) in the adhesion molecule CD18. Neutrophils from BLAD cattle have impaired expression of the beta2 integrin (CD11a,b,c/CD18) of the leukocyte adhesion molecule. Abnormalities in a wide spectrum of adherence dependent functions of leukocytes have been fully characterized. Cattle affected with BLAD have severe ulcers on oral mucous membranes, severe periodontitis, loss of teeth, chronic pneumonia and recurrent or chronic diarrhea. Affected cattle die at an early age due to the infectious complications. Holstein bulls, including carrier sires that had a mutant BLAD gene in heterozygote were controlled from dairy cattle for a decade. The control of BLAD in Holstein cattle by publishing the genotypes and avoiding the mating between BLAD carriers was found to be successful. This paper provides an overview of the genetic disease BLAD with reference to the disease in Holstein cattle.

  6. CD Nomenclature 2015: Human Leukocyte Differentiation Antigen Workshops as a Driving Force in Immunology.

    PubMed

    Engel, Pablo; Boumsell, Laurence; Balderas, Robert; Bensussan, Armand; Gattei, Valter; Horejsi, Vaclav; Jin, Bo-Quan; Malavasi, Fabio; Mortari, Frank; Schwartz-Albiez, Reinhard; Stockinger, Hannes; van Zelm, Menno C; Zola, Heddy; Clark, Georgina

    2015-11-15

    CD (cluster of differentiation) Ags are cell surface molecules expressed on leukocytes and other cells relevant for the immune system. CD nomenclature has been universally adopted by the scientific community and is officially approved by the International Union of Immunological Societies and sanctioned by the World Health Organization. It provides a unified designation system for mAbs, as well as for the cell surface molecules that they recognize. This nomenclature was established by the Human Leukocyte Differentiation Antigens Workshops. In addition to defining the CD nomenclature, these workshops have been instrumental in identifying and determining the expression and function of cell surface molecules. Over the past 30 y, the data generated by the 10 Human Leukocyte Differentiation Antigens Workshops have led to the characterization and formal designation of more than 400 molecules. CD molecules are commonly used as cell markers, allowing the identification and isolation of leukocyte populations, subsets, and differentiation stages. mAbs against these molecules have proven to be essential for biomedical research and diagnosis, as well as in biotechnology. More recently, they have been recognized as invaluable tools for the treatment of several malignancies and autoimmune diseases. In this article, we describe how the CD nomenclature was established, present the official updated list of CD molecules, and provide a rationale for their usefulness in the 21st century.

  7. 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.

  8. Indium-111 leukocyte imaging in patients with rheumatoid arthritis

    SciTech Connect

    Uno, K.; Matsui, N.; Nohira, K.; Suguro, T.; Kitakata, Y.; Uchiyama, G.; Miyoshi, T.; Uematsu, S.; Inoue, S.; Arimizu, N.

    1986-03-01

    This study evaluates the usefulness of labeled leukocyte imaging in patients with rheumatoid arthritis. In 33 patients, the incidence of pain and swelling in 66 wrist joints and 66 knee joints was compared with the accumulation of (/sup 111/In)leukocytes. No accumulation of (/sup 111/In)leukocytes was seen in any of the patients' wrists (0/12) or knee joints (0/14) when both pain and swelling were absent. In contrast, 93% (25/27) of wrist joints and 80% (24/30) of knee joints with both pain and swelling were positive by (/sup 111/In)leukocyte scintigraphy. There was little correlation between the stage of the disease, as determined by radiography, and (/sup 111/In)leukocyte accumulation. This study suggests that (/sup 111/In)leukocyte imaging may be a reliable procedure for monitoring the activity of rheumatoid arthritis, especially for confirming the lack of an ongoing inflammatory response.

  9. Erythrocyte and leukocyte: two partners in bacteria killing.

    PubMed

    Minasyan, Hayk A

    2014-01-01

    Leukocytes can't perform phagocytosis in blood stream. Blood velocity prevents phagocytosis because there is no time for leukocyte to recognize and catch bacteria. Bloodstream clearance from pathogens is performed by erythrocytes. During motion in bloodstream erythrocytes become charged by triboelectric effect. This charge attracts bacteria and fixes them on the surface of erythrocyte, then bacteria are engulfed and killed by hemoglobin oxygen. In bloodstream, leukocyte thin-wrinkled elastic membrane can't be charged by triboelectric effect and so leukocyte can't catch bacteria by means of electrostatic attraction force. Leukocytes engulf and kill bacteria out of blood circulatory system: in tissues, lymph nodes, slow velocity lymph, etc. Erythrocyte and leukocyte are bactericidal partners: the first kills bacteria in bloodstream, the second kills them locally, out of blood circulation.

  10. FAK and PAX-illin get involved in leukocyte diapedesis.

    PubMed

    Luscinskas, Francis W

    2012-02-01

    A major focus of researchers studying leukocyte recruitment has been to identify and understand how cell surface endothelial adhesion molecules, cell-to-cell junctional protein complexes, secreted chemokines and chemoattractants, and the vessel basement membrane structure organization coordinate the process of leukocyte recruitment. As research expands beyond the components initially identified as being necessary for leukocyte recruitment, attention has turned to the structures that regulate endothelial cell-to-matrix adhesion. In this issue of the European Journal of Immunology, Parsons et al. [Eur. J. Immunol. 2012. 42: 436-446] identify new players in the regulation of neutrophil diapedesis (transendothelial migration), namely the focal adhesion proteins, paxillin and focal adhesion kinase (FAK). While understudied, and indeed previously underappreciated, in leukocyte diapedesis, this Commentary discusses how the work by Parsons et al. implicates FAK and paxillin in the proximal (leukocyte rolling) and distal (diapedesis) steps of the multistep adhesion cascade of leukocyte recruitment.

  11. [Role of "leukocyte adhesion molecules" in early periodontal disease].

    PubMed

    Vierucci, S

    1992-01-01

    The purpose of this paper is to focus on functional characteristics of leukocyte adhesion molecules, on their localization and specific ligands. In fact, leukocyte chemotaxis and adhesion to endothelium is an essential step in promoting adequate immune response to bacterial infections. Since periodontal health is highly dependent on neutrophil function against the microbial dental plaque, defects in chemotaxis and adhesion of leukocytes to endothelium often result in severe, early onset periodontitis. Furthermore, oral lesions may be the only clinical manifestation of neutrophil impairment.

  12. Passenger leukocytes and microchimerism: what role in tolerance induction?

    PubMed

    Wood, Kathryn J

    2003-05-15

    The role of passenger leukocytes in determining the outcome after transplantation is complex. In some settings, donor-derived passenger leukocytes can initiate graft rejection, whereas in others they contribute to graft acceptance. Both donor and recipient factors contribute to this potential dual role. Understanding the interaction between passenger leukocytes and the recipient's immune system, particularly after liver transplantation, may provide important clues for developing novel strategies for inducing specific unresponsiveness to donor alloantigens.

  13. Leukocyte opioid receptors mediate analgesia via Ca(2+)-regulated release of opioid peptides.

    PubMed

    Celik, Melih Ö; Labuz, Dominika; Henning, Karen; Busch-Dienstfertig, Melanie; Gaveriaux-Ruff, Claire; Kieffer, Brigitte L; Zimmer, Andreas; Machelska, Halina

    2016-10-01

    Opioids are the most powerful analgesics. As pain is driven by sensory transmission and opioid receptors couple to inhibitory G proteins, according to the classical concept, opioids alleviate pain by activating receptors on neurons and blocking the release of excitatory mediators (e.g., substance P). Here we show that analgesia can be mediated by opioid receptors in immune cells. We propose that activation of leukocyte opioid receptors leads to the secretion of opioid peptides Met-enkephalin, β-endorphin and dynorphin A (1-17), which subsequently act at local neuronal receptors, to relieve pain. In a mouse model of neuropathic pain induced by a chronic constriction injury of the sciatic nerve, exogenous agonists of δ-, μ- and κ-opioid receptors injected at the damaged nerve infiltrated by opioid peptide- and receptor-expressing leukocytes, produced analgesia, as assessed with von Frey filaments. The analgesia was attenuated by pharmacological or genetic inactivation of opioid peptides, and by leukocyte depletion. This decrease in analgesia was restored by the transfer of wild-type, but not opioid receptor-lacking leukocytes. Ex vivo, exogenous opioids triggered secretion of opioid peptides from wild-type immune cells isolated from damaged nerves, which was diminished by blockade of Gαi/o or Gβγ (but not Gαs) proteins, by chelator of intracellular (but not extracellular) Ca(2+), by blockers of phospholipase C (PLC) and inositol 1,4,5-trisphosphate (IP3) receptors, and was partially attenuated by protein kinase C inhibitor. Similarly, the leukocyte depletion-induced decrease in exogenous opioid analgesia was re-established by transfer of immune cells ex vivo pretreated with extracellular Ca(2+) chelator, but was unaltered by leukocytes pretreated with intracellular Ca(2+) chelator or blockers of Gαi/o and Gβγ proteins. Thus, both ex vivo opioid peptide release and in vivo analgesia were mediated by leukocyte opioid receptors coupled to the G

  14. A role for leukocyte-endothelial adhesion mechanisms in epilepsy

    PubMed Central

    Fabene, Paolo F.; Mora, Graciela Navarro; Martinello, Marianna; Rossi, Barbara; Merigo, Flavia; Ottoboni, Linda; Bach, Simona; Angiari, Stefano; Benati, Donatella; Chakir, Asmaa; Zanetti, Lara; Schio, Federica; Osculati, Antonio; Marzola, Pasquina; Nicolato, Elena; Homeister, Jonathon W.; Xia, Lijun; Lowe, John B.; McEver, Rodger P.; Osculati, Francesco; Sbarbati, Andrea; Butcher, Eugene C.; Constantin, Gabriela

    2009-01-01

    The mechanisms involved in the pathogenesis of epilepsy, a chronic neurological disorder that affects approximately 1 percent of the world population, are not well understood1–3. Using a mouse model of epilepsy, we show that seizures induce elevated expression of vascular cell adhesion molecules and enhanced leukocyte rolling and arrest in brain vessels mediated by the leukocyte mucin P-selectin glycoprotein ligand-1 (PSGL-1) and leukocyte integrins α4β1 and αLβ2. Inhibition of leukocyte-vascular interactions either with blocking antibodies, or in mice genetically deficient in functional PSGL-1, dramatically reduced seizures. Treatment with blocking antibodies following acute seizures prevented the development of epilepsy. Neutrophil depletion also inhibited acute seizure induction and chronic spontaneous recurrent seizures. Blood-brain barrier (BBB) leakage, which is known to enhance neuronal excitability, was induced by acute seizure activity but was prevented by blockade of leukocyte-vascular adhesion, suggesting a pathogenetic link between leukocyte-vascular interactions, BBB damage and seizure generation. Consistent with potential leukocyte involvement in the human, leukocytes were more abundant in brains of epileptics than of controls. Our results suggest leukocyte-endothelial interaction as a potential target for the prevention and treatment of epilepsy. PMID:19029985

  15. Activation of Poly(ADP-Ribose) Polymerase by Myocardial Ischemia and Coronary Reperfusion in Human Circulating Leukocytes

    PubMed Central

    Tóth-Zsámboki, Emese; Horváth, Eszter; Vargova, Katarina; Pankotai, Eszter; Murthy, Kanneganti; Zsengellér, Zsuzsanna; Bárány, Tamás; Pék, Tamás; Fekete, Katalin; Kiss, Róbert Gábor; Préda, István; Lacza, Zsombor; Gerö, Domokos; Szabó, Csaba

    2006-01-01

    Reactive free radical and oxidant production leads to DNA damage during myocardial ischemia/reperfusion. Consequent overactivation of poly(ADP-ribose) polymerase (PARP) promotes cellular energy deficit and necrosis. We hypothesized that PARP is activated in circulating leukocytes in patients with myocardial infarction and reperfusion during primary percutaneous coronary intervention (PCI). In 15 patients with ST segment elevation acute myocardial infarction, before and after primary PCI and 24 and 96 h later, we determined serum hydrogen peroxide concentrations, plasma levels of the oxidative DNA adduct 8-hydroxy-2′-deoxyguanosine (8OHdG), tyrosine nitration, PARP activation, and translocation of apoptosis-inducing factor (AIF) in circulating leukocytes. Plasma 8OHdG levels and leukocyte tyrosine nitration were rapidly increased by PCI. Similarly, poly(ADP-ribose) content of the leukocytes increased in cells isolated just after PCI, indicating immediate PARP activation triggered by reperfusion of the myocardium. In contrast, serum hydrogen peroxide concentrations and the translocation of AIF gradually increased over time and were most pronounced at 96 h. Reperfusion-related oxidative/nitrosative stress triggers DNA damage, which leads to PARP activation in circulating leukocytes. Translocation of AIF and lipid peroxidation occurs at a later stage. These results represent the first direct demonstration of PARP activation in human myocardial infarction. Future work is required to test whether pharmacological inhibition of PARP may offer myocardial protection during primary PCI. PMID:17225870

  16. Activation of poly(ADP-ribose) polymerase by myocardial ischemia and coronary reperfusion in human circulating leukocytes.

    PubMed

    Tóth-Zsámboki, Emese; Horváth, Eszter; Vargova, Katarina; Pankotai, Eszter; Murthy, Kanneganti; Zsengellér, Zsuzsanna; Bárány, Tamás; Pék, Tamás; Fekete, Katalin; Kiss, Róbert Gábor; Préda, István; Lacza, Zsombor; Gerö, Domokos; Szabó, Csaba

    2006-01-01

    Reactive free radical and oxidant production leads to DNA damage during myocardial ischemia/reperfusion. Consequent overactivation of poly(ADP-ribose) polymerase (PARP) promotes cellular energy deficit and necrosis. We hypothesized that PARP is activated in circulating leukocytes in patients with myocardial infarction and reperfusion during primary percutaneous coronary intervention (PCI). In 15 patients with ST segment elevation acute myocardial infarction, before and after primary PCI and 24 and 96 h later, we determined serum hydrogen peroxide concentrations, plasma levels of the oxidative DNA adduct 8-hydroxy-2'-deoxyguanosine (8OHdG), tyrosine nitration, PARP activation, and translocation of apoptosis-inducing factor (AIF) in circulating leukocytes. Plasma 8OHdG levels and leukocyte tyrosine nitration were rapidly increased by PCI. Similarly, poly(ADP-ribose) content of the leukocytes increased in cells isolated just after PCI, indicating immediate PARP activation triggered by reperfusion of the myocardium. In contrast, serum hydrogen peroxide concentrations and the translocation of AIF gradually increased over time and were most pronounced at 96 h. Reperfusion-related oxidative/nitrosative stress triggers DNA damage, which leads to PARP activation in circulating leukocytes. Translocation of AIF and lipid peroxidation occurs at a later stage. These results represent the first direct demonstration of PARP activation in human myocardial infarction. Future work is required to test whether pharmacological inhibition of PARP may offer myocardial protection during primary PCI.

  17. A study to evaluate the effect of nootropic drug-piracetam on DNA damage in leukocytes and macrophages.

    PubMed

    Singh, Sarika; Goswami, Poonam; Swarnkar, Supriya; Singh, Sheelendra Pratap; Wahajuddin; Nath, Chandishwar; Sharma, Sharad

    2011-11-27

    Piracetam is a nootropic drug that protects neurons in neuropathological and age-related diseases and the activation and modulation of peripheral blood cells in patients with neuropathological conditions is well known. Therefore, in the present study, in vivo, ex vivo, and in vitro tests were conducted to investigate the effect of piracetam on leukocytes and macrophages. Lipopolysaccharide (LPS) causes oxidative DNA damage; thus, in the present study, LPS was used as a tool to induce DNA damage. In vivo experiments were conducted on Sprague Dawley rats, and piracetam (600mg/kg, oral) was provided for five consecutive days. On the fifth day, a single injection of LPS (10mg/kg, i.p.) was administered. Three hours after LPS injection, blood leukocytes and peritoneal macrophages were collected and processed, and a variety of different assays were conducted. Ex vivo treatments were performed on isolated rat blood leukocytes, and in vitro experiments were conducted on rat macrophage cell line J774A.1. Cell viability and the level of reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and DNA damage were estimated in untreated (control) and piracetam-, LPS- and LPS+piracetam-treated leukocytes and macrophages. In vivo experiments revealed that rats pretreated with piracetam were significantly protected against LPS-induced increases in ROS levels and DNA damage. Ex vivo isolated leukocytes and J774A.1 cells treated with LPS exhibited augmented ROS levels and DNA damage, which were attenuated with piracetam treatment. Thus, the present study revealed the salutary effect of piracetam against LPS-induced oxidative stress and DNA damage in leukocytes and macrophages.

  18. Interaction of activated leukocytes with polymeric microspheres.

    PubMed

    Ayhan, H; Pişkin, E

    1997-12-01

    Three types of polymeric particles with different surface wettabilities, i.e., poly(methylmethacrylate) (PMMA), poly(methylmethacrylate-hydroxyethylmethacrylate) (P(MMA/HEMA)) and poly(methylmethacrylate)/poly(vinyl alcohol) PMMA/PVAL with a diameter of 1.5 microm were produced in this study These particles were incubated with blood samples obtained both from three patients undergoing cardiopulmonary bypass. In the blood samples taken before the bypass operations, there was considerable phagocytosis and/or adhesion of the PMMA particles, i.e., 14+/-4 particles per monocyte and 11+/-3 particles per neutrophil. While there was almost no phagocytosis and/or adhesion of the P(MMA/HEMA) and PMMA/PVAL particles. In the blood samples which were taken during bypass operations, phagocytosis and/or adhesion of PMMA microspheres increased significantly. The P(MMA/HEMA) and/or PMMA/PVAL particles adhered, or were even phagocytosed by the activated leukocytes in this case. Leukocytes activated during the bypass operations gradually returned to normal in about 24 h.

  19. Characterization of rag1 mutant zebrafish leukocytes

    PubMed Central

    Petrie-Hanson, Lora; Hohn, Claudia; Hanson, Larry

    2009-01-01

    Background Zebrafish may prove to be one of the best vertebrate models for innate immunology. These fish have sophisticated immune components, yet rely heavily on innate immune mechanisms. Thus, the development and characterization of mutant and/or knock out zebrafish are critical to help define immune cell and immune gene functions in the zebrafish model. The use of Severe Combined Immunodeficient (SCID) and recombination activation gene 1 and 2 mutant mice has allowed the investigation of the specific contribution of innate defenses in many infectious diseases. Similar zebrafish mutants are now being used in biomedical and fish immunology related research. This report describes the leukocyte populations in a unique model, recombination activation gene 1-/- mutant zebrafish (rag1 mutants). Results Differential counts of peripheral blood leukocytes (PBL) showed that rag1 mutants had significantly decreased lymphocyte-like cell populations (34.7%) compared to wild-types (70.5%), and significantly increased granulocyte populations (52.7%) compared to wild-types (17.6%). Monocyte/macrophage populations were similar between mutants and wild-types, 12.6% and 11.3%, respectively. Differential leukocyte counts of rag1 mutant kidney hematopoietic tissue showed a significantly reduced lymphocyte-like cell population (8%), a significantly increased myelomonocyte population (57%), 34.8% precursor cells, and 0.2% thrombocytes, while wild-type hematopoietic kidney tissue showed 29.4% lymphocytes/lymphocyte-like cells, 36.4% myelomonocytes, 33.8% precursors and 0.5% thrombocytes. Flow cytometric analyses of kidney hematopoietic tissue revealed three leukocyte populations. Population A was monocytes and granulocytes and comprised 34.7% of the gated cells in rag1 mutants and 17.6% in wild-types. Population B consisted of hematopoietic precursors, and comprised 50% of the gated cells for rag1 mutants and 53% for wild-types. Population C consisted of lymphocytes and lymphocyte

  20. Isolation of inflammatory cells from human tumours.

    PubMed

    Polak, Marta E

    2011-01-01

    Inflammatory cells are present in many tumours, and understanding their function is of increasing importance, particularly to studies of tumour immunology. The tumour-infiltrating leukocytes encompass a variety of cell types, e.g. T lymphocytes, macrophages, dendritic cells, NK cells, and mast cells. Choice of the isolation method greatly depends on the tumour type and the leukocyte subset of interest, but the protocol usually includes tissue disaggregation and cell enrichment. We recommend density centrifugation for initial enrichment, followed by specific magnetic bead negative or positive panning with leukocyte and tumour cell selective antibodies.

  1. Social regulation of gene expression in human leukocytes

    PubMed Central

    Cole, Steve W; Hawkley, Louise C; Arevalo, Jesusa M; Sung, Caroline Y; Rose, Robert M; Cacioppo, John T

    2007-01-01

    Background Social environmental influences on human health are well established in the epidemiology literature, but their functional genomic mechanisms are unclear. The present study analyzed genome-wide transcriptional activity in people who chronically experienced high versus low levels of subjective social isolation (loneliness) to assess alterations in the activity of transcription control pathways that might contribute to increased adverse health outcomes in social isolates. Results DNA microarray analysis identified 209 genes that were differentially expressed in circulating leukocytes from 14 high- versus low-lonely individuals, including up-regulation of genes involved in immune activation, transcription control, and cell proliferation, and down-regulation of genes supporting mature B lymphocyte function and type I interferon response. Promoter-based bioinformatic analyses showed under-expression of genes bearing anti-inflammatory glucocorticoid response elements (GREs; p = 0.032) and over-expression of genes bearing response elements for pro-inflammatory NF-κB/Rel transcription factors (p = 0.011). This reciprocal shift in pro- and anti-inflammatory signaling was not attributable to differences in circulating cortisol levels, or to other demographic, psychological, or medical characteristics. Additional transcription control pathways showing differential activity in bioinformatic analyses included the CREB/ATF, JAK/STAT, IRF1, C/EBP, Oct, and GATA pathways. Conclusion These data provide the first indication that human genome-wide transcriptional activity is altered in association with a social epidemiological risk factor. Impaired transcription of glucocorticoid response genes and increased activity of pro-inflammatory transcription control pathways provide a functional genomic explanation for elevated risk of inflammatory disease in individuals who experience chronically high levels of subjective social isolation. PMID:17854483

  2. The effects of stress on the enzymes of peripheral leukocytes

    NASA Technical Reports Server (NTRS)

    Leise, E. M.; Gray, I.

    1973-01-01

    Previous work showed an early response of rabbit and human leukocyte enzymes to the stress of bacterial infection. Since these represented a mixed population of leukocytes and since polymorphonuclear leukocytes (PMN) increased in these preparations, it was necessary to establish whether the observed increase in lactate dehydrenase (LDH) and protein was the result of an increase in any one particular cell type or in all cells. The need for the development of a simple reproducible method for the differential separation of peripheral leukocytes for the furtherance of our own studies was apparent. It was also becoming increasingly apparent that morphologically similar cells, such as small lymphocytes (L) and macrophages, were capable of different biological functions. A dextran gradient centrifugation method was developed which has provided an easily reproducible technique for separating L from PMN. During the course of this work, in which over 250 rabbits were examined, the pattern of daily leukocyte protein and enzyme variation became increasingly more apparent. This information could have some impact on future work with leukocyte enzymes, by our group and by other workers. The differences in normal protein and enzyme levels maintained by some individuals, and some inbred strains, were evaluated and reported separately. It has been shown that one type of leukocyte may react more to a given stress than other leukocytes.

  3. 21 CFR 864.7675 - Leukocyte peroxidase test.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Leukocyte peroxidase test. 864.7675 Section 864... peroxidase test. (a) Identification. A leukocyte peroxidase test is a device used to distinguish certain... peroxidase activity as evidenced by staining. The results of this test are used in the differential...

  4. Degradation of Thyroid Hormones by Phagocytosing Human Leukocytes

    PubMed Central

    Klebanoff, Seymour J.; Green, William L.

    1973-01-01

    Thyroxine (T4) and triiodothyronine (T9) are rapidly degraded by a purified preparation of myeloperoxidase (MPO) and H2O2 with the formation of iodide and material which remains at the origin on paper chromatography. Deiodination by MPO and H2O2 occurs more readily at pH 7.0 than at pH 5.0 in contrast to iodination by this system which is known to occur more readily at pH 5.0 than at pH 7.0. Degradation is inhibited by azide, cyanide, ascorbic acid, and propylthiouracil. Methimazole stimulates deiodination by MPO and H2O2 but inhibits this reaction when MPO is replaced by lactoperoxidase or horseradish peroxidase. Intact human leukocytes, in the resting state, degrade T4 and T3 slowly: degradation, however, is increased markedly during phagocytosis of preopsonized particles. Serum inhibits this reaction. T3 can be detected as a minor product of T4 degradation. Proteolytic digestion of the reaction products increases the recovery of monoiodotyrosine. The fixation of iodine in the cytoplasm of leukocytes which contain ingested bacteria was detected radioautographically. Chronic granulomatous disease leukocytes, which are deficient in H2O2 formation, degrade T4 and T3 poorly during phagocytosis. MPO-deficient leukocytes degrade the thyroid hormones at a slower rate than do normal leukocytes although considerable degradation is still observed. Azide, cyanide, ascorbic acid, and propylthiouracil which inhibit certain peroxidasecatalyzed reactions inhibit degradation by normal leukocytes; however, inhibition is incomplete. Formation of iodinated origin material is inhibited to a greater degree by azide, cyanide, and propylthiouracil than is deiodination. Methimazole inhibits the formation of iodinated origin material by both normal and MPO-deficient leukocytes. However, deiodination by normal leukocytes is stimulated and that of MPO-deficient leukocytes is unaffected by methimazole. Hypoxia inhibits the degradation of T4 and T3 by untreated normal or MPO

  5. Penetration of equine leukocytes by merozoites of Sarcocystis neurona.

    PubMed

    Lindsay, David S; Mitchell, Sheila M; Yang, Jibing; Dubey, J P; Gogal, Robert M; Witonsky, Sharon G

    2006-06-15

    Horses are considered accidental hosts for Sarcocystis neurona and they often develop severe neurological disease when infected with this parasite. Schizont stages develop in the central nervous system (CNS) and cause the neurological lesions associated with equine protozoal myeloencephalitis. The present study was done to examine the ability of S. neurona merozoites to penetrate and develop in equine peripheral blood leukocytes. These infected host cells might serve as a possible transport mechanism into the CNS. S. neurona merozoites penetrated equine leukocytes within 5 min of co-culture. Infected leukocytes were usually monocytes. Infected leukocytes were present up to the final day of examination at 3 days. Up to three merozoites were present in an infected monocyte. No development to schizont stages was observed. All stages observed were in the host cell cytoplasm. We postulate that S. neurona merozoites may cross the blood brain barrier hidden inside leukocytes. Once inside the CNS these merozoites can egress and invade additional cells and cause encephalitis.

  6. Isolation of Human Basophils.

    PubMed

    Schroeder, John T; Bieneman, Anja P

    2016-02-02

    Isolating human basophils from blood has long been hampered by the fact that these granulocytes represent just 1% or less of the circulating leukocyte population. We describe herein laboratory protocols that have been refined over the past ∼25 years that now enable investigators to prepare basophils for use in a variety of assays to assess the in vitro biology of these immune cells, both in IgE -dependent and -independent responses.

  7. Antibiotic proteins of human polymorphonuclear leukocytes.

    PubMed Central

    Gabay, J E; Scott, R W; Campanelli, D; Griffith, J; Wilde, C; Marra, M N; Seeger, M; Nathan, C F

    1989-01-01

    Nine polypeptide peaks with antibiotic activity were resolved from human polymorphonuclear leukocyte azurophil granule membranes. All but 1 of the 12 constituent polypeptides were identified by N-terminal sequence analysis. Near quantitative recovery of protein and activity permitted an assessment of the contribution of each species to the overall respiratory-burst-independent antimicrobial capacity of the cell. Three uncharacterized polypeptides were discovered, including two broad-spectrum antibiotics. One of these, a defensin that we have designated human neutrophil antimicrobial peptide 4, was more potent than previously described defensins but represented less than 1% of the total protein. The other, named azurocidin, was abundant and comparable to bactericidal permeability-increasing factor in its contribution to the killing of Escherichia coli. Images PMID:2501794

  8. Association between Snoring and Leukocyte Telomere Length

    PubMed Central

    Shin, Chol; Yun, Chang-Ho; Yoon, Dae Wui; Baik, Inkyung

    2016-01-01

    Study Objectives: Data on the association between snoring and telomere length, an indicator of biological aging, are very limited. Moreover, no polysomnography (PSG) studies on this association in a general population have been conducted. Our study aimed to evaluate the association between snoring and leukocyte telomere length (LTL) using PSG and a questionnaire. Methods: A cross-sectional PSG study embedded in a population-based cohort from the Korean Genome Epidemiology Study was conducted in 2010–2013. During the same period, questionnaire-based interviews, blood collection, and relative LTL assays were conducted. A total of 887 Korean men and women aged 50–79 y with an apnea-hypopnea index (AHI) < 15 determined in the PSG study were included in the study. Results: We observed that the percentage of time spent snoring during sleep (% time spent snoring) assessed by PSG was inversely associated with LTL even after adjusting for potential risk factors and AHI. In the linear regression association between tertiles of percentage of time spent snoring and log-transformed LTL, coefficient estimates (P value) were −0.076 (< 0.05) for the second tertile and −0.084 (< 0.01) for the third tertile compared with the bottom tertile. When LTL was compared according to snoring status determined using PSG and questionnaire information, both primary snorers and those with mild sleep apnea (5 ≤ AHI < 15) had shorter LTL than nonsnorers. Conclusions: Our findings suggest that snoring may influence telomere attrition independent of sleep apnea. Citation: Shin C, Yun CH, Yoon DW, Baik I. Association between snoring and leukocyte telomere length. SLEEP 2016;39(4):767–772. PMID:26715224

  9. Leukocyte CD11a expression and granulocyte activation during experimental myocardial ischemia and long lasting reperfusion

    PubMed Central

    Lantos, János; Grama, László; Orosz, Tamás; Temes, Gyula; Rőth, Elizabeth

    2001-01-01

    BACKGROUND: Myocardial ischemia and reperfusion are accompanied by leukocyte activation and expression of surface adhesion molecules, which induce pathological interactions between endothelial cells and circulating neutrophils, leading to tissue damage. While the dynamics of these processes have been well defined during acute reperfusion, there is very little information regarding long lasting reperfusion. OBJECTIVES: To investigate neutrophil granulocyte (PMN) activation and the CD11a expression of leukocytes during myocardial ischemia and reperfusion for four weeks. ANIMALS AND METHODS: The left anterior descending coronary artery was occluded for 1 h in six dogs, followed by reperfusion for four weeks. Peripheral blood samples were collected before the operation, at the end of ischemia, at 5 and 60 min of reperfusion, and on postoperative days 1, 2, 3, 7, 14, 21 and 28. Sham operation on four dogs served as control. Leukocyte expression of CD11a was measured by flow cytometry. Superoxide radical production of isolated PMNs was determined spectrophotometrically. RESULTS: Granulocyte CD11a expression increased while the superoxide radical-producing capacity decreased significantly by the third postoperative day. Sham operation produced similar alterations in these parameters during the first postoperative week. From the second postoperative week, however, granulocyte radical production and adhesion molecule expression were higher in the ischemic animals. CONCLUSIONS: The exhaustion of PMN radical production and maximal CD11a expression during the first postoperative week are probably due to the surgical trauma caused by thoracotomy, but increased granulocyte function during later reperfusion indicates prolonged healing of injured myocardium. PMID:20428266

  10. Genotoxic potential of TiO2 on bottlenose dolphin leukocytes.

    PubMed

    Bernardeschi, Margherita; Guidi, Patrizia; Scarcelli, Vittoria; Frenzilli, Giada; Nigro, Marco

    2010-01-01

    Titanium dioxide is extensively used in a variety of products, including industrial materials and cosmetics. Studies mainly performed on human cell lines and in vivo exposure on experimental animals have raised concern about the toxic effects of ultrafine titanium dioxide; however, scarce information is available about its impact on aquatic life. The aim of this article was to assess the genotoxic potential of TiO(2) (anatase and rutile) on bottlenose dolphin leukocytes. Blood samples were obtained from four male and one female specimens reared at the Adriatic SeaWorld "Oltremare" (Riccione, Italy). Leukocytes were isolated by the lyses procedure and in vitro exposed to TiO(2) in RPMI. Experimental solutions were sonicated immediately before dosing the cells. Three exposure times (4, 24 and 48 h) and three doses (20, 50 and 100 microg/ml) were tested. Genotoxicity was detected by the single-cell gel electrophoresis (or comet assay) at pH > or = 13, assessing single/double-strand breaks and alkali-labile sites. Cytotoxicity was also detected by the Trypan blue exclusion method. Results showed that both the crystalline forms of TiO(2) were genotoxic for bottlenose dolphin leukocytes, with a statistically significant increase of DNA fragmentation after exposure to 50 and 100 microg/ml for 24 and 48 h. Although preliminary, these are the first data regarding the genetic susceptibility of toothed cetaceans toward an "emerging" pollutant, such as TiO(2) particles.

  11. A three-dimensional atlas of human dermal leukocytes, lymphatics, and blood vessels.

    PubMed

    Wang, Xiao-Nong; McGovern, Naomi; Gunawan, Merry; Richardson, Connor; Windebank, Martin; Siah, Tee-Wei; Lim, Hwee-Ying; Fink, Katja; Li, Jackson L Yao; Ng, Lai G; Ginhoux, Florent; Angeli, Veronique; Collin, Matthew; Haniffa, Muzlifah

    2014-04-01

    Dendritic cells (DCs), macrophages (Mφ), and T cells are major components of the skin immune system, but their interstitial spatial organization is poorly characterized. Using four-channel whole-mount immunofluorescence staining of the human dermis, we demonstrated the three-dimensional distribution of CD31(+) blood capillaries, LYVE-1(+) lymphatics, discrete populations of CD11c(+) myeloid DCs, FXIIIa(+) Mφ, and lymphocytes. We showed phenotypic and morphological differences in situ between DCs and Mφ. DCs formed the first dermal cellular layer (0-20 μm beneath the dermoepidermal junction), Mφ were located deeper (40-60 μm), and CD3(+) lymphocytes were observed throughout (0-60 μm). Below this level, DCs, T cells, and the majority of Mφ formed stable perivascular sheaths. Whole-mount imaging revealed the true extent of dermal leukocytes previously underestimated from cross-section views. The total area of apical dermis (0-30 μm) contained approximately 10-fold more myeloid DCs than the entire blood volume of an average individual. Surprisingly, <1% of dermal DCs occupied lymphatics in freshly isolated skin. Dermal DCs rapidly accumulated within lymphatics, but Mφ remained fixed in skin explants cultured ex vivo. The leukocyte architecture observed in normal skin was distorted in inflammation and disease. These studies illustrate the micro-anatomy of dermal leukocytes and provide further insights into their functional organization.

  12. Expression of cell adhesion molecules, chemokines and chemokine receptors involved in leukocyte traffic in rats undergoing autoimmune orchitis.

    PubMed

    Guazzone, V A; Jacobo, P; Denduchis, B; Lustig, L

    2012-05-01

    The testis is considered an immunologically privileged site where germ cell antigens are protected from autoimmune attack. Yet in response to infections, inflammatory diseases, or trauma, there is an influx of leukocytes to testicular interstitium. Interactions between endothelial cells (EC) and circulating leukocytes are implicated in the initiation and evolution of inflammatory processes. Chemokines are a family of chemoattractant cytokines characterized by their ability to both recruit and activate cells. Thus, we investigated the expression of CCL3, its receptors, and adhesion molecules CD31 and CD106 in an in vivo model of experimental autoimmune orchitis (EAO). In EAO, the highest content of CCL3 in testicular fluid coincides with onset of the disease. However, CCL3 released in vitro by testicular macrophages is higher during the immunization period. The specific chemokine receptors, CCR1 and CCR5, were expressed by testicular monocytes/macrophages and an increased number of CCR5+ cells was associated with the degree of testicular lesion. EC also play an essential role by facilitating leukocyte recruitment via their ability to express cell surface adhesion molecules that mediate interactions with leukocytes in the bloodstream. Rats with EAO showed a significant increase in the percentage of CD31+ EC that upregulate the expression of CD106. The percentage of leukocytes isolated from peripheral blood and lymph nodes expressing CD49d (CD106 ligand) also increases during orchitis. These data suggest that cell adhesion molecules, in conjunction with chemokines, contribute to the formation of a chemotactic gradient within the testis, causing the leukocyte infiltration characteristic of EAO histopathology.

  13. Monitoring of leukocyte cytomegalovirus DNA in bone marrow transplant recipients by nested PCR.

    PubMed Central

    Yuen, K Y; Lo, S K; Chiu, E K; Wong, S S; Lau, Y L; Liang, R; Chan, T K; Ng, M H

    1995-01-01

    A nested PCR assay for the detection of human cytomegalovirus (CMV) DNA was evaluated by weekly monitoring of blood samples taken from 101 bone marrow transplant (BMT) recipients. When peripheral blood leukocytes were used as the source of CMV DNA, even a modified assay with stringent temperature-cycling conditions was as sensitive as the standard assay. The sensitivity, specificity, and positive predictive value of two consecutively positive leukocytic PCR results with this modified assay in predicting CMV disease of 101 patients submitting 1,441 peripheral blood leukocyte samples were found to be 92.1, 63.5, and 60.3%, respectively. The positive predictive value of patients' seropositivity for CMV was 40%, while that of viremia was 72%. However, viremia followed CMV disease by a median of 1.5 days, while the first leukocytic positive PCR assay preceded disease by a median of 14 days. By use of the criteria of two consecutively positive PCR results instead of recipient CMV seropositivity for starting preemptive ganciclovir treatment, 38 of the 43 recipients with isolated single positive or negative assays (groups I and II) would be spared unnecessary ganciclovir treatment. Moreover, two other findings support the use of antiviral prophylaxis before engraftment in high-risk cases and subsequent preemptive treatment of patients with two consecutively positive PCR assays. First, for 7.9% of 76 patients with positive assays (groups II and III), the first positive PCR assay occurred before engraftment, which implied the presence of viral DNA in the blood (DNAemia) soon after transplantation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8567878

  14. Adhesion of polymorphonuclear leukocytes to endothelium enhances the efficiency of detoxification of oxygen-free radicals.

    PubMed Central

    Hoover, R. L.; Robinson, J. M.; Karnovsky, M. J.

    1987-01-01

    Polymorphonuclear leukocytes can produce active oxygen species such as hydrogen peroxide and superoxide under various conditions. Because these substances can be toxic to cells, it is possible that the interaction between the circulating leukocytes and the blood vessel wall, either in normal circulation or during the acute inflammatory response, could damage the endothelial lining. Using an in vitro system of cultured endothelial cells and isolated polymorphonuclear leukocytes, we have measured the levels of detectable superoxide when neutrophils are attached to either endothelial monolayers or to plastic. Our results show that the levels of superoxide, on a per-cell basis, are lower when the neutrophils are attached to endothelium than when attached to plastic, even if the neutrophils are stimulated with phorbol myristate acetate. This is also reflected in data showing that no injury occurs to the endothelial cells, as measured by 51Cr release, under these same conditions. When endothelial cells are pretreated with an inhibitor of superoxide dismutase, diethyldithiocarbamate, the levels of superoxide detected are the same for neutrophils stimulated on plastic and those on the endothelial monolayer, suggesting that endothelial superoxide dismutase may remove a portion of the neutrophil-generated superoxide from the detection system. Further evidence for the role of endothelium in destroying superoxide is suggested by results that show that the level of detectable superoxide released from neutrophils attached to formalin-fixed endothelial monolayers is the same as that for neutrophils attached to plastic. It is important to note that with the inhibitor of superoxide dismutase present, the endothelial monolayers do not display enhanced 51Cr release under the conditions employed. When both endothelial catalase and glutathione reductase are inhibited, we detect increased 51Cr release from endothelial cells in response to stimulated neutrophils. Our results show that

  15. Optimization and Pharmacological Validation of a Leukocyte Migration Assay in Zebrafish Larvae for the Rapid In Vivo Bioactivity Analysis of Anti-Inflammatory Secondary Metabolites

    PubMed Central

    Vicet-Muro, Liliana; Wilches-Arizábala, Isabel María; Esguerra, Camila V.; de Witte, Peter A. M.; Crawford, Alexander D.

    2013-01-01

    Over the past decade, zebrafish (Danio rerio) have emerged as an attractive model for in vivo drug discovery. In this study, we explore the suitability of zebrafish larvae to rapidly evaluate the anti-inflammatory activity of natural products (NPs) and medicinal plants used in traditional medicine for the treatment of inflammatory disorders. First, we optimized a zebrafish assay for leukocyte migration. Inflammation was induced in four days post-fertilization (dpf) zebrafish larvae by tail transection and co-incubation with bacterial lipopolysaccharides (LPS), resulting in a robust recruitment of leukocytes to the zone of injury. Migrating zebrafish leukocytes were detected in situ by myeloperoxidase (MPO) staining, and anti-inflammatory activity was semi-quantitatively scored using a standardized scale of relative leukocyte migration (RLM). Pharmacological validation of this optimized assay was performed with a panel of anti-inflammatory drugs, demonstrating a concentration-responsive inhibition of leukocyte migration for both steroidal and non-steroidal anti-inflammatory drugs (SAIDs and NSAIDs). Subsequently, we evaluated the bioactivity of structurally diverse NPs with well-documented anti-inflammatory properties. Finally, we further used this zebrafish-based assay to quantify the anti-inflammatory activity in the aqueous and methanolic extracts of several medicinal plants. Our results indicate the suitability of this LPS-enhanced leukocyte migration assay in zebrafish larvae as a front-line screening platform in NP discovery, including for the bioassay-guided isolation of anti-inflammatory secondary metabolites from complex NP extracts. PMID:24124487

  16. Antimicrobial mechanisms against Acinetobacter calcoaceticus of rat polymorphonuclear leukocyte granule extract.

    PubMed Central

    Loeffelholz, M J; Modrzakowski, M C

    1988-01-01

    The antimicrobial mechanisms of rat polymorphonuclear leukocyte granule extract and isolated extract fractions against Acinetobacter calcoaceticus were examined. Crude granule extract and a fraction containing low-molecular-weight cationic peptides (peak D) reduced the viability of A. calcoaceticus and inhibited the uptake of radiolabeled macromolecule precursors by cells. The inhibitory activity observed with peak D was not as great as that of crude granule extract containing equivalent amounts of peak D protein. Crude extract also inhibited incorporation of uracil into trichloroacetic acid-precipitable material, while no isolated fraction, including peak D, had any substantial effect on incorporation. The antimicrobial activities of crude granule extract were more sensitive to boiling than those of isolated peak D. Preincubation of A. calcoaceticus with either crude granule extract or a fraction (peak B) possessing proteolytic activity but lacking any antimicrobial activity caused cells to become sensitive to a subinhibitory concentration of actinomycin D, suggesting that granule extract and peak B increase the outer membrane permeability of A. calcoaceticus. The antimicrobial granule extract fraction, peak D, did not affect outer membrane permeability. These results suggest that rat polymorphonuclear leukocyte granule extract reduces the viability of A. calcoaceticus by inhibiting the transport and incorporation of macromolecule precursors and that either whole granule extract is required for complete antimicrobial activity or an unidentified component is responsible for antimicrobial activity in addition to peak D. The granule extract activity that increases outer membrane permeability does not appear to be directly responsible for the observed decrease in viability. PMID:2449397

  17. Uptake of indium-111-labeled leukocytes by brain metastasis

    SciTech Connect

    Balachandran, S.; Husain, M.M.; Adametz, J.R.; Pallin, J.S.; Angtuaco, T.L.; Boyd, C.M.

    1987-04-01

    Uptake of indium-labeled leukocytes was seen in two cases of histologically proven brain metastasis. In one, this led to misdiagnosis of the lesion as an abscess. On histological evaluation, a large number of white blood cells or macrophages was seen at the neoplastic sites. Reasons for leukocyte accumulation around metastatic brain neoplasms are discussed. In contrast to the current reports that indium-labeled leukocyte scans can differentiate intracranial infection from tumor, these cases demonstrate their lack of specificity in the detection of brain abscess.

  18. Hypothyroidism modifies lipid composition of polymorphonuclear leukocytes.

    PubMed

    Coria, Mariela J; Carmona Viglianco, Yamila V; Marra, Carlos A; Gomez-Mejiba, Sandra E; Ramirez, Dario C; Anzulovich, Ana C; Gimenez, Maria S

    2012-01-01

    Thyroid hormones are important regulators of lipid metabolism. Polymorphonuclear leukocytes (PMN) are essential components of innate immune response. Our goal was to determine whether hypothyroidism affects lipid metabolism in PMN cells. Wistar rats were made hypothyroid by administrating 0.1 g/L 6-propyl-2-thiouracil (PTU) in drinking water during 30 days. Triacylglycerides (TG), cholesterol and phospholipids were determined in PMN and serum by conventional methods. The mRNA expression of LDL receptor (LDL-R), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoAR), sterol regulatory element binding protein 2 (SREBP-2), and diacylglycerol acyltransferase 2 (DGAT-2) were quantified by Real-Time PCR. Cellular neutral lipids were identified by Nile red staining. We found hypothyroidism decreases serum TG whereas it increases them in PMN. This result agrees with those observed in Nile red preparations, however DAGT-2 expression was not modified. Cholesterol synthesizing enzyme HMGCoAR mRNA and protein was reduced in PMN of hypothyroid rats. As expected, cholesterol content decreased in the cells although it increased in serum. Hypothyroidism also reduced relative contents of palmitic, stearic, and arachidonic acids, whereas increased the myristic, linoleic acids, and the unsaturation index in PMN. Thus, hypothyroidism modifies PMN lipid composition. These findings would emphasize the importance of new research to elucidate lipid-induced alterations in specific function(s) of PMN.

  19. Chemotactic peptide receptor modulation in polymorphonuclear leukocytes

    PubMed Central

    1980-01-01

    The binding of the chemotactic peptide N- formylnorleucylleucylphenylalanine (FNLLP) to its receptor on rabbit polymorphonuclear leukocytes (PMNs) modulates the number of available peptide receptors. Incubation with FNLLP decreases subsequent binding capacity, a phenomenon that has been termed receptor down regulation. Down regulation of the chemotactic peptide receptor is concentration dependent in both the rate and extent of receptor loss. The dose response parallels that of FNLLP binding to the recptor. The time- course is rapid; even at concentrations of FNLLP as low as 3 x 10(-9) M, the new equilibrium concentration of receptors is reached within 15 min. Down regulation is temperature dependent, but does occur even at 4 degrees C. Concomitant with down regulation, some of the peptide becomes irreversibly cell associated. At 4 degrees C, there is a small accumulation of nondissociable peptide that rapidly reaches a plateau. At higher temperatures, accumulation of nondissociable peptide continues after the rceptor number has reached equilibrium, and the amount accumulated can exceed the initial number of receptors by as much as 300%. The dose response of peptide uptake at 37 degrees C reflects that of binding, suggesting that it is receptor mediated. This uptake may occur via a pinocytosis mechanism. Although PMNs have not been considered to be pinocytic, the addition of FNLLP causes a fourfold stimulation of the rate of pinocytosis as measured by the uptake of [3H]sucrose. PMID:7391138

  20. Neutrophil Leukocyte: Combustive Microbicidal Action and Chemiluminescence.

    PubMed

    Allen, Robert C

    2015-01-01

    Neutrophil leukocytes protect against a varied and complex array of microbes by providing microbicidal action that is simple, potent, and focused. Neutrophils provide such action via redox reactions that change the frontier orbitals of oxygen (O2) facilitating combustion. The spin conservation rules define the symmetry barrier that prevents direct reaction of diradical O2 with nonradical molecules, explaining why combustion is not spontaneous. In burning, the spin barrier is overcome when energy causes homolytic bond cleavage producing radicals capable of reacting with diradical O2 to yield oxygenated radical products that further participate in reactive propagation. Neutrophil mediated combustion is by a different pathway. Changing the spin quantum state of O2 removes the symmetry restriction to reaction. Electronically excited singlet molecular oxygen ((1)O2(*)) is a potent electrophilic reactant with a finite lifetime that restricts its radius of reactivity and focuses combustive action on the target microbe. The resulting exergonic dioxygenation reactions produce electronically excited carbonyls that relax by light emission, that is, chemiluminescence. This overview of neutrophil combustive microbicidal action takes the perspectives of spin conservation and bosonic-fermionic frontier orbital considerations. The necessary principles of particle physics and quantum mechanics are developed and integrated into a fundamental explanation of neutrophil microbicidal metabolism.

  1. Neutrophil Leukocyte: Combustive Microbicidal Action and Chemiluminescence

    PubMed Central

    Allen, Robert C.

    2015-01-01

    Neutrophil leukocytes protect against a varied and complex array of microbes by providing microbicidal action that is simple, potent, and focused. Neutrophils provide such action via redox reactions that change the frontier orbitals of oxygen (O2) facilitating combustion. The spin conservation rules define the symmetry barrier that prevents direct reaction of diradical O2 with nonradical molecules, explaining why combustion is not spontaneous. In burning, the spin barrier is overcome when energy causes homolytic bond cleavage producing radicals capable of reacting with diradical O2 to yield oxygenated radical products that further participate in reactive propagation. Neutrophil mediated combustion is by a different pathway. Changing the spin quantum state of O2 removes the symmetry restriction to reaction. Electronically excited singlet molecular oxygen (1O2*) is a potent electrophilic reactant with a finite lifetime that restricts its radius of reactivity and focuses combustive action on the target microbe. The resulting exergonic dioxygenation reactions produce electronically excited carbonyls that relax by light emission, that is, chemiluminescence. This overview of neutrophil combustive microbicidal action takes the perspectives of spin conservation and bosonic-fermionic frontier orbital considerations. The necessary principles of particle physics and quantum mechanics are developed and integrated into a fundamental explanation of neutrophil microbicidal metabolism. PMID:26783542

  2. Influence of Magnetite Nanoparticles on Human Leukocyte Activity

    NASA Astrophysics Data System (ADS)

    Džarová, Anežka; Dubničková, Martina; Závišová, Vlasta; Koneracká, Martina; Kopčanský, Peter; Gojzewski, Hubert; Timko, Milan

    2010-12-01

    Chemically synthesized magnetite particles coated by sodium oleate and PEG (MNP), and magnetosomes (MS) influence the process of phagocytosis and the metabolic activity (lysozyme and peroxidase activity) in leukocytes. Lysozyme activity is oxygen-independent liquidation mechanisms of engulfed microorganism, peroxidase activity is an oxygen-dependent mechanism. Both tested types of nanoparticles lysed leukocyte cells during incubation. MNP at concentrations of 10 and 20 μg/mL lysed almost all leukocytes and their cell viability was in the 14±0.05% range. On the other hand MS begin to influence leukocytes activity at the concentration of 1 μg/ml and this influence grows with increasing concentration up to 20 μg/ml. MS are more suitable for biological applications than MNP which are more aggressive material than MS. MS should not be used above 10 μg/mL.

  3. Genetics Home Reference: leukocyte adhesion deficiency type 1

    MedlinePlus

    ... DR. Leukocyte adhesion deficiency type 1: an important consideration in the clinical differential diagnosis of prepubertal periodontitis. ... Leeuwen K, Köker MY, Parvaneh N, Fischer A, Law SK, Klein N, Tezcan FI, Unal E, Patiroglu ...

  4. Leukocyte Agglomeration Reaction in Diagnosis of Allergy Reactions from Antibiotics,

    DTIC Science & Technology

    tested in a clinic on 80 patients with serious allergic anamnesis . The results of the studies indicate that the leukocyte agglomeration reaction is a highly sensitive immunological indicator of hypersensitivity to antibiotics.

  5. Carp thrombocyte phagocytosis requires activation factors secreted from other leukocytes.

    PubMed

    Nagasawa, Takahiro; Somamoto, Tomonori; Nakao, Miki

    2015-10-01

    Thrombocytes are nucleated blood cells in non-mammalian vertebrates, which were recently focused on not only as hemostatic cells but also as immune cells with potent phagocytic activities. We have analyzed the phagocytic activation mechanisms in common carp (Cyprinus carpio) thrombocytes. MACS-sorted mAb(+) thrombocytes showed no phagocytic activity even in the presence of several stimulants. However, remixing these thrombocytes with other anti-thrombocyte mAb(-) leukocyte populations restored their phagocytic activities, indicating that carp thrombocyte phagocytosis requires an appropriate exogenous stimulation. Culture supernatant from anti-thrombocyte mAb(-) leukocytes harvested after PMA or LPS stimulation, but not culture supernatant from unstimulated leukocytes, could activate thrombocyte phagocytosis. This proposed mechanism of thrombocyte phagocytosis activation involving soluble factors produced by activated leukocytes suggests that thrombocyte activation is restricted to areas proximal to injured tissues, ensuring suppression of excessive thrombocyte activation and a balance between inflammation and tissue repair.

  6. PLATELET–LEUKOCYTE INTERACTIONS IN CARDIOVASCULAR DISEASE AND BEYOND

    PubMed Central

    Totani, Licia; Evangelista, Virgilio

    2010-01-01

    Platelet–leukocyte interactions define a basic cell process that is characterized by the exchange of signals between platelets and different types of leukocytes, and that bridges two fundamental physiopathological events: atherothrombosis and immune–inflammatory reactions. When this process takes place at the site of atherosclerotic plaque development or at the site of endothelial injury, platelet-dependent leukocyte recruitment and activation contributes to the inflammatory reaction of the vessel wall, which accounts for the exacerbation of atherosclerosis, and for intimal hyperplasia and plaque instability. Moreover, platelet–leukocyte interactions might have a key role in modulating a wide array of responses of both the innate and adaptive immune systems, thus contributing to the pathogenesis of inflammatory diseases and tissue damage, as well as to host defense. PMID:21071701

  7. The use of inert gas xenon for cryopreservation of leukocytes.

    PubMed

    Laptev, D S; Polezhaeva, T V; Zaitseva, O O; Khudyakov, A N; Solomina, O N; Utemov, S V

    2014-06-01

    We studied the possibility of cryopreservation of human blood nuclear cells under protection with inert gas xenon. A method for inducing clathrate anabiosis of leukocytes was developed that preserved the cells for practical use in biology and medicine.

  8. Osteomyelitis complicating fracture: pitfalls of /sup 111/In leukocyte scintigraphy

    SciTech Connect

    Kim, E.E.; Pjura, G.A.; Lowry, P.A.; Gobuty, A.H.; Traina, J.F.

    1987-05-01

    /sup 111/In-labeled leukocyte imaging has shown greater accuracy and specificity than alternative noninvasive methods in the detection of uncomplicated osteomyelitis. Forty patients with suspected osteomyelitis complicating fractures (with and without surgical intervention) were evaluated with /sup 111/In-labeled leukocytes. All five patients with intense focal uptake, but only one of 13 with no uptake, had active osteomyelitis. However, mild to moderate /sup 111/In leukocyte uptake, observed in 22 cases, indicated the presence of osteomyelitis in only four of these; the other false-positive results were observed in noninfected callus formation, heterotopic bone formation, myositis ossificans, and sickle-cell disease. These results suggest that /sup 111/In-labeled leukocyte imaging is useful for the evaluation of suspected osteomyelitis complicating fracture but must be used in conjunction with clinical and radiographic correlation to avoid false-positive results.

  9. Targeting vascular and leukocyte communication in angiogenesis, inflammation and fibrosis.

    PubMed

    Kreuger, Johan; Phillipson, Mia

    2016-02-01

    Regulation of vascular permeability, recruitment of leukocytes from blood to tissue and angiogenesis are all processes that occur at the level of the microvasculature during both physiological and pathological conditions. The interplay between microvascular cells and leukocytes during inflammation, together with the emerging roles of leukocytes in the modulation of the angiogenic process, make leukocyte-vascular interactions prime targets for therapeutics to potentially treat a wide range of diseases, including pathological and dysfunctional vessel growth, chronic inflammation and fibrosis. In this Review, we discuss how the different cell types that are present in and around microvessels interact, cooperate and instruct each other, and in this context we highlight drug targets as well as emerging druggable processes that can be exploited to restore tissue homeostasis.

  10. Characteristics of histamine-induced leukocyte rolling in the undisturbed microcirculation of the rat mesentery

    PubMed Central

    Yamaki, Kohji; Thorlacius, Henrik; Xie, Xun; Lindbom, Lennart; Hedqvist, Per; Raud, Johan

    1998-01-01

    histamine by 52%, while both H2-receptor antagonists were completely inactive. However, the combination of cimetidine and diphenhydramine reduced the histamine-induced PMNL rolling by 82%. Furthermore, in contrast to an H3-receptor agonist, challenge with either the H1-receptor agonist 2-thiazolylethylamine or two different H2-receptor agonists (impromidine, dimaprit) was sufficient to provoke significant venular PMNL accumulation. Treatment with the nitric oxide-synthase inhibitor L-NAME did not affect the histamine-induced PMNL rolling. On the other hand, 3 h pretreatment with dexamethasone reduced the PMNL response to histamine by 73%, and flow cytometric analysis showed that the dexamethasone treatment almost completely inhibited binding of soluble P-selectin to rat isolated PMNLs. We conclude that initial leukocyte recruitment after mast cell activation in the rat mesentery is critically dependent on histamine release. The cellular response to histamine was specifically due to PMNL rolling, involved activation of both H1- and H2-receptors, and lasted for 2–3 h. Moreover, the histamine-induced PMNL rolling was not dependent on nitric oxide synthesis, but was sensitive to glucocorticoid treatment, possibly via inhibition of expression or function of leukocytic P-selectin ligand(s). PMID:9504378

  11. Platelet-mediated adhesion facilitates leukocyte sequestration in hypoxia-reoxygenated microvessels.

    PubMed

    Zheng, Senfeng; Cao, Yanting; Zhang, Wenjian; Liu, Honglin; You, Jia; Yin, Yiqing; Lou, Jinning; Li, Chenghui

    2016-03-01

    Leukocyte transendothelial migration and sequestration are two distinct outcomes following leukocyte adhesion to endothelium during ischemia-reperfusion injury, in which platelets may play a pivotal role. In the present study, we established an in vitro hypoxia-reoxygenation model to mimic ischemia-reperfusion injury and found platelet pre-incubation significantly increased leukocyte adhesion to endothelial cells after hyoxia-reoxygenation (over 67%). Blockade of endothelial-cell-expressed adhesion molecules inhibited leukocyte direct adhesion to endothelial cells, while platelet-mediated leukocyte adhesion was suppressed by blockade of platelet-expressed adhesion molecules. Further experiments revealed platelets acted as a bridge to mediate leukocyte adhesion, and platelet-mediated adhesion was the predominant pattern in the presence of platelets. However, platelet pre-incubation significantly suppressed leukocyte transendothelial migration after hypoxia-reoxygenation (over 31%), which could be aggravated by blockade of endothelial-cell-expressed adhesion molecules, but alleviated by blockade of platelet- expressed adhesion molecules. This would indicate that platelet-mediated adhesion disrupted leukocyte transendothelial migration. An in vivo mesenteric ischemia-reperfusion model demonstrated leukocyte transfusion alone caused mild leukocyte adhesion to reperfused vessels and subsequent leukocyte infiltration, while simultaneous leukocyte and platelet transfusion led to massive leukocyte adhesion and sequestration within reperfused microvessels. Our studies revealed platelets enhanced leukocyte adhesion to endothelial cells, but suppressed leukocyte transendothelial migration. Overall, this leads to leukocyte sequestration in hypoxia-reoxygenated microvessels.

  12. Enhanced leukotriene synthesis in leukocytes of atopic and asthmatic subjects.

    PubMed Central

    Sampson, A P; Thomas, R U; Costello, J F; Piper, P J

    1992-01-01

    1. We have investigated the capacities of peripheral leukocytes from atopic asthmatic (AA) (n = 7), atopic non-asthmatic (AN) (n = 7), and normal (N) (n = 7) subjects to generate the bronchoconstrictor and proinflammatory mediators leukotrienes (LTs) B4 and C4. 2. Mixed leukocyte preparations containing 61-84% neutrophils, 2.4-15% eosinophils, and 13-29% mononuclear cells were incubated in vitro at 37 degrees C in the presence of calcium ionophore A23187. Synthesis of LTB4 and LTC4 was quantitated by radioimmunoassay. 3. Both in dose-response experiments (0-10 microM A23187 for 5 min), and in time-course investigations (2 microM A23187 for 0-30 min), the mixed leukocytes of the AA and AN subjects generated on average 4- to 5-fold more LTB4 and 3- to 5-fold more LTC4 than the normal leukocytes (P less than 0.01 in all cases; ANOVA). 4. This enhanced LT synthesis by the AN and AA leukocytes was not due to differences in the counts of leukocyte sub-types, or to altered rates of LT catabolism between the subject groups. 5. LTB4 synthesis correlated significantly with LTC4 synthesis in the leukocytes of the AN and AA subjects (r = 0.81, n = 14, P less than 0.01), but not in those of the normal subjects (r = 0.19, n = 7, P greater than 0.05). 6. Our results demonstrate an up-regulation of the leukotriene synthetic pathway in the circulating leukocytes of atopic non-asthmatic and atopic asthmatic subjects, which may have important implications in the pathophysiology of asthma and allergy. PMID:1576069

  13. Thiamin deficiency effects on rat leukocyte pyruvate decarboxylation rates.

    PubMed

    Hathcock, J N

    1978-02-01

    Thiamin status usually is assessed by urinary excretion of thiamin or by exogenous thiamin pyrophosphate (TPP) stimulation of erythrocyte transketolase activity. Because of the possible great utility of a biologically and chemically sensitive alternative method for thiamin status assessment, studies were made of rat leukocyte pyruvate decarboxylation activity in thiamin deficiency. Pyruvate decarboxylation rates were determined by assaying 14CO2 produced by leukocytes from 1-14C-pyruvic acid in vitro. Reaction conditions were 5 mumoles pyruvic acid, 2.2 X 10(4) DPM 1-14C-pyruvic acid, leukocytes from 5 ml whole blood, 50 mumoles NaH2PO4, 5 mumoles MgSO4, and 1 mumole MnSO4 at pH 7.4 in 1 ml reaction volume at 25 C. Four weeks of thiamin deficiency decreased leukocyte pyruvate decarboxylation rates and markedly increased the TPP effect on this reaction. Dual weekly assays in the same rats showed that 21 days of thiamin deficiency significantly increased the TPP effect on leukocyte pyruvate decarboxylation rates. In contrast, the TPP effect on erythrocyte transketolase activity was significantly increased after only 7 days of thiamin deficiency. Erythrocyte transketolase is more sensitive than leukocyte pyruvate decarboxylation rate to early thiamin deficiency in rats.

  14. Physiological levels of testosterone kill salmonid leukocytes in vitro

    USGS Publications Warehouse

    Slater, C.H.; Schreck, C.B.

    1997-01-01

    Adult spring chinook salmon (Oncorhynchus tshawytscha) elaborate high plasma concentrations of testosterone during sexual maturation, and these levels of testosterone have been shown to reduce the salmonid immune response in vitro. Our search for the mechanism of testosterone's immunosuppressive action has led to the characterization of an androgen receptor in salmonid leukocytes. In the present study we examined the specific effects that testosterone had on salmonid leukocytes. Direct counts of viable leukocytes after incubation with and without physiological levels of testosterone demonstrate a significant loss of leukocytes in cultures exposed to testosterone. At least 5 days of contact with testosterone was required to produce significant immunosuppression and addition of a 'conditioned media' (supernatant from proliferating lymphocytes not exposed to testosterone) did not reverse the immunosuppressive effects of testosterone. These data lead us to conclude that testosterone may exert its immunosuppressive effects by direct action on salmonid leukocytes, through the androgen receptor described, and that this action leads to the death of a significant number of these leukocytes.

  15. Insights into psychosis risk from leukocyte microRNA expression

    PubMed Central

    Jeffries, C D; Perkins, D O; Chandler, S D; Stark, T; Yeo, E; Addington, J; Bearden, C E; Cadenhead, K S; Cannon, T D; Cornblatt, B A; Mathalon, D H; McGlashan, T H; Seidman, L J; Walker, E F; Woods, S W; Glatt, S J; Tsuang, M

    2016-01-01

    Dysregulation of immune system functions has been implicated in schizophrenia, suggesting that immune cells may be involved in the development of the disorder. With the goal of a biomarker assay for psychosis risk, we performed small RNA sequencing on RNA isolated from circulating immune cells. We compared baseline microRNA (miRNA) expression for persons who were unaffected (n=27) or who, over a subsequent 2-year period, were at clinical high risk but did not progress to psychosis (n=37), or were at high risk and did progress to psychosis (n=30). A greedy algorithm process led to selection of five miRNAs that when summed with +1 weights distinguished progressed from nonprogressed subjects with an area under the receiver operating characteristic curve of 0.86. Of the five, miR-941 is human-specific with incompletely understood functions, but the other four are prominent in multiple immune system pathways. Three of those four are downregulated in progressed vs. nonprogressed subjects (with weight -1 in a classifier function that increases with risk); all three have also been independently reported as downregulated in monocytes from schizophrenia patients vs. unaffected subjects. Importantly, these findings passed stringent randomization tests that minimized the risk of conclusions arising by chance. Regarding miRNA–miRNA correlations over the three groups, progressed subjects were found to have much weaker miRNA orchestration than nonprogressed or unaffected subjects. If independently verified, the leukocytic miRNA biomarker assay might improve accuracy of psychosis high-risk assessments and eventually help rationalize preventative intervention decisions. PMID:27959328

  16. C-type natriuretic peptide inhibits leukocyte recruitment and platelet-leukocyte interactions via suppression of P-selectin expression

    NASA Astrophysics Data System (ADS)

    Scotland, Ramona S.; Cohen, Marc; Foster, Paul; Lovell, Matthew; Mathur, Anthony; Ahluwalia, Amrita; Hobbs, Adrian J.

    2005-10-01

    The multifaceted process of immune cell recruitment to sites of tissue injury is key to the development of an inflammatory response and involved in the pathogenesis of numerous cardiovascular disorders. We recently identified C-type natriuretic peptide (CNP) as an important endothelium-derived mediator that regulates vascular tone and protects against myocardial ischemia/reperfusion injury. Herein, we investigated whether CNP inhibits leukocyte recruitment and platelet aggregation and thereby exerts a potential antiinflammatory influence on the blood vessel wall. We assessed the effects of CNP on leukocyte-endothelial cell interactions in mouse mesenteric postcapillary venules in vivo in animals with high basal leukocyte activation (endothelial nitric oxide synthase knockout mice, eNOS-/-) or under acute inflammatory conditions (induced by interleukin-1 or histamine). CNP suppressed basal leukocyte rolling in eNOS-/- mice in a rapid, reversible, and concentration-dependent manner. These effects of CNP were mimicked by the selective natriuretic peptide receptor-C agonist cANF4-23. CNP also suppressed leukocyte rolling induced by IL-1 or histamine, inhibited platelet-leukocyte interactions, and prevented thrombin-induced platelet aggregation of human blood. Furthermore, analysis of human umbilical vein endothelial cells, leukocytes, and platelets revealed that CNP selectively attenuates expression of P-selectin. Thus, CNP is a modulator of acute inflammation in the blood vessel wall characterized by leukocyte and platelet activation. These antiinflammatory effects appear to be mediated, at least in part, via suppression of P-selectin expression. These observations suggest that endothelial CNP might maintain an anti-atherogenic influence on the blood vessel wall and represent a target for therapeutic intervention in inflammatory cardiovascular disorders. endothelium | natriuretic peptide receptor type C | atherosclerosis | thrombosis

  17. Telomere shortening in leukocyte subpopulations in depression

    PubMed Central

    2014-01-01

    Background Telomere shortening is a normal age-related process. However, premature shortening of telomeres in leukocytes – as has been reported in depression – may increase the risk for age-related diseases. While previous studies investigated telomere length in peripheral blood mononuclear cells (PBMCs) as a whole, this study investigated specific changes in the clonal composition of white blood cells of the adaptive immune system (CD4+ helper and CD8+ cytotoxic T lymphocytes, and CD20+ B lymphocytes). Methods Forty-four females with a history of unipolar depression were investigated and compared to fifty age-matched female controls. Telomere lengths were compared between three groups: 1) individuals with a history of depression but currently no clinically relevant depressive symptoms, 2) individuals with a history of depression with relevant symptoms of depression, and 3) healthy age-matched controls. Telomere length was assessed using quantitative fluorescence in situ hybridization (qFISH). Results Both groups with a history of unipolar depression (with and without current depressive symptoms) showed significantly shorter telomeres in all three lymphocyte subpopulations. The effect was stronger in CD8+ and CD20+ cells than in CD4+ cells. Individuals with a history of depression and with (without) current symptoms exhibited a CD8+ telomere length shortening corresponding to an age differential of 27.9 (25.3) years. Conclusions A history of depression is associated with shortened telomeres in the main effector populations of the adaptive immune system. Shorter telomeres seem to persist in individuals with lifetime depression independently of the severity of depressive symptoms. CD8+ cytotoxic T cells and CD20+ B cells seem to be particularly affected in depression. The total number of depressive episodes did not influence telomere length in the investigated adaptive immune cell populations. PMID:24996455

  18. The filterability of leukocytes in undiluted blood.

    PubMed

    Cook, A M; Evans, S A; Jones, J G

    1998-01-01

    A filtrometer is described for measuring the flow of fluids through microfilters. The flow of Newtonian fluids through the filters can be predicted from the diameter, length and number of pores. There are no physical artefacts such as turbulent flow or a significant lag period before steady-state flow is achieved. The instrument has been used as a viscometer and has been used to record and analyse the flow of undiluted blood through 5 microns polycarbonate filters. The calculated viscosity of Newtonian fluids agrees well with those measured by a more conventional viscometer (Ostwald). Flow profiles of blood have been analysed to give both the numbers and the flow properties of a small population of slow leukocytes which equate numerically with the monocytes. They are subdivided into three distinct sub-populations, according to their rheological properties, and these are termed SL1, SL2 and PB. The concentration of these cells, in blood, are 0.12 +/- 0.02 x 10(6) ml-1, 0.11 +/- 0.02 x 10(6) ml-1, 0.09 +/- 0.02 x 10(6) ml-1 in young females aged about 25 years. The transit time of these cells, through 5 microns pores, is 34.8 +/- 1.4 s, 147.5 +/- 2.5 s and > 300 s, respectively. Analysis of blood from older men (53-79 years) gives essentially the same results although the concentration of SL1 is slightly higher at 0.19 +/- 0.09 x 10(6) ml-1.

  19. Adhesion of leukocytes to dermal endothelial cells is induced after single-dose, but reduced after repeated doses of UVA.

    PubMed

    Heckmann, M; Pirthauer, M; Plewig, G

    1997-12-01

    Approximately 20-50% of ultraviolet A (UVA) irradiation delivered to the skin surface may reach the human dermal microvascular endothelial cells (HDMEC) that play a pivotal role in cellular inflammatory tissue; however, the pathophysiologic role of HDMEC in UVA-induced skin changes is largely unknown. Based on previous in vivo and in vitro studies revealing UVA-induced expression of endothelial adhesion molecules, we studied isolated HDMEC under various conditions in order to further delineate the impact of UVA on these cells. The expression of cell adhesion molecules was determined by flow cytometry and the resulting changes of stable adhesion of leukocytes to endothelial cells were quantitated for granulocytes, lymphocytes, and monocytes using a newly developed multicellular adhesion assay. Additionally, antibody blocking experiments were performed to delineate the role of individual cell adhesion molecules in UVA-induced leukocyte adherence. High-dose polychromatic UVA (25 J per cm2, maximal emission at 375 nm) induced intercellular adhesion molecule-1 and E-selectin with different kinetics but correlating the adhesion of leukocyte subsets. This effect subsided, however, in the course of 3-6 daily applied UVA doses. Moreover, pro-inflammatory cytokine challenge by tumor necrosis factor-alpha and interleukin-1-alpha resulted in significantly weaker induction of intercellular adhesion molecule-1 and E-selectin in repeatedly UVA-exposed HDMEC. Differential quantitation of peripheral blood derived granulocytes, lymphocytes, and monocytes revealed reduced adhesion particularly of lymphocytes followed by monocytes and granulocytes compared with leukocyte adhesion to nonirradiated but cytokine-stimulated HDMEC. It is concluded that UVA substantially influences endothelial cell adhesion molecules expression and thus directly interferes with leukocyte adhesion to endothelial cells. Divergent UVA-induced effects in this respect can be attributed to the mode of UV exposure

  20. Extravasation of leukocytes in comparison to tumor cells

    PubMed Central

    Strell, Carina; Entschladen, Frank

    2008-01-01

    The multi-step process of the emigration of cells from the blood stream through the vascular endothelium into the tissue has been termed extravasation. The extravasation of leukocytes is fairly well characterized down to the molecular level, and has been reviewed in several aspects. Comparatively little is known about the extravasation of tumor cells, which is part of the hematogenic metastasis formation. Although the steps of the process are basically the same in leukocytes and tumor cells, i.e. rolling, adhesion, transmigration (diapedesis), the molecules that are involved are different. A further important difference is that leukocyte interaction with the endothelium changes the endothelial integrity only temporarily, whereas tumor cell interaction leads to an irreversible damage of the endothelial architecture. Moreover, tumor cells utilize leukocytes for their extravasation as linkers to the endothelium. Thus, metastasis formation is indirectly susceptible to localization signals that are literally specific for the immune system. We herein compare the extravasation of leukocytes and tumor cells with regard to the involved receptors and the localization signals that direct the cells to certain organs and sites of the body. PMID:19055814

  1. Platelets Guide Leukocytes to Their Sites of Extravasation

    PubMed Central

    Puhr-Westerheide, Daniel; Pörnbacher, Michaela; Lauber, Kirsten; Krombach, Fritz; Reichel, Christoph Andreas

    2016-01-01

    Effective immune responses require the directed migration of leukocytes from the vasculature to the site of injury or infection. How immune cells “find” their site of extravasation remains largely obscure. Here, we identified a previously unrecognized role of platelets as pathfinders guiding leukocytes to their exit points in the microvasculature: upon onset of inflammation, circulating platelets were found to immediately adhere at distinct sites in venular microvessels enabling these cellular blood components to capture neutrophils and, in turn, inflammatory monocytes via CD40-CD40L-dependent interactions. In this cellular crosstalk, ligation of PSGL-1 by P-selectin leads to ERK1/2 MAPK-dependent conformational changes of leukocyte integrins, which promote the successive extravasation of neutrophils and monocytes to the perivascular tissue. Conversely, blockade of this cellular partnership resulted in misguided, inefficient leukocyte responses. Our experimental data uncover a platelet-directed, spatiotemporally organized, multicellular crosstalk that is essential for effective trafficking of leukocytes to the site of inflammation. PMID:27152726

  2. L1 adhesion molecule on mouse leukocytes: regulation and involvement in endothelial cell binding.

    PubMed

    Hubbe, M; Kowitz, A; Schirrmacher, V; Schachner, M; Altevogt, P

    1993-11-01

    L1 is a cell surface glycoprotein of the immunoglobulin superfamily which was initially shown to mediate adhesion between neural cells. Recently we have reported that L1 is expressed by bone marrow cells and the majority of mature lymphocytes (Kowitz et al., Eur. J. Immunol. 1992. 22: 1199-1205). To analyze the function of L1 on leukocytes we studied its regulation following cell activation. In vitro activation of B lymphocytes with lipopolysaccharide or T lymphocytes with phorbol 12-myristate 13-acetate/Ca2+ ionophore, concanavalin A or anti-CD3 monoclonal antibody as well as in vivo activation of V beta 8+ T cells with staphylococcal enterotoxin B (SEB) revealed a down-regulation of L1 within 48 h. A rapid loss of L1 expression was seen when mouse neutrophils were activated with PMA alone. This rapid loss paralleled the shedding of L-selectin. We also studied a possible role of L1 in the binding of leukocytes to endothelial cells. ESb-MP lymphoma cells with a high expression of L1 (L1hi) could bind to bend3 endothelioma cells without prior activation with inflammatory cytokines. The interaction was inhibited by anti-L1 antibodies. In contrast, ESb-MP cells with low L1 expression (L1lo) were only marginally bound. Latex beads coated with affinity-isolated L1 antigen were also able to bind to the endothelioma cells in a specific fashion. The binding of ESb-MP lymphoma cells required Ca2+ and Mg2+ ions and was sensitive to cold temperature. Since the endothelioma cells did not express L1 the binding mechanism studied here is distinct from the established L1-L1 homotypic interaction. It is possible that the novel L1-mediated adhesion pathway involves an unidentified ligand and could play a role in leukocyte migration.

  3. Definition of leukocyte subsets in primate central nervous system by polychromatic flow cytometry.

    PubMed

    Bischoff, Tanja; Stahl-Hennig, Christiane; Mätz-Rensing, Kerstin; Koutsilieri, Eleni; Sopper, Sieghart

    2011-06-01

    Haematopoietic immune cell populations play an important role in the pathogenesis of numerous neurological disorders. To better understand the function of resident mononuclear phagocytes and migrating leukocytes in the central nervous system (CNS), the definition of these populations in healthy individuals is crucial. Therefore, the composition of CNS-associated leukocytes, isolated from macaque brain tissue, was assessed using multicolor flow cytometry. We established a combination of antibodies directed against nine different antigens that enabled a precise classification of all major immune cell populations in a single tube. Macrophages, dendritic cells (DCs), B and T lymphocytes, and natural killer (NK) cells were differentiated in CNS and peripheral blood. Additionally, microglia cells were detected in the brain. Using this antibody combination also allowed the discrimination of functionally different subsets among the distinct immunocyte populations, for example, CD8 positive cytotoxic T lymphocytes. About 95% of the leukocytes in the brain are microglia cells. Two additional myeloid cell populations, CD14 positive macrophages and CD11c-positive DCs, were also identified. In contrast to blood, where macrophages are more abundant, DCs outnumbered macrophages in the brain. Among lymphocytes, proportions of CD20 positive B lymphocytes were decreased, and T lymphocytes as well as NK cells were increased in brain compared to blood. Significant changes were also detected for macrophage and T-cell subpopulations. The nonexclusive expression of certain surface makers on different cell populations demanded a simultaneous classification of all intrathecal immune cells. Knowing their exact composition offers new insights on interaction and regulation in inflammatory processes and will be instrumental to monitor alterations in the course of neurological diseases.

  4. Inflamed leukocyte-mimetic nanoparticles for molecular imaging of inflammation.

    PubMed

    Chen, Xiaoyue; Wong, Richard; Khalidov, Ildar; Wang, Andrew Y; Leelawattanachai, Jeerapond; Wang, Yi; Jin, Moonsoo M

    2011-10-01

    Dysregulated host inflammatory response causes many diseases, including cardiovascular and neurodegenerative diseases, cancer, and sepsis. Sensitive detection of the site of inflammation will, therefore, produce a wide-ranging impact on disease diagnosis and treatment. We hypothesized that nanoprobes designed to mimic the molecular interactions occurring between inflamed leukocytes and endothelium may possess selectivity toward diverse host inflammatory responses. To incorporate inflammation-sensitive molecular interactions, super paramagnetic iron oxide nanoparticles were conjugated with integrin lymphocyte function-associated antigen (LFA)-1 I domain, engineered to mimic activated leukocytes in physiology. Whole body optical and magnetic resonance imaging in vivo revealed that leukocyte-mimetic nanoparticles localized preferentially to the vasculature within and in the invasive front of the tumor, as well as to the site of acute inflammation. This study explored in vivo detection of tumor-associated vasculature with systemically injected inflammation-specific nanoparticles, presenting a possibility of tumor detection by inflamed tumor microenvironment.

  5. Inhibition of human polymorphonuclear leukocyte chemotaxis by oxygenated sterol compounds

    SciTech Connect

    Gordon, L.I.; Bass, J.; Yachnin, S.

    1980-07-01

    When preincubated with certain oxygenated sterol compounds in lipoprotein-depleted serum (20% (vol/vol)), human polymorphonuclear leukocytes show inhibition of chemotaxis toward the synthetic dipeptide N-formylmethionylphenylalinine without alteration of random movement or loss of cell viability. These effects can occur at sterol concentrations as low as 6.25 ..mu..M and after as little as 5 min of preincubation, but they are increased at higher concentrations and longer preincubation times. The inhibition can be almost completely reversed by preincubation in lipoprotein-replete serum (human AB serum, 20% (vol/vol)) and may be partially corrected by addition of free cholesterol (0.125 mM) to the medium. These effects are unlikely to be due to inhibition of cellular sterol synthesis, competition for chemotaxin membrane binding sites, or deactivation of the leukocytes but they may be a consequence of insertion of the sterol molecule into the leukocyte plasma membranes.

  6. Flow cytometric analysis of leukocytes and reticulocytes stained with proflavine.

    PubMed

    Sagawa, H; Tatsumi, N

    1997-12-01

    Proflavine, an acridine analog for industrial use, was used to stain blood cells. A drop of blood treated with ethylenediaminetetraacetic acid-2K was mixed with a 0.00001% solution of the dye and observed immediately by fluorescence microscopy with a green filter. Leukocytes, platelets, and reticulocytes were stained but mature red blood cells were not. Chromatin in the nuclei of all leukocytes and nucleoli of lymphocytes and monocytes had greenish-yellow fluorescence, and the kind of cell could be identified by the tone and intensity of this color. Granules in granulocytes were in green. Reticular fine-granular or granulofibrous structures in the reticulocytes were brownish. The proflavine could be used routinely in clinical laboratories because this single stain makes possible simultaneous differentiation of leukocytes and counting of reticulocytes.

  7. Improving diagnosis of appendicitis. Early autologous leukocyte scanning.

    PubMed

    DeLaney, A R; Raviola, C A; Weber, P N; McDonald, P T; Navarro, D A; Jasko, I

    1989-10-01

    A prospective nonrandomized study investigating the accuracy and utility of autologous leukocyte scanning in the diagnosis of apendicitis was performed. One hundred patients in whom the clinical diagnosis of appendicitis was uncertain underwent indium 111 oxyquinoline labelling of autologous leukocytes and underwent scanning 2 hours following reinjection. Of 32 patients with proved appendicitis, three scans revealed normal results (false-negative rate, 0.09). Of 68 patients without appendicitis, three scans had positive results (false-positive rate, 0.03; sensitivity, 0.91; specificity, 0.97; predictive value of positive scan, 0.94; predictive value of negative scan, 0.96; and overall accuracy, 0.95). Scan results altered clinical decisions in 19 patients. In 13 cases, the scan produced images consistent with diagnoses other than appendicitis, expediting appropriate management. Early-imaging111 In oxyquinoline autologous leukocyte scanning is a practical and highly accurate adjunct for diagnosing appendicitis.

  8. Therapeutic hypothermia impacts leukocyte kinetics after cardiac arrest

    PubMed Central

    Dufner, Matthias C.; Andre, Florian; Stiepak, Jan; Zelniker, Thomas; Chorianopoulos, Emmanuel; Preusch, Michael; Katus, Hugo A.

    2016-01-01

    Background Patients admitted to the hospital after primarily successful cardiopulmonary resuscitation (CPR) are at a very high risk for neurologic deficits and death. Targeted temperature management (TTM) for mild therapeutic hypothermia has been shown to improve survival compared to standard treatment. Acute cardiovascular events, such as myocardial infarction (MI), are a major cause for cardiac arrest (CA) in patients who undergo CPR. Recent findings have demonstrated the importance and impact of the leukocyte response following acute MI. Methods In this retrospective, single center study we enrolled 169 patients with CA due to non-traumatic causes and primarily successful CPR. A total of 111 subjects (66%) underwent TTM aiming for a target temperature of 32–34 °C. Results Analysis of 30 day follow up showed a significantly improved survival of all patients who received TTM compared to patients without hypothermia (P=0.0001). Furthermore TTM was an independent variable of good neurological outcome after 6 months (P=0.0030). Therapeutic hypothermia was found to be beneficial independent of differences in age and sex between both groups. While a higher rate of pneumonia was observed with TTM, this diagnosis had no additional impact on survival or neurological outcome. The beneficial effect on mortality remained significant in patients with the diagnosis of an acute cardiac event (P=0.0145). Next, we evaluated the kinetics of leukocytes in this group over the course of 7 days after CA. At presentation, patients showed a mean level of 16.5±6.7 of leukocytes per microliter. While this level stayed stable in the group of patients without hypothermia, patients who received TTM showed a significant decline of leukocyte levels resulting in significantly lower numbers of leukocytes on days 3 and 5 after CPR. Interestingly, these differences in leukocyte counts remained beyond the time period of TTM while C-reactive protein (CRP) levels were suppressed only during

  9. Toxins–Useful Biochemical Tools for Leukocyte Research

    PubMed Central

    Cubillos, Susana; Norgauer, Johannes; Lehmann, Katja

    2010-01-01

    Leukocytes are a heterogeneous group of cells that display differences in anatomic localization, cell surface phenotype, and function. The different subtypes include e.g., granulocytes, monocytes, dendritic cells, T cells, B cells and NK cells. These different cell types represent the cellular component of innate and adaptive immunity. Using certain toxins such as pertussis toxin, cholera toxin or clostridium difficile toxin, the regulatory functions of Gαi, Gαs and small GTPases of the Rho family in leukocytes have been reported. A summary of these reports is discussed in this review. PMID:22069594

  10. The Effect of Hemiscorpius lepturus (Scorpionida: Hemiscorpiidae) Venom on Leukocytes and the Leukocyte Subgroups in Peripheral Blood of Rat

    PubMed Central

    Ghafourian, Mehri; Ganjalikhanhakemi, Neda; Hemmati, Ali Asghar; Dehghani, Rouhullah; Kooti, Wesam

    2016-01-01

    Background: The aim of this study was to investigate the effect of Hemiscorpius lepturus venom on leukocytes and the leukocyte subgroups in peripheral blood of rat. Methods: In this experimental study, sixty N-Mari rats were divided into three groups of 20 rats. Then the rats in each group were divided into four subgroups based on the blood sampling time that was 2, 6, 24 and 48 hours after the venom injection, respectively. The control group did not receive anything, however, the first and the second experimental groups received 0.1 and 0.01mg/kg of venom, subcutaneously. In accordance with a designated four sampling times, the blood sampling was carried out in three groups. After RBC lysis, the leukocytes and leukocyte sub-populations were determined and counted using appropriate hematological standard methods. Results: The leukocyte and the neutrophil count at two (P<0.05), six (P<0.01) and 24 (P<0.05) hours after the venom injection showed a significant decline compared with the control group, this decrease was significant at the dose of 0.1 mg/kg until 48 hours after the venom injection (P<0.05). The lymphocyte count showed a significant decline throughout the all hours of the experiment, compared with the control group (P<0.05). Conclusion: Leukocytes are probably affected by the cytotoxicity effect of the H. lepturus venom in a dose-dependent manner. This could be a wakeup call for the medical staff to perform quick and accurate treatment in the least time possible. PMID:27308274

  11. Induction of expression of iNOS by N-nitrosodimethylamine (NDMA) in human leukocytes.

    PubMed

    Ratajczak-Wrona, Wioletta; Jablonska, Ewa; Jablonski, Jakub; Marcinczyk, Magdalena

    2009-01-01

    The aim of this study was to assess the influence of N-nitrosodimethylamine (NDMA) on expression of inducible nitric oxide synthase (iNOS), as well as production of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) by human neutrophils (PMN) and peripheral blood mononuclear cells (PBMC), and the participation of the p38 MAPK kinase in this process. Furthermore, the ability of neutrophils to release superoxide anion was determined. The influence of N-nitrosodimethylamine on iNOS expression was determined in isolated PMN and PBMC cells from peripheral blood of healthy individuals. The mononuclear cells showed higher sensitivity to NDMA. Moreover, cytotoxic effect of NDMA can be influenced in some way by the impact of this xenobiotic on nitric oxide and superoxide anion release from human leukocytes. Furthermore, increased generation of these radicals by human leukocytes suggest that neutrophils and mononuclear cells that are exposed to NDMA activity can play a key role in endogenous NDMA generation. However the relationship between iNOS expression and phospho-p38 MAPK in neutrophils and mononuclear cells shows that p38 MAPK pathway participates in induction of iNOS expression in the presence of NDMA.

  12. Peripheral Blood Leukocytes Interleukin-1 Beta (IL-1β) Cytokine Hyper-Reactivity in Chronic Periodontitis

    PubMed Central

    Sakalauskiene, Jurgina; Giedrimiene, Dalia; Gleiznys, Darius; Gleiznys, Alvydas; Gleizniene, Rymante; Vitkauskiene, Astra

    2016-01-01

    Background Levels of pro-inflammatory cytokine (IL-1β) released by peripheral blood leukocyte medium (PBLM), isolated from chronic periodontitis patients (P) before therapy and matched to controls, were determined in the presence or absence of non-opsonized Escherichia coli and Staphylococcus aureus. Material/Methods In this investigation, 26 patients with untreated, severe, generalized, chronic periodontitis and 26 healthy subjects (H) were enrolled. Periodontal status was assessed by measuring bleeding on probing (BOP), clinical attachment loss (CAL), probing pocket depth (PPD), and Ramfjord index (PDI). The levels of IL-1β (μg/ml) were assayed by a standard Immunoenzymetric Assay Diasource IL-1β ELISA kit in PBLM. Results Our study showed that the values of IL-1β levels in PBLM of the P group (stimulated with non-opsonized E. coli and S. aureus) were significantly higher than in the analogous medium of H group subjects (P<0.001). All correlations between the cytokine levels of IL-1β in the samples of PBLM (stimulated with non-opsonized E. coli and S. aureus) and clinical parameters such as BOP, PPD, CAL, and PDI were significantly higher in the group of patients with periodontitis. Conclusions Levels of IL-1β secreted by leukocytes may help measure severe, generalized, chronic periodontitis, and can be predictive of future detrimental clinical sequelae associated with chronic periodontitis. PMID:27847385

  13. Improved survival of newborns receiving leukocyte transfusions for sepsis

    SciTech Connect

    Cairo, M.S.; Rucker, R.; Bennetts, G.A.; Hicks, D.; Worcester, C.; Amlie, R.; Johnson, S.; Katz, J.

    1984-11-01

    To determine the role of polymorphonuclear (PMN) leukocyte transfusions in neonates with sepsis, 23 consecutive newborns were prospectively randomly selected during an 18-month period in a treatment plan to receive polymorphonuclear leukocyte transfusions with supportive care or supportive care alone. Thirteen neonates received transfusions every 12 hours for a total of five transfusions. Each transfusion consisting of 15 mL/kg of polymorphonuclear leukocytes was subjected to 1,500 rads of radiation. The polymorphonuclear leukocytes were obtained by continuous-flow centrifugation leukapheresis and contained 0.5 to 1.0 X 10(9) granulocytes per 15 mL with less than 10% lymphocytes. Positive findings on blood cultures were obtained in 14/23 patients and seven were randomly selected for each treatment group. Absolute granulocyte counts were less than 1,500/microL in 13 patients but tibial bone marrow examinations revealed that the neutrophil supply pool was depleted in only three patients. The survival was significantly greater in the treatment group compared with the group that did not receive transfusions.

  14. Recent advances in microscopic techniques for visualizing leukocytes in vivo

    PubMed Central

    Jain, Rohit; Tikoo, Shweta; Weninger, Wolfgang

    2016-01-01

    Leukocytes are inherently motile and interactive cells. Recent advances in intravital microscopy approaches have enabled a new vista of their behavior within intact tissues in real time. This brief review summarizes the developments enabling the tracking of immune responses in vivo. PMID:27239292

  15. Leukocyte driven-decidual angiogenesis in early pregnancy

    PubMed Central

    Lima, Patricia DA; Zhang, Jianhong; Dunk, Caroline; Lye, Stephen J; Anne Croy, B

    2014-01-01

    Successful pregnancy and long-term, post-natal maternal and offspring cardiac, vascular and metabolic health require key maternal cardiovascular adaptations over gestation. Within the pregnant decidualizing uterus, coordinated vascular, immunological and stromal cell changes occur. Considerable attention has been given to the roles of uterine natural killer (uNK) cells in initiating decidual spiral arterial remodeling, a process normally completed by mid-gestation in mice and in humans. However, leukocyte roles in much earlier, region specific, decidual vascular remodeling are now being defined. Interest in immune cell-promoted vascular remodeling is driven by vascular aberrations that are reported in human gestational complications such as infertility, recurrent spontaneous abortion, preeclampsia (PE) and fetal growth restriction. Appropriate maternal cardiovascular responses during pregnancy protect mothers and their children from later cardiovascular disease risk elevation. One of the earliest uterine responses to pregnancy in species with hemochorial placentation is stromal cell decidualization, which creates unique niches for angiogenesis and leukocyte recruitment. In early decidua basalis, the aspect of the implantation site that will cradle the developing placenta and provide the major blood vessels to support mature placental functions, leukocytes are greatly enriched and display specialized properties. UNK cells, the most abundant leukocyte subset in early decidua basalis, have angiogenic abilities and are essential for normal early decidual angiogenesis. The regulation of uNK cells and their roles in determining maternal and progeny cardiovascular health over pregnancy and postpartum are discussed. PMID:25066422

  16. Uptake of radiolabeled leukocytes in prosthetic graft infection

    SciTech Connect

    Serota, A.I.; Williams, R.A.; Rose, J.G.; Wilson, S.E.

    1981-07-01

    The utility of radionuclide labeled leukocytes in the demonstration of infection within vascular prostheses was examined. The infrarenal aorta was replaced with a 3 cm Dacron graft in 12 dogs. On the third postoperative day, six of the animals received an intravenous injection of 10(8) Staphylococcus aureus. Labeled leukocyte scans were performed at postoperative days one and three, and then weekly for 8 weeks with indium-111 and technetium-99 labeled autologous leukocytes. When scans showed focal uptake of isotope in the area of prosthetic material, the grafts were aseptically excised and cultured on mannitol-salt agar. Both control and infected animals had retroperitoneal isotope activity in the immediate postoperative period that disappeared by the end of the first week. By the eighth postoperative week, all of the animals that received the bacteremic challenge had both radionuclide concentration in the region of the vascular prosthesis and S. aureus cultured subsequently from the perigraft tissues. None of the control animals had either radionuclide or bacteriologic evidence of infection at the eighth postoperative week. The radiolabeled leukocyte scan is a highly sensitive and specific technique, clinically applicable for the diagnosis of vascular prosthetic infections.

  17. Differential MSC activation leads to distinct mononuclear leukocyte binding mechanisms

    NASA Astrophysics Data System (ADS)

    Kota, Daniel J.; Dicarlo, Bryan; Hetz, Robert A.; Smith, Philippa; Cox, Charles S.; Olson, Scott D.

    2014-04-01

    Advances in the field of Multipotent Mesenchymal Stromal cell (MSC) biology have demonstrated that MSCs can improve disease outcome when `activated' to exert immunomodulatory effects. However, the precise mechanisms modulating MSC-immune cells interactions remain largely elusive. In here, we activated MSC based on a recent polarization paradigm, in which MSCs can be polarized towards a pro- or anti-inflammatory phenotype depending on the Toll-like receptor stimulated, to dissect the mechanisms through which MSCs physically interact with and modulate leukocytes in this context. Our data show that MSCs activated through the Toll-like receptor (TLR) 4 pathway increased VCAM-1 and ICAM-1 dependent binding of leukocytes. On the other hand, TLR3 stimulation strongly increases leukocytes affinity to MSC comparatively, through the formation of cable-like hyaluronic acid structures. In addition, TLR4 activation elicited secretion of pro-inflammatory mediators by MSCs, whereas TLR3-activated MSCs displayed a milder pro-inflammatory phenotype, similar to inactivated MSCs. However, the differently activated MSCs maintained their ability to suppress leukocyte activation at similar levels in our in vitro model, and this immunomodulatory property was shown here to be partially mediated by prostaglandin. These results reinforce the concept that alternate activation profiles control MSC responses and may impact the therapeutic use of MSCs.

  18. 21 CFR 864.7675 - Leukocyte peroxidase test.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Leukocyte peroxidase test. 864.7675 Section 864.7675 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7675...

  19. 21 CFR 864.7675 - Leukocyte peroxidase test.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Leukocyte peroxidase test. 864.7675 Section 864.7675 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7675...

  20. 21 CFR 864.7675 - Leukocyte peroxidase test.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Leukocyte peroxidase test. 864.7675 Section 864.7675 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7675...

  1. Medical Applications of Leukocyte Surface Molecules—the CD molecules

    PubMed Central

    Zola, Heddy

    2006-01-01

    Leukocytes are the cells of the immune system and are centrally involved in defense against infection, in autoimmune disease, allergy, inflammation, and in organ graft rejection. Lymphomas and leukemias are malignancies of leukocytes, and the immune system is almost certainly involved in most other cancers. Each leukocyte expresses a selection of cell surface glycoproteins and glycolipids which mediate its interaction with antigen, with other components of the immune system, and with other tissues. It is therefore not surprising that the leukocyte surface molecules (CD molecules) have provided targets for diagnosis and therapy. Among the “celebrities” are CD20, a target for lymphoma therapeutic antibodies which earns $2 billion annually (and makes a significant difference to lymphoma patients), and CD4, the molecule used by the human immunodeficiency virus (HIV) as an entry portal into cells of the immune system. This short review provides a background to the CD molecules and antibodies against them, and summarizes research, diagnostic, and therapeutic applications of antibodies against these molecules. PMID:17380197

  2. Leukocyte driven-decidual angiogenesis in early pregnancy.

    PubMed

    Lima, Patricia D A; Zhang, Jianhong; Dunk, Caroline; Lye, Stephen J; Croy, B Anne

    2014-11-01

    Successful pregnancy and long-term, post-natal maternal and offspring cardiac, vascular and metabolic health require key maternal cardiovascular adaptations over gestation. Within the pregnant decidualizing uterus, coordinated vascular, immunological and stromal cell changes occur. Considerable attention has been given to the roles of uterine natural killer (uNK) cells in initiating decidual spiral arterial remodeling, a process normally completed by mid-gestation in mice and in humans. However, leukocyte roles in much earlier, region specific, decidual vascular remodeling are now being defined. Interest in immune cell-promoted vascular remodeling is driven by vascular aberrations that are reported in human gestational complications such as infertility, recurrent spontaneous abortion, preeclampsia (PE) and fetal growth restriction. Appropriate maternal cardiovascular responses during pregnancy protect mothers and their children from later cardiovascular disease risk elevation. One of the earliest uterine responses to pregnancy in species with hemochorial placentation is stromal cell decidualization, which creates unique niches for angiogenesis and leukocyte recruitment. In early decidua basalis, the aspect of the implantation site that will cradle the developing placenta and provide the major blood vessels to support mature placental functions, leukocytes are greatly enriched and display specialized properties. UNK cells, the most abundant leukocyte subset in early decidua basalis, have angiogenic abilities and are essential for normal early decidual angiogenesis. The regulation of uNK cells and their roles in determining maternal and progeny cardiovascular health over pregnancy and postpartum are discussed.

  3. Effect of Low Dose Gamma Irradiation together with Lipid A on Human Leukocytes Activities In Vitro

    NASA Astrophysics Data System (ADS)

    Belyakova, E.; Dubnickova, M.; Boreyko, A.

    2010-01-01

    The influence of gamma irradiation and of Lipid A from Escherichia coli on phagocytosis, lyzosyme and peroxidase activities of human leukocytes, in vitro was investigated. Leukocytes samples were irradiated with 1 and 5 Gy, respectively. The number of irradiated leukocytes was decreased in the irradiated samples. Only samples with additive Lipid A were not damaged by irradiation. The Lipid A had positive influence on biological activities of the irradiated leukocytes.

  4. Antipseudomonal agents exhibit differential pharmacodynamic interactions with human polymorphonuclear leukocytes against established biofilms of Pseudomonas aeruginosa.

    PubMed

    Chatzimoschou, Athanasios; Simitsopoulou, Maria; Antachopoulos, Charalampos; Walsh, Thomas J; Roilides, Emmanuel

    2015-04-01

    Pseudomonas aeruginosa is the most common pathogen infecting the lower respiratory tract of cystic fibrosis (CF) patients, where it forms tracheobronchial biofilms. Pseudomonas biofilms are refractory to antibacterials and to phagocytic cells with innate immunity, leading to refractory infection. Little is known about the interaction between antipseudomonal agents and phagocytic cells in eradication of P. aeruginosa biofilms. Herein, we investigated the capacity of three antipseudomonal agents, amikacin (AMK), ceftazidime (CAZ), and ciprofloxacin (CIP), to interact with human polymorphonuclear leukocytes (PMNs) against biofilms and planktonic cells of P. aeruginosa isolates recovered from sputa of CF patients. Three of the isolates were resistant and three were susceptible to each of these antibiotics. The concentrations studied (2, 8, and 32 mg/liter) were subinhibitory for biofilms of resistant isolates, whereas for biofilms of susceptible isolates, they ranged between sub-MIC and 2 × MIC values. The activity of each antibiotic alone or in combination with human PMNs against 48-h mature biofilms or planktonic cells was determined by XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay. All combinations of AMK with PMNs resulted in synergistic or additive effects against planktonic cells and biofilms of P. aeruginosa isolates compared to each component alone. More than 75% of CAZ combinations exhibited additive interactions against biofilms of P. aeruginosa isolates, whereas CIP had mostly antagonistic interaction or no interaction with PMNs against biofilms of P. aeruginosa. Our findings demonstrate a greater positive interaction between AMK with PMNs than that observed for CAZ and especially CIP against isolates of P. aeruginosa from the respiratory tract of CF patients.

  5. Lack of direct DNA damage in human blood leukocytes and lymphocytes after in vitro exposure to high power microwave pulses.

    PubMed

    Chemeris, N K; Gapeyev, A B; Sirota, N P; Gudkova, O Yu; Tankanag, A V; Konovalov, I V; Buzoverya, M E; Suvorov, V G; Logunov, V A

    2006-04-01

    Currently, the potential genotoxicity of high power microwave pulses (HPMP) is not clear. Using the alkaline single cell gel electrophoresis assay, also known as the alkaline comet assay, we studied the effects of HPMP (8.8 GHz, 180 ns pulse width, peak power 65 kW, pulse repetition frequency 50 Hz) on DNA of human whole-blood leukocytes and isolated lymphocytes. The cell suspensions were exposed to HPMP for 40 min in a rectangular waveguide. The average SAR calculated from the temperature kinetics was about 1.6 kW/kg (peak SAR was about 300 MW/kg). The steady-state temperature rise in the 50 microl samples exposed to HPMP was 3.5 +/- 0.1 degrees C. In independent experiments, we did not find any statistically significant DNA damage manifested immediately after in vitro HPMP exposure of human blood leukocytes or lymphocytes or after HPMP exposure of leukocytes subsequently incubated at 37 degrees C for 30 min. Our results indicate that HPMP under the given exposure conditions did not induce DNA strand breaks, alkali-labile sites, and incomplete excision repair sites, which could be detected by the alkaline comet assay.

  6. Reduced antibody-dependent cellular cytotoxicity to herpes simplex virus-infected cells of salivary polymorphonuclear leukocytes and inhibition of peripheral blood polymorphonuclear leukocyte cytotoxicity by saliva.

    PubMed

    Ashkenazi, M; Kohl, S

    1990-06-15

    Blood polymorphonuclear leukocytes (BPMN) have been shown to mediate antibody-dependent cellular cytotoxicity (ADCC) against HSV-infected cells. Although HSV infections are frequently found in the oral cavity, the ADCC capacity of salivary PMN (SPMN) has not been studied, mainly because methods to isolate SPMN were not available. We have recently developed a method to isolate SPMN, and in this study have evaluated their ADCC activity against HSV-infected cells. SPMN were obtained by repeated washings of the oral cavity, and separated from epithelial cells by nylon mesh filtration. ADCC was quantitatively determined by 51Cr release from HSV-infected Chang liver cells. SPMN in the presence of antibody were able to destroy HSV-infected cells, but SPMN were much less effective in mediating ADCC than BPMN (3.4% vs 40.7%, p less than 0.0001). In the presence of antiviral antibody, SPMN were able to adhere to HSV-infected cells, but less so than BPMN (34% vs 67%), and specific antibody-induced adherence was significantly lower in SPMN (p less than 0.04). The spontaneous adherence to HSV-infected cells was higher for SPMN than BPMN. SPMN demonstrated up-regulation of the adhesion glycoprotein CD18, but down-regulation of the FcRIII receptor. Incubation with saliva decreased ADCC capacity of BPMN, up-regulated CD18 expression, and down-regulated FcRIII expression.

  7. The effects of space flight on polymorphonuclear leukocyte response experiment MA-032

    NASA Technical Reports Server (NTRS)

    Martin, R. R.

    1976-01-01

    In a series of studies performed at intervals from 30 day before flight to 30 days after recovery, blood samples were obtained from the three astronauts of the Apollo Soyuz Test Project and from eight control subjects. To determine the effects of space flight on polymorphonuclear leukocytes, tests were performed on blood samples obtained as quickly as possible after splashdown and on the day following recovery. The astronauts' inhalation of propellant gases and the inception of corticosteroid therapy 1 day after recovery provided an additional opportunity to investigate the possible effects of these factors on leukocyte function. Data were obtained during each time period on the total leukocyte count, differential count, leukocyte adhesion, leukocyte migration and chemotaxis, phagocytosis, and histochemical staining for leukocyte acid and alkaline phosphatase. These observations present a variety of in vitro correlates to white blood cell function within the body. Taken together, they serve as a reasonable approximation of the effects of space flight on leukocyte function.

  8. Endothelial actin-binding proteins and actin dynamics in leukocyte transendothelial migration.

    PubMed

    Schnoor, Michael

    2015-04-15

    The endothelium is the first barrier that leukocytes have to overcome during recruitment to sites of inflamed tissues. The leukocyte extravasation cascade is a complex multistep process that requires the activation of various adhesion molecules and signaling pathways, as well as actin remodeling, in both leukocytes and endothelial cells. Endothelial adhesion molecules, such as E-selectin or ICAM-1, are connected to the actin cytoskeleton via actin-binding proteins (ABPs). Although the contribution of receptor-ligand interactions to leukocyte extravasation has been studied extensively, the contribution of endothelial ABPs to the regulation of leukocyte adhesion and transendothelial migration remains poorly understood. This review focuses on recently published evidence that endothelial ABPs, such as cortactin, myosin, or α-actinin, regulate leukocyte extravasation by controlling actin dynamics, biomechanical properties of endothelia, and signaling pathways, such as GTPase activation, during inflammation. Thus, ABPs may serve as targets for novel treatment strategies for disorders characterized by excessive leukocyte recruitment.

  9. Influence of erythrocyte aggregation on leukocyte margination in postcapillary expansions: A lattice Boltzmann analysis

    NASA Astrophysics Data System (ADS)

    Sun, Chenghai; Munn, Lance L.

    2006-03-01

    Leukocyte rolling on the vascular endothelium requires initial contact between the circulating leukocytes in the blood and the vessel wall. Although specific adhesion mechanisms are involved in leukocyte-endothelium interactions, adhesion patterns in vivo suggest other rheological mechanisms are involved as well. Previous studies have proposed that the abundance of leukocyte rolling in postcapillary venules is due to interactions between red blood cells and leukocytes as they enter capillary expansions as well as red blood cell (RBC) aggregation. We have established a lattice Boltzmann approach to analyze the interactions of RBC aggregates and leukocytes as they flow through a postcapillary expansion. The lattice Boltzmann technique provides the complete solution of the flow field and quantification of the particle-particle forces. Our results show that RBC aggregation strongly influences leukocyte-endothelium interactions.

  10. Endothelial tetraspanin microdomains regulate leukocyte firm adhesion during extravasation.

    PubMed

    Barreiro, Olga; Yáñez-Mó, María; Sala-Valdés, Mónica; Gutiérrez-López, María Dolores; Ovalle, Susana; Higginbottom, Adrian; Monk, Peter N; Cabañas, Carlos; Sánchez-Madrid, Francisco

    2005-04-01

    Tetraspanins associate with several transmembrane proteins forming microdomains involved in intercellular adhesion and migration. Here, we show that endothelial tetraspanins relocalize to the contact site with transmigrating leukocytes and associate laterally with both intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Alteration of endothelial tetraspanin microdomains by CD9-large extracellular loop (LEL)-glutathione S-transferase (GST) peptides or CD9/CD151 siRNA oligonucleotides interfered with ICAM-1 and VCAM-1 function, preventing lymphocyte transendothelial migration and increasing lymphocyte detachment under shear flow. Heterotypic intercellular adhesion mediated by VCAM-1 or ICAM-1 was augmented when expressed exogenously in the appropriate tetraspanin environment. Therefore, tetraspanin microdomains have a crucial role in the proper adhesive function of ICAM-1 and VCAM-1 during leukocyte adhesion and transendothelial migration.

  11. Microfluidic platform for the quantitative analysis of leukocyte migration signatures.

    PubMed

    Boneschansker, Leo; Yan, Jun; Wong, Elisabeth; Briscoe, David M; Irimia, Daniel

    2014-09-03

    Leukocyte migration into tissues is characteristic of inflammation. It is usually measured in vitro as the average displacement of populations of cells towards a chemokine gradient, not acknowledging other patterns of cell migration. Here, we designed and validated a microfluidic migration platform to simultaneously analyse four qualitative migration patterns: chemoattraction, -repulsion, -kinesis and -inhibition, using single-cell quantitative metrics of direction, speed, persistence and fraction of cells responding. We find that established chemokines, complement component 5a and IL-8 induce chemoattraction and repulsion in equal proportions, resulting in the dispersal of cells. These migration signatures are characterized by high persistence and speed and are independent of the chemokine dose or receptor expression. Furthermore, we find that twice as many T lymphocytes migrate away than towards stromal cell-derived factor 1 and their directional migration patterns are not persistent. Overall, our platform helps discover migratory signature responses and uncovers an avenue for precise characterization of leukocyte migration and therapeutic modulators.

  12. MICROFLUIDIC PLATFORM FOR THE QUANTITATIVE ANALYSIS OF LEUKOCYTE MIGRATION SIGNATURES

    PubMed Central

    Wong, Elisabeth; Briscoe, David M.; Irimia, Daniel

    2014-01-01

    Leukocyte migration into tissues is characteristic of inflammation. It is usually measured in vitro as the average displacement of populations of cells towards a chemokine gradient, not acknowledging other patterns of cell migration. Here, we designed and validated a microfluidic migration platform to simultaneously analyze four qualitative migration patterns: chemo-attraction, -repulsion, -kinesis and -inhibition, using single-cell quantitative metrics of direction, speed, persistence, and fraction of cells responding. We find that established chemokines C5a and IL-8 induce chemoattraction and repulsion in equal proportions, resulting in the dispersal of cells. These migration signatures are characterized by high persistence and speed and are independent of the chemokine dose or receptor expression. Furthermore, we find that twice as many T-lymphocytes migrate away than towards SDF-1 and their directional migration patterns are not persistent. Overall, our platform characterizes migratory signature responses and uncovers an avenue for precise characterization of leukocyte migration and therapeutic modulators. PMID:25183261

  13. Imaging Cytometry of Human Leukocytes with Third Harmonic Generation Microscopy

    PubMed Central

    Wu, Cheng-Ham; Wang, Tzung-Dau; Hsieh, Chia-Hung; Huang, Shih-Hung; Lin, Jong-Wei; Hsu, Szu-Chun; Wu, Hau-Tieng; Wu, Yao-Ming; Liu, Tzu-Ming

    2016-01-01

    Based on third-harmonic-generation (THG) microscopy and a k-means clustering algorithm, we developed a label-free imaging cytometry method to differentiate and determine the types of human leukocytes. According to the size and average intensity of cells in THG images, in a two-dimensional scatter plot, the neutrophils, monocytes, and lymphocytes in peripheral blood samples from healthy volunteers were clustered into three differentiable groups. Using these features in THG images, we could count the number of each of the three leukocyte types both in vitro and in vivo. The THG imaging-based counting results agreed well with conventional blood count results. In the future, we believe that the combination of this THG microscopy-based imaging cytometry approach with advanced texture analysis of sub-cellular features can differentiate and count more types of blood cells with smaller quantities of blood. PMID:27845443

  14. Segmentation of leukocytes and erythrocytes in blood smear images.

    PubMed

    Bergen, Tobias; Steckhan, Dirk; Wittenberg, Thomas; Zerfass, Thorsten

    2008-01-01

    Differential blood count is a standard method in hematological laboratory diagnosis. In the course of developing a computer-assisted microscopy system for the generation of differential blood counts, the detection and segmentation of white and red blood cells forms an essential step and its exactness is a fundamental prerequisite for the effectiveness of the subsequent classification step. We propose a method for the exact segmentation of leukocytes and erythrocytes in a simultaneous and cooperative way. We combine pixel-wise classification with template matching to locate erythrocytes and use a level-set approach in order to get the exact cell contours of leukocyte nucleus and plasma regions as well as erythrocyte regions. An evaluation comparing the performance of the algorithm to the manual segmentation performed by several persons yielded good results.

  15. Imaging Cytometry of Human Leukocytes with Third Harmonic Generation Microscopy

    NASA Astrophysics Data System (ADS)

    Wu, Cheng-Ham; Wang, Tzung-Dau; Hsieh, Chia-Hung; Huang, Shih-Hung; Lin, Jong-Wei; Hsu, Szu-Chun; Wu, Hau-Tieng; Wu, Yao-Ming; Liu, Tzu-Ming

    2016-11-01

    Based on third-harmonic-generation (THG) microscopy and a k-means clustering algorithm, we developed a label-free imaging cytometry method to differentiate and determine the types of human leukocytes. According to the size and average intensity of cells in THG images, in a two-dimensional scatter plot, the neutrophils, monocytes, and lymphocytes in peripheral blood samples from healthy volunteers were clustered into three differentiable groups. Using these features in THG images, we could count the number of each of the three leukocyte types both in vitro and in vivo. The THG imaging-based counting results agreed well with conventional blood count results. In the future, we believe that the combination of this THG microscopy-based imaging cytometry approach with advanced texture analysis of sub-cellular features can differentiate and count more types of blood cells with smaller quantities of blood.

  16. Antiviral activities of hybrids of two major human leukocyte interferons.

    PubMed Central

    Weck, P K; Apperson, S; Stebbing, N; Gray, P W; Leung, D; Shepard, H M; Goeddel, D V

    1981-01-01

    Four hybrid human leukocyte interferon (LeIF or IFN-alpha) genes have been constructed by in vitro recombination of LeIF-A (IFN-alpha 2) and LeIF-D (IFN-alpha 1) genes at common restriction endonuclease sites located within their coding regions. These hybrid genes have been expressed in E. coli under trp promoter control. The interferons produced [LeIF-AD (BglII), -AD (PvuII), -DA (BglII), -DA (PvuII)] have antiviral properties distinct from the parental molecules LeIF-A and -D, varying considerably in their abilities to inhibit plaque formation by different viruses in a range of mammalian cells. All six of the cloned LeIFs exhibit the heat stability, pH 2 stability and antigenic specificity of natural leukocyte interferons. PMID:6171779

  17. Indium-111 leukocyte scintigraphy in Wegener's granulomatosis involving the spleen

    SciTech Connect

    Morayati, S.J.; Fink-Bennett, D.

    1986-12-01

    Indium-111-labeled leukocyte scintigraphy was performed on a 44-yr-old man to exclude an occult abscess. Four- and twenty-four-hour images of the abdomen revealed splenic photopenia except for a rim of activity medially. A subsequent computed tomography (CT) study demonstrated necrosis or hemorrhage of the spleen except for a medial rim. Exploratory laparotomy demonstrated necrotizing vasculitis with granuloma formation consistent with Wegener's granulomatosis and a rim of viable splenic tissue corresponding to the radionuclide and CT studies.

  18. Characterization of uterine leukocyte infiltration in gilts after artificial insemination.

    PubMed

    Rozeboom, K J; Troedsson, M H; Crabo, B G

    1998-11-01

    The objective of this study was to characterize the uterine leukocyte influx after artificial insemination (AI). After detection of oestrus with a boar at intervals of 1.5 h, seventy-two gilts were randomly assigned to a 2 x 3 x 4 factorial arrangement. AI was performed with 100 ml extended semen containing 5 x 10(9) spermatozoa (semen; n = 36) or 100 ml VSP semen extender (extender; n = 36) at one of three times after detection of oestrus: 12, 24 or 36 h (n = 24/time). The uterus was lavaged at 6, 12, 18 or 24 h (n = 18/time) after AI to determine the total number of uterine leukocytes. In addition, uterine lavage was performed on nine untreated gilts immediately after the detection of oestrus to establish a baseline number of leukocytes. The leukocyte response in all samples consisted predominately (92-99%) of polymorphonuclear neutrophilic granulocytes (PMNs). The mean number of PMNs recovered from the uteri of gilts treated with semen was greater than in gilts treated with extender and in untreated gilts (P < 0.01). The greatest number of PMNs in semen-treated gilts was found 12 h after AI (P < 0.01), and this number was sustained for 24 h. In contrast, the number of uterine PMNs recovered from extender-treated gilts reached a peak at 6 h and had declined by 12 h after AI (P < 0.05). It was concluded that an extensive influx of PMNs into the uterus is a normal sequence to AI. The consequences and importance of semen-induced uterine leukocytosis needs further investigation.

  19. Influence of light sources on the migration of polymorphonuclear leukocytes

    NASA Astrophysics Data System (ADS)

    DellaVecchia, Michael A.; Beard, Richard B.; Dai, Xiaoyan

    1995-05-01

    In the process of inflammation, leukocytes must travel from the intraluminal space of the capillary to the interstitial space in order to reach the site of the inflammation. The two major populations of mature human leukocytes based on the morphology are the polymorphonuclear leukocytes (PMN), and mononuclear leukocytes (MNL). Previous research on PMNs and MNLs at the Biomedical Engineering and Science Institute of Drexel University have shown that their migration can be markedly enhanced by excitation with electric and magnetic fields. This presentation demonstrates that the migration of PMNs under excitation of photons is enhanced in the red light region of (lambda) equals 660 nm and inhibited in the green light region of (lambda) equals 565 nm. There is an intensity threshold at which red light enhances migration and an intensity threshold at which green light inhibits migration. In these experiments the Boyden technique was used with the distance of the cell migration through a cellulose filter measured in terms of the leading edge. The comparison of the relative value of the distance to cell migration under a light to cell migration without a light stimulus was recorded as a cytokinetic index, K.I.. K.I. is a measure of the cytokinesis which is the progress of the cell movement in which the migration is enhanced by substances in the cell environment irrespective of a concentration gradient. The cytotactic index is a measure of cytotaxis which is the directional movement along a chemical gradient formed by a chemotactic factor. A Russian pulsed commercial laser biostimulator in the near infrared wavelength above an intensity threshold enhances PMN migration. Intermittent green and red stimulators below the intensity threshold markedly influence the cytokinetic index of PMNs while above the intensity threshold, this influence is deminished.

  20. Indium-111 leukocyte localization in infected prosthetic graft

    SciTech Connect

    Purnell, G.L.; Walker, C.W.; Allison, J.W.; Dalrymple, G.V. )

    1990-08-01

    Infective endocarditis can be difficult to prove, even in the face of strong clinical suspicion. A case in which standard methods of diagnosis failed to demonstrate endocarditis in a patient with recurrent Staphylococcus aureus bacteremia and porcine aortic valve is reported. An In-111 labelled leukocyte SPECT study demonstrated uptake in the aortic root and leaflets, and autopsy demonstrated vegetations on the leaflets. In-111 may prove useful in demonstrating endocarditis in patients with prosthetic valve infection.

  1. Labeled leukocyte scans for detection of retained polyurethane foam

    SciTech Connect

    Ellenberger, P.; Graham, W.P. 3d.; Manders, E.K.; Basarab, R.M.

    1986-01-01

    Complete removal of an infected polyurethane-covered breast prosthesis is difficult, and retained tissue-embedded foam can form a nidus for persistent infection. Scanning the chest wall after administration of indium-111 oxine-labeled autogenous leukocytes will locate areas of infection around retained fragments of foam, thereby facilitating their removal and allowing eventual successful reconstruction. This technique may deserve wider application for locating infected foreign bodies in a variety of patient problems.

  2. Myxoma and vaccinia viruses bind differentially to human leukocytes.

    PubMed

    Chan, Winnie M; Bartee, Eric C; Moreb, Jan S; Dower, Ken; Connor, John H; McFadden, Grant

    2013-04-01

    Myxoma virus (MYXV) and vaccinia virus (VACV), two distinct members of the family Poxviridae, are both currently being developed as oncolytic virotherapeutic agents. Recent studies have demonstrated that ex vivo treatment with MYXV can selectively recognize and kill contaminating cancerous cells from autologous bone marrow transplants without perturbing the engraftment of normal CD34(+) hematopoietic stem and progenitor cells. However, the mechanism(s) by which MYXV specifically recognizes and eliminates the cancer cells in the autografts is not understood. While little is known about the cellular attachment factor(s) exploited by MYXV for entry into any target cells, VACV has been shown to utilize cell surface glycosaminoglycans such as heparan sulfate (HS), the extracellular matrix protein laminin, and/or integrin β1. We have constructed MYXV and VACV virions tagged with the Venus fluorescent protein and compared their characteristics of binding to various human cancer cell lines as well as to primary human leukocytes. We report that the binding of MYXV or VACV to some adherent cell lines could be partially inhibited by heparin, but laminin blocked only VACV binding. In contrast to cultured fibroblasts, the binding of MYXV and VACV to a wide spectrum of primary human leukocytes could not be competed by either HS or laminin. Additionally, MYXV and VACV exhibited very different binding characteristics against certain select human leukocytes, suggesting that the two poxviruses utilize different cell surface determinants for the attachment to these cells. These results indicate that VACV and MYXV can exhibit very different oncolytic tropisms against some cancerous human leukocytes.

  3. Leukocyte Trafficking to the Small Intestine and Colon.

    PubMed

    Habtezion, Aida; Nguyen, Linh P; Hadeiba, Husein; Butcher, Eugene C

    2016-02-01

    Leukocyte trafficking to the small and large intestines is tightly controlled to maintain intestinal immune homeostasis, mediate immune responses, and regulate inflammation. A wide array of chemoattractants, chemoattractant receptors, and adhesion molecules expressed by leukocytes, mucosal endothelium, epithelium, and stromal cells controls leukocyte recruitment and microenvironmental localization in intestine and in the gut-associated lymphoid tissues (GALTs). Naive lymphocytes traffic to the gut-draining mesenteric lymph nodes where they undergo antigen-induced activation and priming; these processes determine their memory/effector phenotypes and imprint them with the capacity to migrate via the lymph and blood to the intestines. Mechanisms of T-cell recruitment to GALT and of T cells and plasmablasts to the small intestine are well described. Recent advances include the discovery of an unexpected role for lectin CD22 as a B-cell homing receptor GALT, and identification of the orphan G-protein-coupled receptor 15 (GPR15) as a T-cell chemoattractant/trafficking receptor for the colon. GPR15 decorates distinct subsets of T cells in mice and humans, a difference in species that could affect translation of the results of mouse colitis models to humans. Clinical studies with antibodies to integrin α4β7 and its vascular ligand mucosal vascular addressin cell adhesion molecule 1 are proving the value of lymphocyte trafficking mechanisms as therapeutic targets for inflammatory bowel diseases. In contrast to lymphocytes, cells of the innate immune system express adhesion and chemoattractant receptors that allow them to migrate directly to effector tissue sites during inflammation. We review the mechanisms for innate and adaptive leukocyte localization to the intestinal tract and GALT, and discuss their relevance to human intestinal homeostasis and inflammation.

  4. Myxoma and Vaccinia Viruses Bind Differentially to Human Leukocytes

    PubMed Central

    Chan, Winnie M.; Bartee, Eric C.; Moreb, Jan S.; Dower, Ken; Connor, John H.

    2013-01-01

    Myxoma virus (MYXV) and vaccinia virus (VACV), two distinct members of the family Poxviridae, are both currently being developed as oncolytic virotherapeutic agents. Recent studies have demonstrated that ex vivo treatment with MYXV can selectively recognize and kill contaminating cancerous cells from autologous bone marrow transplants without perturbing the engraftment of normal CD34+ hematopoietic stem and progenitor cells. However, the mechanism(s) by which MYXV specifically recognizes and eliminates the cancer cells in the autografts is not understood. While little is known about the cellular attachment factor(s) exploited by MYXV for entry into any target cells, VACV has been shown to utilize cell surface glycosaminoglycans such as heparan sulfate (HS), the extracellular matrix protein laminin, and/or integrin β1. We have constructed MYXV and VACV virions tagged with the Venus fluorescent protein and compared their characteristics of binding to various human cancer cell lines as well as to primary human leukocytes. We report that the binding of MYXV or VACV to some adherent cell lines could be partially inhibited by heparin, but laminin blocked only VACV binding. In contrast to cultured fibroblasts, the binding of MYXV and VACV to a wide spectrum of primary human leukocytes could not be competed by either HS or laminin. Additionally, MYXV and VACV exhibited very different binding characteristics against certain select human leukocytes, suggesting that the two poxviruses utilize different cell surface determinants for the attachment to these cells. These results indicate that VACV and MYXV can exhibit very different oncolytic tropisms against some cancerous human leukocytes. PMID:23388707

  5. Leukocyte trafficking: Can we bring the fight to the tumor?

    PubMed

    Pachynski, Russell; Nazha, Jonathon; Kohrt, Holbrook

    2016-03-01

    Control of leukocyte trafficking plays a critical role in the establishment of effective immune responses. It is now well established that the number or ratio of effector to suppressor immune cells within the tumor microenvironment can significantly impact tumor growth and clinical outcomes. Recently approved immunotherapies by the FDA, and those in development, aim to stimulate effector immune cell function. For example, many checkpoint inhibitors seek to stimulate an immune response to tumors by reversing T-cell exhaustion. However, activation of the immune response outside the tumor microenvironment can lead to sometimes fatal immune-mediated adverse events -- the result of "on-target, off-tumor" effects. Thus, control of localization of these activated effector cells remains a critical component of optimizing tumor response while minimizing immune-mediated adverse events. Chemokines and chemoattractants, along with their receptors on immune cells, govern leukocyte trafficking; thus, understanding their expression pattern in the context of the tumor microenvironment and developing approaches to favorably alter those should lead to improved efficacy of current immunotherapeutics. This review highlights the background of cancer immunotherapy, leukocyte trafficking, and some novel approaches being utilized to optimize recruitment of effector immune cells into the tumor microenvironment. Future combinatorial immunotherapy should incorporate therapeutics aimed at 1) favorably altering the tumor microenvironment, 2) activating effector immune cells, and 3) optimizing effector cell trafficking into tumors.

  6. Imaging leukocytes in vivo with third harmonic generation microscopy

    NASA Astrophysics Data System (ADS)

    Tsai, Cheng-Kun; Chen, Chien-Kuo; Chen, Yu-Shing; Wu, Pei-Chun; Hsieh, Tsung-Yuan; Liu, Han-Wen; Yeh, Chiou-Yueh; Lin, Win-Li; Chia, Jean-San; Liu, Tzu-Ming

    2013-02-01

    Without a labeling, we demonstrated that lipid granules in leukocytes have distinctive third harmonic generation (THG) contrast. Excited by a 1230nm femtosecond laser, THG signals were generated at a significantly higher level in neutrophils than other mononuclear cells, whereas signals in agranular lymphocytes were one order smaller. These characteristic THG features can also be observed in vivo to trace the newly recruited leukocytes following lipopolysaccharide (LPS) challenge. Furthermore, using video-rate THG microscopy, we also captured images of blood cells in human capillaries. Quite different from red-blood-cells, every now and then, round and granule rich blood cells with strong THG contrast appeared in circulation. The corresponding volume densities in blood, evaluated from their frequencies of appearance and the velocity of circulation, fall within the physiological range of human white blood cell counts. These results suggested that labeling-free THG imaging may provide timely tracing of leukocyte movement and hematology inspection without disturbing the normal cellular or physiological status.

  7. Exposure to mercury alters early activation events in fish leukocytes.

    PubMed Central

    MacDougal, K C; Johnson, M D; Burnett, K G

    1996-01-01

    Although fish in natural populations may carry high body burdens of both organic and inorganic mercury, the effects of this divalent metal on such lower vertebrates is poorly understood. In this report, inorganic mercury in the form of mercuric chloride (HgCl2) is shown to produce both high-dose inhibition and low-dose activation of leukocytes in a marine teleost fish, Sciaenops ocellatus. Concentrations of inorganic mercury > or = 10 microM suppressed DNA synthesis and induced rapid influx of radiolabeled calcium, as well as tyrosine phosphorylation of numerous cellular proteins. Lower concentrations (0.1-1 microM) of HgCl2 that activated cell growth also induced a slow sustained rise in intracellular calcium in cells loaded with the calcium indicator dye fura-2, but did not produce detectable tyrosine phosphorylation of leukocyte proteins. These studies support the possibility that subtoxic doses of HgCl2 may inappropriately activate teleost leukocytes, potentially altering the processes that regulate the magnitude and specificity of the fish immune response to environmental pathogens. Images Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. PMID:8930553

  8. Phenotypic characterization of leukocytes in prenatal human dermis.

    PubMed

    Schuster, Christopher; Vaculik, Christine; Prior, Marion; Fiala, Christian; Mildner, Michael; Eppel, Wolfgang; Stingl, Georg; Elbe-Bürger, Adelheid

    2012-11-01

    The adult human skin harbors a variety of leukocytes providing immune surveillance and host defense, but knowledge about their ontogeny is scarce. In this study we investigated the number and phenotype of leukocytes in prenatal human skin (dermal dendritic cells (DDCs), macrophages, T cells (including FoxP3(+) regulatory T cells), and mast cells) to unravel their derivation and to get a clue as to their putative function in utero. By flow cytometry and immunofluorescence, we found a distinction between CD206(+)CD1c(+)CD11c(+) DDCs and CD206(+)CD209(+)CD1c(-) skin macrophages by 9 weeks estimated gestational age (EGA). T cells appear at the end of the first trimester, expressing CD3 intracytoplasmatically. During midgestation, CD3(+)FoxP3(-) and CD3(+)FoxP3(+) cells can exclusively be found in the dermis. Similarly, other leukocytes such as CD117(+) (c-kit) mast cells were not identified before 12-14 weeks EGA and only slowly acquire a mature phenotype during gestation. Our data show at which time point during gestation antigen-presenting cells, T cells, and mast cells populate the human dermis and provide a step forward to a better understanding of the development of the human skin immune system.

  9. Comparison of photonic and electromagnetic effects on the human leukocyte

    NASA Astrophysics Data System (ADS)

    DellaVecchia, Michael A.; Beard, Richard B.; Feng, D.; Dai, Xiaoyan; Pourrezaei, Kambiz; Priezzhev, Alexander V.

    1998-06-01

    The dielectric and magnetic influence on human cells have been widely studied previously by the authors. Recently, the effects of energy in the visible electromagnetic spectrum have been investigated. In this subsequent study, the photonic effects on the in vitro migration of the polymorphonuclear and mononuclear leukocytes are compared with the corresponding electromagnetic field effects. Dielectric spectra of the polymorph in the 300 KHz to 400 KHz and 700 KHz to 800 KHz range have been measured. At frequencies of 350 KHz and 720 KHz an increase in the migration of the polymorphonuclear leukocyte have been observed. This stimulation was attributed to the charges on the nuclear surface. Recent preliminary data have shown a similar increased migration in the 20 MHz range. Photonic studies have indicated an enhanced migration for the polymorphonuclear leukocytes at a wavelength of 660 nm (red) and an inhibited migration at 565 nm (green). The photonic effects were postulated to be the results of a biochemical interaction rather than a membranous surface charge displacement secondary to an electric field. The migration of the white blood cells were measurement via the Boyden chamber technique and expressed in terms of a cytokinetic index which expresses the cellular movement independent of its environmental concentration gradient.

  10. Tracking flow of leukocytes in blood for drug analysis

    NASA Astrophysics Data System (ADS)

    Basharat, Arslan; Turner, Wesley; Stephens, Gillian; Badillo, Benjamin; Lumpkin, Rick; Andre, Patrick; Perera, Amitha

    2011-03-01

    Modern microscopy techniques allow imaging of circulating blood components under vascular flow conditions. The resulting video sequences provide unique insights into the behavior of blood cells within the vasculature and can be used as a method to monitor and quantitate the recruitment of inflammatory cells at sites of vascular injury/ inflammation and potentially serve as a pharmacodynamic biomarker, helping screen new therapies and individualize dose and combinations of drugs. However, manual analysis of these video sequences is intractable, requiring hours per 400 second video clip. In this paper, we present an automated technique to analyze the behavior and recruitment of human leukocytes in whole blood under physiological conditions of shear through a simple multi-channel fluorescence microscope in real-time. This technique detects and tracks the recruitment of leukocytes to a bioactive surface coated on a flow chamber. Rolling cells (cells which partially bind to the bioactive matrix) are detected counted, and have their velocity measured and graphed. The challenges here include: high cell density, appearance similarity, and low (1Hz) frame rate. Our approach performs frame differencing based motion segmentation, track initialization and online tracking of individual leukocytes.

  11. Interaction of Candida albicans with Human Leukocytes and Serum1

    PubMed Central

    Lehrer, Robert I.; Cline, Martin J.

    1969-01-01

    A quantitative assay of candidacidal activity based on differential staining of non-viable Candida albicans by methylene blue was developed and applied to studies of leukocytes from normal individuals and patients with fungal and other infections. Serum factors were necessary for optimal phagocytosis of C. albicans but lacked direct candidacidal activity. Normal human neutrophils (38 studies) killed 29.0 ± 7.4% of ingested C. albicans in 1 hr. Eosinophils and monocytes killed a smaller percentage. Neutrophil candidacidal activity did not require protein or ribonucleic acid synthesis by the leukocyte but was inhibited by anaerobic conditions, potassium cyanide, and colchicine. Leukocytes of a patient with hereditary myeloperoxidase deficiency and of three children with chronic granulomatous disease phagocytized C. albicans normally, yet failed to kill them. Our data suggest that the neutrophil can play an important role in resistance to Candida infection and that the lysosomal enzyme myeloperoxidase and its oxidant substrate hydrogen peroxide are the major participants in neutrophil candidacidal activity. Images PMID:4182532

  12. Macrophage recognition of ICAM-3 on apoptotic leukocytes.

    PubMed

    Moffatt, O D; Devitt, A; Bell, E D; Simmons, D L; Gregory, C D

    1999-06-01

    Cells undergoing apoptosis are cleared rapidly by phagocytes, thus preventing tissue damage caused by loss of plasma membrane integrity. In this study, we show that the surface of leukocytes is altered during apoptosis such that the first Ig-like domain of ICAM-3 (CD50) can participate in the recognition and phagocytosis of the apoptotic cells by macrophages. Macrophage recognition of apoptotic cell-associated ICAM-3 was demonstrated both on leukocytes and, following transfection of exogenous ICAM-3, on nonleukocytes. The change in ICAM-3 was a consistent consequence of apoptosis triggered by various stimuli, suggesting that it occurs as part of a final common pathway of apoptosis. Alteration of ICAM-3 on apoptotic cells permitting recognition by macrophages resulted in a switch in ICAM-3-binding preference from the prototypic ICAM-3 counterreceptor, LFA-1, to an alternative macrophage receptor. Using mAbs to block macrophage/apoptotic cell interactions, we were unable to obtain evidence that either the alternative ICAM-3 counterreceptor alpha d beta 2 or the apoptotic cell receptor alpha v beta 3 was involved in the recognition of ICAM-3. By contrast, mAb blockade of macrophage CD14 inhibited ICAM-3-dependent recognition of apoptotic cells. These results show that ICAM-3 can function as a phagocytic marker of apoptotic leukocytes on which it acquires altered macrophage receptor-binding activity.

  13. Tenocytes, pro-inflammatory cytokines and leukocytes: a relationship?

    PubMed Central

    Al-Sadi, Onays; Schulze-Tanzil, Gundula; Kohl, Benjamin; Lohan, Anke; Lemke, Marion; Ertel, Wolfgang; John, Thilo

    2011-01-01

    Summary Leukocyte derived pro-inflammatory mediators could be involved in tendon healing and scar formation. Hence, the effect of autologous leukocytes (PBMCs, peripheral blood mononuclear cells and neutrophils) on primary rabbit Achilles tenocytes gene expression was tested in insert assisted co-cultures. Subsequently, tenocytes gene expression of extra-cellular matrix (ECM) components (type I collagen, decorin, fibronectin), the cell-ECM receptor β1-integrin, the angiogenic factor myodulin, ECM degrading matrix-metalloproteinase (MMP)1 and pro-inflammatory cytokines (interleukin [IL]-1β, tumour necrosis factor [TNFα] and IL-6) was analysed. The only significant effect of leukocytes on tenocytes ECM genes expression was a suppression of type I collagen by neutrophils combined with TNFα stimulation. The same effect could be observed analysing the β1-integrin and myodulin gene expression. However, PBMCs up-regulated significantly cytokine and MMP1 gene expression in tenocytes. These in vitro results suggest that mononuclear cells could present an exogenic stimulus for the induction of pro-inflammatory and catabolic mediators in tendon. PMID:23738251

  14. DNA methylation map in circulating leukocytes mirrors subcutaneous adipose tissue methylation pattern: a genome-wide analysis from non-obese and obese patients

    PubMed Central

    Crujeiras, A. B.; Diaz-Lagares, A.; Sandoval, J.; Milagro, F. I.; Navas-Carretero, S.; Carreira, M. C.; Gomez, A.; Hervas, D.; Monteiro, M. P.; Casanueva, F. F.; Esteller, M.; Martinez, J. A.

    2017-01-01

    The characterization of the epigenetic changes within the obesity-related adipose tissue will provide new insights to understand this metabolic disorder, but adipose tissue is not easy to sample in population-based studies. We aimed to evaluate the capacity of circulating leukocytes to reflect the adipose tissue-specific DNA methylation status of obesity susceptibility. DNA samples isolated from subcutaneous adipose tissue and circulating leukocytes were hybridized in the Infinium HumanMethylation 450 BeadChip. Data were compared between samples from obese (n = 45) and non-obese (n = 8–10) patients by Wilcoxon-rank test, unadjusted for cell type distributions. A global hypomethylation of the differentially methylated CpG sites (DMCpGs) was observed in the obese subcutaneous adipose tissue and leukocytes. The overlap analysis yielded a number of genes mapped by the common DMCpGs that were identified to reflect the obesity state in the leukocytes. Specifically, the methylation levels of FGFRL1, NCAPH2, PNKD and SMAD3 exhibited excellent and statistically significant efficiencies in the discrimination of obesity from non-obesity status (AUC > 0.80; p < 0.05) and a great correlation between both tissues. Therefore, the current study provided new and valuable DNA methylation biomarkers of obesity-related adipose tissue pathogenesis through peripheral blood analysis, an easily accessible and minimally invasive biological material instead of adipose tissue. PMID:28211912

  15. Flow cytometric assay for analysis of cytotoxic effects of potential drugs on human peripheral blood leukocytes

    NASA Astrophysics Data System (ADS)

    Nieschke, Kathleen; Mittag, Anja; Golab, Karolina; Bocsi, Jozsef; Pierzchalski, Arkadiusz; Kamysz, Wojciech; Tarnok, Attila

    2014-03-01

    Toxicity test of new chemicals belongs to the first steps in the drug screening, using different cultured cell lines. However, primary human cells represent the human organism better than cultured tumor derived cell lines. We developed a very gentle toxicity assay for isolation and incubation of human peripheral blood leukocytes (PBL) and tested it using different bioactive oligopeptides (OP). Effects of different PBL isolation methods (red blood cell lysis; Histopaque isolation among others), different incubation tubes (e.g. FACS tubes), anticoagulants and blood sources on PBL viability were tested using propidium iodide-exclusion as viability measure (incubation time: 60 min, 36°C) and flow cytometry. Toxicity concentration and time-depended effects (10-60 min, 36 °C, 0-100 μg /ml of OP) on human PBL were analyzed. Erythrocyte lysis by hypotonic shock (dH2O) was the fastest PBL isolation method with highest viability (>85%) compared to NH4Cl-Lysis (49%). Density gradient centrifugation led to neutrophil granulocyte cell loss. Heparin anticoagulation resulted in higher viability than EDTA. Conical 1.5 mL and 2 mL micro-reaction tubes (both polypropylene (PP)) had the highest viability (99% and 97%) compared to other tubes, i.e. three types of 5.0 mL round-bottom tubes PP (opaque-60%), PP (blue-62%), Polystyrene (PS-64%). Viability of PBL did not differ between venous and capillary blood. A gentle reproducible preparation and analytical toxicity-assay for human PBL was developed and evaluated. Using our assay toxicity, time-course, dose-dependence and aggregate formation by OP could be clearly differentiated and quantified. This novel assay enables for rapid and cost effective multiparametric toxicological screening and pharmacological testing on primary human PBL and can be adapted to high-throughput-screening.°z

  16. Altered Immunogenicity of Donor Lungs via Removal of Passenger Leukocytes Using Ex Vivo Lung Perfusion.

    PubMed

    Stone, J P; Critchley, W R; Major, T; Rajan, G; Risnes, I; Scott, H; Liao, Q; Wohlfart, B; Sjöberg, T; Yonan, N; Steen, S; Fildes, J E

    2016-01-01

    Passenger leukocyte transfer from the donor lung to the recipient is intrinsically involved in acute rejection. Direct presentation of alloantigen expressed on donor leukocytes is recognized by recipient T cells, promoting acute cellular rejection. We utilized ex vivo lung perfusion (EVLP) to study passenger leukocyte migration from donor lungs into the recipient and to evaluate the effects of donor leukocyte depletion prior to transplantation. For this purpose, female pigs received male left lungs either following 3 h of EVLP or retrieved using standard protocols. Recipients were monitored for 24 h and sequential samples were collected. EVLP-reduced donor leukocyte transfer into the recipient and migration to recipient lymph nodes was markedly reduced. Recipient T cell infiltration of the donor lung was significantly diminished via EVLP. Donor leukocyte removal during EVLP reduces direct allorecognition and T cell priming, diminishing recipient T cell infiltration, the hallmark of acute rejection.

  17. Effect of (1→3),(1→6)-β-glucan on in vitro production of cytokines by leukocytes of patients with periodontitis.

    PubMed

    Akramas, Laimis; Akramienė, Dalia; Sakalauskienė, Jurgina; Kubilius, Ričardas; Gleiznys, Alvydas

    2012-01-01

    BACKGROUND. The aim of the study was to investigate the effect of (1→3), (1→6)-β-glucan on the production of interleukin 10 (IL-10) and tumor necrosis factor α (TNF-α) in vitro by peripheral blood leukocytes of patients with periodontitis. MATERIAL AND METHODS. In total, 20 patients suffering from untreated severe chronic generalized periodontitis were enrolled in this study. Periodontitis was confirmed by clinical and radiologic examination. Besides, 20 periodontally healthy patients served as a control group. Peripheral venous blood was sampled from the patients, and isolated leukocytes were treated with (1→3),(1→6)-β-glucan from yeast at different concentrations. The levels of IL-10 and TNF-α secreted by the leukocytes unstimulated and stimulated with unopsonized E. coli in vitro were determined by the enzyme amplified sensitivity immunoassay method. RESULTS. Our data showed that (1→3),(1→6)-β-glucan induced a significant decrease (P<0.05) in the TNF-α level and a significant increase (P<0.001) in the IL-10 level in the media of unstimulated and stimulated leukocytes of the patients with periodontitis in comparison with those of the healthy subjects. CONCLUSIONS. The present in vitro study showed that (1→3),(1→6)-β-glucan modulated the response of leukocytes of the patients with periodontitis differently in comparison with those of the healthy subjects. It increased the release of IL-10, which is protective of the tooth-supporting tissues in patients with periodontal disease, but decreased the release of TNF-α, which is mainly responsible for the destruction of the tooth-supporting tissues during periodontal disease.

  18. Imaging of Leukocyte Trafficking in Alzheimer’s Disease

    PubMed Central

    Pietronigro, Enrica; Zenaro, Elena; Constantin, Gabriela

    2016-01-01

    Alzheimer’s disease (AD) is the most common neurodegenerative disorder and is characterized by a progressive decline of cognitive functions. The neuropathological features of AD include amyloid beta (Aβ) deposition, intracellular neurofibrillary tangles derived from the cytoskeletal hyperphosphorylated tau protein, amyloid angiopathy, the loss of synapses, and neuronal degeneration. In the last decade, inflammation has emerged as a key feature of AD, but most studies have focused on the role of microglia-driven neuroinflammation mechanisms. A dysfunctional blood–brain barrier has also been implicated in the pathogenesis of AD, and several studies have demonstrated that the vascular deposition of Aβ induces the expression of adhesion molecules and alters the expression of tight junction proteins, potentially facilitating the transmigration of circulating leukocytes. Two-photon laser scanning microscopy (TPLSM) has become an indispensable tool to dissect the molecular mechanisms controlling leukocyte trafficking in the central nervous system (CNS). Recent TPLSM studies have shown that vascular deposition of Aβ in the CNS promotes intraluminal neutrophil adhesion and crawling on the brain endothelium and also that neutrophils extravasate in the parenchyma preferentially in areas with Aβ deposits. These studies have also highlighted a role for LFA-1 integrin in neutrophil accumulation in the CNS of AD-like disease models, revealing that LFA-1 inhibition reduces the corresponding cognitive deficit and AD neuropathology. In this article, we consider how current imaging techniques can help to unravel new inflammation mechanisms in the pathogenesis of AD and identify novel therapeutic strategies to treat the disease by interfering with leukocyte trafficking mechanisms. PMID:26913031

  19. Theileria-transformed bovine leukocytes have cancer hallmarks.

    PubMed

    Tretina, Kyle; Gotia, Hanzel T; Mann, David J; Silva, Joana C

    2015-07-01

    The genus Theileria includes tick-transmitted apicomplexan parasites of ruminants with substantial economic impact in endemic countries. Some species, including Theileria parva and Theileria annulata, infect leukocytes where they induce phenotypes that are shared with some cancers, most notably immortalization, hyperproliferation, and dissemination. Despite considerable research into the affected host signaling pathways, the parasite proteins directly responsible for these host phenotypes remain unknown. In this review we outline current knowledge on the manipulation of host cells by transformation-inducing Theileria, and we propose that comparisons between cancer biology and host-Theileria interactions can reveal chemotherapeutic targets against Theileria-induced pathogenesis based on cancer treatment approaches.

  20. The Study of Leukocyte Functions in a Rotating Wall Vessel

    NASA Technical Reports Server (NTRS)

    Trial, JoAnn

    1998-01-01

    The objective of this study was to investigate the behavior of leukocytes under free-fall conditions in a rotating wall vessel. In such a vessel, the tendency of a cell to fall in response to gravity is opposed by the rotation of the vessel and the culture medium within, keeping the cells in suspension without fluid shear. Previous reports indicated that such functions as lymphocyte migration through collagen matrix or monocyte cytokine secretion are altered under these conditions, and these changes correlate with similar functional defects of cultured cells seen during spaceflight.

  1. Longitudinal evaluation of leukocyte transcripts in killer whales (Orcinus Orca)

    USGS Publications Warehouse

    Sitt, Tatjana; Bowen, Lizabeth; Lee, Chia-Shan; Blanchard, Myra; McBain, James; Dold, Christopher; Stott, Jeffrey L.

    2016-01-01

    Early identification of illness and/or presence of environmental and/or social stressors in free-ranging and domestic cetaceans is a priority for marine mammal health care professionals. Incorporation of leukocyte gene transcript analysis into the diagnostic tool kit has the potential to augment classical diagnostics based upon ease of sample storage and shipment, inducible nature and well-defined roles of transcription and associated downstream actions. Development of biomarkers that could serve to identify “insults” and potentially differentiate disease etiology would be of great diagnostic value. To this end, a modest number of peripheral blood leukocyte gene transcripts were selected for application to a domestic killer whale population with a focus on broad representation of inducible immunologically relevant genes. Normalized leukocyte transcript values, longitudinally acquired from 232 blood samples derived from 26 clinically healthy whales, were not visibly influenced temporally nor by sex or the specific Park in which they resided. Stability in leukocyte transcript number during periods of health enhances their potential use in diagnostics through identification of outliers. Transcript levels of two cytokine genes, IL-4 and IL-17, were highly variable within the group as compared to the other transcripts. IL-4 transcripts were typically absent. Analysis of transcript levels on the other genes of interest, on an individual animal basis, identified more outliers than were visible when analyzed in the context of the entire population. The majority of outliers (9 samples) were low, though elevated transcripts were identified for IL-17 from 2 animals and one each for Cox-2 and IL-10. The low number of outliers was not unexpected as sample selection was intentionally directed towards animals that were clinically healthy at the time of collection. Outliers may reflect animals experiencing subclinical disease that is transient and self-limiting. The

  2. Longitudinal evaluation of leukocyte transcripts in killer whales (Orcinus Orca).

    PubMed

    Sitt, Tatjana; Bowen, Lizabeth; Lee, Chia-Shan; Blanchard, Myra T; McBain, James; Dold, Christopher; Stott, Jeffrey L

    2016-07-01

    Early identification of illness and/or presence of environmental and/or social stressors in free-ranging and domestic cetaceans is a priority for marine mammal health care professionals. Incorporation of leukocyte gene transcript analysis into the diagnostic tool kit has the potential to augment classical diagnostics based upon ease of sample storage and shipment, inducible nature and well-defined roles of transcription and associated downstream actions. Development of biomarkers that could serve to identify "insults" and potentially differentiate disease etiology would be of great diagnostic value. To this end, a modest number of peripheral blood leukocyte gene transcripts were selected for application to a domestic killer whale population with a focus on broad representation of inducible immunologically relevant genes. Normalized leukocyte transcript values, longitudinally acquired from 232 blood samples derived from 26 clinically healthy whales, were not visibly influenced temporally nor by sex or the specific Park in which they resided. Stability in leukocyte transcript number during periods of health enhances their potential use in diagnostics through identification of outliers. Transcript levels of two cytokine genes, IL-4 and IL-17, were highly variable within the group as compared to the other transcripts. IL-4 transcripts were typically absent. Analysis of transcript levels on the other genes of interest, on an individual animal basis, identified more outliers than were visible when analyzed in the context of the entire population. The majority of outliers (9 samples) were low, though elevated transcripts were identified for IL-17 from 2 animals and one each for Cox-2 and IL-10. The low number of outliers was not unexpected as sample selection was intentionally directed towards animals that were clinically healthy at the time of collection. Outliers may reflect animals experiencing subclinical disease that is transient and self-limiting. The immunologic

  3. Goat cathelicidin-2 is secreted by blood leukocytes regardless of lipopolysaccharide stimulation.

    PubMed

    Srisaikham, Supreena; Suksombat, Wisitiporn; Yoshimura, Yukinori; Isobe, Naoki

    2016-03-01

    It has been reported that goat cathelicidin-2, an antimicrobial peptide, localizes in leukocytes and is present in milk. Here, we examined whether cathelicidin-2 is secreted by leukocytes. Different concentrations (10(5)-10(8) cells/mL) of blood leukocytes were cultured for 0-48 h with or without lipopolysaccharide (LPS). After culture, the concentrations of cathelicidin-2 in the conditioned media were measured. Blood was collected from male goats 0-24 h after the intravenous injection of Escherichia coli O111:B4 LPS. The plasma cathelicidin-2 concentrations were determined and the blood leukocytes immunostained with anti-cathelicidin-2 antibody to calculate the proportion of cathelicidin-2-positive cells in the total leukocytes. When higher concentrations of leukocytes were cultured, the cathelicidin-2 concentrations in the media increased significantly, whereas the addition of LPS to the media caused no further increase. The plasma cathelicidin-2 concentrations did not increase with time after LPS infusion. The proportion of cathelicidin-2-positive cells in the total leukocytes was significantly reduced 1 h after LPS injection compared with that at 0 h, but increased again at 6 h and thereafter. These results suggest that cathlicidin-2 is secreted by leukocytes even without LPS stimulation, whereas LPS may be required for cathelicidin-2-containing leukocytes to be recruited from the blood to tissues showing inflammation.

  4. Passive recruitment of circulating leukocytes into capillary sprouts from existing capillaries in a microfluidic system.

    PubMed

    Forouzan, Omid; Burns, Jennie M; Robichaux, Jennifer L; Murfee, Walter L; Shevkoplyas, Sergey S

    2011-06-07

    Recent evidence implicating leukocytes in angiogenesis raises the question of whether leukocytes and other cells circulating with the blood in microvascular networks can home to capillary sprouts intraluminally. This study describes an investigation of leukocyte trafficking in sprouting capillaries fabricated using soft lithography. The leukocytes passing with whole blood through existing capillaries were able to enter microfabricated capillary sprouts of variable length and sprouting angle due to the mechanical interaction with red blood cells (RBCs) at the sprouting bifurcation, in spite of the complete absence of blood flow through the blind-ended sprouts or any chemoattractants. The RBCs formed "comet tails" (the densely packed cellular trains forming behind leukocytes as they move through narrow capillaries) and effectively pushed leukocytes into the microfabricated sprouts while bypassing them at the sprouting bifurcation. Individual sprouts filled with several leukocytes, as wells as RBCs and platelets, were observed. The results of this study suggest that (i) blood cells are likely present in capillary sprouts throughout their development, (ii) leukocytes and other circulating cells may use this mechanism to home to capillary sprouts intraluminally for direct engraftment, and (iii) tissues may use this phenomenon as another mechanism for local recruitment of leukocytes from the blood stream.

  5. The Prognostic Values of Leukocyte Rho Kinase Activity in Acute Ischemic Stroke

    PubMed Central

    Cheng, Cheng-I.; Lin, Yu-Chun; Tsai, Tzu-Hsien; Lin, Hung-Sheng; Liou, Chia-Wei; Chang, Wen-Neng; Lu, Cheng-Hsien; Yuen, Chun-Man; Yip, Hon-Kan

    2014-01-01

    Objective. It has been reported that leukocyte ROCK activity is elevated in patients after ischemic stroke, but it is unclear whether leukocyte ROCK activity is associated with clinical outcomes following acute stroke events. The objective of this study is to investigate if leukocyte ROCK activity can predict the outcomes in patients with acute ischemic stroke. Materials and Methods. We enrolled 110 patients of acute ischemic stroke and measured the leukocyte ROCK activity and plasma level of inflammatory cytokines to correlate the clinical outcomes of these patients. Results. The leukocyte ROCK activity at 48 hours after admission in acute ischemic stroke patients was higher as compared to a risk-matched population. The leukocyte ROCK activity significantly correlated with National Institute of Health Stroke Scale (NIHSS) difference between admission and 90 days after stroke event. Kaplan-Meier survival estimates showed lower stroke-free survival during follow-up period in patients with high leukocyte ROCK activity or plasma hsCRP level. Leukocyte ROCK activity independently predicted the recurrent stroke in patients with atherosclerotic stroke. Conclusions. This study shows elevated leukocyte ROCK activity in patients with ischemic stroke as compared to risk-matched subjects and is an independent predictor for recurrent stroke. PMID:24716192

  6. How leukocytes trigger opening and sealing of gaps in the endothelial barrier

    PubMed Central

    Goswami, Debashree; Vestweber, Dietmar

    2016-01-01

    The entry of leukocytes into tissues requires well-coordinated interactions between the immune cells and endothelial cells which form the inner lining of blood vessels. The molecular basis for recognition, capture, and adhesion of leukocytes to the endothelial apical surface is well studied. This review will focus on recent advances in our understanding of events following the firm interaction of leukocytes with the inner surface of the blood vessel wall. We will discuss how leukocytes initiate the transmigration (diapedesis) process, trigger the opening of gaps in the endothelial barrier, and eventually move through this boundary. PMID:27703663

  7. Fcγ Receptor Heterogeneity in Leukocyte Functional Responses

    PubMed Central

    Rosales, Carlos

    2017-01-01

    Antibodies participate in defense of the organism from all types of pathogens, including viruses, bacteria, fungi, and protozoa. IgG antibodies recognize their associated antigen via their two Fab portions and are in turn recognized though their Fc portion by specific Fcγ receptors (FcγRs) on the membrane of immune cells. Multiple types and polymorphic variants of FcγR exist. These receptors are expressed in many cells types and are also redundant in inducing cell responses. Crosslinking of FcγR on the surface of leukocytes activates several effector functions aimed toward the destruction of pathogens and the induction of an inflammatory response. In the past few years, new evidence on how the particular IgG subclass and the glycosylation pattern of the antibody modulate the IgG–FcγR interaction has been presented. Despite these advances, our knowledge of what particular effector function is activated in a certain cell and in response to a specific type of FcγR remains very limited today. On one hand, each immune cell could be programmed to perform a particular cell function after FcγR crosslinking. On the other, each FcγR could activate a particular signaling pathway leading to a unique cell response. In this review, I describe the main types of FcγRs and our current view of how particular FcγRs activate various signaling pathways to promote unique leukocyte functions. PMID:28373871

  8. Leukocyte responses to immobilized patterns of CXCL8.

    PubMed

    Girrbach, Maria; Rink, Ina; Ladnorg, Tatjana; Azucena, Carlos; Heißler, Stefan; Haraszti, Tamás; Schepers, Ute; Schmitz, Katja

    2016-06-01

    The attachment of neutrophils to the endothelial surface and their migration towards the site of inflammation following chemokine gradients play an essential role in the innate immune response. Chemokines adhere to glycosaminoglycans on the endothelial surface to be detected by leukocytes and trigger their movement along surface- bound gradients in a process called haptotaxis. In assays to systematically study the response of leukocytes to surface-bound compounds both the spatial arrangement of the compound as well as the mode of immobilization need to be controlled. In this study microcontact printing was employed to create patterns of hydrophobic or functionalized thiols on gold-coated glass slides and CXCL8 was immobilized on the thiol coated areas using three different strategies. Human neutrophils adhered to the CXCL8-coated lines but not to the PEG-coated background. We could show that more cells adhered to CXCL8 adsorbed to hydrophobic octadecanethiol than on CXCL8 covalently bound to amino undecanethiol or CXCL8 specifically bound to immobilized heparin on aminothiol. Likewise general cell activity such as lamellipodia formation and random migration were most pronounced for CXCL8 adsorbed on a hydrophobic surface which may be attributed to the larger amounts of protein immobilized on this type of surface.

  9. Strategies that Target Leukocyte Traffic in IBD: Recent Developments

    PubMed Central

    Rivera-Nieves, Jesús

    2015-01-01

    Purpose of review We review the most recent developments regarding the targeting of molecules involved in the traffic of leukocytes for the treatment of IBD. Recent Findings We discuss the most important findings of one published phase II trial that targeted the β7 integrin (Etrolizumab), two phase II trials that targeted the α4β7 integrin ligand: Mucosal Addressin Cell Adhesion Molecule-1 (MAdCAM-1, PF-00547659), a phase II targeting the chemokine IP-10 (CXCL10) in Crohn’s and a phase II trial that targeted the sphingosine-1-phosphate receptor-1 (S1P1): ozanimod in patients with ulcerative colitis (UC). Summary Targeting molecules involved in leukocyte traffic has recently become an effective and safe strategy for the treatment of IBD. Novel approaches now not only target the integrins on the lymphocyte surface, but also its endothelial ligand: MAdCAM-1. As with vedolizumab, antibodies against MAdCAM-1 appear most effective in ulcerative colitis rather than in Crohn’s. Targeting chemokines or their receptors does not appear to have the same efficacy as those that target the most stable integrin:immunoglobulin superfamily interactions between the lymphocyte and endothelium. Preliminary results also suggest that the sphingosine-1-phosphate pathway might also be targeted therapeutically in IBD, no longer with parenterally administered antibodies but with orally administered small molecules. PMID:26398681

  10. Electromagnetic wave emitting products and "Kikoh" potentiate human leukocyte functions.

    PubMed

    Niwa, Y; Iizawa, O; Ishimoto, K; Jiang, X; Kanoh, T

    1993-09-01

    Tourmaline (electric stone, a type of granite stone), common granite stone, ceramic disks, hot spring water and human palmar energy (called "Kikoh" in Japan and China), all which emit electromagnetic radiation in the far infrared region (wavelength 4-14 microns). These materials were thus examined for effects on human leukocyte activity and on lipid peroxidation of unsaturated fatty acids. It was revealed that these materials significantly increased intracellular calcium ion concentration, phagocytosis, and generation of reactive oxygen species in neutrophils, and the blastogenetic response of lymphocytes to mitogens. Chemotactic activity by neutrophils was also enhanced by exposure to tourmaline and the palm of "Kikohshi" i.e., a person who heals professionally by the laying on of hands. Despite the increase in reactive oxygen species generated by neutrophils, lipid peroxidation from unsaturated fatty acid was markedly inhibited by these four materials. The results suggest that materials emitting electromagnetic radiation in the far infrared range, which are widely used in Japan for cosmetic, therapeutic, and preservative purposes, appear capable of potentiating leukocyte functions without promoting oxidative injury.

  11. Early and delayed indium 111 leukocyte imaging in Crohn's disease

    SciTech Connect

    Navab, F.; Boyd, C.M.; Diner, W.C.; Subramani, R.; Chan, C.

    1987-10-01

    Twenty-seven patients with Crohn's disease were studied for the presence and location of activity by both early (4 h) and delayed (18-24 h) indium 111 leukocyte imaging. The results were compared with other parameters of disease activity including Crohn's disease activity index, barium studies, and endoscopy. There was a correlation between early images and Crohn's disease activity index (r = 0.78) and between delayed images and index (r = 0.82). Based upon the corresponding Crohn's disease activity index, the sensitivity of early and delayed imaging was 81.0% and 95.2%, respectively. Specificity of early and delayed imaging was 75.0% and 87.0%, respectively. Presence of activity on the early and delayed imaging agreed with activity on barium studies and colonoscopy in approximately 80% of cases. Correlation of location of disease by leukocyte imaging and x-ray was observed in 58.9% of early scans and 55.0% of delayed scans. Correlation of the location of disease by imaging and endoscopy was observed in 71.4% of early and 75.0% of delayed studies. Because of the possibility of occurrence of false-negative results in early images, delayed imaging should always be included in evaluation of disease activity in patients with Crohn's disease who are suspected of having mild activity. Delayed imaging is not required if the early imaging study clearly shows activity.

  12. Electromagnetic wave emitting products and ``Kikoh'' potentiate human leukocyte functions

    NASA Astrophysics Data System (ADS)

    Niwa, Yukie; Iizawa, Osamu; Ishimoto, Koichi; Jiang, Xiaoxia; Kanoh, Tadashi

    1993-09-01

    Tourmaline (electric stone, a type of granite stone), common granite stone, ceramic disks, hot spring water and human palmar energy (called “Kikoh” in Japan and China), all which emit electromagnetic radiation in the far infrared region (wavelength 4 14 µm). These materials were thus examined for effects on human leukocyte activity and on lipid peroxidation of unsaturated fatty acids. It was revealed that these materials significantly increased intracellular calcium ion concentration, phagocytosis, and generation of reactive oxygen species in neutrophils, and the blastogenetic response of lymphocytes to mitogens. Chemotactic activity by neutrophils was also enhanced by exposure to tourmaline and the palm of “Kikohshi” i.e., a person who heals professionally by the laying on of hands. Despite the increase in reactive oxygen species generated by neutrophils, lipid peroxidation from unsaturated fatty acid was markedly inhibited by these four materials. The results suggest that materials emitting electromagnetic radiation in the far infrared range, which are widely used in Japan for cosmetic, therapeutic, and preservative purposes, appear capable of potentiating leukocyte functions without promoting oxidative injury.

  13. Detecting apoptosis of leukocytes in mouse lymph nodes.

    PubMed

    Gómez-Cabañas, Laura; Delgado-Martín, Cristina; López-Cotarelo, Pilar; Escribano-Diaz, Cristina; Alonso-C, Luis M; Riol-Blanco, Lorena; Rodríguez-Fernández, José Luis

    2014-05-01

    Although there are multiple methods for analyzing apoptosis in cultured cells, methodologies for analyzing apoptosis in vivo are sparse. In this protocol, we describe how to detect apoptosis of leukocytes in mouse lymph nodes (LNs) via the detection of apoptotic caspases. We have previously used this protocol to study factors that modulate dendritic cell (DC) survival in LNs; however, it can also be used to analyze other leukocytes that migrate to the LNs. DCs labeled with a fluorescent cell tracker are subcutaneously injected in the posterior footpads of mice. Once the labeled DCs reach the popliteal LN (PLN), the animals are intravenously injected with FLIVO, a permeant fluorescent reagent that selectively marks active caspases and consequently apoptotic cells. Explanted PLNs are then examined under a two-photon microscope to look for the presence of apoptotic cells among the DCs injected. The protocol requires 6-6.5 h for preparation and analysis plus an additional 34-40 h to allow apoptosis of the injected DCs in the PLN.

  14. Ethylene formation by polymorphonuclear leukocytes. Role of myeloperoxidase

    PubMed Central

    1978-01-01

    Ethylene formation from the thioethers, beta-methylthiopropionaldehyde (methional) and 2-keto-4-thiomethylbutyric acid by phagocytosing polymorphonuclear leukocytes (PMNs) was found to be largely dependent on myeloperoxidase (MPO). Conversion was less than 10% of normal when MPO-deficient PMNs were employed; formation by normal PMNs was inhibited by the peroxidase inhibitors, azide, and cyanide, and a model system consisting of MPO, H2O2, chloride (or bromide) and EDTA was found which shared many of the properties of the predominant PMN system. MPO-independent mechanisms of ethylene formation were also identified. Ethylene formation from methional by phagocytosing eosinophils and by H2O2 in the presence or absence of catalase was stimulated by azide. The presence of MPO-independent, azide-stimulable systems in the PMN preparations was suggested by the azide stimulation of ethylene formation from methional when MPO-deficient leukocytes were employed. Ethylene formation by dye-sensitized photooxidation was also demonstrated and evidence obtained for the involvement of singlet oxygen (1O2). These findings are discussed in relation to the participation of H2O2, hydroxyl radicals, the superoxide anion and 1O2 in the formation of ethylene by PMNs and by the MPO model system. PMID:212502

  15. Leukocyte subsets and neutrophil function after short-term spaceflight

    NASA Technical Reports Server (NTRS)

    Stowe, R. P.; Sams, C. F.; Mehta, S. K.; Kaur, I.; Jones, M. L.; Feeback, D. L.; Pierson, D. L.

    1999-01-01

    Changes in leukocyte subpopulations and function after spaceflight have been observed but the mechanisms underlying these changes are not well defined. This study investigated the effects of short-term spaceflight (8-15 days) on circulating leukocyte subsets, stress hormones, immunoglobulin levels, and neutrophil function. At landing, a 1.5-fold increase in neutrophils was observed compared with preflight values; lymphocytes were slightly decreased, whereas the results were variable for monocytes. No significant changes were observed in plasma levels of immunoglobulins, cortisol, or adrenocorticotropic hormone. In contrast, urinary epinephrine, norepinephrine, and cortisol were significantly elevated at landing. Band neutrophils were observed in 9 of 16 astronauts. Neutrophil chemotactic assays showed a 10-fold decrease in the optimal dose response after landing. Neutrophil adhesion to endothelial cells was increased both before and after spaceflight. At landing, the expression of MAC-1 was significantly decreased while L-selectin was significantly increased. These functional alterations may be of clinical significance on long-duration space missions.

  16. Immunosuppressive lignans isolated from Saururus chinensis.

    PubMed

    Park, Shin-Young; Lee, Sung Hak; Choi, Woo Hyuck; Koh, Eun Mi; Seo, Jee Hee; Ryu, Shi Yong; Kim, Young Sup; Kwon, Dae Young; Koh, Woo Suk

    2007-06-01

    Bioactivity-guided fractionation of Saururus chinensis (Saururaceae) using a lymphoproliferation assay led us to isolate 5 lignans (compounds 1 - 5). Compounds 1 - 5 were identified as sauchinone, (-)-saucerneol, saucerneol C, manassantin A, and manassantin B, respectively, by spectroscopic analyses. The immunosuppressive activities of the active compounds were evaluated using lymphoproliferation, mixed leukocyte response, and Th1/Th2 cytokine assays. The relative potency was in the order: manassantin A, B > (-)-saucerneol > saucerneol C > sauchinone.

  17. Fragmentation of gelatin-bound fibronectin (Fn) by inflammatory polymorphonuclear leukocytes (PMNL): A role for leukocyte elastase

    SciTech Connect

    Daudi, I.; Gudewicz, P.W.; Saba, T.M.; Cho, E.; Lewis, M. )

    1990-02-26

    Fragmentation of lung matrix Fn by proteases released from activated leukocytes sequestered in the lung has been implicated in lung vascular injury. The authors determined if Fn bound to a denatured collagen (gelatin) surface was susceptible to degradation by inflammatory PMNLs. Tissue culture wells coated with 1.5% denatured collagen (2 ml/well) prior to the addition of rat peritonal exudate cells, harvested 16 hours after i.p. sterile casein. Inflammatory PMNLs (1 {times} 10{sup 6}) stimulated with zymosan (1 mg) released 3 times more {sup 125}I-Fn into the culture media (DMEM) during a 4 hour incubation as compared to unstimulated PMNLs (2885{plus minus}95 cpm vs 1027{plus minus}82 cpm/100 ul). {sup 125}I-Fn released by stimulated PMNLs was markedly blocked by addition of a leukocyte elastase inhibitor (AAPVCK), moderately blocked by a trypsin inhibitor (TLCK), and not blocked by a thrombin inhibitor (Hirudin). Western blot analysis demonstrated fragmentation of released Fn. Thus, Fn complexed with denatured collagen is susceptible to proteolysis by stimulated inflammatory PMNLs. This may contribute to lung vascular injury with sepsis and intravascular coagulation which elicit sequestration of activated PMNLs in the lung.

  18. Leukocyte cell surface proteinases: regulation of expression, functions, and mechanisms of surface localization.

    PubMed

    Owen, Caroline A

    2008-01-01

    A number of proteinases are expressed on the surface of leukocytes including members of the serine, metallo-, and cysteine proteinase superfamilies. Some proteinases are anchored to the plasma membrane of leukocytes by a transmembrane domain or a glycosyl phosphatidyl inositol (GPI) anchor. Other proteinases bind with high affinity to classical receptors, or with lower affinity to integrins, proteoglycans, or other leukocyte surface molecules. Leukocyte surface levels of proteinases are regulated by: (1) cytokines, chemokines, bacterial products, and growth factors which stimulate synthesis and/or release of proteinases by cells; (2) the availability of surface binding sites for proteinases; and/or (3) internalization or shedding of surface-bound proteinases. The binding of proteinases to leukocyte surfaces serves many functions including: (1) concentrating the activity of proteinases to the immediate pericellular environment; (2) facilitating pro-enzyme activation; (3) increasing proteinase stability and retention in the extracellular space; (4) regulating leukocyte function by proteinases signaling through cell surface binding sites or other surface proteins; and (5) protecting proteinases from inhibition by extracellular proteinase inhibitors. There is strong evidence that membrane-associated proteinases on leukocytes play critical roles in wound healing, inflammation, extracellular matrix remodeling, fibrinolysis, and coagulation. This review will outline the biology of membrane-associated proteinases expressed by leukocytes and their roles in physiologic and pathologic processes.

  19. Ultrastructural characterization of macrophage-like mononuclear leukocytes in human astrocytic tumors.

    PubMed

    Arismendi-Morillo, Gabriel; Castellano-Ramírez, Alan; Medina, Zulamita

    2010-12-01

    The aim of this study was to describe the ultrastructural features of macrophage-like mononuclear leukocytes associated with human astrocytic tumors. Tumoral biopsies of 10 patients with a pathological diagnosis of astrocytic tumor by means of transmission electron microscopy were examined. The macrophage-like mononuclear leukocyte shows ultrastructural characteristics related with the physiologic phenotype of the alternatively activated macrophage (M2), localized principally around of tumoral vasculature and tumor milieu; classically activated macrophages (M1) in surrounding necrosis areas were observed. The presence of these two ultrastructural kinds of macrophage-like mononuclear leukocytes into different areas of the tumor denotes that cellular response of TAMs is dependent of microenvironment stimuli in different parts of a tumor. The process of transvascular emigration of monocyte/macrophage-like mononuclear leukocytes into tumor is presented. The preponderance of alternatively activated macrophage-like mononuclear leukocytes suggests disequilibrium between pro-tumoral leukocytes and anti-tumoral leukocytes. Therefore, macrophage polarization toward anti-tumoral macrophage-like mononuclear leukocytes would be a potential target for therapeutic manipulation in human astrocytic tumors.

  20. Receptor cleavage and P-selectin-dependent reduction of leukocyte adhesion in the spontaneously hypertensive rat

    PubMed Central

    Chen, Angela Y.; Ha, Jessica N.; DeLano, Frank A.; Schmid-Schönbein, Geert W.

    2012-01-01

    The SHR, a genetic model for hypertension and the metabolic syndrome, has attenuated leukocyte adhesion to the postcapillary endothelium by an unknown mechanism. Based on recent evidence of elevated levels of MMPs in plasma and on microvascular endothelium of the SHR with cleavage of several receptor types, we hypothesize that the reduced leukocyte-endothelial interaction is a result of enhanced proteolytic cleavage of P-selectin on the postcapillary endothelium and PSGL-1 on leukocytes. The attenuated rolling interactions of SHR leukocytes with the endothelium were restored by chronic treatment with a broad-spectrum MMP inhibitor (CGS) for 24 weeks. The SHR MMP levels, in plasma and mesentery, as well as the systolic blood pressure, decreased significantly with treatment. In the SHR mesentery, labeling of P-selectin in the postcapillary venules by immunohistochemistry demonstrated, on average, a 31% lower extracellular P-selectin density compared with the normotensive WKY. A significantly lower extracellular PSGL-1 density on the membranes of SHR neutrophils compared with the WKY also supported our hypothesis. In vivo stimulation of the mesenteric postcapillary venules with histamine demonstrated that the SHR had an attenuated response, as measured by leukocyte rolling velocity on the endothelium. The reduced P-selectin and PSGL-1 density, on SHR postcapillary endothelium and on SHR leukocytes, respectively, was restored significantly by chronic MMP inhibition. The impaired ability of SHR leukocytes to reduce rolling velocity upon inflammatory stimulation led to fewer firmly adhered leukocytes to the endothelium as a contributor to immune suppression. PMID:22566571

  1. Colonic localization of indium-111 labeled leukocytes in active Behcet's disease

    SciTech Connect

    Harre, R.G.; Conrad, G.R.; Seabold, J.E.

    1988-06-01

    A patient with known Behcet's disease demonstrated intense colonic localization of In-111 labeled leukocytes. Gastrointestinal involvement had not been previously manifested, but extensive colonic inflammation was documented by endoscopy. This case illustrates the utility of In-111 labeled leukocyte imaging for detecting active bowel disease in a debilitated patient with documented Behcet's vasculitis.

  2. The method of selection of leukocytes in images of preparations of peripheral blood and bone marrow

    NASA Astrophysics Data System (ADS)

    Zakharenko, Y. V.; Nikitaev, V. G.; Polyakov, E. V.; Seldyukov, S. O.

    2017-01-01

    Study of the segmentation method on the basis of histogram analysis for the selection of leukocytes in the images of blood and bone marrow in the diagnosis of acute leukemia was conducted in this paper. Method of filtering was offered to eliminate the artifacts, resulting from the selection of leukocytes.

  3. Are isolated wetlands isolated?

    USGS Publications Warehouse

    Smith, Loren M.; Euliss, Ned H.; Haukos, David A.

    2011-01-01

    While federal regulations during the past 10 years have treated isolated wetlands as unconnected to aquatic resources protected by the Clean Water Act, they provide critical ecosystem services to society that extend well beyond their wetland boundaries. The authors offer well-documented examples from the scientific literature on some of the ecosystem services provided by isolated wetlands to society and other ecosystems.

  4. Social stress up-regulates inflammatory gene expression in the leukocyte transcriptome via β-adrenergic induction of myelopoiesis

    PubMed Central

    Powell, Nicole D.; Sloan, Erica K.; Bailey, Michael T.; Arevalo, Jesusa M. G.; Miller, Gregory E.; Chen, Edith; Kobor, Michael S.; Reader, Brenda F.; Sheridan, John F.; Cole, Steven W.

    2013-01-01

    Across a variety of adverse life circumstances, such as social isolation and low socioeconomic status, mammalian immune cells have been found to show a conserved transcriptional response to adversity (CTRA) involving increased expression of proinflammatory genes. The present study examines whether such effects might stem in part from the selective up-regulation of a subpopulation of immature proinflammatory monocytes (Ly-6chigh in mice, CD16− in humans) within the circulating leukocyte pool. Transcriptome representation analyses showed relative expansion of the immature proinflammatory monocyte transcriptome in peripheral blood mononuclear cells from people subject to chronic social stress (low socioeconomic status) and mice subject to repeated social defeat. Cellular dissection of the mouse peripheral blood mononuclear cell transcriptome confirmed these results, and promoter-based bioinformatic analyses indicated increased activity of transcription factors involved in early myeloid lineage differentiation and proinflammatory effector function (PU.1, NF-κB, EGR1, MZF1, NRF2). Analysis of bone marrow hematopoiesis confirmed increased myelopoietic output of Ly-6chigh monocytes and Ly-6cintermediate granulocytes in mice subject to repeated social defeat, and these effects were blocked by pharmacologic antagonists of β-adrenoreceptors and the myelopoietic growth factor GM-CSF. These results suggest that sympathetic nervous system-induced up-regulation of myelopoiesis mediates the proinflammatory component of the leukocyte CTRA dynamic and may contribute to the increased risk of inflammation-related disease associated with adverse social conditions. PMID:24062448

  5. Local Oxidative and Nitrosative Stress Increases in the Microcirculation during Leukocytes-Endothelial Cell Interactions

    PubMed Central

    Kar, Saptarshi; Kavdia, Mahendra

    2012-01-01

    Leukocyte-endothelial cell interactions and leukocyte activation are important factors for vascular diseases including nephropathy, retinopathy and angiopathy. In addition, endothelial cell dysfunction is reported in vascular disease condition. Endothelial dysfunction is characterized by increased superoxide (O2•−) production from endothelium and reduction in NO bioavailability. Experimental studies have suggested a possible role for leukocyte-endothelial cell interaction in the vessel NO and peroxynitrite levels and their role in vascular disorders in the arterial side of microcirculation. However, anti-adhesion therapies for preventing leukocyte-endothelial cell interaction related vascular disorders showed limited success. The endothelial dysfunction related changes in vessel NO and peroxynitrite levels, leukocyte-endothelial cell interaction and leukocyte activation are not completely understood in vascular disorders. The objective of this study was to investigate the role of endothelial dysfunction extent, leukocyte-endothelial interaction, leukocyte activation and superoxide dismutase therapy on the transport and interactions of NO, O2•− and peroxynitrite in the microcirculation. We developed a biotransport model of NO, O2•− and peroxynitrite in the arteriolar microcirculation and incorporated leukocytes-endothelial cell interactions. The concentration profiles of NO, O2•− and peroxynitrite within blood vessel and leukocytes are presented at multiple levels of endothelial oxidative stress with leukocyte activation and increased superoxide dismutase accounted for in certain cases. The results showed that the maximum concentrations of NO decreased ∼0.6 fold, O2•− increased ∼27 fold and peroxynitrite increased ∼30 fold in the endothelial and smooth muscle region in severe oxidative stress condition as compared to that of normal physiologic conditions. The results show that the onset of endothelial oxidative stress can cause an increase

  6. Role of in-111 labeled leukocyte scintigraphy in the diagnosis of intracerebral lesions

    SciTech Connect

    Palestro, C.J.; Swyer, A.J.; Kim, C.K.; Muzinic, M.; Goldsmith, S.J. )

    1991-05-01

    The differential diagnosis of intracerebral enhancing lesions on contrast computed tomography includes tumors, abscesses, and cerebrovascular accidents. Particularly important is the differentiation between tumor and abscess. While In-111 labeled leukocyte imaging is an accurate test for identification of foci of infection in general, the role of this procedure in the evaluation of the intracranial lesion is not well established. We undertook a retrospective review of 16 patients with contrast enhancing intracerebral lesions identified on computed tomography, who were also studied with labeled leukocyte imaging. Final diagnoses were: abscess (n = 2), primary brain tumor (n = 6), metastasis (n = 4), dermoid cyst (n = 1), and cerebral infarct (n = 3). There were two positive labeled leukocyte studies; both were cerebral abscesses. No labeled leukocyte activity was identified in any of the tumors or infarcts. We conclude that In-111 labeled leukocyte imaging is an accurate method of differentiating infectious from noninfectious causes of intracerebral lesions identified on computed tomography.

  7. Tc-99m labeled leukocytes: preparation and use in identification of abscess and tissue rejection

    SciTech Connect

    Farid, N.A.; White, S.M.; Heck, L.L.; Van Hove, E.D.

    1983-09-01

    A simple and reproducible method for the preparation and labeling of leukocytes with Tc-99m has been developed. Leukocytes were separated from blood, incubated with stannous pyrophosphate, and then with 20-30 mCi (740-1110 MBq) of /sup 99m/TcO-4. In leukocytes separated from human blood, the labeling efficiency was 81% +/- 6% (n . 4). Experiments on dogs with abscesses showed accumulation of the Tc-99m-labeled leukocytes in the infected sites, indicating the viability of the labeled leukocytes. Additional studies showed that rat lymphocytes that were labeled with Tc-99m, using the same technique, localized in heart transplant tissue that was being rejected.

  8. Tc-99m labeled leukocytes: preparation and use in identification of abscess and tissue rejection

    SciTech Connect

    Farid, N.A.; White, S.M.; Heck, L.L.; Van Hove, E.D.

    1983-09-01

    A simple and reproducible method for the preparation and labeling of leukocytes with Tc-99m, has been developed. Leukocytes were separated from blood, incubated with stannous pyrophosphate, and then with 20-30 mCi (740-1110 M Bq) of /sup 99m/TcO/sub 4//sup -/. In leukocytes separated from human blood, the labeling efficiency was 81% +/- 6% (n=4). Experiments on dogs with abscesses showed accumulation of the Tc-99m-labeled leukocytes in the infected sites, indicating the viability of the labeled leukocytes. Additional studies showed that rat lymphocytes that were labeled with Tc-99m, using the same technique, localized in heart transplant tissue that was being rejected.

  9. Four-part leukocyte differential count based on sheathless microflow cytometer and fluorescent dye assay.

    PubMed

    Shi, Wendian; Guo, Luke; Kasdan, Harvey; Tai, Yu-Chong

    2013-04-07

    Leukocyte differential count is one of the most frequently ordered clinical tests in hospitals. This paper reports a point-of-care test for the leukocyte count by using a microflow cytometer and a fluorescent dye assay. The dye assay relied on fluorescent detection alone to count leukocytes in blood and to identify leukocyte subtypes. By combining the fluorescent assay with a sheathless microflow design, the proposed method achieved a minimal sample volume by eliminating excessive dilution and sheath flow. In this paper, a four-part leukocyte differential count including lymphocyte, monocyte, neutrophil and eosinophil was demonstrated, and the whole test consumed only a small amount of blood (5 μL) and reagents (68 μL in total). The merits of minimal sample volume, long reagent shelf life and portable instrument made this method optimal for point-of-care applications.

  10. N-Formylmethionyl Peptide Receptors on Equine Leukocytes Initiate Secretion but not Chemotaxis

    NASA Astrophysics Data System (ADS)

    Snyderman, Ralph; Pike, Marilyn C.

    1980-07-01

    The chemotaxis of leukocytes appears to be initiated by the binding of chemotactic factors to the surface of these cells. N-Formylated peptides induce chemotaxis and lysosomal enzyme secretion of leukocytes; because these peptides are available in a purified radiolabeled form, they have been useful in the characterization of receptors for chemotactic factors. Equine polymorphonuclear leukocytes secrete lysosomal enzymes but do not exhibit chemotaxis in response to the N-formylated peptides, even though they have a high-affinity cell surface receptor for these agents. The specificity of the equine receptor resembles the specificity of the receptor on chemotactically responsive leukocytes from other species. Equine polymorphonuclear leukocytes may thus be an excellent model for the study of the events that lead to a biological response following receptor occupancy.

  11. Inhibition of leukocyte-type 12-lipoxygenase by guava tea leaves prevents development of atherosclerosis.

    PubMed

    Takahashi, Yoshitaka; Otsuki, Akemi; Mori, Yoshiko; Kawakami, Yuki; Ito, Hideyuki

    2015-11-01

    Oxidation of low-density lipoprotein (LDL) is one of the crucial steps for atherosclerosis development, and an essential role of leukocyte-type 12-lipoxygenase expressed in macrophages in this process has been demonstrated. The biochemical mechanism of the oxidation of circulating LDL by leukocyte-type 12-lipoxygenase in macrophages has been proposed. The major ingredients in guava tea leaves which inhibited the catalytic activity of leukocyte-type 12-lipoxygenase were quercetin and ethyl gallate. Administration of extracts from guava tea leaves to apoE-deficient mice significantly attenuated atherogenic lesions in the aorta and aortic sinus. We recently showed that Qing Shan Lu Shui inhibited the catalytic activity of leukocyte-type 12-lipoxygenase. The major components inhibiting the enzyme contained in Qing Shan Lu Shui were identified to be novel monoterpene glycosides. The anti-atherogenic effect of the tea leaves might be attributed to the inhibition of leukocyte-type 12-lipoxygenase by these components.

  12. Human Leukocyte Antigen Diversity: A Southern African Perspective

    PubMed Central

    Tshabalala, Mqondisi; Mellet, Juanita; Pepper, Michael S.

    2015-01-01

    Despite the increasingly well-documented evidence of high genetic, ethnic, and linguistic diversity amongst African populations, there is limited data on human leukocyte antigen (HLA) diversity in these populations. HLA is part of the host defense mechanism mediated through antigen presentation to effector cells of the immune system. With the high disease burden in southern Africa, HLA diversity data is increasingly important in the design of population-specific vaccines and the improvement of transplantation therapeutic interventions. This review highlights the paucity of HLA diversity data amongst southern African populations and defines a need for information of this kind. This information will support disease association studies, provide guidance in vaccine design, and improve transplantation outcomes. PMID:26347896

  13. Nerve growth factor: stimulation of polymorphonuclear leukocyte chemotaxis in vitro.

    PubMed Central

    Gee, A P; Boyle, M D; Munger, K L; Lawman, M J; Young, M

    1983-01-01

    Topical application of mouse nerve growth factor (NGF) to superficial skin wounds of mice has previously been shown to accelerate the rate of wound contraction. Results of the present study reveal that NGF in the presence of plasma is also chemotactic for human polymorphonuclear leukocytes in vitro, and the concentration of NGF required for this effect is similar to that which stimulates ganglionic neurite outgrowth. This property does not arise from liberation of the C5a fragment of complement, nor does it require the known enzymic activity of NGF. (NGF inactivated with diisopropyl fluorophosphate is equally active.) We conclude that NGF can display biological effects on cells of nonneural origin and function, and this feature might play a role in the early inflammatory response to injury. PMID:6580641

  14. Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial.

    PubMed

    Madurantakam, Parthasarathy; Yoganarasimha, Suyog; Hasan, Fadi K

    2015-09-29

    Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.

  15. Chronic stress, leukocyte subpopulations, and humoral response to latent viruses

    SciTech Connect

    McKinnon, W.; Weisse, C.S.; Reynolds, C.P.; Bowles, C.A.; Baum, A. )

    1989-01-01

    Psychological stress has been shown to affect immune system status and function, but most studies of this relationship have focused on acute stress and/or laboratory situations. The present study compared total numbers of leukocytes and lymphocyte subpopulations (determined by flow cytometry) and antibody titers to latent and nonlatent viruses among a group of chronically stressed individuals living near the damaged Three Mile Island (TMI) nuclear power plant with those of a demographically comparable control group. Urinary catecholamine and cortisol levels were also examined. Residents of the TMI area exhibited greater numbers of neutrophils, which were positively correlated with epinephrine levels. The TMI group also exhibited fewer B lymphocytes, T-suppressor/cytotoxic lymphocytes, and natural killer cells. Antibody titers to herpes simplex were significantly different across groups as well, whereas titers to nonlatent rubella virus as well as IgG and IgM levels were comparable.

  16. Flow cytofluorometric monitoring of leukocyte apoptosis in experimental cholera

    NASA Astrophysics Data System (ADS)

    Lotsmanova, Ekaterina Y.; Kravtsov, Alexander L.; Livanova, Ludmila F.; Kobkova, Irina M.; Kuznetsov, Oleg S.; Shchukovskaya, Tatyana N.; Smirnova, Nina I.; Kutyrev, Vladimir V.

    2003-10-01

    Flow cytofluorometric DNA analysis was applied to determine of the relative contents of proliferative (more then 2C DNA per cell) and apoptotic (less then 2C DNA per cell) leukocytes in blood of adult rabbits, challenged with 10,000 times the 50 % effective dose of Vibrio cholerae virulent strain by the RITARD technique. It has been shown that irreversible increase the percentage of cells carrying DNA in the degradation stage brings to disbalance between the genetically controlled cell proliferation and apoptosis that leads to animal death from the cholera infection. Such fatal changes were not observed in challenging of immunized animals that were not died. Thus received data show that the flow cytofluorometric measurements may be used for detection of transgressions in homeostasis during acute infection diseases, for outlet prognosis of the cholera infection.

  17. Automated leukocyte processing by microfluidic deterministic lateral displacement.

    PubMed

    Civin, Curt I; Ward, Tony; Skelley, Alison M; Gandhi, Khushroo; Peilun Lee, Zendra; Dosier, Christopher R; D'Silva, Joseph L; Chen, Yu; Kim, MinJung; Moynihan, James; Chen, Xiaochun; Aurich, Lee; Gulnik, Sergei; Brittain, George C; Recktenwald, Diether J; Austin, Robert H; Sturm, James C

    2016-12-01

    We previously developed a Deterministic Lateral Displacement (DLD) microfluidic method in silicon to separate cells of various sizes from blood (Davis et al., Proc Natl Acad Sci 2006;103:14779-14784; Huang et al., Science 2004;304:987-990). Here, we present the reduction-to-practice of this technology with a commercially produced, high precision plastic microfluidic chip-based device designed for automated preparation of human leukocytes (white blood cells; WBCs) for flow cytometry, without centrifugation or manual handling of samples. After a human blood sample was incubated with fluorochrome-conjugated monoclonal antibodies (mAbs), the mixture was input to a DLD microfluidic chip (microchip) where it was driven through a micropost array designed to deflect WBCs via DLD on the basis of cell size from the Input flow stream into a buffer stream, thus separating WBCs and any larger cells from smaller cells and particles and washing them simultaneously. We developed a microfluidic cell processing protocol that recovered 88% (average) of input WBCs and removed 99.985% (average) of Input erythrocytes (red blood cells) and >99% of unbound mAb in 18 min (average). Flow cytometric evaluation of the microchip Product, with no further processing, lysis or centrifugation, revealed excellent forward and side light scattering and fluorescence characteristics of immunolabeled WBCs. These results indicate that cost-effective plastic DLD microchips can speed and automate leukocyte processing for high quality flow cytometry analysis, and suggest their utility for multiple other research and clinical applications involving enrichment or depletion of common or rare cell types from blood or tissue samples. © 2016 International Society for Advancement of Cytometry.

  18. Cytoplasmic sequestration of p53 promotes survival in leukocytes transformed by Theileria.

    PubMed

    Haller, D; Mackiewicz, M; Gerber, S; Beyer, D; Kullmann, B; Schneider, I; Ahmed, J S; Seitzer, U

    2010-05-27

    The function of the p53 protein as the central effector molecule of the p53 apoptotic pathway was investigated in a reversible model of epigenetic transformation. The infection of bovine leukocytes by the intracellular protozoan parasite Theileria annulata results in parasite-dependent transformation and proliferation of the host cells. We found p53 to be largely localized in the host cell cytoplasm and associated with the parasite membrane of isolated schizonts. Curing infected cells of the parasite with the theilericidal drug buparvaquone resulted in a time-dependent translocation of p53 into the host cell nucleus and the upregulation of the proapoptotic Bax and Apaf-1 and the downregulation of the anti-apoptotic Bcl-2 proteins. Although buparvaquone treatment led to apoptosis of the host cell, inhibition of either p53 or Bax significantly reduced buparvaquone-induced apoptosis of the transformed cells. Thus, the p53 apoptotic pathway of host cells is not induced by infection and transformation with Theileria by a mechanism involving cytoplasmic sequestration of p53. The close association of host cell p53 with the parasite membrane implies that the parasite either interacts directly with p53 or mediates cytoplasmic sequestration of p53 by interacting with other host cell proteins regulating p53 localization.

  19. Smooth muscle cell phenotype alters cocultured endothelial cell response to biomaterial-pretreated leukocytes.

    PubMed

    Rose, Stacey L; Babensee, Julia E

    2008-03-01

    Model in vitro culturing systems were developed to analyze roles of biomaterial-induced leukocyte activation on endothelial cell (EC) and smooth muscle cell (SMC) phenotype, and their crosstalk. Isolated monocytes or neutrophils were pretreated with model biomaterial beads and applied directly to "more secretory" (cultured in media containing 5% fetal bovine serum) or forced contractile (serum and growth factor starved) human aortic SMCs (HASMCs), or to the human aortic EC (HAEC) surface of HAEC/HASMC cocultures (HASMC phenotype varied to be "more or less secretory") for 5 or 24 h of static culture. Surface expression of proinflammatory [ICAM-1, VCAM-1, E-selectin], procoagulant (tissue factor), and anticoagulant (thrombomodulin) markers, as well as HAEC proliferation, were assessed by flow cytometry. Incubation of HAEC with biomaterial-pretreated monocytes (and neutrophils to lesser degree) suppressed HAEC proliferation and induced a proinflammatory/procoagulant HAEC phenotype. This HAEC phenotype was amplified in coculture with "more secretory" HASMCs and subdued in coculture with "less secretory" HASMCs. Direct incubation of biomaterial-pretreated monocytes or neutrophils with "more secretory" HASMCs further increased HASMC ICAM-1 and tissue factor expression. Direct incubation of biomaterial-pretreated monocytes or neutrophils with forced contractile HASMCs upregulated ICAM-1, VCAM-1, and tissue factor expression above the presence of serum-containing media alone.

  20. LINE-1 Methylation Levels in Leukocyte DNA and Risk of Renal Cell Cancer

    PubMed Central

    Liao, Linda M.; Brennan, Paul; van Bemmel, Dana M.; Zaridze, David; Matveev, Vsevolod; Janout, Vladimir; Kollarova, Hellena; Bencko, Vladimir; Navratilova, Marie; Szeszenia-Dabrowska, Neonila; Mates, Dana; Rothman, Nathaniel; Boffetta, Paolo; Chow, Wong-Ho; Moore, Lee E.

    2011-01-01

    Purpose Leukocyte global DNA methylation levels are currently being considered as biomarkers of cancer susceptibility and have been associated with risk of several cancers. In this study, we aimed to examine the association between long interspersed nuclear elements (LINE-1) methylation levels, as a biomarker of global DNA methylation in blood cell DNA, and renal cell cancer risk. Experimental Design LINE-1 methylation of bisulfite-converted genomic DNA isolated from leukocytes was quantified by pyrosequencing measured in triplicate, and averaged across 4 CpG sites. A total of 328 RCC cases and 654 controls frequency-matched(2∶1) on age(±5years), sex and study center, from a large case-control study conducted in Central and Eastern Europe were evaluated. Results LINE-1 methylation levels were significantly higher in RCC cases with a median of 81.97% (interquartile range[IQR]: 80.84–83.47) compared to 81.67% (IQR: 80.35–83.03) among controls (p = 0.003, Wilcoxon). Compared to the lowest LINE-1 methylation quartile(Q1), the adjusted ORs for increasing methylation quartiles were as follows: OR(Q2) = 1.84(1.20−2.81), OR(Q3) = 1.72(1.11−2.65) and OR(Q4) = 2.06(1.34−3.17), with a p-trend = 0.004. The association was stronger among current smokers (p-trend<0.001) than former or never smokers (p-interaction = 0.03). To eliminate the possibility of selection bias among controls, the relationship between LINE-1 methylation and smoking was evaluated and confirmed in a case-only analysis, as well. Conclusions Higher levels of LINE-1 methylation appear to be positively associated with RCC risk, particularly among current smokers. Further investigations using both post- and pre-diagnostic genomic DNA is warranted to confirm findings and will be necessary to determine whether the observed differences occur prior to, or as a result of carcinogenesis. PMID:22076155

  1. Blood and intrauterine leukocyte profile and function in dairy cows that spontaneously recovered from postpartum endometritis.

    PubMed

    Mateus, Luísa; Lopes da Costa, L; Carvalho, H; Serra, P; Robalo Silva, J

    2002-06-01

    The profile and function of blood and uterine leukocytes were evaluated in 14 dairy cows that spontaneously recovered from postpartum endometritis (mild, n=6 and heavy, n=8; general health not affected). From a minimum of 2 weeks before parturition until 6 weeks postpartum, blood samples were obtained twice weekly for leukocyte counts and leukogram determination and once weekly for flow cytometry assessment of polimorphonuclear neutrophils (PMN) phagocytic capacity and oxidative burst activity. Uterine fluid-stained smears, obtained twice weekly from parturition until fluid was present in the uterus, were used for determination of the percentage of PMN, of phagocytizing PMN (phago-PMN) and of the mean number of phagocyted bacteria per phagocytizing PMN (phagocytic index; PI). Uterine swabs were obtained twice weekly from parturition until 35 days postpartum for bacteriological examination. The time of endometritis diagnosis was similar in cows with mild or heavy endometritis but the latter cows had a significantly longer persistence of the infection and of the isolation of Gram-negative anaerobes from the uterus. However, the effect of group (mild versus heavy) was not significant for all the blood and uterine parameters analysed. The effect of sampling day (within group effect) was significant (p<0.01 to p<0.00001) for all parameters, except for the blood monocyte count and the blood PMN phagocytic capacity, in which only a tendency for significance was observed (p<0.1). The effect of the interaction group x sampling day was significant only for the blood monocyte count. The phago-PMN and the PI were significantly correlated (r=0.70, p<0.001). A significant correlation was also observed between the uterine fluid phago-PMN and the blood PMN oxidative burst activity (r=-0.41, p<0.05). At the spontaneous recovery, the blood PMN oxidative burst activity was significantly higher (p<0.05) and the percentage of intrauterine phago-PMN and the PI were significantly lower

  2. Transcript origin analysis identifies antigen-presenting cells as primary targets of socially regulated gene expression in leukocytes

    PubMed Central

    Cole, Steven W.; Hawkley, Louise C.; Arevalo, Jesusa M. G.; Cacioppo, John T.

    2011-01-01

    To clarify the biological rationale for social regulation of gene expression, this study sought to identify the specific immune cell types that are transcriptionally sensitive to subjective social isolation (loneliness). Using reference distributions for the expression of each human gene in each major leukocyte subtype, we mapped the cellular origin of transcripts found to be differentially expressed in the circulating immune cells from chronically lonely individuals. Loneliness-associated genes derived primarily from plasmacytoid dendritic cells, monocytes, and, to a lesser extent, B lymphocytes. Those dynamics reflected per-cell changes in the expression of inducible genes and related more strongly to the subjective experience of loneliness than to objective social network size. Evolutionarily ancient myeloid antigen-presenting cells appear to have evolved a transcriptional sensitivity to socioenvironmental conditions that may allow them to shift basal gene expression profiles to counter the changing microbial threats associated with hostile vs. affine social conditions. PMID:21300872

  3. In vivo and in vitro effects of dexamethasone on leukocyte migration in the rat adjuvant arthritis model

    SciTech Connect

    Thieme, T.R.; Mirkovich, A.; Maloney, P.; Goodwin, D.A.

    1982-12-01

    When polymorphonuclear leukocytes (PMNs) and mononuclear cells were isolated from the blood of dexamethasone-treated normal rats, in vitro mononuclear cell migration was inhibited and PMN migration was stimulated in comparison to controls. Inflammogen-induced PMNs showed inhibited cell migration due to dexamethasone treatment. Gamma camera imaging was then used to detect cells in vivo after labeling with /sup 111/In. When the dexamethasone-treated blood cells were injected into adjuvant arthritis diseased rats, mononuclear cells showed depressed migration into the inflamed paws, while PMNs showed stimulated migration into the inflamed paws in comparison to controls. When the recipient adjuvant arthritic animals were treated with dexamethasone, both normal mononuclear cell and normal PMN migration to the inflamed paws were inhibited.

  4. A multiscale SPH particle model of the near-wall dynamics of leukocytes in flow.

    PubMed

    Gholami, Babak; Comerford, Andrew; Ellero, Marco

    2014-01-01

    A novel multiscale Lagrangian particle solver based on SPH is developed with the intended application of leukocyte transport in large arteries. In such arteries, the transport of leukocytes and red blood cells can be divided into two distinct regions: the bulk flow and the near-wall region. In the bulk flow, the transport can be modeled on a continuum basis as the transport of passive scalar concentrations. Whereas in the near-wall region, specific particle tracking of the leukocytes is required and lubrication forces need to be separately taken into account. Because of large separation of spatio-temporal scales involved in the problem, simulations of red blood cells and leukocytes are handled separately. In order to take the exchange of leukocytes between the bulk fluid and the near-wall region into account, solutions are communicated through coupling of conserved quantities at the interface between these regions. Because the particle tracking is limited to those leukocytes lying in the near-wall region only, our approach brings considerable speedup to the simulation of leukocyte circulation in a test geometry of a backward-facing step, which encompasses many flow features observed in vivo.

  5. Single Cell Analysis of Leukocyte Protease Activity Using Integrated Continuous-Flow Microfluidics.

    PubMed

    Jing, Tengyang; Lai, Zhangxing; Wu, Lidan; Han, Jongyoon; Lim, Chwee Teck; Chen, Chia-Hung

    2016-12-06

    Leukocytes are the essential cells of the immune system that protect the human body against bacteria, viruses, and other foreign invaders. Secretory products of individual leukocytes, such as matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase (ADAMs), are critical for regulating the inflammatory response and mediating host defense. Conventional single cell analytical methods, such as flow cytometry for cellular surface biomarker studies, are insufficient for performing functional assays of the protease activity of individual leukocytes. Here, an integrated continuous-flow microfluidic assay is developed to effectively detect secretory protease activity of individual viable leukocytes. Leukocytes in blood are first washed on-chip with defined buffer to remove background activity, followed by encapsulating individual leukocytes with protease sensors in water-in-oil droplets and incubating for 1 h to measure protease secretion. With this design, single leukocyte protease profiles under naive and phorbol 12-myristate 13-acetate (PMA)-stimulated conditions are reliably measured. It is found that PMA treatment not only elevates the average protease activity level but also reduces the cellular heterogeneity in protease secretion, which is important in understanding immune capability and the disease condition of individual patients.

  6. Distribution of leukocyte subtypes in the sheep ovary after laser drilling.

    PubMed

    Tozawa, H; Brännström, M; Petrucco, O; Walker, S; Chambers, H; Pascoe, V; Norman, R J

    1995-03-01

    The distribution of leukocyte subtypes in the sheep ovary following laser drilling of the ovarian capsule was examined to understand a possible mechanism by which this treatment promotes ovulation in polycystic ovarian syndrome. Ovaries were removed from sheep at different time-points following laparoscopic laser drilling and immunohistochemical detection of leukocyte subtypes, using specific monoclonal antibodies; standard histological staining was performed. Migration of leukocytes into the laser-drilled site was observed as early as 6 h after laser drilling and the total number of leukocytes in the site was found to increase up to the 12th day after surgery. In the earlier period, polymorphonuclear leukocytes were the dominant leukocyte subtypes, while macrophages and lymphocytes were the major cellular components on the 12th day and later. These results show that the tissue changes in the ovary following laser drilling are consistent with a local inflammatory reaction. The prolonged appearance of numerous macrophages following the acute inflammatory phase could lead to the secretion of cytokines and other substances suggested to be important in promoting ovulation. These data indicate that part of the effectiveness of the laser drilling in polycystic ovarian syndrome may be attributable to the secretory products of these leukocytes.

  7. A high-throughput microfluidic approach for 1000-fold leukocyte reduction of platelet-rich plasma

    PubMed Central

    Xia, Hui; Strachan, Briony C.; Gifford, Sean C.; Shevkoplyas, Sergey S.

    2016-01-01

    Leukocyte reduction of donated blood products substantially reduces the risk of a number of transfusion-related complications. Current ‘leukoreduction’ filters operate by trapping leukocytes within specialized filtration material, while allowing desired blood components to pass through. However, the continuous release of inflammatory cytokines from the retained leukocytes, as well as the potential for platelet activation and clogging, are significant drawbacks of conventional ‘dead end’ filtration. To address these limitations, here we demonstrate our newly-developed ‘controlled incremental filtration’ (CIF) approach to perform high-throughput microfluidic removal of leukocytes from platelet-rich plasma (PRP) in a continuous flow regime. Leukocytes are separated from platelets within the PRP by progressively syphoning clarified PRP away from the concentrated leukocyte flowstream. Filtrate PRP collected from an optimally-designed CIF device typically showed a ~1000-fold (i.e. 99.9%) reduction in leukocyte concentration, while recovering >80% of the original platelets, at volumetric throughputs of ~1 mL/min. These results suggest that the CIF approach will enable users in many fields to now apply the advantages of microfluidic devices to particle separation, even for applications requiring macroscale flowrates. PMID:27775049

  8. Effect of leukocyte filtration on the P-selectin expression of apheresis platelets.

    PubMed

    Xie, Z T; Chen, C; Zhang, S H; Yang, H M; Tao, Z H

    2015-06-01

    The aim of this study was to investigate the effect of leukocyte filtration on the P-selectin (CD62P) surface expression of apheresis platelets during the retention period. Ten bags of apheresis platelets stored for 1 day (0-24 h) and 10 bags of apheresis platelets stored for 2 days (24-48 h) were used for leukocyte filtration (experimental group). Ten bags of apheresis platelets with the corresponding retention periods but without filtration were used as a negative control (control group). Thereafter, 100 μL of platelet suspensions from apheresis platelets with or without leukocyte filtration were sampled before and after leukocyte filtration for the detection of CD62P surface expression by flow cytometry. No statistical difference in the CD62P surface expression of apheresis platelets was observed before and after leukocyte filtration (P > 0.05), neither did the CD62P surface expression exhibit any change among the different retention periods. Leukocyte filtration does not affect the CD62P surface expression of apheresis platelets stored for up to 2 days, which indicates that leukocyte filtration does not damage the activation of apheresis platelets within the retention period.

  9. Exercise and leukocyte interchange among central circulation, lung, spleen, and muscle ☆

    PubMed Central

    Adams, Gregory R.; Zaldivar, Frank P.; Nance, Dwight M.; Kodesh, Einat; Radom-Aizik, Shlomit; Cooper, Dan M.

    2015-01-01

    Circulating leukocytes increase rapidly with exercise then quickly decrease when the exercise ends. We tested whether exercise acutely led to bidirectional interchange of leukocytes between the circulation and the lung, spleen, and active skeletal muscle. To accomplish this it was necessary to label a large number of immune cells (granulocytes, monocytes, and lymphocytes) in a way that resulted in minimal perturbation of cell function. Rats were injected intravenously with a single bolus of carboxyfluorescein diacetate succinamidyl ester (CFSE) dye which is rapidly and irreversibly taken up by circulating cells. The time course of the disappearance of labeled cells and their reappearance in the circulation following exercise was determined via flow cytometry. The majority of circulating leukocytes were labeled at 4 h. post-injection and this proportion slowly declined out to 120 h. At both 24 and 120 h, running resulted in an increase in the proportion of labeled leukocytes in the circulation. Analysis of the skeletal muscle, spleen and lung indicated that labeled leukocytes had accumulated in those tissues and were mobilized to the circulation in response to exercise. This indicates that there is an ongoing exchange of leukocytes between the circulation and tissues and that exercise can stimulate their redistribution. Exchange was slower with muscle than with spleen and lung, but in all cases, influenced by exercise. Exercise bouts redistribute leukocytes between the circulation and the lung, spleen and muscle. The modulatory effects of exercise on the immune system may be regulated in part by the systemic redistribution of immune cells. PMID:21238578

  10. A high-throughput microfluidic approach for 1000-fold leukocyte reduction of platelet-rich plasma.

    PubMed

    Xia, Hui; Strachan, Briony C; Gifford, Sean C; Shevkoplyas, Sergey S

    2016-10-24

    Leukocyte reduction of donated blood products substantially reduces the risk of a number of transfusion-related complications. Current 'leukoreduction' filters operate by trapping leukocytes within specialized filtration material, while allowing desired blood components to pass through. However, the continuous release of inflammatory cytokines from the retained leukocytes, as well as the potential for platelet activation and clogging, are significant drawbacks of conventional 'dead end' filtration. To address these limitations, here we demonstrate our newly-developed 'controlled incremental filtration' (CIF) approach to perform high-throughput microfluidic removal of leukocytes from platelet-rich plasma (PRP) in a continuous flow regime. Leukocytes are separated from platelets within the PRP by progressively syphoning clarified PRP away from the concentrated leukocyte flowstream. Filtrate PRP collected from an optimally-designed CIF device typically showed a ~1000-fold (i.e. 99.9%) reduction in leukocyte concentration, while recovering >80% of the original platelets, at volumetric throughputs of ~1 mL/min. These results suggest that the CIF approach will enable users in many fields to now apply the advantages of microfluidic devices to particle separation, even for applications requiring macroscale flowrates.

  11. A high-throughput microfluidic approach for 1000-fold leukocyte reduction of platelet-rich plasma

    NASA Astrophysics Data System (ADS)

    Xia, Hui; Strachan, Briony C.; Gifford, Sean C.; Shevkoplyas, Sergey S.

    2016-10-01

    Leukocyte reduction of donated blood products substantially reduces the risk of a number of transfusion-related complications. Current ‘leukoreduction’ filters operate by trapping leukocytes within specialized filtration material, while allowing desired blood components to pass through. However, the continuous release of inflammatory cytokines from the retained leukocytes, as well as the potential for platelet activation and clogging, are significant drawbacks of conventional ‘dead end’ filtration. To address these limitations, here we demonstrate our newly-developed ‘controlled incremental filtration’ (CIF) approach to perform high-throughput microfluidic removal of leukocytes from platelet-rich plasma (PRP) in a continuous flow regime. Leukocytes are separated from platelets within the PRP by progressively syphoning clarified PRP away from the concentrated leukocyte flowstream. Filtrate PRP collected from an optimally-designed CIF device typically showed a ~1000-fold (i.e. 99.9%) reduction in leukocyte concentration, while recovering >80% of the original platelets, at volumetric throughputs of ~1 mL/min. These results suggest that the CIF approach will enable users in many fields to now apply the advantages of microfluidic devices to particle separation, even for applications requiring macroscale flowrates.

  12. Attenuation of leukocyte adhesion by recombinant TNF-binding protein after hemorrhagic shock in the rat.

    PubMed

    Maier, Marcus; Ströbele, Hubert; Voges, Jaqueline; Bauer, Clemens; Marzi, Ingo

    2003-05-01

    Ischemia/reperfusion injury involves a large number of humoral and cellular mediators that activate leukocytes that subsequently migrate to local tissues. Tumor necrosis factor (TNF)-alpha may be one of the most important mediators of this post-shock inflammatory response. In this study, we investigated the influence of a recombinant Type I (55 kDa) TNF-binding protein (TNF-BP) on leukocyte-endothelial interactions in the liver after hemorrhagic shock. Hemorrhagic shock was induced in female Sprague-Dawley rats (40 mmHg for 90 min) and a standardized resuscitation regimen was applied. At the time of resuscitation, animals were treated intravenously with either TNF-BP 4 mg/kg or placebo. The liver microcirculation was investigated using intravital fluorescence microscopy and immunohistochemistry at 5 h and 48 h after reperfusion. At 5 h, treatment with TNF-BP significantly reduced temporary leukocyte adhesion in the liver sinusoids as well as mean adhesion time of leukocytes in the hepatic central vein. In contrast, after 48 h, permanent leukocyte adhesion in the central hepatic vein was significantly reduced in the group receiving TNF-BP, whereas temporary leukocyte adhesion and mean adhesion time did not differ between the two groups. Both types of leukocyte adhesion, rolling adhesion after 5 h and firm adhesion after 48 h, were reduced in the group treated with TNF-BP, thereby suggesting a long-lasting anti-inflammatory effect.

  13. Association of weight regain with specific methylation levels in the NPY and POMC promoters in leukocytes of obese men: a translational study.

    PubMed

    Crujeiras, Ana B; Campion, Javier; Díaz-Lagares, Angel; Milagro, Fermin I; Goyenechea, Estíbaliz; Abete, Itziar; Casanueva, Felipe F; Martínez, J Alfredo

    2013-09-10

    Specific methylation of appetite-related genes in leukocytes could serve as a useful biomarker to predict weight regain after an energy restriction program. We aimed to evaluate whether the pre-intervention DNA methylation patterns involved in the epigenetic control of appetite-regulatory genes in leukocytes are associated with the weight regain process. Eighteen men who lost ≥5% of body weight after an 8-week nutritional intervention were categorized as "regainers" (≥10% weight regain) and "non-regainers" (<10% weight regain) 32weeks after stopping dieting. At baseline, leukocytes were isolated and DNA was analyzed for epigenetic methylation patterns of appetite-related gene promoters by MALDI-TOF mass spectrometry. Regainers showed higher methylation levels than non-regainers in proopiomelanocortin (POMC) CpG sites +136bp and +138bp (fold change from non-regainers=26%; p=0.020) and lower methylation of the whole analyzed region of neuropeptide Y (NPY; fold change from non-regainers=-22%; p=0.033), as well as of several individual NPY-promoter CpG sites. Importantly, total baseline NPY methylation was associated with weight-loss regain (r=-0.76; p<0.001), baseline plasma ghrelin levels (r=0.60; p=0.011) and leptin/ghrelin ratio (r=-0.52; p=0.046). Lower methylation levels of POMC CpG sites +136bp and +138bp were associated with success in weight-loss maintenance (odds ratio=0.042 [95% CI 0.01-0.57]; p=0.018), whereas lower total methylation levels in NPY promoter were associated with higher risk of weight regain (odds ratio=14.0 [95% CI 1.13-172]; p=0.039). Therefore, the study of leukocyte methylation levels reflects a putative epigenetic regulation of NPY and POMC, which might be implicated in the weight regain process and be used as biomarkers for predicting weight regain after dieting.

  14. The role of platelets in the recruitment of leukocytes during vascular disease.

    PubMed

    Ed Rainger, G; Chimen, Myriam; Harrison, Matthew J; Yates, Clara M; Harrison, Paul; Watson, Stephen P; Lordkipanidzé, Marie; Nash, Gerard B

    2015-01-01

    Besides their role in the formation of thrombus during haemostasis, it is becoming clear that platelets contribute to a number of other processes within the vasculature. Indeed, the integrated function of the thrombotic and inflammatory systems, which results in platelet-mediated recruitment of leukocytes, is now considered to be of great importance in the propagation, progression and pathogenesis of atherosclerotic disease of the arteries. There are three scenarios by which platelets can interact with leukocytes: (1) during haemostasis, when platelets adhere to and are activated on sub-endothelial matrix proteins exposed by vascular damage and then recruit leukocytes to a growing thrombus. (2) Platelets adhere to and are activated on stimulated endothelial cells and then bridge blood borne leukocytes to the vessel wall and. (3) Adhesion between platelets and leukocytes occurs in the blood leading to formation of heterotypic aggregates prior to contact with endothelial cells. In the following review we will not discuss leukocyte recruitment during haemostasis, as this represents a physiological response to tissue trauma that can progress, at least in its early stages, in the absence of inflammation. Rather we will deal with scenarios 2 and 3, as these pathways of platelet-leukocyte interactions are important during inflammation and in chronic inflammatory diseases such as atherosclerosis. Indeed, these interactions mean that leukocytes possess means of adhesion to the vessel wall under conditions that may not normally be permissive of leukocyte-endothelial cell adhesion, meaning that the disease process may be able to bypass the regulatory pathways which would ordinarily moderate the inflammatory response.

  15. Study of terahertz-radiation-induced DNA damage in human blood leukocytes

    SciTech Connect

    Angeluts, A A; Esaulkov, M N; Kosareva, O G; Solyankin, P M; Shkurinov, A P; Gapeyev, A B; Pashovkin, T N; Matyunin, S N; Nazarov, M M; Cherkasova, O P

    2014-03-28

    We have carried out the studies aimed at assessing the effect of terahertz radiation on DNA molecules in human blood leukocytes. Genotoxic testing of terahertz radiation was performed in three different oscillation regimes, the blood leukocytes from healthy donors being irradiated for 20 minutes with the mean intensity of 8 – 200 μW cm{sup -2} within the frequency range of 0.1 – 6.5 THz. Using the comet assay it is shown that in the selected regimes such radiation does not induce a direct DNA damage in viable human blood leukocytes. (biophotonics)

  16. Study of terahertz-radiation-induced DNA damage in human blood leukocytes

    NASA Astrophysics Data System (ADS)

    Angeluts, A. A.; Gapeyev, A. B.; Esaulkov, M. N.; Kosareva, O. G.; Matyunin, S. N.; Nazarov, M. M.; Pashovkin, T. N.; Solyankin, P. M.; Cherkasova, O. P.; Shkurinov, A. P.

    2014-03-01

    We have carried out the studies aimed at assessing the effect of terahertz radiation on DNA molecules in human blood leukocytes. Genotoxic testing of terahertz radiation was performed in three different oscillation regimes, the blood leukocytes from healthy donors being irradiated for 20 minutes with the mean intensity of 8 - 200 μW cm-2 within the frequency range of 0.1 - 6.5 THz. Using the comet assay it is shown that in the selected regimes such radiation does not induce a direct DNA damage in viable human blood leukocytes.

  17. Integrin activation by P-Rex1 is required for selectin-mediated slow leukocyte rolling and intravascular crawling.

    PubMed

    Herter, Jan M; Rossaint, Jan; Block, Helena; Welch, Heidi; Zarbock, Alexander

    2013-03-21

    Integrin activation is essential for the function of leukocytes. Impaired integrin activation on leukocytes is the hallmark of the leukocyte adhesion deficiency syndrome in humans, characterized by impaired leukocyte recruitment and recurrent infections. In inflammation, leukocytes collect different signals during the contact with the microvasculature, which activate signaling pathways leading to integrin activation and leukocyte recruitment. We report the role of P-Rex1, a Rac-specific guanine nucleotide exchanging factor, in integrin activation and leukocyte recruitment. We find that P-Rex1 is required for inducing selectin-mediated lymphocyte function-associated antigen-1 (LFA-1) extension that corresponds to intermediate affinity and induces slow leukocyte rolling, whereas P-Rex1 is not involved in the induction of the high-affinity conformation of LFA-1 obligatory for leukocyte arrest. Furthermore, we demonstrate that P-Rex1 is involved in Mac-1-dependent intravascular crawling. In vivo, both LFA-1-dependent slow rolling and Mac-1-dependent crawling are defective in P-Rex1(-/-) leukocytes, whereas chemokine-induced arrest and postadhesion strengthening remain intact in P-Rex1-deficient leukocytes. Rac1 is involved in E-selectin-mediated slow rolling and crawling. In vivo, in an ischemia-reperfusion-induced model of acute kidney injury, abolished selectin-mediated integrin activation contributed to decreased neutrophil recruitment and reduced kidney damage in P-Rex1-deficient mice. We conclude that P-Rex1 serves distinct functions in LFA-1 and Mac-1 activation.

  18. Fluorescence imaging microscopy of leukocytes-endothelium interaction in rat mesenteric microcirculation after endotoxin injection: role of inhaled nitric oxide

    NASA Astrophysics Data System (ADS)

    Mordon, Serge R.; Neviere, Remi; Marechal, Xavier-Marie; Buys, Bruno; Dhelin, Guy; Lesage, Jean C.; Mathieu, D.; Guery, Benoit; Chopin, Claude

    1999-02-01

    The adhesion of leukocytes to microvascular endothelium has been recognized as an important factor in the development of multiple organ dysfunction after a septic insult. We tested the hypothesis whether inhaled NO would reduce leukocyte rolling and / or leukocyte adhesion in the mesenteric venule preparation in endotoxemic rats. This study was performed with fluorescence imaging microscopy using a closed chamber for in vivo mesentery visualization. Leukocytes were selectively stained with acridine red. Compared to saline, endotoxemia was associated with increases in the flux of rolling leukocytes and in adherent and emigrated leukocytes. Inhaled nitric oxide treatment had no effects on leukocyte behavior in saline treated rats, whereas it reduced adherent and emigrated leukocytes in endotoxin-treated rats. In conclusion, we demonstrated that endotoxemia-induced leukocyte infiltration was related to an increase in the number of rolling leukocytes and subsequent adhesion and emigration in the mesenteric venule. Our results clearly showed that inhaled NO reduces leukocyte adhesion and transmigration in mesenteric venule of endotoxemic rats presumably by interfering with specific cell adhesion molecules.

  19. EDU pretreatment decreases polymorphonuclear leukocyte migration into rat lung airways.

    PubMed

    Bassett, D J; Elbon, C L; Ishii, Y; Yang, H; Otterbein, L; Boswell, G A; Kerr, J S

    1994-07-01

    Pretreatment with the heterocyclic compound EDU (N-[2-(2-oxo-1-imidazolindinyl)ethyl]-N'-phenylurea) has previously been shown to reduce polymorphonuclear leukocyte (PMN) infiltration into the airways of ozone-exposed rats. The present study further examined the effects of 1 and 2 days EDU pretreatment on rat lung inflammatory responses by determining PMN infiltration in response to intratracheal instillation with the chemoattractant formyl-norleucine-leucine-phenylalanine (fNLP). Maximal recovery of PMNs by bronchoalveolar lavage was observed 4 hr after fNLP instillation with no alteration in the numbers of recoverable macrophages and lymphocytes. Although 1-day pretreatment with EDU did not affect PMN recovery from fNLP-instilled rat lungs, 2 days of EDU pretreatment prevented PMN infiltration as indicated by PMN recoveries that were similar to those obtained from saline-instilled lungs. Measurements of lung-marginated and interstitial pools of inflammatory cells using collagenase tissue digestion demonstrated no effect of 2 days EDU pretreatment. Although 2 days EDU pretreatment alone did not alter blood PMN content, lung permeability, and the lavage recoveries of inflammatory cells, blood PMN responses to chemotactic stimuli in vitro were impaired. In addition, EDU was shown to directly inhibit PMN chemotaxis and superoxide anion generation in vitro. These data demonstrated that EDU acts by interfering with PMN activation and migration rather than by decreasing PMN availability. EDU, by modulating the inflammatory response, represents a useful compound for preventing PMN-associated amplification of acute lung injuries.

  20. Impact of Dehydroepiandrosterone Sulfate on Newborn Leukocyte Telomere Length

    PubMed Central

    Liu, Han; Zhou, Guangdi; Chen, Qian; Ouyang, Fengxiu; Little, Julian; Zhang, Jun; Chen, Dan

    2017-01-01

    The newborn setting of leukocyte telomere length (LTL) likely has important implications for telomere dynamics over the lifespan. However, its determinants are poorly understood. Hormones play an important role during pregnancy and delivery. We hypothesized that exposure to hormones may impact the fetal telomere biology system. To test this hypothesis, cortisol, estradiol, dehydroepiandrosterone sulfate (DHEAS) and reactive oxygen species (ROS) were measured in cord blood of 821 newborns from a prospective study. After accounting for the effects of potential determinants of newborn LTL, a 10-fold increase in DHEAS concentration was associated with a 0.021 increase in T/S ratio of newborn LTL (95% confidence interval: 0.009–0.034, P = 0.0008). For newborns who fell in the lowest quartile of DHEAS level, the mean newborn LTL was estimated to be approximately 2.0% shorter than the newborns in the highest DHEAS concentration quartile (P = 0.0014). However, no association was found between newborn LTL and cortisol or estradiol. As expected, newborns with higher ROS level (ROS > 260 mol/L) had lower LTL compared to that with lower ROS level (ROS ≤ 260 mol/L) (P = 0.007). There was also an inverse relationship between DHEAS and ROS (P < 1×10−4). Our findings suggest that exposure to DHEAS may exert a “programming” effect on the newborn telomere biology system. PMID:28186106

  1. Leukocyte Homing, Fate, and Function Are Controlled by Retinoic Acid

    PubMed Central

    Guo, Yanxia; Brown, Chrysothemis; Ortiz, Carla; Noelle, Randolph J.

    2015-01-01

    Although vitamin A was recognized as an “anti-infective vitamin” over 90 years ago, the mechanism of how vitamin A regulates immunity is only beginning to be understood. Early studies which focused on the immune responses in vitamin A-deficient (VAD) animals clearly demonstrated compromised immunity and consequently increased susceptibility to infectious disease. The active form of vitamin A, retinoic acid (RA), has been shown to have a profound impact on the homing and differentiation of leukocytes. Both pharmacological and genetic approaches have been applied to the understanding of how RA regulates the development and differentiation of various immune cell subsets, and how RA influences the development of immunity versus tolerance. These studies clearly show that RA profoundly impacts on cell- and humoral-mediated immunity. In this review, the early findings on the complex relationship between VAD and immunity are discussed as well as vitamin A metabolism and signaling within hematopoietic cells. Particular attention is focused on how RA impacts on T-cell lineage commitment and plasticity in various diseases. PMID:25540140

  2. Uptake and intracellular activity of fluconazole in human polymorphonuclear leukocytes.

    PubMed Central

    Pascual, A; García, I; Conejo, C; Perea, E J

    1993-01-01

    The penetration of fluconazole into human polymorphonuclear leukocytes (PMNs) and tissue culture epithelial cells (McCoy) was evaluated. At different extracellular concentrations (0.5 to 10 mg/liter), fluconazole reached cell-associated concentrations greater than the extracellular ones in either human PMNs (intracellular concentration to extracellular concentration ratio, > or = 2.2) or McCoy cells (intracellular concentration to extracellular concentration ratio, > or = 1.3). The uptake of fluconazole by PMNs was rapid and reversible but was not energy dependent. The intracellular penetration of fluconazole was not affected by environmental pH or temperature. Ingestion of opsonized zymosan and opsonized Candida albicans did not significantly increase the amount of PMN-associated fluconazole. At therapeutic extracellular concentrations, the intracellular activity of fluconazole against C. albicans in PMNs was significantly lower than that of amphotericin B. It was concluded that fluconazole reaches high intracellular concentrations within PMNs but shows moderate activity against intracellular C. albicans in vitro. PMID:8452347

  3. Plasminogen activator inhibitor-2 (PAI-2) in eosinophilic leukocytes.

    PubMed

    Swartz, Jonathan M; Byström, Jonas; Dyer, Kimberly D; Nitto, Takeaki; Wynn, Thomas A; Rosenberg, Helene F

    2004-10-01

    Plasminogen activator inhibitor-2 (PAI-2) as a potential eosinophil protein was inferred from our gene microarray study of mouse eosinophilopoiesis. Here, we detect 47 kDa intracellular and approximately 60 kDa secretory forms of PAI-2 in purified human eosinophil extracts. PAI-2 is present at variable concentrations in eosinophil lysates, ranging from 30 to 444 ng/10(6) cells, with a mean of 182 ng/10(6) cells from 10 normal donors, which is the highest per-cell concentration among all leukocyte subtypes evaluated. Enzymatic assay confirmed that eosinophil-derived PAI-2 is biologically active and inhibits activation of its preferred substrate, urokinase. Immunohistochemical and immunogold staining demonstrated PAI-2 localization in eosinophil-specific granules. Immunoreactive PAI-2 was detected in extracellular deposits in and around the eosinophil-enriched granuloma tissue encapsulating the parasitic egg in livers of wild-type mice infected with the helminthic parasite Schistosoma mansoni. Among the possibilities, we consider a role for eosinophil-derived PAI-2 in inflammation and remodeling associated with parasitic infection as well as allergic airways disease, respiratory virus infection, and host responses to tumors and metastasis in vivo.

  4. Leukocyte esterase-nitrite and bioluminescence assays as urine screens.

    PubMed Central

    Males, B M; Bartholomew, W R; Amsterdam, D

    1985-01-01

    The 1-min leukocyte esterase (LE)-nitrite test (Chemstrip 9; Biodynamics, Division of Boehringer Mannheim Biochemicals, Indianapolis, Ind.) and a bioluminescence assay (Monolight centrifugation method; Analytical Luminescence Laboratory, Inc., San Diego, Calif.) were tested for their efficacy as urine screens among 453 patients at a tertiary-care teaching hospital. Both methods had the capacity to exclude significant bacteriuria (greater than or equal to 10(5) CFU/ml) when compared with the results of conventional culture methods, with predictive values of 99 and 93%, respectively, for a negative test. Bioluminescence was the more accurate nonculture method used. Sensitivity and specificity values were 97 and 71%, respectively, for bioluminescence, 82 and 60%, respectively, for LE with nitrite, and 72 and 64%, respectively, for LE without nitrite. At reduced levels of bacteriuria less than 10(5) CFU/ml), the sensitivities of LE-nitrite and bioluminescence were decreased but comparable. The addition of protein and blood test results in the Chemstrip 9, along with LE-nitrite as bacteriuria indicators, were unsatisfactory because of the large numbers of false-positive results attributed to protein and blood determinations. LE activity as detected by the LE test was a poor predictor of significant bacteriuria in both male and female patients. The sensitivity (71%) and specificity (57%) of the LE test in male patients were significantly lower than those previously reported and varied with the patient population studied. PMID:3935662

  5. Affinity-tuning leukocyte integrin for development of safe therapeutics

    NASA Astrophysics Data System (ADS)

    Park, Spencer

    Much attention has been given to the molecular and cellular pathways linking inflammation with cancer and the local tumor environment to identify new target molecules that could lead to improved diagnosis and treatment. Among the many molecular players involved in the complex response, central to the induction of inflammation is intercellular adhesion molecule (ICAM)-1, which is of particular interest for its highly sensitive and localized expression in response to inflammatory signals. ICAM-1, which has been implicated to play a critical role in tumor progression in various types of cancer, has also been linked to cancer metastases, where ICAM-1 facilitates the spread of metastatic cancer cells to secondary sites. This unique expression profile of ICAM-1 throughout solid tumor microenvironment makes ICAM-1 an intriguing molecular target, which holds great potential as an important diagnostic and therapeutic tool. Herein, we have engineered the ligand binding domain, or the inserted (I) domain of a leukocyte integrin, to exhibit a wide range of monovalent affinities to the natural ligand, ICAM-1. Using the resulting I domain variants, we have created drug and gene delivery nanoparticles, as well as targeted immunotherapeutics that have the ability to bind and migrate to inflammatory sites prevalent in tumors and the associated microenvironment. Through the delivery of diagnostic agents, chemotherapeutics, and immunotherapeutics, the following chapters demonstrate that the affinity enhancements achieved by directed evolution bring the affinity of I domains into the range optimal for numerous applications.

  6. Automated real-time measurements of leukocyte chemotaxis.

    PubMed

    Hadjout, Nacima; Yin, Xiuyun; Knecht, David A; Lynes, Michael A

    2007-03-30

    We have previously described an automated system (ECIS/taxis) for measuring chemotactic movement of Dictyostelium amoebae in a folic acid gradient [Hadjout, N., Laevsky, G., Knecht, D.A. and Lynes, M.A., 2001. Automated real-time measurement of chemotactic cell motility. Biotechniques 31, 1130-1138.]. In the ECIS/taxis system, cells migrate in an under-agarose environment, and their position is monitored by determining the impedance change caused by cells crawling onto the surface of an electrode. In this report, we show that chemotaxis of primary and immortalized leukocytes in response to complement (C5a) could be measured using the ECIS/taxis system. Several modifications to the design of the target electrode were tested, and a linear electrode perpendicular to the direction of movement was found to increase the sensitivity and reliability of the assay. Using the optimized ECIS/taxis assay, the dose response of neutrophils and WBC 265-9C cells was established and compared to the Boyden chamber assay. The ECIS/taxis assay system can be used to compare the movement of different cell types, to assess the effect of complex chemotactic gradients, or to determine the effects of pharmaceuticals on chemotactic motility.

  7. Extracellular release of antimicrobial defensins by human polymorphonuclear leukocytes.

    PubMed Central

    Ganz, T

    1987-01-01

    Human polymorphonuclear leukocytes (PMN) contain three antimicrobial and cytotoxic peptides which belong to a family of mammalian granulocyte peptides named defensins. To determine their potential availability for extracellular microbicidal or cytotoxic events, we quantified the extracellular release of defensins after stimulation of human PMN with phorbol myristate acetate and opsonized zymosan. As determined by enzyme immunoassay and confirmed by polyacrylamide gel electrophoresis and densitometry, 10(6) human PMN contained 4 to 5 micrograms of defensins. After stimulation with a high concentration of phorbol myristate acetate (1 microgram/ml), about 8% of PMN defensins were found in the media. Release of defensins correlated best with the release of azurophil granule marker beta-glucuronidase or elastase and poorly with the release of either the specific granule marker lactoferrin or cytoplasmic lactate dehydrogenase. Phagocytosis of opsonized zymosan resulted in the extracellular release of less than 3% of PMN defensins. The factors responsible for less release of defensins into media relative to the release of other azurophil granule proteins may include heterogeneity of azurophil granules and the affinity of defensins for cellular surfaces and opsonized particles. In vivo, defensins are most likely to reach effective microbicidal or cytotoxic concentrations in PMN-rich exudates (pus), in confined environments of the phagolysosomes, or in intercellular clefts between PMN and their targets. PMID:3643886

  8. Effects of lead on the killing mechanisms of polymorphonuclear leukocytes

    SciTech Connect

    Silberstein, C.F.

    1984-01-01

    The effects of lead on the killing mechanisms of rat polymorphonuclear leukocytes (PMN) were investigated, using male Long-Evans rats exposed to 1% lead acetate in the drinking water for varying periods of time to achieve blood lead levels ranging from 20-200 ..mu..g/dl. Studies of PMN bacterial and fungal killing activity, chemotaxis and phagocytosis demonstrated that: 1) bactericidal activity of PMN from rats exposed to lead was not altered; 2) chemotactic activity remained within normal limits; 3) the phagocytic ability of the PMN also remained unaltered. In addition to these normal findings, one major abnormality was demonstrated: a significant decrease in the ability of PMN from rats exposed to lead to kill Candida albicans. This defect was not related to age or to length of exposure. It could not be produced by addition of lead to the test system in vitro. Further investigation revealed significant decreases in PMN glucose-6-phosphate dehydrogenase, catalase, and myeloperoxidase activities. These data support two possible mechanisms for the abnormal fungicidal activity of PMN from lead-exposed rats: decrease in ability to reduce oxygen to active metabolites, or reduction in myeloperoxidase activity due to diminshed synthesis of the heme moiety required for its function.

  9. Inhibitory leukocyte immunoglobulin-like receptors in cancer development.

    PubMed

    Zhang, FeiFei; Zheng, JunKe; Kang, XunLei; Deng, Mi; Lu, ZhiGang; Kim, Jaehyup; Zhang, ChengCheng

    2015-12-01

    Inhibitory leukocyte immunoglobulin-like receptors (LILRB1-5) signal through immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their intracellular domains and recruit phosphatases protein tyrosine phosphatase, non-receptor type 6 (PTPN6, SHP-1), protein tyrosine phosphatase, non-receptor type 6 (PTPN6, SHP-2), or Src homology 2 domain containing inositol phosphatase (SHIP) to negatively regulate immune cell activation. These receptors are known to play important regulatory roles in immune and neuronal functions. Recent studies demonstrated that several of these receptors are expressed by cancer cells. Importantly, they may directly regulate development, drug resistance, and relapse of cancer, and the activity of cancer stem cells. Although counterintuitive, these findings are consistent with the generally immune-suppressive and thus tumor-promoting roles of the inhibitory receptors in the immune system. This review focuses on the ligands, expression pattern, signaling, and function of LILRB family in the context of cancer development. Because inhibition of the signaling of certain LILRBs directly blocks cancer growth and stimulates immunity that may suppress tumorigenesis, but does not disturb normal development, LILRB signaling pathways may represent ideal targets for treating hematological malignancies and perhaps other tumors.

  10. Uptake of antibiotics by human polymorphonuclear leukocyte cytoplasts

    SciTech Connect

    Hand, W.L.; King-Thompson, N.L. , Decatur, GA )

    1990-06-01

    Enucleated human polymorphonuclear leukocytes (PMN cytoplasts), which have no nuclei and only a few granules, retain many of the functions of intact neutrophils. To better define the mechanisms and intracellular sites of antimicrobial agent accumulation in human neutrophils, we studied the antibiotic uptake process in PMN cytoplasts. Entry of eight radiolabeled antibiotics into PMN cytoplasts was determined by means of a velocity gradient centrifugation technique. Uptakes of these antibiotics by cytoplasts were compared with our findings in intact PMN. Penicillin entered both intact PMN and cytoplasts poorly. Metronidazole achieved a concentration in cytoplasts (and PMN) equal to or somewhat less than the extracellular concentration. Chloramphenicol, a lipid-soluble drug, and trimethoprim were concentrated three- to fourfold by cytoplasts. An unusual finding was that trimethroprim, unlike other tested antibiotics, was accumulated by cytoplasts more readily at 25 degrees C than at 37 degrees C. After an initial rapid association with cytoplasts, cell-associated imipenem declined progressively with time. Clindamycin and two macrolide antibiotics (roxithromycin, erythromycin) were concentrated 7- to 14-fold by cytoplasts. This indicates that cytoplasmic granules are not essential for accumulation of these drugs. Adenosine inhibited cytoplast uptake of clindamycin, which enters intact phagocytic cells by the membrane nucleoside transport system. Roxithromycin uptake by cytoplasts was inhibited by phagocytosis, which may reduce the number of cell membrane sites available for the transport of macrolides. These studies have added to our understanding of uptake mechanisms for antibiotics which are highly concentrated in phagocytes.

  11. Delay discounting, genetic sensitivity, and leukocyte telomere length.

    PubMed

    Yim, Onn-Siong; Zhang, Xing; Shalev, Idan; Monakhov, Mikhail; Zhong, Songfa; Hsu, Ming; Chew, Soo Hong; Lai, Poh San; Ebstein, Richard P

    2016-03-08

    In a graying world, there is an increasing interest in correlates of aging, especially those found in early life. Leukocyte telomere length (LTL) is an emerging marker of aging at the cellular level, but little is known regarding its link with poor decision making that often entails being overly impatient. Here we investigate the relationship between LTL and the degree of impatience, which is measured in the laboratory using an incentivized delay discounting task. In a sample of 1,158 Han Chinese undergraduates, we observe that steeper delay discounting, indexing higher degree of impatience, is negatively associated with LTL. The relationship is robust after controlling for health-related variables, as well as risk attitude-another important determinant of decision making. LTL in females is more sensitive to impatience than in males. We then asked if genes possibly modulate the effect of impatient behavior on LTL. The oxytocin receptor gene (OXTR) polymorphism rs53576, which has figured prominently in investigations of social cognition and psychological resources, and the estrogen receptor β gene (ESR2) polymorphism rs2978381, one of two gonadal sex hormone genes, significantly mitigate the negative effect of impatience on cellular aging in females. The current results contribute to understanding the relationship between preferences in decision making, particularly impatience, and cellular aging, for the first time to our knowledge. Notably, oxytocin and estrogen receptor polymorphisms temper accelerated cellular aging in young females who tend to make impatient choices.

  12. Delay discounting, genetic sensitivity, and leukocyte telomere length

    PubMed Central

    Yim, Onn-Siong; Zhang, Xing; Shalev, Idan; Monakhov, Mikhail; Zhong, Songfa; Hsu, Ming; Chew, Soo Hong; Lai, Poh San; Ebstein, Richard P.

    2016-01-01

    In a graying world, there is an increasing interest in correlates of aging, especially those found in early life. Leukocyte telomere length (LTL) is an emerging marker of aging at the cellular level, but little is known regarding its link with poor decision making that often entails being overly impatient. Here we investigate the relationship between LTL and the degree of impatience, which is measured in the laboratory using an incentivized delay discounting task. In a sample of 1,158 Han Chinese undergraduates, we observe that steeper delay discounting, indexing higher degree of impatience, is negatively associated with LTL. The relationship is robust after controlling for health-related variables, as well as risk attitude—another important determinant of decision making. LTL in females is more sensitive to impatience than in males. We then asked if genes possibly modulate the effect of impatient behavior on LTL. The oxytocin receptor gene (OXTR) polymorphism rs53576, which has figured prominently in investigations of social cognition and psychological resources, and the estrogen receptor β gene (ESR2) polymorphism rs2978381, one of two gonadal sex hormone genes, significantly mitigate the negative effect of impatience on cellular aging in females. The current results contribute to understanding the relationship between preferences in decision making, particularly impatience, and cellular aging, for the first time to our knowledge. Notably, oxytocin and estrogen receptor polymorphisms temper accelerated cellular aging in young females who tend to make impatient choices. PMID:26903639

  13. Leukocyte deformability: finite element modeling of large viscoelastic deformation.

    PubMed

    Dong, C; Skalak, R

    1992-09-21

    An axisymmetric deformation of a viscoelastic sphere bounded by a prestressed elastic thin shell in response to external pressure is studied by a finite element method. The research is motivated by the need for understanding the passive behavior of human leukocytes (white blood cells) and interpreting extensive experimental data in terms of the mechanical properties. The cell at rest is modeled as a sphere consisting of a cortical prestressed shell with incompressible Maxwell fluid interior. A large-strain deformation theory is developed based on the proposed model. General non-linear, large strain constitutive relations for the cortical shell are derived by neglecting the bending stiffness. A representation of the constitutive equations in the form of an integral of strain history for the incompressible Maxwell interior is used in the formulation of numerical scheme. A finite element program is developed, in which a sliding boundary condition is imposed on all contact surfaces. The mathematical model developed is applied to evaluate experimental data of pipette tests and observations of blood flow.

  14. Leukocyte tyrosine kinase functions in pigment cell development.

    PubMed

    Lopes, Susana S; Yang, Xueyan; Müller, Jeanette; Carney, Thomas J; McAdow, Anthony R; Rauch, Gerd-Jörg; Jacoby, Arie S; Hurst, Laurence D; Delfino-Machín, Mariana; Haffter, Pascal; Geisler, Robert; Johnson, Stephen L; Ward, Andrew; Kelsh, Robert N

    2008-03-07

    A fundamental problem in developmental biology concerns how multipotent precursors choose specific fates. Neural crest cells (NCCs) are multipotent, yet the mechanisms driving specific fate choices remain incompletely understood. Sox10 is required for specification of neural cells and melanocytes from NCCs. Like sox10 mutants, zebrafish shady mutants lack iridophores; we have proposed that sox10 and shady are required for iridophore specification from NCCs. We show using diverse approaches that shady encodes zebrafish leukocyte tyrosine kinase (Ltk). Cell transplantation studies show that Ltk acts cell-autonomously within the iridophore lineage. Consistent with this, ltk is expressed in a subset of NCCs, before becoming restricted to the iridophore lineage. Marker analysis reveals a primary defect in iridophore specification in ltk mutants. We saw no evidence for a fate-shift of neural crest cells into other pigment cell fates and some NCCs were subsequently lost by apoptosis. These features are also characteristic of the neural crest cell phenotype in sox10 mutants, leading us to examine iridophores in sox10 mutants. As expected, sox10 mutants largely lacked iridophore markers at late stages. In addition, sox10 mutants unexpectedly showed more ltk-expressing cells than wild-type siblings. These cells remained in a premigratory position and expressed sox10 but not the earliest neural crest markers and may represent multipotent, but partially-restricted, progenitors. In summary, we have discovered a novel signalling pathway in NCC development and demonstrate fate specification of iridophores as the first identified role for Ltk.

  15. Acoustic and photoacoustic microscopy imaging of single leukocytes

    NASA Astrophysics Data System (ADS)

    Strohm, Eric M.; Moore, Michael J.; Kolios, Michael C.

    2016-03-01

    An acoustic/photoacoustic microscope was used to create micrometer resolution images of stained cells from a blood smear. Pulse echo ultrasound images were made using a 1000 MHz transducer with 1 μm resolution. Photoacoustic images were made using a fiber coupled 532 nm laser, where energy losses through stimulated Raman scattering enabled output wavelengths from 532 nm to 620 nm. The laser was focused onto the sample using a 20x objective, and the laser spot co-aligned with the 1000 MHz transducer opposite the laser. The blood smear was stained with Wright-Giemsa, a common metachromatic dye that differentially stains the cellular components for visual identification. A neutrophil, lymphocyte and a monocyte were imaged using acoustic and photoacoustic microscopy at two different wavelengths, 532 nm and 600 nm. Unique features in each imaging modality enabled identification of the different cell types. This imaging method provides a new way of imaging stained leukocytes, with applications towards identifying and differentiating cell types, and detecting disease at the single cell level.

  16. Detection of a prosthetic aortic valvular abscess with indium-111-labeled leukocytes

    SciTech Connect

    Oates, E.; Sarno, R.C.

    1988-10-01

    An unsuspected annular abscess at the base of a prosthetic aortic valve in a patient with endocarditis was identified by indium-111-labeled leukocyte scintigraphy alone. This highly sensitive and specific technique expediently demonstrated the surgically proven inflammatory focus.

  17. Endothelial CD99 signals through soluble adenylyl cyclase and PKA to regulate leukocyte transendothelial migration

    PubMed Central

    Watson, Richard L.; Buck, Jochen; Levin, Lonny R.; Winger, Ryan C.; Wang, Jing; Arase, Hisashi

    2015-01-01

    CD99 is a critical regulator of leukocyte transendothelial migration (TEM). How CD99 signals during this process remains unknown. We show that during TEM, endothelial cell (EC) CD99 activates protein kinase A (PKA) via a signaling complex formed with the lysine-rich juxtamembrane cytoplasmic tail of CD99, the A-kinase anchoring protein ezrin, and soluble adenylyl cyclase (sAC). PKA then stimulates membrane trafficking from the lateral border recycling compartment to sites of TEM, facilitating the passage of leukocytes across the endothelium. Pharmacologic or genetic inhibition of EC sAC or PKA, like CD99 blockade, arrests neutrophils and monocytes partway through EC junctions, in vitro and in vivo, without affecting leukocyte adhesion or the expression of relevant cellular adhesion molecules. This is the first description of the CD99 signaling pathway in TEM as well as the first demonstration of a role for sAC in leukocyte TEM. PMID:26101266

  18. Expression analysis of psychological stress-associated genes in peripheral blood leukocytes.

    PubMed

    Morita, Kyoko; Saito, Toshiro; Ohta, Masayuki; Ohmori, Tetsuro; Kawai, Kaori; Teshima-Kondo, Shigetada; Rokutan, Kazuhito

    In this study, we have developed a microarray including 1467 cDNAs that were selected to specifically measure stress response in peripheral blood leukocytes. Venous blood was collected from 10 graduate students 2 h before and 2 or 24 h after an open presentation for their Ph.D. The mRNA levels in leukocytes were compared with those prepared 4 weeks before the presentation. Hierarchical cluster showed that distinct groups of genes uniformly changed their expression values in response to the stress. Bayesian t test identified significantly up-regulated 49 genes and down-regulated 21 genes. Most of them are categorized into cytokines, cytokine receptors, growth- or apoptosis-related molecules, and heat shock proteins, suggesting that stressful life events trigger acute responses in leukocytes. Our results suggest that gene expression profile in peripheral blood leukocytes may be a potentially useful method for the assessment of complex stress responses.

  19. Endothelial CD99 signals through soluble adenylyl cyclase and PKA to regulate leukocyte transendothelial migration.

    PubMed

    Watson, Richard L; Buck, Jochen; Levin, Lonny R; Winger, Ryan C; Wang, Jing; Arase, Hisashi; Muller, William A

    2015-06-29

    CD99 is a critical regulator of leukocyte transendothelial migration (TEM). How CD99 signals during this process remains unknown. We show that during TEM, endothelial cell (EC) CD99 activates protein kinase A (PKA) via a signaling complex formed with the lysine-rich juxtamembrane cytoplasmic tail of CD99, the A-kinase anchoring protein ezrin, and soluble adenylyl cyclase (sAC). PKA then stimulates membrane trafficking from the lateral border recycling compartment to sites of TEM, facilitating the passage of leukocytes across the endothelium. Pharmacologic or genetic inhibition of EC sAC or PKA, like CD99 blockade, arrests neutrophils and monocytes partway through EC junctions, in vitro and in vivo, without affecting leukocyte adhesion or the expression of relevant cellular adhesion molecules. This is the first description of the CD99 signaling pathway in TEM as well as the first demonstration of a role for sAC in leukocyte TEM.

  20. Inflammation after Ischemic Stroke: The Role of Leukocytes and Glial Cells

    PubMed Central

    Kim, Jong Youl; Park, Joohyun; Chang, Ji Young; Kim, Sa-Hyun

    2016-01-01

    The immune response after stroke is known to play a major role in ischemic brain pathobiology. The inflammatory signals released by immune mediators activated by brain injury sets off a complex series of biochemical and molecular events which have been increasingly recognized as a key contributor to neuronal cell death. The primary immune mediators involved are glial cells and infiltrating leukocytes, including neutrophils, monocytes and lymphocyte. After ischemic stroke, activation of glial cells and subsequent release of pro- and anti-inflammatory signals are important for modulating both neuronal cell damage and wound healing. Infiltrated leukocytes release inflammatory mediators into the site of the lesion, thereby exacerbating brain injury. This review describes how the roles of glial cells and circulating leukocytes are a double-edged sword for neuroinflammation by focusing on their detrimental and protective effects in ischemic stroke. Here, we will focus on underlying characterize of glial cells and leukocytes under inflammation after ischemic stroke. PMID:27790058

  1. Appearance of acute gouty arthritis on indium-111-labeled leukocyte scintigraphy

    SciTech Connect

    Palestro, C.J.; Vega, A.; Kim, C.K.; Swyer, A.J.; Goldsmith, S.J. )

    1990-05-01

    Indium-111-labeled leukocyte scintigraphy was performed on a 66-yr-old male with polyarticular acute gouty arthritis. Images revealed intense labeled leukocyte accumulation in a pattern indistinguishable from septic arthritis, in both knees and ankles, and the metatarsophalangeal joint of both great toes, all of which were involved in the acute gouty attack. Joint aspirate as well as blood cultures were reported as no growth; the patient was treated with intravenous colchicine and ACTH for 10 days with dramatic improvement noted. Labeled leukocyte imaging, repeated 12 days after the initial study, revealed near total resolution of joint abnormalities, concordant with the patient's clinical improvement. This case demonstrates that while acute gouty arthritis is a potential pitfall in labeled leukocyte imaging, in the presence of known gout, it may provide a simple, objective, noninvasive method of evaluating patient response to therapy.

  2. Vitamin C Prevents Cigarette Smoke-Induced Leukocyte Aggregation and Adhesion to Endothelium in vivo

    NASA Astrophysics Data System (ADS)

    Lehr, Hans-Anton; Frei, Balz; Arfors, Karl-E.

    1994-08-01

    A common feature of cigarette-smoke (CS)-associated diseases such as atherosclerosis and pulmonary emphysema is the activation, aggregation, and adhesion of leukocytes to micro- and macrovascular endothelium. A previous study, using a skinfold chamber model for intravital fluorescence microscopy in awake hamsters, has shown that exposure of hamsters to the smoke generated by one research cigarette elicits the adhesion of fluorescently labeled leukocytes to the endothelium of arterioles and small venules. By the combined use of intravital microscopy and scanning electron microscopy, we now demonstrate in the same animal model that (i) CS-induced leukocyte adhesion is not confined to the microcirculation, but that leukocytes also adhere singly and in clusters to the aortic endothelium; (ii) CS induces the formation in the bloodstream of aggregates between leukocytes and platelets; and (iii) CS-induced leukocyte adhesion to micro- and macrovascular endothelium and leukocyte-platelet aggregate formation are almost entirely prevented by dietary or intravenous pretreatment with the water-soluble antioxidant vitamin C (venules, 21.4 ± 11.0 vs. 149.6 ± 38.7 leukocytes per mm^2, P < 0.01; arterioles, 8.5 ± 4.2 vs. 54.3 ± 21.6 leukocytes per mm^2, P < 0.01; aortas, 0.8 ± 0.4 vs. 12.4 ± 5.6 leukocytes per mm^2, P < 0.01; means ± SD of n = 7 animals, 15 min after CS exposure). No inhibitory effect was observed by pretreatment of the animals with the lipid-soluble antioxidants vitamin E or probucol. The protective effects of vitamin C on CS-induced leukocyte adhesion and aggregation were seen at vitamin C plasma levels (55.6 ± 22.2 μM, n = 7) that can easily be reached in humans by dietary means or supplementation, suggesting that vitamin C effectively contributes to protection from CS-associated cardiovascular and pulmonary diseases in humans.

  3. 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

  4. Lytic Effects of Serum and Mononuclear Leukocytes on Oral Epithelial Cells in Recurrent Aphthous Stomatitis.

    DTIC Science & Technology

    1984-06-07

    Publiqation 3-32to4-84 RECURRENT APHTHOUS STOMATITIS 6. PERFORMING ORG. REPORT NUMBER 7. AUTHOR(*) S. CONTRACT OR GRANT NUMBER(e) PAUL R. BURNETT AND DAVID...vitro cytolytic reactions correlating with recur- rent aphthous stomatitis (RAS). The cytolytic effects of sera and mononuclear leukocytes from...the attached manuscript entitled "Lytic Effects of Serum and Mononuclear Leukocytes on Oral Epithelial Cells in Recurrent Aphthous Stomatitis ." 2. If

  5. Indium-111 leukocyte scanning. False-negative study in a renal abscess

    SciTech Connect

    Kumar, R.; Bedi, D.G.; Fawcett, H.D.; Winsett, M.Z.; Fagan, C.J.

    1986-04-01

    A 33-year-old man had clinical features of a right renal abscess. Results of excretory urography and ultrasonography showed a focal complex mass lesion in the right kidney. An In-111 leukocyte scan failed to detect the right renal abscess, which later was aspirated under CT guidance and explored surgically. The role of In-111 leukocyte imaging in the detection of intra-abdominal abscesses, with limitations of the procedure, is discussed.

  6. Indium-111 leukocyte scintigraphic detection of myocardial abscess formation in patients with endocarditis

    SciTech Connect

    Cerqueira, M.D.; Jacobson, A.F.

    1989-05-01

    Myocardial abscess formation in patients with bacterial endocarditis in most clinical settings, especially in patients with prosthetic valves, is a primary indicator for surgical valve replacement. We report the detection of myocardial abscesses using /sup 111/In leukocyte scintigraphy in three patients with prosthetic or native valve endocarditis and nondiagnostic echocardiograms. Leukocyte scintigraphy may allow identification of myocardial abscess formation earlier than other imaging modalities.

  7. The antioxidant effect of free bilirubin on cumene-hydroperoxide treated human leukocytes.

    PubMed

    Yesilkaya, A; Altinayak, R; Korgun, D K

    2000-07-01

    To examine the antioxidant effect of bilirubin (BR) on leukocyte, we treated leukocytes obtained from healthy subjects with an oxidant and various concentrations of BR. High concentrations of BR decreased thiobarbituric acid reactive substances (TBARS) and catalase activities, increased superoxide dismutase (SOD) activity, but had no effect on glutathione (GSH) concentration. Our results showed that under physiological conditions, BR has an antioxidant effect only in high concentrations.

  8. In-111 labeled leukocyte scintigraphy in a case of multifocal candidiasis

    SciTech Connect

    Palestro, C.J.; Vega, A.; Kim, C.K.; Goldsmith, S.J. )

    1990-06-01

    The value of indium-111 labeled leukocyte scintigraphy for the diagnosis of infection in the general population is well documented; there is less information available on its role in the evaluation of the immunocompromised patient. In this study, leukocyte scintigraphy was performed on a 31-year-old immunocompromised woman who had a possible intra-abdominal abscess. No abscess was detected, but intense oral, esophageal, gastric, and vaginal uptake was observed. Candida infection was histologically confirmed at all four sites.

  9. Clinical Associations of Leukocyte Telomere Length in a Cohort of Repatriated Prisoners of War

    DTIC Science & Technology

    2014-07-01

    to 01-07-2014 4 . TITLE AND SUBTITLE Clinical Associations of Leukocyte Telomere Length in a Cohort of Repatriated Prisoners of War 5a. CONTRACT...biomarker of aging and disease ( 4 ). However, these commercially available blood tests utilize the readily accessible leukocyte telomere length (LTL...added LTL measurements to its laboratory panel in 2011. This preliminary study was undertaken to determine if a commercial 4 RPW TELOMERE LENGTH LTL

  10. In-111-leukocyte scintigraphy for detection of infection associated with peritoneal dialysis catheters

    SciTech Connect

    Kipper, S.L.; Steiner, R.W.; Witztum, K.F.; Basarab, R.M.; Kipper, M.S.; Halpern, S.E.; Ashburn, W.L.

    1984-05-01

    In-111-labeled leukocytes were administered to 13 patients on continuous ambulatory peritoneal dialysis in order to locate catheter-associated infections. Using a marker to indicate the catheter exit site, infections of the catheter tunnel were correctly identified prior to surgery in 4 patients with relapsing peritonitis and infections of the exit site were diagnosed in 5 out of 7 patients. The authors conclude that In-111-leukocyte scintigraphy appears to be accurate in diagnosing peritoneal infections of the dialysis catheter tunnel.

  11. Advantage of indium-111 leukocytes over ultrasound in imaging an infected renal cyst

    SciTech Connect

    Fortner, A.; Taylor, A. Jr.; Alazraki, N.; Datz, F.L.

    1986-07-01

    Indium-111-labeled leukocyte scanning is a highly sensitive and specific method of detecting abscesses. This report describes a patient with polycystic kidneys and a single infected cyst. Ultrasound could not determine which cyst was infected, but the infected cyst could be localized by (/sup 111/In)leukocyte imaging in conjunction with a (/sup 99m/Tc)DMSA renal scan. The two radionuclide studies were used to identify an infected renal cyst and direct ultrasound guided aspiration.

  12. Observing a fictitious stressful event: haematological changes, including circulating leukocyte activation.

    PubMed

    Mian, Rubina; Shelton-Rayner, Graham; Harkin, Brendan; Williams, Paul

    2003-03-01

    The aim of this study was to assess the effect of watching a psychological stressful event on the activation of leukocytes in healthy human volunteers. Blood samples were obtained from 32 healthy male and female subjects aged between 20 and 26 years before, during and after either watching an 83-minute horror film that none of the subjects had previously seen (The Texas Chainsaw Massacre, 1974) or by sitting quietly in a room (control group). Total differential cell counts, leukocyte activation as measured by the nitroblue tetrazolium (NBT) test, heart rate and blood pressure (BP) measurements were taken at defined time points. There were significant increases in peripheral circulating leukocytes, the number of activated circulating leukocytes, haemoglobin (Hb) concentration and haematocrit (Hct) in response to the stressor. These were accompanied by significant increases in heart rate, systolic and diastolic BP (P<0.05 from baseline). This is the first reported study on the effects of observing a psychologically stressful, albeit fictitious event on circulating leukocyte numbers and the state of leukocyte activation as determined by the nitrotetrazolium test.

  13. Chemokine-leukocyte interactions. The voodoo that they do so well.

    PubMed

    Taub, D D

    1996-12-01

    Leukocyte recruitment from the circulation into inflammatory tissues requires a series of soluble and cell-bound signals between the responding leukocyte and vascular endothelial barrier. Chemotactic factors are believed to be responsible for this selective adhesion and transmigration. A superfamily of small, soluble, structurally-related molecules called 'chemokines' have been identified and shown to selectively promote the rapid adhesion and chemotaxis of a variety of leukocyte subtypes both in vivo and in vitro. Chemokines are produced by almost every cell type in the body in response to a number of inflammatory signals, in particular those which activate leukocyte-endothelial cell interactions. These molecules also appear to play important roles in hematopoesis, cellular activation, and leukocyte effector functions. In addition, chemokines have been found in the tissues of a variety of disease states characterized by distinct leukocytic infiltrates, including rheumatoid arthritis, sepsis, atherosclerosis, asthma, psoriasis, ischemia/reperfusion injury, HIV replication, and a variety of pulmonary disease states. This review will primarily focus on the role of chemokines in cell adhesion and trafficking as well as their role as effector molecules.

  14. Dark chocolate consumption improves leukocyte adhesion factors and vascular function in overweight men.

    PubMed

    Esser, Diederik; Mars, Monica; Oosterink, Els; Stalmach, Angelique; Müller, Michael; Afman, Lydia A

    2014-03-01

    Flavanol-enriched chocolate consumption increases endothelium-dependent vasodilation. Most research so far has focused on flow-mediated dilation (FMD) only; the effects on other factors relevant to endothelial health, such as inflammation and leukocyte adhesion, have hardly been addressed. We investigated whether consumption of regular dark chocolate also affects other markers of endothelial health, and whether chocolate enrichment with flavanols has additional benefits. In a randomized double-blind crossover study, the effects of acute and of 4 wk daily consumption of high flavanol chocolate (HFC) and normal flavanol chocolate (NFC) on FMD, augmentation index (AIX), leukocyte count, plasma cytokines, and leukocyte cell surface molecules in overweight men (age 45-70 yr) were investigated. Sensory profiles and motivation scores to eat chocolate were also collected. Findings showed that a 4 wk chocolate intake increased FMD by 1%, which was paralleled by a decreased AIX of 1%, decreased leukocyte cell count, decreased plasma sICAM1 and sICAM3, and decreased leukocyte adhesion marker expression (P<0.05 for time effect), with no difference between HFC and NFC consumption. Flavanol enrichment did affect taste and negatively affected motivation to consume chocolate. This study provides new insights on how chocolate affects endothelial health by demonstrating that chocolate consumption, besides improving vascular function, also lowers the adherence capacity of leukocytes in the circulation.

  15. Immunosuppressive drugs affect high-mannose/hybrid N-glycans on human allostimulated leukocytes.

    PubMed

    Pocheć, Ewa; Bocian, Katarzyna; Ząbczyńska, Marta; Korczak-Kowalska, Grażyna; Lityńska, Anna

    2015-01-01

    N-glycosylation plays an important role in the majority of physiological and pathological processes occurring in the immune system. Alteration of the type and abundance of glycans is an element of lymphocyte differentiation; it is also common in the development of immune-mediated inflammatory diseases. The N-glycosylation process is very sensitive to different environmental agents, among them the pharmacological environment of immunosuppressive drugs. Some results show that high-mannose oligosaccharides have the ability to suppress different stages of the immune response. We evaluated the effects of cyclosporin A (CsA) and rapamycin (Rapa) on high-mannose/hybrid-type glycosylation in human leukocytes activated in a two-way mixed leukocyte reaction (MLR). CsA significantly reduced the number of leukocytes covered by high-mannose/hybrid N-glycans, and the synergistic action of CsA and Rapa led to an increase of these structures on the remaining leukocytes. This is the first study indicating that β1 and β3 integrins bearing high-mannose/hybrid structures are affected by Rapa and CsA. Rapa taken separately and together with CsA changed the expression of β1 and β3 integrins and, by regulating the protein amount, increased the oligomannose/hybrid-type N-glycosylation on the leukocyte surface. We suggest that the changes in the glycosylation profile of leukocytes may promote the development of tolerance in transplantation.

  16. Highly specific blockade of CCR5 inhibits leukocyte trafficking and reduces mucosal inflammation in murine colitis.

    PubMed

    Mencarelli, Andrea; Cipriani, Sabrina; Francisci, Daniela; Santucci, Luca; Baldelli, Franco; Distrutti, Eleonora; Fiorucci, Stefano

    2016-08-05

    Targeted disruption of leukocyte trafficking to the gut represents a promising approach for the treatment of inflammatory bowel diseases (IBDs). CCR5, the shared receptor for MIP1α and β and RANTES, is expressed by multiple leukocytes. Here, we aimed to determine the role of CCR5 in mediating leukocyte trafficking in models of colitis, and evaluate the therapeutic potential of maraviroc, an orally active CCR5 antagonist used in the treatment of CCR5-tropic HIV. Acute and chronic colitis were induced by administration of DSS or TNBS to wild-type and CCR5(-/-) mice or adoptive transfer of splenic naïve CD4(+) T-cells from wild type or CCR5(-/-) mice into RAG-1(-/-). CCR5 gene ablation reduced the mucosal recruitment and activation of CCR5-bearing CD4(+) and CD11b(+) leukocytes, resulting in profound attenuation of signs and symptoms of inflammation in the TNBS and transfer models of colitis. In the DSS/TNBS colitis and in the transfer model, maraviroc attenuated development of intestinal inflammation by selectively reducing the recruitment of CCR5 bearing leukocytes. In summary, CCR5 regulates recruitment of blood leukocytes into the colon indicating that targeting CCR5 may offer therapeutic options in IBDs.

  17. Highly specific blockade of CCR5 inhibits leukocyte trafficking and reduces mucosal inflammation in murine colitis

    PubMed Central

    Mencarelli, Andrea; Cipriani, Sabrina; Francisci, Daniela; Santucci, Luca; Baldelli, Franco; Distrutti, Eleonora; Fiorucci, Stefano

    2016-01-01

    Targeted disruption of leukocyte trafficking to the gut represents a promising approach for the treatment of inflammatory bowel diseases (IBDs). CCR5, the shared receptor for MIP1α and β and RANTES, is expressed by multiple leukocytes. Here, we aimed to determine the role of CCR5 in mediating leukocyte trafficking in models of colitis, and evaluate the therapeutic potential of maraviroc, an orally active CCR5 antagonist used in the treatment of CCR5-tropic HIV. Acute and chronic colitis were induced by administration of DSS or TNBS to wild-type and CCR5−/− mice or adoptive transfer of splenic naïve CD4+ T-cells from wild type or CCR5−/− mice into RAG-1−/−. CCR5 gene ablation reduced the mucosal recruitment and activation of CCR5-bearing CD4+ and CD11b+ leukocytes, resulting in profound attenuation of signs and symptoms of inflammation in the TNBS and transfer models of colitis. In the DSS/TNBS colitis and in the transfer model, maraviroc attenuated development of intestinal inflammation by selectively reducing the recruitment of CCR5 bearing leukocytes. In summary, CCR5 regulates recruitment of blood leukocytes into the colon indicating that targeting CCR5 may offer therapeutic options in IBDs. PMID:27492684

  18. Seasonal changes in cortisol sensitivity and glucocorticoid receptor affinity and number in leukocytes of coho salmon

    USGS Publications Warehouse

    Maule, Alec G.; Schreck, Carl B.; Sharpe, Cameron

    1993-01-01

    To determine if there were organ-specific changes in immune responses or immune-endocrine interaction, we monitored in vitro immune response, cortisol sensitivity and number and affinity of glucocorticoid receptors (GR) in leukocytes from freshwater-adapted juvenile coho salmon (Oncorhynchus kisutch) during the physiological changes that prepare them to enter the marine environment. During this period, absolute immune response declined, but splenic leukocytes generated more antibody-producing cells than did cells from anterior kidney. Splenic leukocytes were initially more sensitive to the suppressive effects of cortisol and had fewer GR than leukocytes from the anterior kidney. Leukocytes from the anterior kidney were initially insensitive to cortisol but developed sensitivity at about the same time as the dissociation constant and number of GR increased. In vitro incubation of anterior kidney leukocytes in cortisol altered GR variables when experiments were conducted during March through September but not during November through February. In some years, changes in GR or immune responses were correlated with plasma cortisol titers, but in other years there was no correlation. Thus, the exact relation between cortisol, GR and immune response in anadromous salmonids is unclear and other factors are involved.

  19. Diagnosis of osteomyelitis of the foot in diabetic patients: Value of 111In-leukocyte scintigraphy

    SciTech Connect

    Larcos, G.; Brown, M.L.; Sutton, R.T. )

    1991-09-01

    The noninvasive diagnosis of osteomyelitis of the foot in diabetic patients with currently available radiologic and radionuclide imaging techniques is often difficult. Recently, 111In-labeled leukocyte scintigraphy has been proposed as an attractive alternative. Accordingly, the authors retrospectively reviewed 51 111In-labeled leukocyte scans, 49 technetium-99m bone scans, and 49 plain radiographs obtained in 51 adults with diabetes in whom osteomyelitis of the foot was suspected. The sensitivity and specificity of these techniques were evaluated in all patients, as well as in a subgroup of 11 patients with neuroarthropathy. Results with 111In-labeled leukocyte scans were also examined in subsets of patients with soft-tissue ulcers (n = 35) and those receiving antibiotics during investigation (n = 20). Confirmation or exclusion of osteomyelitis was made surgically in 28 patients and clinically in 23. Fourteen patients had osteomyelitis. Bone scans were most sensitive (93%) but least specific (43%); plain radiographs were most specific (83%) but least sensitive (43%). 111In-labeled leukocyte scans were both sensitive (79%) and specific (78%), and remained useful in patients with neuroarthropathy, soft-tissue ulcers, and antibiotic treatment. Poor spatial resolution contributed to the false-negative and false-positive 111In-labeled leukocyte scans, suggesting that this technique should not be interpreted independent of other tests. 111In-labeled leukocyte scans are a valuable diagnostic tool for the diagnosis of pedal osteomyelitis in diabetic patients.

  20. Stress-induced enhancement of leukocyte trafficking into sites of surgery or immune activation

    NASA Astrophysics Data System (ADS)

    Viswanathan, Kavitha; Dhabhar, Firdaus S.

    2005-04-01

    Effective immunoprotection requires rapid recruitment of leukocytes into sites of surgery, wounding, infection, or vaccination. In contrast to immunosuppressive chronic stressors, short-term acute stressors have immunoenhancing effects. Here, we quantify leukocyte infiltration within a surgical sponge to elucidate the kinetics, magnitude, subpopulation, and chemoattractant specificity of an acute stress-induced increase in leukocyte trafficking to a site of immune activation. Mice acutely stressed before sponge implantation showed 200-300% higher neutrophil, macrophage, natural killer cell, and T cell infiltration than did nonstressed animals. We also quantified the effects of acute stress on lymphotactin- (LTN; a predominantly lymphocyte-specific chemokine), and TNF-- (a proinflammatory cytokine) stimulated leukocyte infiltration. An additional stress-induced increase in infiltration was observed for neutrophils, in response to TNF-, macrophages, in response to TNF- and LTN, and natural killer cells and T cells in response to LTN. These results show that acute stress initially increases trafficking of all major leukocyte subpopulations to a site of immune activation. Tissue damage-, antigen-, or pathogen-driven chemoattractants subsequently determine which subpopulations are recruited more vigorously. Such stress-induced increases in leukocyte trafficking may enhance immunoprotection during surgery, vaccination, or infection, but may also exacerbate immunopathology during inflammatory (cardiovascular disease or gingivitis) or autoimmune (psoriasis, arthritis, or multiple sclerosis) diseases. chemokine | psychophysiological stress | surgical sponge | wound healing | lymphotactin

  1. Rho-Kinase Activation in Leukocytes Plays a Pivotal Role in Myocardial Ischemia/Reperfusion Injury

    PubMed Central

    Kitano, Katsunori; Usui, Soichiro; Ootsuji, Hiroshi; Takashima, Shin-ichiro; Kobayashi, Daisuke; Murai, Hisayoshi; Furusho, Hiroshi; Nomura, Ayano; Kaneko, Shuichi; Takamura, Masayuki

    2014-01-01

    The Rho/Rho-kinase pathway plays an important role in many cardiovascular diseases such as hypertension, atherosclerosis, heart failure, and myocardial infarction. Although previous studies have shown that Rho-kinase inhibitors reduce ischemia/reperfusion (I/R) injury and cytokine production, the role of Rho-kinase in leukocytes during I/R injury is not well understood. Mice were subjected to 30-min ischemia and reperfusion. Rho-kinase activity was significantly greater in leukocytes subjected to myocardial I/R compared to the sham-operated mice. Administration of fasudil, a Rho-kinase inhibitor, significantly reduced the I/R-induced expression of the proinflammatory cytokines interleukin (IL)-6, C-C motif chemoattractant ligand 2 (CCL2), and tumor necrosis factor (TNF)-α, in leukocytes, compared with saline as the vehicle. Furthermore, fasudil decreased I/R-induced myocardial infarction/area at risk (IA) and I/R-induced leukocyte infiltration in the myocardium. Interestingly, IA in fasudil-administered mice with leukocyte depletion was similar to that in fasudil-administered mice. I/R also resulted in remarkable increases in the mRNA expression levels of the proinflammatory cytokines TNF-α, IL-6, and CCL2 in the heart. Inhibition of Rho-kinase activation in leukocytes has an important role in fasudil-induced cardioprotective effects. Hence, inhibition of Rho-kinase may be an additional therapeutic intervention for the treatment of acute coronary syndrome. PMID:24638037

  2. Cell-stiffness-induced mechanosignaling - a key driver of leukocyte transendothelial migration.

    PubMed

    Schaefer, Antje; Hordijk, Peter L

    2015-07-01

    The breaching of cellular and structural barriers by migrating cells is a driving factor in development, inflammation and tumor cell metastasis. One of the most extensively studied examples is the extravasation of activated leukocytes across the vascular endothelium, the inner lining of blood vessels. Each step of this leukocyte transendothelial migration (TEM) process is regulated by distinct endothelial adhesion receptors such as the intercellular adhesion molecule 1 (ICAM1). Adherent leukocytes exert force on these receptors, which sense mechanical cues and transform them into localized mechanosignaling in endothelial cells. In turn, the function of the mechanoreceptors is controlled by the stiffness of the endothelial cells and of the underlying substrate representing a positive-feedback loop. In this Commentary, we focus on the mechanotransduction in leukocytes and endothelial cells, which is induced in response to variations in substrate stiffness. Recent studies have described the first key proteins involved in these mechanosensitive events, allowing us to identify common regulatory mechanisms in both cell types. Finally, we discuss how endothelial cell stiffness controls the individual steps in the leukocyte TEM process. We identify endothelial cell stiffness as an important component, in addition to locally presented chemokines and adhesion receptors, which guides leukocytes to sites that permit TEM.

  3. Differential Alterations in Host Peripheral Polymorphonuclear Leukocyte Chemiluminescence During the Course of Bacterial and Viral Infections

    PubMed Central

    McCarthy, James P.; Bodroghy, Robert S.; Jahrling, Peter B.; Sobocinski, Philip Z.

    1980-01-01

    Previous studies have shown that stimulation of the oxidative metabolism in polymorphonuclear leukocytes (PMN) by in vitro phagocytosis of various microorganisms results in photon emission, termed chemiluminescence (CL). Studies were conducted to determine whether bacterial and viral infections induce enhanced basal endogenous host peripheral PMN CL in the absence of in vitro phagocytic stimulation. Nonimmune rats and guinea pigs as well as immune rats were inoculated with various doses (105 to 107) of live vaccine strain Francisella tularensis (per 100 g of body weight). In addition, nonimmune guinea pigs were inoculated with 40,000 plaque-forming units of Pichinde virus. Luminol-assisted endogenous PMN CL was measured at various time intervals after inoculation of microorganisms. Enhanced endogenous PMN CL was detected as early as the appearance of fever (12 h) in nonimmune animals infected with F. tularensis. Addition of sodium azide, N-ethylmaleimide, superoxide dismutase, or catalase to the CL reaction mixture containing PMN from infected animals significantly decreased the CL response. Immune rats challenged with F. tularensis exhibited resistance to infection and a decreased PMN CL compared with nonimmune rats 24 and 48 h after inoculation. However, the CL response from immune rats was significantly elevated, compared with control values. In contrast to the results obtained with the model bacterial infection, PMN isolated from guinea pigs inoculated with Pichinde virus failed to exhibit enhanced CL, compared with controls, despite significant viremia and fever. Results suggest that enhanced endogenous CL during bacterial infection occurs through mechanisms involving increased PMN oxidative metabolism and the subsequent generation of microbicidal forms of oxygen. Further, measurement of endogenous PMN CL may have diagnostic and prognostic value in infectious diseases. PMID:7228389

  4. Porcine mononuclear leukocyte nuclear thyroid hormone receptors: Effects of cold exposure on receptor kinetics

    SciTech Connect

    D'Alesandro, M.; Reed, L.; Malik, M.; Quesada, M.; Hesslink, R.; Castro, S.; Homer, L.; Young, B. Univ. of Alberta, Edmonton )

    1991-03-11

    Changes in kinetic characteristics of the triiodothyronine (T{sub 3}) receptor may be a mechanism involved in the thermoregulatory action of T{sub 3} at the nuclear level. To study this, the authors analyzed changes in T{sub 3} nuclear receptor kinetics in cold exposed swine and compared them with similar animals housed at thermoneutral temperature. Receptors were from isolated nuclear extracts of circulating mononuclear leukocytes (MNL). Scatchard analysis indicates the presence of a single class of binding sites. The authors were unable to detect differences in the equilibrium dissociation constant (Kd) or the maximum binding capacity (MBC, fmol/up DNA) between the two groups. The Kd for T{sub 3} in the control group was 1.17 {plus minus} 0.11 nmol/L and 1.25 {plus minus} 0.19 nmol/L in the cold exposed group. The MBC was 0.43 {plus minus} 0.04 fmol/ug DNA in the control group and 0.40 {plus minus} 0.06 fmol/L in the cold exposed group. In competition studies using thyroid hormone analogues, 10{sup {minus}7} M reverse T{sub 3} and 3,5-diiodothyronine resulted in approximately 50% displacement from the porcine receptor. TRIAC and L-T{sub 4} had no effect at 10{sup {minus}7} M. The porcine values for both Kd and MBC are similar to those previously reported for human MNL. Although T{sub 3} production and serum T{sub 3} values in the cold exposed group are nearly double the control group (Reed et al., FASEB 1991), continuous short-term cold exposure had no significant effect on MNL nuclear T{sub 3} receptor kinetics.

  5. Relative Telomere Repeat Mass in Buccal and Leukocyte-Derived DNA

    PubMed Central

    Finnicum, Casey T.; Dolan, Conor V.; Willemsen, Gonneke; Weber, Zachary M.; Petersen, Jason L.; Beck, Jeffrey J.; Codd, Veryan; Boomsma, Dorret I.; Davies, Gareth E.; Ehli, Erik A.

    2017-01-01

    Telomere length has garnered interest due to the potential role it may play as a biomarker for the cellular aging process. Telomere measurements obtained from blood-derived DNA are often used in epidemiological studies. However, the invasive nature of blood draws severely limits sample collection, particularly with children. Buccal cells are commonly sampled for DNA isolation and thus may present a non-invasive alternative for telomere measurement. Buccal and leukocyte derived DNA obtained from samples collected at the same time period were analyzed for telomere repeat mass (TRM). TRM was measured in buccal-derived DNA samples from individuals for whom previous TRM data from blood samples existed. TRM measurement was performed by qPCR and was normalized to the single copy 36B4 gene relative to a reference DNA sample (K562). Correlations between TRM from blood and buccal DNA were obtained and also between the same blood DNA samples measured in separate laboratories. Using the classical twin design, TRM heritability was estimated (N = 1892, MZ = 1044, DZ = 775). Buccal samples measured for TRM showed a significant correlation with the blood-1 (R = 0.39, p < 0.01) and blood-2 (R = 0.36, p < 0.01) samples. Sex and age effects were observed within the buccal samples as is the norm within blood-derived DNA. The buccal, blood-1, and blood-2 measurements generated heritability estimates of 23.3%, 47.6% and 22.2%, respectively. Buccal derived DNA provides a valid source for the determination of TRM, paving the way for non-invasive projects, such as longitudinal studies in children. PMID:28125671

  6. Respiratory burst facilitates the digestion of Escherichia coli killed by polymorphonuclear leukocytes.

    PubMed Central

    Weiss, J; Kao, L; Victor, M; Elsbach, P

    1987-01-01

    We examined factors that may limit degradation of bacterial protein of Escherichia coli S15 killed by polymorphonuclear leukocytes (PMN). Both human and rabbit PMN degraded up to 40% of [14C]amino acid-labeled protein of ingested and killed E. coli in 2 h as determined by loss of acid-precipitable radioactivity. In contrast, equally bactericidal broken-PMN preparations or isolated granules degraded only about 10% of bacterial protein regardless of pH. To determine whether activation of the respiratory burst contributes to digestion, we compared degradation by intact PMN in room air and under N2. Depletion of O2 by N2 flushing had no effect on the bactericidal activity of either human or rabbit PMN but reduced degradation by approximately 50%. Protein degradation during phagocytosis was also reduced in the presence of cyanide or azide, inhibitors of myeloperoxidase (MPO). PMN of two patients with chronic granulomatous disease ingested and killed E. coli S15 as well as did normal PMN but degraded bacterial protein as did normal PMN incubated under N2. The low degradative activity of PMN disrupted by sonication could be raised to nearly the level of intact PMN incubated in room air by preincubation of the PMN with 10(-7) M formyl-methionyl-leucyl-phenylalanine (fMLP) before sonication and by pretreatment of E. coli with MPO. Depletion of O2 or chloride during these preincubations with formyl-methionyl-leucyl-phenylalanine respectively, virtually abolished and markedly diminished stimulation of bacterial protein degradation. We conclude that enhanced MPO-mediated O2 metabolism of intact PMN plays a role in the digestion of killed E. coli. PMID:3305366

  7. Involvement of CD44 in leukocyte recruitment to the rat testis in experimental autoimmune orchitis.

    PubMed

    Guazzone, V A; Denduchis, B; Lustig, L

    2005-05-01

    Experimental autoimmune orchitis (EAO) is characterized by an interstitial mononuclear cell infiltrate and a severe lesion of the seminiferous tubules with germ cells that undergo apoptosis and sloughing. The aim of this study was to determine the role of CD44 in testicular leukocyte recruitment in EAO. The biological functions of CD44 have been attributed to the generation of a functionally active hyaluronan-binding phenotype. Orchitis was induced in Sprague-Dawley adult rats by active immunization with an emulsion of testicular homogenate and complete Freund's adjuvant using Bordetella pertussis as co-adjuvant. Control rats (C) injected with saline and adjuvants and normal (N) untreated rats were also studied. CD44 expression was analyzed by flow cytometry in peripheral blood mononuclear cells (PBMC) and lymph node cells isolated from rats at different times after the first immunization. We observed an increase in the mean fluorescence intensity of both samples in the C and experimental (E) groups only after the immunization period. A significant decrease in percentage of CD44+PBMC and in mean fluorescence intensity was observed in rats with orchitis compared with the C group. By in vitro hyaluronic acid-binding assay we demonstrated that the percentage of PBMC adhesion was higher in the E group compared with the C and N groups. By immunohistochemistry, we observed a significant increase in the number of CD44+cells in the testicular interstitium of rats with severe orchitis compared with the N and C groups. These results suggested that the CD44 molecule is involved in the homing of lymphomonocytes into the testes of rats with autoimmune orchitis.

  8. Production of reactive oxygen species by man-made vitreous fibres in human polymorphonuclear leukocytes.

    PubMed

    Ruotsalainen, M; Hirvonen, M R; Luoto, K; Savolainen, K M

    1999-06-01

    Human polymorphonuclear leukocytes (PMNL) or erythrocytes, isolated from human blood, were exposed to graded doses of asbestos (chrysotile), quartz, or man-made vitreous fibres (MMVF), i.e. refractory ceramic fibres (RCF), glasswool, or rockwool fibres. None of the MMVF affected either the viability of PMNL, as measured by trypan blue exclusion test, or induced haemolysis, whereas the positive controls, quartz and chrysotile, dose-dependently induced haemolysis in PMNL. MMVF did not increase the release of lactate dehydrogenase (LDH) from the PMNL, whereas the positive controls, chrysotile and quartz, induced a marked and dose-dependent release of LDH. When PMNL were exposed to MMVF, some of the fibre types slightly increased the levels of free intracellular calcium ([Ca2+]i) within the cells in a manner similar to that induced by chrysotile or quartz. All MMVF induced a dose-dependent production of reactive oxygen species (ROS) in PMNL, with RCF-induced production of ROS being the most marked. Production of ROS by MMVF seemed to depend on the availability of extracellular calcium because it could be attenuated with a Ca2+ channel blocker, verapamil, or a Ca2+ chelating agent, EGTA. Production of ROS may be a common pathway through which PMNL respond to MMVF-induced cell activation, but alterations of levels of free intracellular Ca2+ do not seem to be an absolute prerequisite for this effect. Fibre length seemed not to be an important factor in affecting the ability of MMVF to induce ROS production in PMNL. However, the balance between different elements in the fibre seemed importantly to affect the biological activity of a fibre.

  9. Release of platelet activating factor in rabbits with antibody-mediated injury of the lung: the role of leukocytes and of pulmonary endothelial cells.

    PubMed

    Camussi, G; Pawlowski, I; Bussolino, F; Caldwell, P R; Brentjens, J; Andres, G

    1983-10-01

    This paper describes the release of platelet-activating factor (PAF) into the circulation of rabbits with acute pulmonary injury induced by antibody reacting with pulmonary endothelium. Eight rabbits were injected i.v. with 2 mg/kg of body weight of goat anti-rabbit lung angiotensin-converting enzyme gamma-globulin (GtARbACE). All animals developed acute pneumonitis, characterized by severe endothelial damage, accumulation of polymorphonuclear leukocytes (PMN) and platelets (Plt) in the lumina of alveolar capillaries, and deposits of goat IgG and rabbit C3 along alveolar capillary walls. Six of the rabbits died from acute pulmonary edema. PAF was detected in the plasma of all animals within 5 min after injection of GtARbACE. Five other rabbits were depleted of leukocytes by nitrogen mustard and then injected with 2 mg/kg of body weight of GtARbACE. In three of these rabbits release of PAF was demonstrated, though in amounts smaller than in non-leukocyte-depleted rabbits; all three animals died from pulmonary edema. After injection of 0.03 mg/kg of body weight of GtARbACE in six additional rabbits, three of them leukocyte-depleted, small amounts of PAF were detected in the circulation. None of these six rabbits died of pulmonary edema. PAF release was not observed in ten rabbits injected i.v. with 2 or 0.03 mg/kg of body weight of normal goat gamma-globulin. In separate experiments in vitro, incubation of isolated lung or thoracic aorta with GtARbACE resulted in deposits of goat IgG along endothelia and significant release of PAF. PAF was also released from endothelial cells removed from thoracic aorta by cellulose acetate paper and then incubated with GtARbACE. When segments of thoracic aorta were stripped of endothelium and then incubated with GtARbACE, PAF release could not be shown. The data obtained are consistent with the interpretation that PAF released into the circulation after binding of GtARbACE to the endothelia of lung and aorta originates from

  10. Thyrotropin Receptor Epitope and Human Leukocyte Antigen in Graves’ Disease

    PubMed Central

    Inaba, Hidefumi; De Groot, Leslie J.; Akamizu, Takashi

    2016-01-01

    Graves’ disease (GD) is an organ-specific autoimmune disease, and thyrotropin (TSH) receptor (TSHR) is a major autoantigen in this condition. Since the extracellular domain of human TSHR (TSHR-ECD) is shed into the circulation, TSHR-ECD is a preferentially immunogenic portion of TSHR. Both genetic factors and environmental factors contribute to development of GD. Inheritance of human leukocyte antigen (HLA) genes, especially HLA-DR3, is associated with GD. TSHR-ECD protein is endocytosed into antigen-presenting cells (APCs), and processed to TSHR-ECD peptides. These peptide epitopes bind to HLA-class II molecules, and subsequently the complex of HLA-class II and TSHR-ECD epitope is presented to CD4+ T cells. The activated CD4+ T cells secrete cytokines/chemokines that stimulate B-cells to produce TSAb, and in turn hyperthyroidism occurs. Numerous studies have been done to identify T- and B-cell epitopes in TSHR-ECD, including (1) in silico, (2) in vitro, (3) in vivo, and (4) clinical experiments. Murine models of GD and HLA-transgenic mice have played a pivotal role in elucidating the immunological mechanisms. To date, linear or conformational epitopes of TSHR-ECD, as well as the molecular structure of the epitope-binding groove in HLA-DR, were reported to be related to the pathogenesis in GD. Dysfunction of central tolerance in the thymus, or in peripheral tolerance, such as regulatory T cells, could allow development of GD. Novel treatments using TSHR antagonists or mutated TSHR peptides have been reported to be effective. We review and update the role of immunogenic TSHR epitopes and HLA in GD, and offer perspectives on TSHR epitope specific treatments. PMID:27602020

  11. Reduced Arylsulfatase B Activity in Leukocytes from Cystic Fibrosis Patients

    PubMed Central

    Sharma, Girish; Burke, Jenifer; Bhattacharyya, Sumit; Sharma, Neha; Katyal, Shivani; Park, R. Lucy; Tobacman, Joanne

    2013-01-01

    Summary The enzyme Arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) removes 4-sulfate groups from chondroitin-4-sulfate and dermatan sulfate and is required for the degradation of these sulfated glycosaminoglycans (GAGs). Since these GAGs accumulate in patients with Cystic Fibrosis (CF), we investigated the activity of ARSB in leukocytes of patients with CF, to consider if reduced activity of ARSB might contribute to the pathophysiology of CF. Previous cell-based experiments had demonstrated that when the deficiency of the cystic fibrosis transmembrane regulator (CFTR) was corrected in bronchial epithelial cells, the ARSB activity increased significantly. De-identified, citrated blood samples were collected from 16 children with cystic fibrosis and 31 control subjects, seen in the Pediatric Clinic at Rush University Medical Center. Polymorphonuclear (PMN) and mononuclear cell (MC) populations were separated by density gradient, and blinded determinations of ARSB activity were performed using the exogenous substrate 4-methylumbilliferyl sulfate. Interleukin-6 was measured in the plasma samples by ELISA. ARSB activity was significantly less in the PMN and MC from the CF patients than controls (p<0.0001, unpaired t-test, two-tailed). Interleukin-6 levels in plasma were significantly greater in the CF population (p<0.001). Mean age, age range, and male:female ratio of CF patients and controls were similar, and no association of ARSB activity with age, gender, or CFTR genotype was evident. Since recombinant human ARSB is used successfully for replacement therapy in Mucopolysaccharidosis VI, it may be useful to restore ARSB activity to normal levels and increase degradation of sulfated GAGs in CF patients. PMID:22550062

  12. Preparation and application of an innovative thrombocyte/leukocyte-enriched plasma to promote tissue repair in chelonians.

    PubMed

    Di Ianni, Francesco; Merli, Elisa; Burtini, Francesca; Conti, Virna; Pelizzone, Igor; Di Lecce, Rosanna; Parmigiani, Enrico; Squassino, Gian Paolo; Del Bue, Maurizio; Lucarelli, Enrico; Ramoni, Roberto; Grolli, Stefano

    2015-01-01

    Platelet concentrates are widely used in mammalian regenerative medicine to improve tissue healing. Chelonians (Testudines) would benefit from the application of thrombocyte preparations to regenerate damaged tissues, since traumatic injuries are leading causes of morbidity and mortality for both wild-living and domesticated animals. The aim of this study was to establish a protocol that optimized the recovery of the thrombocytes from blood samples and to show the efficacy of thrombocyte-enriched plasma in chelonians. Peripheral blood samples were obtained from Testudo spp. (n = 12) and Trachemys scripta elegans (n = 10). Blood cells were fractionated by sodium diatrizoate-sodium polysucrose density gradient using a two-step centrifugation protocol. Thrombocytes and leukocytes were isolated and resuspended to obtain thrombocyte-leucocyte rich plasma (TLRP). The mean recovery of leukocytes and thrombocytes was 48.9% (±4.0 SEM, n = 22) of the whole blood cell content. No statistically significant difference was observed between blood samples collected from different turtle species. The ability of TLRP to form a gel was evaluated by adding variable concentrations of calcium gluconate at room temperature and at 37°C. A reliable and consistent clotting of the TLRP was obtained in glass tubes and dishes by adding 5-20% v/v of a 100 mg/ml solution of calcium gluconate. Furthermore, in order to test the clinical efficacy of TLRP, a preliminary evaluation was performed on four turtles (Testudo spp.) with traumatic injuries. In all the four animals, a successful clinical outcome was observed. The results demonstrated that a thrombocyte-enriched plasma, comparable to mammalian platelet rich plasma, can be prepared from chelonian blood samples. Furthermore, although the low number of cases presented does not allow definitive conclusions from a clinical point of view, their outcome suggests that TLRP application could be further investigated to improve the healing process of

  13. Preparation and Application of an Innovative Thrombocyte/Leukocyte-Enriched Plasma to Promote Tissue Repair in Chelonians

    PubMed Central

    Di Ianni, Francesco; Merli, Elisa; Burtini, Francesca; Conti, Virna; Pelizzone, Igor; Di Lecce, Rosanna; Parmigiani, Enrico; Squassino, Gian Paolo; Del Bue, Maurizio; Lucarelli, Enrico; Ramoni, Roberto; Grolli, Stefano

    2015-01-01

    Platelet concentrates are widely used in mammalian regenerative medicine to improve tissue healing. Chelonians (Testudines) would benefit from the application of thrombocyte preparations to regenerate damaged tissues, since traumatic injuries are leading causes of morbidity and mortality for both wild-living and domesticated animals. The aim of this study was to establish a protocol that optimized the recovery of the thrombocytes from blood samples and to show the efficacy of thrombocyte-enriched plasma in chelonians. Peripheral blood samples were obtained from Testudo spp. (n = 12) and Trachemys scripta elegans (n = 10). Blood cells were fractionated by sodium diatrizoate-sodium polysucrose density gradient using a two-step centrifugation protocol. Thrombocytes and leukocytes were isolated and resuspended to obtain thrombocyte-leucocyte rich plasma (TLRP). The mean recovery of leukocytes and thrombocytes was 48.9% (±4.0 SEM, n = 22) of the whole blood cell content. No statistically significant difference was observed between blood samples collected from different turtle species. The ability of TLRP to form a gel was evaluated by adding variable concentrations of calcium gluconate at room temperature and at 37°C. A reliable and consistent clotting of the TLRP was obtained in glass tubes and dishes by adding 5-20% v/v of a 100 mg/ml solution of calcium gluconate. Furthermore, in order to test the clinical efficacy of TLRP, a preliminary evaluation was performed on four turtles (Testudo spp.) with traumatic injuries. In all the four animals, a successful clinical outcome was observed. The results demonstrated that a thrombocyte-enriched plasma, comparable to mammalian platelet rich plasma, can be prepared from chelonian blood samples. Furthermore, although the low number of cases presented does not allow definitive conclusions from a clinical point of view, their outcome suggests that TLRP application could be further investigated to improve the healing process of

  14. Polymeric capsule-cushioned leukocyte cell membrane vesicles as a biomimetic delivery platform

    NASA Astrophysics Data System (ADS)

    Gao, Changyong; Wu, Zhiguang; Lin, Zhihua; Lin, Xiankun; He, Qiang

    2016-02-01

    We report a biomimetic delivery of microsized capsule-cushioned leukocyte membrane vesicles (CLMVs) through the conversion of freshly reassembled leukocyte membrane vesicles (LMVs), including membrane lipids and membrane-bound proteins onto the surface of layer-by-layer assembled polymeric multilayer microcapsules. The leukocyte membrane coating was verified by using electron microscopy, a quartz crystal microbalance, dynamic light scattering, and confocal laser scanning microscopy. The resulting CLMVs have the ability to effectively evade clearance by the immune system and thus prolong the circulation time in mice. Moreover, we also show that the right-side-out leukocyte membrane coating can distinctly improve the accumulation of capsules in tumor sites through the molecular recognition of membrane-bound proteins of CLMVs with those of tumor cells in vitro and in vivo. The natural cell membrane camouflaged polymeric multilayer capsules with the immunosuppressive and tumor-recognition functionalities of natural leukocytes provide a new biomimetic delivery platform for disease therapy.We report a biomimetic delivery of microsized capsule-cushioned leukocyte membrane vesicles (CLMVs) through the conversion of freshly reassembled leukocyte membrane vesicles (LMVs), including membrane lipids and membrane-bound proteins onto the surface of layer-by-layer assembled polymeric multilayer microcapsules. The leukocyte membrane coating was verified by using electron microscopy, a quartz crystal microbalance, dynamic light scattering, and confocal laser scanning microscopy. The resulting CLMVs have the ability to effectively evade clearance by the immune system and thus prolong the circulation time in mice. Moreover, we also show that the right-side-out leukocyte membrane coating can distinctly improve the accumulation of capsules in tumor sites through the molecular recognition of membrane-bound proteins of CLMVs with those of tumor cells in vitro and in vivo. The natural

  15. Dynamic changes in circulating leukocytes during the induction of equine laminitis with black walnut extract.

    PubMed

    Hurley, David J; Parks, Robert J; Reber, Adrian J; Donovan, Douglas C; Okinaga, Tatsuyuki; Vandenplas, Michel L; Peroni, John F; Moore, James N

    2006-04-15

    Administration of black walnut heartwood extract (BWHE) via nasogastric tube induces acute laminitis in horses. However, the processes responsible for the development of laminitis, including laminitis induced with BWHE, remain unclear. The results of recent studies indicate that administration of BWHE initiates an inflammatory response in the laminar tissues and that this response may be due to extravasation of activated leukocytes from the circulation. This study examines the effects of BWHE administration on the dynamics of circulating neutrophils and monocytes, and the capacity of blood leukocytes to produce radical oxygen species (ROS) over the time period from administration of BWHE to the development of lameness consistent with Obel grade I laminitis. Individual horses, free of pre-existing musculoskeletal disease, were administered either 6l of BWHE or an equal volume of water at time 0 (T=0). Blood samples were collected prior to dosing and at 1, 2, 3, 4, 6, 8, 10 and 12h after dosing, or until the onset of Obel grade I laminitis. For each sample, total leukocyte counts were determined followed by collection of buffy coats and removal of erythrocytes by hypotonic lysis. Leukocytes were either fixed for flow cytometric assessment of differential counts or maintained in culture to measure endogenous and phorbol ester-induced production of ROS. At each sample time, the number of cells recovered and the flow cytometric differential counts were compared with corresponding total leukocyte counts determined by the Clinical Pathology laboratory. Horses administered BWHE had a significant reduction in circulating leukocytes at 3-4 h relative to values for horses administered the same volume of water. Horses that developed Obel grade I laminitis had a significant reduction in circulating leukocytes when compared to values for horses administered BWHE that did not become lame. Flow cytometric analysis revealed a consistent decrease in the total number of monocytes

  16. Altered expression of adhesion molecules on peripheral blood leukocytes in feline infectious peritonitis.

    PubMed

    Olyslaegers, Dominique A J; Dedeurwaerder, Annelike; Desmarets, Lowiese M B; Vermeulen, Ben L; Dewerchin, Hannah L; Nauwynck, Hans J

    2013-10-25

    Feline infectious peritonitis (FIP) is a fatal, coronavirus-induced systemic disease in domestic and wild felids. The pathology associated with FIP (multifocal granulomatous vasculitis) is considered to be elicited by exaggerated activation and subsequent extravasation of leukocytes. As changes in the expression of adhesion molecules on circulating leukocytes precede their margination and emigration, we reasoned that the expression of leukocyte adhesion molecules may be altered in FIP. In present study, the expression of principal adhesion molecules involved in leukocyte transmigration (CD15s, CD11a, CD11b, CD18, CD49d, and CD54) on peripheral blood leukocytes from cats with naturally occurring FIP (n=15) and controls (n=12) was quantified by flow cytometry using a formaldehyde-based rapid leukocyte preparation technique. T- and B-lymphocytes from FIP patients exhibit higher expression of both subunits (CD11a and CD18) composing the β2 integrin lymphocyte function-associated antigen (LFA)-1. In addition, the expression of the α4 subunit (CD49d) of the β1 integrin very late antigen (VLA)-4 was elevated on B-lymphocytes from FIP patients. The expression of CD11b and CD18, that combine to form the β2 integrin macrophage-1 antigen (Mac-1), was elevated on monocytes, whereas the density of CD49d was reduced on this population in FIP. Granulocytes of FIP cats displayed an increased expression of the α chain of Mac-1 (CD11b). These observations suggest that leukocytes from FIP patients show signs of systemic activation causing them to extravasate into surrounding tissues and ultimately contribute to pyogranuloma formation seen in FIP.

  17. The effect of shear on in vitro platelet and leukocyte material-induced activation.

    PubMed

    Chang, Xiaojian; Gorbet, Maud

    2013-09-01

    The failure to understand the mechanisms of biomaterial-associated thrombosis prevents us from improving the blood compatibility of stents and mechanical heart valves. Blood-material interactions trigger a complex series of events and anticoagulant and anti-platelet therapies are needed to reduce the risks of thrombotic complications with most cardiovascular materials. While material interaction with platelets has been widely studied, little is currently known on material-induced leukocyte activation in the presence of shear. In vitro experiments were performed to assess the effect of flow on blood cell activation induced by medical grade metals, ST316L and TiAl6V4. Blood was circulated in flow chambers preloaded with or without metal wires at shear rates of 100, 500, and 1500 s⁻¹. Platelet and leukocyte activation, leukocyte-platelet aggregation, and tissue factor expression on monocytes were measured by flow cytometry. Metal surfaces were characterized by scanning electron microscopy. Under physiological shear rates, no significant platelet microparticle formation was observed. However, significant CD11b up-regulation, leukocyte-platelet aggregates, and tissue factor expression were observed at 100 s⁻¹. As shear rate increased to 1500 s⁻¹, leukocyte activation reduced to control values. TiAl6V4-induced leukocyte activation was generally lower than that of ST316L. Adhesion significantly decreased with increasing shear rate to 1500 s⁻¹. In blood, increase within physiological shear rates led to a significant reduction in in vitro material-induced leukocyte activation, suggesting that difference between material biocompatibility may be better identified at low shear rates or under pathological shear conditions.

  18. Leukocyte Cell-Derived Chemotaxin 2-Associated Amyloidosis: A Recently Recognized Disease with Distinct Clinicopathologic Characteristics.

    PubMed

    Nasr, Samih H; Dogan, Ahmet; Larsen, Christopher P

    2015-11-06

    Amyloidosis derived from leukocyte cell-derived chemotaxin 2 is a recently recognized form of amyloidosis, and it has already been established as a frequent form of systemic amyloidosis in the United States, with predominant involvement of kidney and liver. The disease has a strong ethnic bias, affecting mainly Hispanics (particularly Mexicans). Additional ethnic groups prone to develop amyloidosis derived from leukocyte cell-derived chemotaxin 2 include Punjabis, First Nations people in British Columbia, and Native Americans. Most patients are elderly who present with chronic renal insufficiency and bland urinary sediment. Proteinuria is variable, being absent altogether in about one third of patients. Liver involvement is frequently an incidental finding. Amyloidosis derived from leukocyte cell-derived chemotaxin 2 deposits shows a characteristic distribution: in the kidney, there is consistent involvement of cortical interstitium, whereas in the liver, there is a preferential involvement of periportal and pericentral vein regions. Concurrent renal disease is frequent, with diabetic nephropathy and IgA nephropathy being the most common. Patient survival is excellent, likely because of the rarity of cardiac involvement, whereas renal survival is guarded, with a median renal survival of 62 months in those without concurrent renal disease. There is currently no efficacious therapy for amyloidosis derived from leukocyte cell-derived chemotaxin 2 amyloidosis. Renal transplantation seems to be a reasonable treatment for patients with advanced renal failure, although the disease may recur in the allograft. The pathogenesis of amyloidosis derived from leukocyte cell-derived chemotaxin 2 amyloidosis has not yet been elucidated. It could be a result of leukocyte cell-derived chemotaxin 2 overexpression by hepatocytes either constitutively (controlled by yet-uncharacterized genetic defects) or secondary to hepatocellular damage. It is critical not to misdiagnose amyloidosis

  19. Cognitive Change during the Life Course and Leukocyte Telomere Length in Late Middle-Aged Men

    PubMed Central

    Rask, Lene; Bendix, Laila; Harbo, Maria; Fagerlund, Birgitte; Mortensen, Erik L.; Lauritzen, Martin J.; Osler, Merete

    2016-01-01

    Importance: Cognitive skills are known to decline through the lifespan with large individual differences. The molecular mechanisms for this decline are incompletely understood. Although leukocyte telomere length provides an index of cellular age that predicts the incidence of age-related diseases, it is unclear whether there is an association between cognitive decline and leukocyte telomere length. Objective: To examine the association between changes in cognitive function during adult life and leukocyte telomere length after adjusting for confounding factors such as education, mental health and life style. Design, Setting, and Participants: Two groups of men with negative (n = 97) and positive (n = 93) change in cognitive performance were selected from a birth cohort of 1985 Danish men born in 1953. Cognitive performance of each individual was assessed at age ~20 and 56 years. Leukocyte telomere length at age ~58 was measured using qPCR. Linear regression models were used to investigate the association between cognitive function and leukocyte telomere length. Results: Men with negative change in cognitive performance during adult life had significantly shorter mean leukocyte telomere length than men with positive change in cognitive performance (unadjusted difference β = −0.09, 95% CI −0.16 to −0.02, p = 0.02). This association remained significant after adjusting for smoking, alcohol consumption, leisure time activity, body mass index (BMI) and cholesterol (adjusted difference β = −0.09, 95% CI −0.17 to −0.01, p = 0.02) but was non-significant after adjusting for smoking, alcohol consumption, leisure time activity, BMI, cholesterol, current cognitive function, depression and education (adjusted difference β = −0.07, 95% CI −0.16 to −0.01, p = 0.08). Conclusion and Relevance: Preclinical cognitive changes may be associated with leukocyte telomere length. PMID:28018213

  20. Relation between leukocyte count, adiposity, and cardiorespiratory fitness in pubertal adolescents

    PubMed Central

    Tenório, Thiago Ricardo dos Santos; Farah, Breno Quintella; Ritti-Dias, Raphael Mendes; Botero, João Paulo; Brito, Daniel Calado; de Moura, Patrícia Muniz Mendes Freire; do Prado, Wagner Luiz

    2014-01-01

    Objective To compare the total and differential leukocyte count in obese and normal-weight adolescents, and to verify their possible relations with cardiorespiratory fitness and adiposity indicators. Methods A cross-sectional study conducted with 139 adolescents (107 obese and 32 normal weight) aged between 13 and 18 years. Cardiorespiratory fitness was determined by direct gas analysis during an incremental treadmill test. Total leukocytes and subsets were estimated by flow cytometry. Body composition was assessed by dual-energy X-ray absorptiometry. The t-test for independent samples was used for comparison between groups. The relation between leukocytes, cardiorespiratory fitness and adiposity indicators was verified by Pearson’s correlation and multiple linear regression (adjusted for age and body mass index) tests. Results Obese adolescents had higher leukocyte (8.12±2.36u/L x 103; p=0.001), neutrophil (4.33±1.86u/L x 103; p=0.002), and monocyte (0.70±0.22u/L x 103; p=0.002) counts compared to the levels of normal weight subjects. After the necessary adjustments, cardiorespiratory fitness had a negative association with leukocytes, neutrophils, and monocytes in boys. Conclusion Obese adolescents had higher total and differential leucocyte count when compared to normal weight individuals. We also observed a weak positive association between adiposity and total leukocyte, monocyte, and neutrophil counts, and in boys, a negative association between cardiorespiratory fitness and total count of leukocytes, monocytes, and neutrophils. PMID:25628191

  1. Semiphysiological model for the time course of leukocytes after varying schedules of 5-fluorouracil in rats.

    PubMed

    Friberg, L E; Freijs, A; Sandström, M; Karlsson, M O

    2000-11-01

    Models of leukopenia after chemotherapy are mainly empirical. To increase the derived models' potential of mechanistic understanding and extrapolation, more physiologically based models are being developed. To date, presented models cannot characterize the often-observed rebound of leukocytes. Therefore, a model able to describe the transient decrease and rebound in leukocytes was developed. Three different dosing regimens of 5-fluorouracil were given to rats. One group received a single dose of 127 mg/kg. The other two groups received two and three injections of 63 mg/kg and 49 mg/kg, respectively, with a 2-day interval. Leukocyte counts were followed for 23 to 25 days after the first dose. Plasma concentrations were determined by high-performance liquid chromatography. Population pharmacokinetic and pharmacodynamic models were developed using NONMEM. 5-Fluorouracil showed one-compartment disposition with capacity-limited elimination. The 49-mg/kg dose injected on three occasions produced the lowest leukocyte count (28% of baseline) and the most prominent rebound of the schedules, despite the fact that the fractionated regimens produced only 52 to 56% of the area under the concentration-time curve from time 0 to infinity in the single-dose group. The final semiphysiological model included two 5-fluorouracil-sensitive and two -insensitive transit compartments as well as a compartment of circulating leukocytes. Second order rate constants from the transit compartments and a negative feedback from the circulating leukocytes to the input of the first sensitive compartment characterized the pronounced changes in leukocyte counts. A posterior predictive check as well as predictions into a new data set showed that our model could well predict the schedule-dependent leukopenic effects of 5-fluorouracil.

  2. A unique secreted adenovirus E3 protein binds to the leukocyte common antigen CD45 and modulates leukocyte functions.

    PubMed

    Windheim, Mark; Southcombe, Jennifer H; Kremmer, Elisabeth; Chaplin, Lucy; Urlaub, Doris; Falk, Christine S; Claus, Maren; Mihm, Janine; Braithwaite, Myles; Dennehy, Kevin; Renz, Harald; Sester, Martina; Watzl, Carsten; Burgert, Hans-Gerhard

    2013-12-10

    The E3 transcription unit of human adenoviruses (Ads) encodes immunomodulatory proteins. Interestingly, the size and composition of the E3 region differs considerably among Ad species, suggesting that distinct sets of immunomodulatory E3 proteins may influence their interaction with the human host and the disease pattern. However, to date, only common immune evasion functions of species C E3 proteins have been described. Here we report on the immunomodulatory activity of a species D-specific E3 protein, E3/49K. Unlike all other E3 proteins that act on infected cells, E3/49K seems to target uninfected cells. Initially synthesized as an 80- to 100-kDa type I transmembrane protein, E3/49K is subsequently cleaved, with the large ectodomain (sec49K) secreted. We found that purified sec49K exhibits specific binding to lymphoid cell lines and all primary leukocytes, but not to fibroblasts or epithelial cells. Consistent with this binding profile and the molecular mass, the sec49K receptor was identified as the cell surface protein tyrosine phosphatase CD45. Antibody-blocking studies suggested that sec49K binds to the membrane proximal domains present in all CD45 isoforms. Functional studies showed that sec49K can suppress the activation and cytotoxicity of natural killer cells as well as the activation, signaling, and cytokine production of T cells. Thus, we have discovered an adenovirus protein that is actively secreted and describe immunomodulatory activities of an E3 protein uniquely expressed by a single Ad species.

  3. Alimentary and respiratory tract lesions in eight medically fragile Holstein cattle with bovine leukocyte adhesion deficiency (BLAD).

    PubMed

    Ackermann, M R; Kehrli, M E; Laufer, J A; Nusz, L T

    1996-05-01

    Lesions in the alimentary tract were studied in eight medically fragile Holstein cattle homozygous for the bovine leukocyte adhesion deficiency (BLAD) allele as determined by polymerase chain reaction and restriction endonuclease analysis. These cattle received institutional medical care but died or were euthanatized because of chronic debilitation associated with diarrhea (6/8) and pneumonia (4/8). The six cattle with diarrhea had acute (n = 3) or chronic (n = 3) intestinal ulcers, but the other two remained relatively healthy for 3 years and did not develop intestinal tract ulcers. Ulcerated areas were present in the small intestine in six animals, and two of these also had ulcers in the large intestine. Ulcers were covered by thick exudates that, in chronic lesions, partially occluded the intestinal lumen. Intramural and serosal fibrosis also contributed to lumen constriction. Pseudomonas aeruginosa was isolated from the intestine of four cattle. Bovine viral disease virus and Salmonella were not isolated from the five cattle that were tested. Respiratory tract lesions consisted of dense infiltrates of neutrophils in bronchi, bronchioles, and alveoli. This study suggests that intestinal lesions are integral to the demise of BLAD cattle that receive intensive medical care and that neutrophils do infiltrate the lung and enter airway lumina, despite the adhesion deficiency.

  4. Infected knee prosthesis: diagnosis with In-111 leukocyte, Tc-99m sulfur colloid, and Tc-99m MDP imaging

    SciTech Connect

    Palestro, C.J.; Swyer, A.J.; Kim, C.K.; Goldsmith, S.J. )

    1991-06-01

    Forty-one possible cases of infected total knee prostheses studied with indium-111-labeled leukocyte scintigraphy were retrospectively reviewed. Twenty-four of the prostheses were studied with technetium-99m methylene diphosphonate bone scintigraphy and 19 with Tc-99m sulfur colloid marrow scintigraphy. Nine prostheses were infected, and 32 were uninfected. The accuracy of combined labeled leukocyte and sulfur colloid marrow imaging (95%) was higher than that of labeled leukocyte scintigraphy alone (78%), bone scintigraphy alone (74%), or combined labeled leukocyte and bone scintigraphy (75%). The authors conclude that combined labeled leukocyte and sulfur colloid imaging is an accurate method for diagnosis of infected knee prostheses. In this series, this technique was superior to labeled leukocyte and bone imaging, alone or in combination.

  5. Dendritic Cells Take up and Present Antigens from Viable and Apoptotic Polymorphonuclear Leukocytes

    PubMed Central

    Alfaro, Carlos; Suarez, Natalia; Oñate, Carmen; Perez-Gracia, Jose L.; Martinez-Forero, Ivan; Hervas-Stubbs, Sandra; Rodriguez, Inmaculada; Perez, Guiomar; Bolaños, Elixabet; Palazon, Asis; de Sanmamed, Miguel Fernandez; Morales-Kastresana, Aizea; Gonzalez, Alvaro; Melero, Ignacio

    2011-01-01

    Dendritic cells (DC) are endowed with the ability to cross-present antigens from other cell types to cognate T cells. DC are poised to meet polymorphonuclear leukocytes (PMNs) as a result of being co-attracted by interleukin-8 (IL-8), for instance as produced by tumor cells or infected tissue. Human monocyte-derived and mouse bone marrow-derived DC can readily internalize viable or UV-irradiated PMNs. Such internalization was abrogated at 4°C and partly inhibited by anti-CD18 mAb. In mice, DC which had internalized PMNs containing electroporated ovalbumin (OVA) protein, were able to cross-present the antigen to CD8 (OT-1) and CD4 (OT-2) TCR-transgenic T cells. Moreover, in humans, tumor cell debris is internalized by PMNs and the tumor-cell material can be subsequently taken up from the immunomagnetically re-isolated PMNs by DC. Importantly, if human neutrophils had endocytosed bacteria, they were able to trigger the maturation program of the DC. Moreover, when mouse PMNs with E. coli in their interior are co-injected in the foot pad with DC, many DC loaded with fluorescent material from the PMNs reach draining lymph nodes. Using CT26 (H-2d) mouse tumor cells, it was observed that if tumor cells are intracellularly loaded with OVA protein and UV-irradiated, they become phagocytic prey of H-2d PMNs. If such PMNs, that cannot present antigens to OT-1 T cells, are immunomagnetically re-isolated and phagocytosed by H-2b DC, such DC productively cross-present OVA antigen determinants to OT-1 T cells. Cross-presentation to adoptively transferred OT-1 lymphocytes at draining lymph nodes also take place when OVA-loaded PMNs (H-2d) are coinjected in the footpad of mice with autologous DC (H-2b). In summary, our results indicate that antigens phagocytosed by short-lived PMNs can be in turn internalized and productively cross-presented by DC. PMID:22206007

  6. Role of CD11/CD18 in shear rate-dependent leukocyte-endothelial cell interactions in cat mesenteric venules.

    PubMed Central

    Perry, M A; Granger, D N

    1991-01-01

    In vivo microscopy was used to assess the relationships among shear rate (and shear stress), leukocyte rolling velocity, and leukocyte adherence in a cat mesentery preparation. Shear rate in individual venules and arterioles of 25-35 microns diameter were varied over a wide range by graded occlusion of an arterial loop. There was a linear decline in leukocyte rolling velocity (Vwbc) as red cell velocity (Vrbc) was reduced. The ratio Vwbc/Vrbc remained constant despite variations in shear stress from 5-25 dyn/cm2. A reduction in shear stress was associated with an increased leukocyte adherence, particularly when Vwbc was reduced below 50 microns/s. Reduction in wall shear rate below 500 s-1 in arterioles allowed 1-3 leukocytes to adhere per 100 microns length of vessel, while venules exposed to the same shear rates had 5-16 adherent leukocytes. In arterioles, leukocyte rolling was only observed at low shear rates. At shear rates less than 250 s-1 leukocyte rolling velocity was faster in arterioles than venules, and the ratio Vwbc/Vrbc for arterioles was 0.08 +/- 0.02, which was fourfold higher than the ratio obtained in venules at similar shear rates. Pretreatment with the CD18-specific antibody (mAb) IB4 increased leukocyte rolling velocity in venules by approximately 20 microns/s at red cell velocities below 2,000 microns/s. mAb IB4 largely prevented the leukocyte adherence to arterioles and venules, and increased the ratio Vwbc/Vrbc observed in venules at low shear elicit a CD18-dependent adhesive interaction between leukocytes and microvascular endothelium, and that differences in shear rates cannot explain the greater propensity for leukocyte rolling and adhesion in venules than arterioles. PMID:1673690

  7. Analysis of cell locomotion. Contact guidance of human polymorphonuclear leukocytes.

    PubMed

    Matthes, T; Gruler, H

    1988-01-01

    The methods of statistical physics have been applied to the analysis of cell movement. Human polymorphonuclear leukocytes were exposed to different surfaces possessing parallel oriented physical structures (scratched glass surface, machine drilled aluminum surface, optical grid and stretched polyethylene foil) and cell migration was observed using time-lapse photography. We demonstrate that in cell migration along physical structures, referred to as contact guidance, two subgroups can be distinguished: 1) The nematic type where the cell size is large in relation to the grid distance of the undulate surface. 2) The smectic type where the cell size is small in relation to the grid distance of the substrate. Nematic contact guidance is characterized by an anisotropic random walk. In all substrates investigated the diffusion process parallel to the lines was faster than the diffusion process perpendicular to them. The angular dependent diffusion coefficient was described by an ellipse. Deviation from a circle defined an apolar order parameter, whose value was about 0.3. The amount of information which the cells collected from, the undulate surface was very low, between 0.1 and 0.2 bits. We demonstrate that cells do not recognize all the details of their surroundings and that their migration can be compared to the "groping around" of a short sighted man. The blurred environment can be described by a mean field whose strength is proportional to the apolar order parameter. It is argued that the anisotropic surface tension is the basic source for nematic contact guidance. Smectic contact guidance is characterized by an anisotropic random walk and is quantified by a density order parameter which is 0.28 in the case of the scratched glass surface of a Neubauer counting chamber. The information which the cells collect from their environment is very low (0.03 bits). The lines seen by the cell can be described by a mean field whose strength is proportional to the density oder

  8. Sialylated, fucosylated ligands for L-selectin expressed on leukocytes mediate tethering and rolling adhesions in physiologic flow conditions

    PubMed Central

    1996-01-01

    Interaction of leukocytes in flow with adherent leukocytes may contribute to their accumulation at sites of inflammation. Using L- selectin immobilized in a flow chamber, a model system that mimics presentation of L-selectin by adherent leukocytes, we characterize ligands for L-selectin on leukocytes and show that they mediate tethering and rolling in shear flow. We demonstrate the presence of L- selectin ligands on granulocytes, monocytes, and myeloid and lymphoid cell lines, and not on peripheral blood T lymphocytes. These ligands are calcium dependent, sensitive to protease and neuraminidase, and structurally distinct from previously described ligands for L-selectin on high endothelial venules (HEV). Differential sensitivity to O-sialo- glycoprotease provides evidence for ligand activity on both mucin-like and nonmucin-like structures. Transfection with fucosyltransferase induces expression of functional L-selectin ligands on both a lymphoid cell line and a nonhematopoietic cell line. L-selectin presented on adherent cells is also capable of supporting tethering and rolling interactions in physiologic shear flow. L-selectin ligands on leukocytes may be important in promoting leukocyte-leukocyte and subsequent leukocyte endothelial interactions in vivo, thereby enhancing leukocyte localization at sites of inflammation. PMID:8909555

  9. Channel catfish (Ictalurus punctatus) leukocytes express estrogen receptor isoforms ERα and ERβ2 and are functionally modulated by estrogens

    USGS Publications Warehouse

    Iwanowicz, Luke R.; Stafford, James L.; Patiño, Reynaldo; Bengten, Eva; Miller, Norman W.; Blazer, Vicki

    2014-01-01

    Estrogens are recognized as modulators of immune responses in mammals and teleosts. While it is known that the effects of estrogens are mediated via leukocyte-specific estrogen receptors (ERs) in humans and mice, leucocyte-specific estrogen receptor expression and the effects of estrogens on this cell population is less explored and poorly understood in teleosts. Here in, we verify that channel catfish (Ictalurus punctaus) leukocytes express ERα and ERβ2. Transcripts of these isoforms were detected in tissue-associated leukocyte populations by PCR, but ERβ2 was rarely detected in PBLs. Expression of these receptors was temporally regulated in PBLs following polyclonal activation by concanavalin A, lipopolysaccharide or alloantigen based on evaluation by quantitative and end-point PCR. Examination of long-term leukocyte cell lines demonstrated that these receptors are differentially expressed depending on leukocyte lineage and phenotype. Expression of ERs was also temporally dynamic in some leukocyte lineages and may reflect stage of cell maturity. Estrogens affect the responsiveness of channel catfish peripheral blood leukocytes (PBLs) to mitogens in vitro. Similarly, bactericidal activity and phorbol 12-myristate 13-acetate induced respiratory burst was modulated by 17β-estradiol. These actions were blocked by the pure ER antagonist ICI 182780 indicating that response is, in part, mediated via ERα. In summary, estrogen receptors are expressed in channel catfish leukocytes and participate in the regulation of the immune response. This is the first time leukocyte lineage expression has been reported in teleost cell lines.

  10. Monocytes initiate a cycle of leukocyte recruitment when cocultured with endothelial cells.

    PubMed

    Tsouknos, Andreas; Nash, Gerard B; Rainger, G Ed

    2003-09-01

    During the development of atherosclerotic plaque, monocytes and T-lymphocytes are recruited to the arterial intima by endothelial cells (EC) lining the vessel. This process is associated with chronic arterial inflammation and requires the activation-dependent expression of adhesion receptors and chemokines on EC. Here we show that monocytes can activate cocultured EC so that they support the adhesion, activation and transmigration of a secondary bolus of flowing peripheral blood monocytes or lymphocytes. The number of adherent leukocytes and their behaviour was comparable to that seen on EC activated with tumour necrosis factor-alpha (TNF-alpha). Depending upon the duration of endothelial cell/monocyte coculture different patterns of adhesion receptors were utilised by leukocytes. After 4 h coculture, antibodies against E-selectin, P-selectin and vascular cell adhesion molecule-1 (VCAM-1) reduced mononuclear leukocyte adhesion. After 24 h coculture, antibodies against E-selectin and VCAM-1 but not P-selectin were effective. Immunofluorescence analysis confirmed that monocyte coculture induced endothelial expression of E-selectin and VCAM-1, while P-selectin was at the limit of detection. We conclude that EC stimulated by monocytes can support the adhesion of flowing mononuclear leukocytes. We hypothesise that this mode of EC activation and leukocyte recruitment could initiate a self-perpetuating cycle of inflammation that could be relevant to atherogenesis and other chronic inflammatory disease states.

  11. Sera from dams of calves with bovine neonatal pancytopenia contain alloimmune antibodies directed against calf leukocytes.

    PubMed

    Pardon, Bart; Stuyven, Edith; Stuyvaert, Sabrina; Hostens, Miel; Dewulf, Jeroen; Goddeeris, Bruno Maria; Cox, Eric; Deprez, Piet

    2011-06-15

    Bovine neonatal pancytopenia (BNP) is a bleeding and pancytopenic syndrome in neonatal calves, which recently emerged all over Europe. The present study tested whether antibodies directed against calf leukocytes are present in sera from known BNP dams. Sera from BNP dams (n=11) were combined with leukocytes from 11 calves (5 BNP survivors and 6 controls). After adding a fluorescein conjugated F(ab')(2) fragment of rabbit anti-bovine IgG (H&L) the level of antibody binding was measured by flow cytometry. As control groups both sera from dams from BNP affected (n=48) as from unaffected (n=54) herds were combined with leukocytes from the same calves. With sera from BNP dams, antibody binding could be visualised by immunofluoresence in both peripheral blood as in bone marrow smears. Mean fluoresence intensity values of all leukocyte subpopulations were significantly higher for the BNP dams compared to both control groups (P<0.01). BNP dams showed significantly more antibody binding on multiple leukocyte subpopulations of both BNP survivors and control calves and this from cut off values of MFI 100 onwards (P<0.01). The BNP survivor calves reacted significantly more often with sera from the BNP dams than the control calves (P<0.01). In conclusion the present study supports the hypothesis that BNP is an immune-mediated disease.

  12. Hydrodynamic forces on a wall-bound leukocyte due to interactions with flowing red cells

    NASA Astrophysics Data System (ADS)

    Isfahani, Amir H. G.; Freund, Jonathan B.

    2011-11-01

    As part of both healthy and pathologically physiological mechanisms sphere-like white blood cells (leukocytes) adhere to the walls of small blood vessels. We use quantitative numerical simulations to compare the forces from flowing red blood cells on a wall-adhered leukocyte to a homogenized model of blood at the same flow conditions. We model the highly flexible red blood cells using a fast O (N log N) boundary integral formulation. These elastic membranes deform substantially but strongly resist surface dilatation. They enclose a higher than plasma viscosity hemoglobin solution. The no-slip condition is enforced on the stationary leukocyte as well as the vessel walls. Vessel diameters of 10 to 20 microns are studied. Different hematocrits, leukocyte shapes, and flow conditions are examined. In vessels comparable to the size of the cells, we show that the particulate character of blood significantly affects the magnitude of the forces that the leukocyte experiences, transiently increasing it well above the homogenized-blood prediction: for example, for a tube hematocrit of 25 % and a spherical protrusion with a diameter 0.75 that of the tube, the average forces are increased by about 40 % and the local forces by more than 100 % relative to those expected for a blood model homogenized by its effective viscosity.

  13. KCNE gene expression is dependent on the proliferation and mode of activation of leukocytes

    PubMed Central

    Solé, Laura; Vallejo-Gracia, Albert; Roig, Sara R.; Serrano-Albarrás, Antonio; Marruecos, Laura; Manils, Joan; Gómez, Diana; Soler, Concepció; Felipe, Antonio

    2013-01-01

    Voltage-dependent K+ (Kv) channels are tightly regulated during the immune system response. Leukocytes have a limited repertoire of Kv channels, whose physiological role is under intense investigation. A functional Kv channel is an oligomeric complex composed of pore-forming and ancillary subunits. The KCNE gene family is a novel group of modulatory Kv channel elements in leukocytes. Here, we characterized the gene expression of KCNEs (1–5) in leukocytes and investigated their regulation during leukocyte proliferation and mode of activation. Murine bone-marrow-derived macrophages, human Jurkat T-lymphocytes and human Raji B-cells were analyzed. KCNEs (1–5) are expressed in all leukocytes lineages. Most KCNE mRNAs show cell cycle-dependent regulation and are differentially regulated under specific insults. Our results further suggest a new and yet undefined physiological role for KCNE subunits in the immune system. Putative associations of these ancillary proteins with Kv channels would yield a wide variety of biophysically and pharmacologically distinct channels that fine-tune the immunological response. PMID:23327879

  14. KCNE gene expression is dependent on the proliferation and mode of activation of leukocytes.

    PubMed

    Solé, Laura; Vallejo-Gracia, Albert; Roig, Sara R; Serrano-Albarrás, Antonio; Marruecos, Laura; Manils, Joan; Gómez, Diana; Soler, Concepció; Felipe, Antonio

    2013-01-01

    Voltage-dependent K (+) (Kv) channels are tightly regulated during the immune system response. Leukocytes have a limited repertoire of Kv channels, whose physiological role is under intense investigation. A functional Kv channel is an oligomeric complex composed of pore-forming and ancillary subunits. The KCNE gene family is a novel group of modulatory Kv channel elements in leukocytes. Here, we characterized the gene expression of KCNEs (1-5) in leukocytes and investigated their regulation during leukocyte proliferation and mode of activation. Murine bone-marrow-derived macrophages, human Jurkat T-lymphocytes and human Raji B-cells were analyzed. KCNEs (1-5) are expressed in all leukocytes lineages. Most KCNE mRNAs show cell cycle-dependent regulation and are differentially regulated under specific insults. Our results further suggest a new and yet undefined physiological role for KCNE subunits in the immune system. Putative associations of these ancillary proteins with Kv channels would yield a wide variety of biophysically and pharmacologically distinct channels that fine-tune the immunological response.

  15. A novel mutation in leukocyte adhesion deficiency type II/CDGIIc.

    PubMed

    Cagdas, Deniz; Yilmaz, Mustafa; Kandemir, Nurgün; Tezcan, Ilhan; Etzioni, Amos; Sanal, Özden

    2014-11-01

    Leukocyte adhesion deficiencies (LAD) are autosomal recessive immunodeficiency syndromes characterized by severe and recurrent bacterial infections, impaired wound healing and leukocytosis. Block in different steps in the leukocyte adhesion cascade causes different types of leukocyte adhesion deficiencies, LAD type I, II and III. In LAD type II, the rolling phase of the leukocyte adhesion cascade is affected due to mutations in the specific fucose transporter GFTP (GDP fucose transporter), causing defect in the biosynthesis of selectin ligands on leukocytes. Thus this syndrome is also called congenital disorder of glycosylation IIc (CGDIIc). LAD II/CGDIIc is very rare and has been diagnosed in nine children to date. Fever, leukocytosis, typical dysmorphic features, growth, psychomotor retardation and the Bombay blood group, are characteristic findings in patients. Here, we describe two Turkish siblings with a novel mutation in GFTP. They both have the characteristic features of the syndrome. The older sibling died of severe bacterial pneumonia at the age of 3 years. The younger sibling, diagnosed at the age of 3 months, responded to high dose oral fucose supplementation. Secundum atrial septal defect which was not described in previously reported patients, but present in both of our patients, may primarily related to the defect in fucosylation.

  16. Reduced platelet-mediated and enhanced leukocyte-mediated fibrinolysis in experimentally induced diabetes in rats

    SciTech Connect

    Winocour, P.D.; Colwell, J.A.

    1985-05-01

    Studies of fibrinolytic activity in diabetes mellitus have produced conflicting results. This may be a result of methodologic insensitivity or of variable contributions of the different blood components to whole blood fibrinolysis. To explore these two possibilities, the authors used a sensitive solid-phase radiometric assay to examine the fibrinolytic activity of whole blood, platelet-rich plasma, leukocytes, and platelet- and leukocyte-poor plasma prepared from control rats and rats with streptozocin-induced diabetes at various times after induction of diabetes. Fibrinolytic activity of whole blood from diabetic rats after 7 days was significantly reduced, and remained reduced after longer durations of diabetes up to 28 days. Platelet-rich plasma from diabetic rats had decreased fibrinolytic activity, which followed the same time course of changes as in whole blood. The platelet contribution to whole blood fibrinolysis was further reduced in vivo after 14 days of diabetes by a reduced whole blood platelet count. In contrast, fibrinolytic activity of leukocytes from diabetic rats became enhanced after 7 days of diabetes. After 49 days of diabetes, the whole blood leukocyte count was reduced, and in vivo would offset the enhanced activity. Plasma fibrinolytic activity was small compared with that of whole blood and was unaltered in diabetic rats. The authors conclude that altered platelet function contributes to decreased fibrinolytic activity of whole blood in diabetic rats, and that this may be partially offset by enhanced leukocyte-mediated fibrinolysis.

  17. A Leukocyte Filter Does Not Provide Further Benefit During Ex Vivo Lung Perfusion.

    PubMed

    Luc, Jessica G Y; Aboelnazar, Nader S; Himmat, Sayed; Hatami, Sanaz; Haromy, Alois; Matsumura, Nobutoshi; Vasanthan, Vishnu; White, Christopher W; Mengel, Michael; Freed, Darren H; Nagendran, Jayan

    2017-02-20

    Normothermic ex vivo lung perfusion (EVLP) allows for assessment and reconditioning of donor lungs. Though a leukocyte filter (LF) is routinely incorporated into the EVLP circuit, its efficacy remains to be determined. Twelve pig lungs were perfused and ventilated ex vivo in a normothermic state for 12 hours. Lungs (n=3) were allocated to 4 groups according to perfusate composition and the presence or absence of a LF in the circuit (acellular ± LF, cellular ± LF). Acceptable physiologic lung parameters were achieved during EVLP; however, increased amounts of pro-inflammatory cytokines (TNF-α, IL-6) and leukocytes in the perfusate were observed despite the presence or absence of a LF. Analysis of cells washed off the LF demonstrates that it trapped leukocytes though was ineffective throughout perfusion as it became saturated over 12 hours. We conclude that there is no objective evidence to support the routine incorporation of a LF during EVLP as it does not provide further benefit and its removal does not appear to cause harm. The lack of hypothesized benefit to a LF may be due to the saturation of the LF with donor leukocytes, leading to similar amounts of circulating leukocytes still present in the perfusate with and without a LF.

  18. Differential migratory properties of mouse, fish, and frog leukocytes treated with agonists of opioid receptors.

    PubMed

    Chadzinska, Magdalena; Plytycz, Barbara

    2004-07-01

    Zymosan-induced peritoneal inflammation was inhibited by morphine co-injection in mice and fish but not in anuran amphibians. In present experiments, an in vitro migration of mouse, goldfish, and frog leukocytes to L15 medium, control serum (S) or zymosan-activated serum (ZAS) was recorded following cell preincubation with L15 or with agonists of mu, delta, or kappa opioid receptors (morphine, deltorphine, or U-50,488H, respectively). In all species, migration of control leukocytes was in the order ZAS > S > L15. Pretreatment with morphine or deltorphine (but not with U-50,488H) enhanced leukocyte migration to L15 and S in each species, while it inhibited migration of mouse and fish (but not frog) leukocytes to ZAS, phenomena reversed by specific antagonists of mu and delta opioid receptors (CTOP or naltrindole, respectively). It seems that final effects of opioids on cell migration are dependent on a species-specific balance between up- and down-regulation of leukocyte migration resulted from interplay between receptors for opioids and chemotactic factors.

  19. Production of superoxide ions by leukocytes of the American alligator (Alligator mississippiensis).

    PubMed

    Merchant, Mark; Williams, Stetson; Hardy, Ross

    2009-01-01

    This study was conducted to characterize the production of superoxide ions by leukocytes in whole blood of the American alligator (Alligator mississippiensis). We used WST-1, a tetrazolium salt which can be reduced to a water-soluble formazan compound with high molar absorptivity at 438 nm, to probe the production of superoxide by alligator leukocytes. Incubation of alligator whole blood with WST-1 resulted in a time- and concentration-dependent increase in absorbance of the plasma at 438 nm. The reduction of WST-1 was inhibited in a concentration-dependent manner by superoxide dismutase, an enzyme that catalyzes the reduction of superoxide to peroxide, confirming that the reduction of WST-1 was due to the presence of superoxide. Treatment of whole blood with nitrotetrazolium blue (NBT) resulted in the staining of heterophils and monocytes, enforcing the idea that that the production of superoxide is due to the presence of leukocytes, and not other blood cell components. It is interesting to note that the production of superoxide by the alligator leukocytes required no external stimulation while human leukocytes must be stimulated with an immunological challenge before producing superoxide. This is the first report of the production of superoxide as an innate immune mechanism in crocodilians.

  20. Uropod elongation is a common final step in leukocyte extravasation through inflamed vessels

    PubMed Central

    Hyun, Young-Min; Sumagin, Ronen; Sarangi, Pranita P.; Lomakina, Elena; Overstreet, Michael G.; Baker, Christina M.; Fowell, Deborah J.; Waugh, Richard E.; Sarelius, Ingrid H.

    2012-01-01

    The efficient trafficking of immune cells into peripheral nonlymphoid tissues is key to enact their protective functions. Despite considerable advances in our understanding of cell migration in secondary lymphoid organs, real-time leukocyte recruitment into inflamed tissues is not well characterized. The conventional multistep paradigm of leukocyte extravasation depends on CD18 integrin–mediated events such as rapid arrest and crawling on the surface of the endothelium and transmigration through the endothelial layer. Using enhanced three-dimensional detection of fluorescent CD18 fusion proteins in a newly developed knockin mouse, we report that extravasating leukocytes (neutrophils, monocytes, and T cells) show delayed uropod detachment and become extremely elongated before complete transmigration across the endothelium. Additionally, these cells deposit CD18+ microparticles at the subendothelial layer before retracting the stretched uropod. Experiments with knockout mice and blocking antibodies reveal that the uropod elongation and microparticle formation are the result of LFA-1–mediated adhesion and VLA-3–mediated cell migration through the vascular basement membrane. These findings suggest that uropod elongation is a final step in the leukocyte extravasation cascade, which may be important for precise regulation of leukocyte recruitment into inflamed tissues. PMID:22711877

  1. Effect of Estragole on Leukocyte Behavior and Phagocytic Activity of Macrophages

    PubMed Central

    Wiirzler, Luiz Alexandre Marques; Silva-Filho, Saulo Euclides; Kummer, Raquel; Pedroso, Raissa Bocchi; Spironello, Ricardo Alexandre; Silva, Expedito Leite; Bersani-Amado, Ciomar Aparecida; Cuman, Roberto Kenji Nakamura

    2014-01-01

    Estragole, a chemical constituent of the essential oils of many aromatic plants, is used as flavoring in beverage and food industries. In vivo and in vitro experimental assays have shown that EST has sedative, anticonvulsant, antioxidant, antimicrobial, and anesthetic activity. In this work, we evaluate the effect of EST on leukocyte behavior and phagocytic activity of macrophages. In the peritonitis model, EST (500 and 750 mg/kg) decreased the infiltration of peritoneal exudate leukocytes. In vitro chemotaxis assay showed that EST (3, 10, 30, and 60 μg/mL) inhibited neutrophil migration toward fMLP. In the in vivo microcirculation assay, EST at doses of 250, 500, and 750 mg/kg significantly reduced the number of rolling and adherent leukocytes and at doses of 250 and 500 mg/kg decreased number of leukocyte migrated to perivascular tissue. The results showed that EST (3, 10, and 30 μg/mL) was able to stimulate the macrophages phagocytosis but only at concentration of 10 μg/mL promoted an increase in nitric oxide (NO) production. In conclusion, this study showed that EST had potential anti-inflammatory effects, likely by inhibiting leukocyte migration and by stimulating macrophages phagocytosis. PMID:25152763

  2. Selection of the best features for leukocytes classification in blood smear microscopic images

    NASA Astrophysics Data System (ADS)

    Sarrafzadeh, Omid; Rabbani, Hossein; Talebi, Ardeshir; Banaem, Hossein Usefi

    2014-03-01

    Automatic differential counting of leukocytes provides invaluable information to pathologist for diagnosis and treatment of many diseases. The main objective of this paper is to detect leukocytes from a blood smear microscopic image and classify them into their types: Neutrophil, Eosinophil, Basophil, Lymphocyte and Monocyte using features that pathologists consider to differentiate leukocytes. Features contain color, geometric and texture features. Colors of nucleus and cytoplasm vary among the leukocytes. Lymphocytes have single, large, round or oval and Monocytes have singular convoluted shape nucleus. Nucleus of Eosinophils is divided into 2 segments and nucleus of Neutrophils into 2 to 5 segments. Lymphocytes often have no granules, Monocytes have tiny granules, Neutrophils have fine granules and Eosinophils have large granules in cytoplasm. Six color features is extracted from both nucleus and cytoplasm, 6 geometric features only from nucleus and 6 statistical features and 7 moment invariants features only from cytoplasm of leukocytes. These features are fed to support vector machine (SVM) classifiers with one to one architecture. The results obtained by applying the proposed method on blood smear microscopic image of 10 patients including 149 white blood cells (WBCs) indicate that correct rate for all classifiers are above 93% which is in a higher level in comparison with previous literatures.

  3. The effect of acupuncture on leukocyte levels in peripheral blood is modified by aspirin.

    PubMed

    Rivas-Vilchis, José Federico; Barrera-Escorcia, Eduardo; Fregoso-Padilla, Martha

    2009-01-01

    It has been shown that acupuncture can modify circulating levels of subpopulations of leukocytes. There have been few investigations on the effect of acupuncture on prostaglandins metabolism. Aspirin is capable of inhibiting the metabolism of prostaglandins and to produce several pharmacological effects. The objective of this study was to determine whether prior administration of aspirin could modify the action of acupuncture on levels of circulating leukocytes. Fourteen healthy males (age: 19-23 years) were recruited from a university student population. This study was a placebo-controlled, prospective, cross-over design. Subjects were randomly assigned into A or B groups. Group A received aspirin 500 mg and group B placebo, after 1 week of a washout period, group A received placebo and group B aspirin. Subjects were given acupuncture with manual needling in GV14 (Dazhui) acupoint 2 hr after receiving medication. The needle was stimulated for 10 sec and was kept in place for 5 min. Leukocytes and their subpopulations were quantified in blood samples taken immediately before and 2 hr after acupuncture treatment. In each subject pre-acupuncture values were compared to those post-acupuncture. The results showed that acupuncture significantly increased overall leukocytes (p=0.006) and neutrophils (p<0.001). Aspirin partially inhibited these effects. The data suggest that the effect of acupuncture on leukocytes may be related to levels of prostaglandins.

  4. Allium sativum L. extract prevents methyl mercury-induced cytotoxicity in peripheral blood leukocytes (LS).

    PubMed

    Abdalla, F H; Bellé, L P; De Bona, K S; Bitencourt, P E R; Pigatto, A S; Moretto, M B

    2010-01-01

    Adenosine deaminase (ADA) is involved in purine metabolism and plays a significant role in the immune system. The focus of this investigation was to examine the effects of low concentrations of organic mercury on ADA activity in human leukocytes and to investigate the relationship between these effects and cell death. We have examined the protective potential effects of Allium sativum extract (GaE) against Methylmercury (MeHg)-induced cytotoxic effects on human leucocytes under in vitro conditions. MeHg (0.05-10 microM) significantly decreased leukocyte viability (58.97% for MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and 51.67% for Alamar Blue (AB) and this decrease was positively correlated to the MeHg-induced inhibition of ADA activity. N-acetylcysteine (NAC) and GaE prevented both the MeHg-induced cytotoxic effects on leukocytes according to MTT and AB assays and the effects on the ADA activity. The present results suggest that the protective effects of GaE against MeHg-induced leukocyte damage is related to the removal of oxidant species generated in the presence of MeHg due to the antioxidant efficacy of garlic constituents. It is important to point out that the intense presence of ADA in Leukocyte suspension (LS) highlights the relevant effects in the immune system and in vitro cytotoxicity of MeHg exposure.

  5. Glycocalyx Degradation Induces a Proinflammatory Phenotype and Increased Leukocyte Adhesion in Cultured Endothelial Cells under Flow

    PubMed Central

    McDonald, Karli K.; Cooper, Scott; Danielzak, Lisa; Leask, Richard L.

    2016-01-01

    Leukocyte adhesion to the endothelium is an early step in the pathogenesis of atherosclerosis. Effective adhesion requires the binding of leukocytes to their cognate receptors on the surface of endothelial cells. The glycocalyx covers the surface of endothelial cells and is important in the mechanotransduction of shear stress. This study aimed to identify the molecular mechanisms underlying the role of the glycocalyx in leukocyte adhesion under flow. We performed experiments using 3-D cell culture models, exposing human abdominal aortic endothelial cells to steady laminar shear stress (10 dynes/cm2 for 24 hours). We found that with the enzymatic degradation of the glycocalyx, endothelial cells developed a proinflammatory phenotype when exposed to uniform steady shear stress leading to an increase in leukocyte adhesion. Our results show an up-regulation of ICAM-1 with degradation compared to non-degraded controls (3-fold increase, p<0.05) and we attribute this effect to a de-regulation in NF-κB activity in response to flow. These results suggest that the glycocalyx is not solely a physical barrier to adhesion but rather plays an important role in governing the phenotype of endothelial cells, a key determinant in leukocyte adhesion. We provide evidence for how the destabilization of this structure may be an early and defining feature in the initiation of atherosclerosis. PMID:27907146

  6. Exposure to Sodium Fluoride Produces Signs of Apoptosis in Rat Leukocytes

    PubMed Central

    Gutiérrez-Salinas, José; Morales-González, José A.; Madrigal-Santillán, Eduardo; Esquivel-Soto, Jaime; Esquivel-Chirino, César; González-Rubio, Manuel García-Luna y; Suástegui-Domínguez, Sigrit; Valadez-Vega, Carmen

    2010-01-01

    Fluoride is naturally present in the earth’s crust and can be found in rocks, coal, and clay; thus, it can be found in small quantities in water, air, plants, and animals. Therefore, humans are exposed to fluoride through food, drinking water, and in the air they breathe. Flouride is essential to maintain bone strength and to protect against dental decay, but if it is absorbed too frequently, it can cause tooth decay, osteoporosis, and damage to kidneys, bones, nerves, and muscles. Therefore, the present work was aimed at determining the effect of intake of sodium fluoride (NaF) as an apoptosis inducer in leukocytes of rats treated for eight weeks with 1 or 50 parts per million (ppm) NaF. Expression of p53, bcl-2, and caspade-3 were used as apoptotic and general metabolism indicators of leukocyte-like indicators of the (INT) oxidation system. Male rats were exposed to NaF (1 and 500 ppm) for eight weeks, and then sacrificed weekly to obtain blood samples. Expression of p53, bcl-2, and caspase-3 were determined in leukocytes by Western blot, and general metabolism of leukocytes was analyzed with a commercial kit. We found changes in the expression of the proteins described, especially when the animals received 50 ppm of NaF. These results indicate that NaF intoxication can be an apoptosis inducer in rat leukocytes treated with the compound for eight weeks. PMID:20957113

  7. Flow cytometric detection of bovine viral diarrhea virus in peripheral blood leukocytes of persistently infected cattle.

    PubMed Central

    Qvist, P; Aasted, B; Bloch, B; Meyling, A; Rønsholt, L; Houe, H

    1990-01-01

    Flow cytometry was investigated for detection of bovine viral diarrhea virus (BVDV) in peripheral blood mononuclear leukocytes of persistently infected cattle. The mononuclear leukocytes were purified by sedimentation in a gradient of Ficoll-Paque, fixed, permeabilized, and then labelled by indirect immunofluorescence using biotinylated immunoglobulins from a porcine antiserum to BVDV. Flow cytometric analysis of blood samples obtained from persistently infected cattle revealed virus in 3.0-21.0% (mean +/- SD, 11.2% +/- 6.4%) of the mononuclear leukocytes. Fluorescent cells were not observed in controls. Flow cytometric detection of BVDV in blood cells of persistently infected bovines is a rapid and objective technique which does not require cell culture facilities. PMID:2174298

  8. A transmigratory cup in leukocyte diapedesis both through individual vascular endothelial cells and between them

    PubMed Central

    Carman, Christopher V.; Springer, Timothy A.

    2004-01-01

    The basic route and mechanisms for leukocyte migration across the endothelium remain poorly defined. We provide definitive evidence for transcellular (i.e., through individual endothelial cells) diapedesis in vitro and demonstrate that virtually all, both para- and transcellular, diapedesis occurs in the context of a novel “cuplike” transmigratory structure. This endothelial structure was comprised of highly intercellular adhesion molecule-1– and vascular cell adhesion molecule-1–enriched vertical microvilli-like projections that surrounded transmigrating leukocytes and drove redistribution of their integrins into linear tracks oriented parallel to the direction of diapedesis. Disruption of projections was highly correlated with inhibition of transmigration. These findings suggest a novel mechanism, the “transmigratory cup”, by which the endothelium provides directional guidance to leukocytes for extravasation. PMID:15504916

  9. Effect of Phenylbutazone on Phagocytosis and Intracellular Killing by Guinea Pig Polymorphonuclear Leukocytes1

    PubMed Central

    Strauss, Robert R.; Paul, Benoy B.; Sbarra, Anthony J.

    1968-01-01

    The anti-inflammatory drug phenylbutazone has been found to inhibit both engulfment and intracellular killing of E. coli by guinea pig peritoneal polymorphonuclear (PMN) leukocytes. The bactericidal activity of leukocytic homogenates was also inhibited by the drug. Addition of the drug at various time intervals to a phagocytic reacting system caused an almost immediate cessation of bactericidal activity. Metabolic studies showed that the drug sharply curtailed glucose-l-14C and 14C-formate oxidation of both resting and phagocytizing PMN leukocytes. These data indicated an effect upon the hexose monophosphate shunt and H2O2 formation. Further investigation showed that the sites of inhibition were on glucose-6-phosphate and 6-phosphogluconate dehydrogenase. These inhibitions resulted in decreased H2O2 production. It is suggested that H2O2 activates lysosomes and subsequently complexes with the lysosomal enzyme, myeloperoxidase. This complex is a potent bactericidal agent in the phagocyte. PMID:4881700

  10. [A leukocyte pattern recognition based on feature fusion in multi-color space].

    PubMed

    Hao, Liangwang; Hong, Wenxue

    2013-10-01

    To solve the ineffective problem of leukocytes classification based on multi-feature fusion in a single color space, we proposed an automatic leukocyte pattern recognition by means of feature fusion with color histogram and texture granular in multi-color space. The interactive performance of three color spaces (RGB, HSV and Lab), two features (color histogram and texture granular) and four similarity measured distance metrics (normalized intersection, Euclidean distance, chi2-metric distance and Mahalanobis distance) were discussed. The optimized classification modes of high precision, extensive universality and low cost to different leukocyte types were obtained respectively, and then the recognition system of tree-integration of the optimized modes was established. The experimental results proved that the performance of the fusion classification was improved by 12.3% at least.

  11. Jon Van Rood: pioneer at the crossroad of human leukocyte antigens and transplantation.

    PubMed

    Jansen, Jan

    2007-04-01

    Jon Van Rood (born in 1926) has made major contributions to the fields of transfusion medicine as well as organ and stem cell transplantation. His group was the first to start unraveling the complexity of the human leukocyte antigen (HLA) system through collaborative studies that used panels of sera and leukocyte samples. Furthermore, using HLA typing, he introduced the first HLA-matched platelet transfusions and developed routine leukocyte depletion as a means to prevent HLA alloimmunization. Van Rood has also been active in the fields of kidney transplantation (Eurotransplant) and stem cell transplantation (Europdonor). He combined scientific laboratory research with application to clinical medicine. He retired from his university position in 1991 but remains active in the field.

  12. TNFα promotes CAR-dependent migration of leukocytes across epithelial monolayers.

    PubMed

    Morton, Penny E; Hicks, Alexander; Ortiz-Zapater, Elena; Raghavan, Swetavalli; Pike, Rosemary; Noble, Alistair; Woodfin, Abigail; Jenkins, Gisli; Rayner, Emma; Santis, George; Parsons, Maddy

    2016-05-19

    Trans-epithelial migration (TEpM) of leukocytes during inflammation requires engagement with receptors expressed on the basolateral surface of the epithelium. One such receptor is Coxsackie and Adenovirus Receptor (CAR) that binds to Junctional Adhesion Molecule-like (JAM-L) expressed on leukocytes. Here we provide the first evidence that efficient TEpM of monocyte-derived THP-1 cells requires and is controlled by phosphorylation of CAR. We show that TNFα acts in a paracrine manner on epithelial cells via a TNFR1-PI3K-PKCδ pathway leading to CAR phosphorylation and subsequent transmigration across cell junctions. Moreover, we show that CAR is hyper-phosphorylated in vivo in acute and chronic lung inflammation models and this response is required to facilitate immune cell recruitment. This represents a novel mechanism of feedback between leukocytes and epithelial cells during TEpM and may be important in controlling responses to pro-inflammatory cytokines in pathological settings.

  13. Temporal leukocyte numbers and granulocyte activation in pulsatile and rotary ventricular assist device patients.

    PubMed

    Woolley, Joshua R; Teuteberg, Jeffrey J; Bermudez, Christian A; Bhama, Jay K; Lockard, Kathleen L; Kormos, Robert L; Wagner, William R

    2014-06-01

    Individual ventricular assist device (VAD) design may affect leukocytes and impact immunity. Few studies have presented leukocyte and infection profiles in VAD patients over the course of the implant period. CD11b (MAC-1) expression on granulocytes is an indicator of activation during inflammation, mediating extravasation and the release of reactive oxygen species in tissue. No reported studies have presented MAC-1 expression on circulating granulocytes in VAD patients. Fifty-six patients implanted at a single center with a HeartMate II (HMII; n = 32), HeartWare (HW; n = 12), or Thoratec pneumatic VAD (PVAD; n = 12) between 1999 and 2011 were followed for 120 days of support. The leukocyte profiles and infectious events of all patients were evaluated; additionally, a subset had MAC-1 expression on circulating granulocytes was measured (HMII n = 9; HW n = 7; PVAD n = 4). All groups exhibited a significant peak in leukocyte numbers at postoperative day (POD) 14 while simultaneously experiencing a significant decrease in hematocrit. HMII patients exhibited a 3.2-fold increase in granulocyte MAC-1 expression at POD 14, and the temporal trend over the implant period differed from that experienced by HW patients. Further, HW patients experienced significantly fewer infection events. Alterations in leukocyte profiles and granulocyte activation experienced by VAD patients appear to be device-specific. Elevations in leukocyte activation may be related to an increased risk for infection, although the specific relationship between these phenomena in this patient group is not known.

  14. [The effect of leukocyte depletion by filtration on the quality of apheresis platelets].

    PubMed

    Yu, Yang; Feng, Qian; Zhang, Ting; Ma, Chun-Ya; Zhang, Xiao-Juan; Ge, Guo-Feng; Lin, Zi-Lin; Pan, Ji-Chun; Wang, De-Qing; Luo, Qun; Tian, Ya-Ping

    2009-08-01

    This study was aimed to investigate the effect of leukocyte depletion by filtration on the quality of apheresis platelets. 20 units of donor apheresis platelets were randomly selected and were preserved with agitation at 20 - 24 degrees C for 24 - 96 hours, then were filtered on polyester flatbed filters. The platelet concentration, mean platelet volume (MPV), volume of apheresis platelets, leukocyte count, pH value, lactate dehydrogenase (LDH) concentration, K(+) concentration and CD62p expression level on surface of platelet membrane, were detected before and after filtration, as well as the rate of leukocyte depletion and platelet loss were calculated. The results showed that the leukocyte count after filtration was remarkably lower than that before filtration (p < 0.001), and the rate of leukocyte depletion was 99.97%. Platelet loss was approximately 8%, and obviously lower than that of the national standard (p < 0.001). MPV, pH value, K(+) and LDH concentration were not significantly different before and after filtration. Compared with platelets before filtration, CD62p expression level after filtration slightly decreased (p > 0.05). CD62p expression on surface of platelet membrane in perfusion fluid obtained from filter plate was obviously higher than that before filtration (p < 0.05). MA of platelet after filtration slightly decreased (p > 0.05). It is concluded that leukocyte and partial activated platelets can be removed efficiently by using polyester flatbed filters, and platelet loss is very low. Filtration does not adversely affect coagulation activity of the platelets in vitro. Apheresis platelets after filtration can fulfil quality requirements to prevent infection of cytomegalovirus and HLA alloimmunization.

  15. Altered leukocyte delivery to specific and nonspecific inflammatory skin lesions following burn injury

    SciTech Connect

    Tchervenkov, J.I.; Latter, D.A.; Psychogios, J.; Christou, N.V.

    1988-05-01

    This study assessed the effect of burn trauma on the in vivo leukocyte cell delivery during the first 24 hr of the delayed type hypersensitivity (DTH) skin test reaction and a bacterial skin abscess. Inbred male Lewis rats sensitized to keyhole limpet hemocyanin (KLH) were given a 30% scald burn or sham burn. Three days later the animals were injected intradermally, at different sites, with 0.3 mg of KLH, 10(8) organisms of S. aureus 502A, and 0.1 cc of saline, at 2 to 24 hr. Leukocytes labelled with Indium-111 oxine(leu-111) were injected intravenously. In sham rats the peak leu-111 influx in the DTH reaction occurred at 2-4 hr while in the abscess it was biphasic with peaks at 3 hr and 6-8 hr. In burn trauma rats there was a markedly increased leu111 peak at 2 hr in both the DTH and abscess reactions followed by a significantly lower than normal leu111 delivery in the late (6-24) hours. This marked early leukocyte influx in burned rats was paralleled by a reduced DTH skin test lesion (8.2 +/- 1.1 mm to 4.2 +/- 1.1 mm) and an increased bacterial abscess (5.1 +/- 1.1 mm to 8.1 +/- 0.9 mm) post burn. There was a direct correlation between leukocyte cell delivery to a DTH reaction and a bacterial abscess (r8 = 0.69, Spearman rank; p less than 0.001). We conclude that burn trauma results in altered leukocyte delivery to inflammatory lesions and the DTH response can be used to assess the ability of a burn trauma host to recruit leukocytes at a site of infection.

  16. Triggering of leukocytes by phase contrast in imaging cytometry with scanning fluorescence microscope (SFM)

    NASA Astrophysics Data System (ADS)

    Bocsi, József; Pierzchalski, Arkadiusz; Marecka, Monika; Malkusch, Wolf; Tárnok, Attila

    2009-02-01

    Slide-based cytometry (SBC) leads to breakthrough in cytometry of cells in tissues, culture and suspension. Carl Zeiss Imaging Solutions' new automated SFM combines imaging with cytometry. A critical step in image analysis is selection of appropriate triggering signal to detect all objects. Without correct target cell definition analysis is hampered. DNA-staining is among the most common triggering signals. However, the majority of DNA-dyes yield massive spillover into other fluorescence channels limiting their application. By microscopy objects of >5μm diameter can be easily detected by phase-contrast signal (PCS) without any staining. Aim was to establish PCS - triggering for cell identification. Axio Imager.Z1 motorized SFM was used (high-resolution digital camera, AxioCam MRm; AxioVision software: automatic multi-channel scanning, analysis). Leukocytes were stained with FITC (CD4, CD8) and APC (CD3) labelled antibodies in combinations using whole blood method. Samples were scanned in three channels (PCS/FITC/APC). Exposition-times for PCS were set as low as possible; the detection efficiency was verified by fluorescence. CD45-stained leukocytes were counted and compared to the number of PCS detected events. Leukocyte subtyping was compared with other cytometers. In focus the PCS of cells showed ring-form that was not optimal for cell definition. Out of focus PCS allows more effective qualitative and quantitative cell analyses. PCS was an accurate triggering signal for leukocytes enabling cell counting and discrimination of leukocytes from platelets. Leukocyte subpopulation frequencies were comparable to those obtained by other cytometers. In conclusion PCS is a suitable trigger-signal not interfering with fluorescence detection.

  17. Mechanisms of transfusion-related acute lung injury (TRALI): anti-leukocyte antibodies.

    PubMed

    Curtis, Brian R; McFarland, Janice G

    2006-05-01

    There is abundant evidence that leukocyte antibodies in blood donor products are somehow involved in transfusion-related acute lung injury (TRALI). Human leukocyte antigen (HLA) class I, HLA class II, and neutrophil-specific antibodies in the plasma of both blood donors and recipients have been implicated in the pathogenesis of TRALI. The case for a relationship between leukocyte antibodies and TRALI is more compelling if concordance between the antigen specificity of the leukocyte antibodies in the donor plasma and the corresponding antigen on the cells of the affected recipient is demonstrated. Such antibody-antigen concordance can be investigated by typing the recipient for the cognate leukocyte antigens or by cross-matching the donor plasma against the recipient's leukocytes. Two proposed pathophysiologic mechanisms for TRALI have received the most attention: the antibody hypothesis and the two-event hypothesis. The final common pathway in all of the proposed pathogenic mechanisms of TRALI is increased pulmonary capillary permeability, which results in movement of plasma into the alveolar space causing pulmonary edema. A typical TRALI serologic workup consists of tests for HLA class I and II and neutrophil-specific antibodies. The use of flow cytometry and HLA-coated microbeads is recommended for detection of HLA antibodies in plasma of implicated blood donors and a combination of the granulocyte agglutination test and granulocyte immunofluorescence test for detection of neutrophil-specific antibodies. Genotyping for class I and II HLA and for a limited number of neutrophil antigens may also be helpful in establishing antibody-antigen concordance.

  18. Junctional Adhesion Molecule C Mediates Leukocyte Adhesion to Rheumatoid Arthritis Synovium

    PubMed Central

    Rabquer, Bradley J.; Pakozdi, Angela; Michel, James E.; Gujar, Bansari S.; Haines, G. Kenneth; Imhof, Beat A.; Koch, Alisa E.

    2010-01-01

    Objective Leukocyte infiltration into the rheumatoid arthritis (RA) synovium is a multistep process in which leukocytes leave the bloodstream and invade the synovial tissue (ST). Leukocyte transendothelial migration and adhesion to RA ST requires adhesion molecules on the surface of endothelial cells and RA ST fibroblasts. This study was undertaken to investigate the role of junctional adhesion molecule C (JAM-C) in mediating leukocyte recruitment and retention in the RA joint. Methods Immunohistologic analysis was performed on RA, osteoarthritis (OA), and normal ST samples to quantify JAM-C expression. Fibroblast JAM-C expression was also analyzed using Western blotting, cell surface enzyme-linked immunosorbent assay, and immunofluorescence. To determine the role of JAM-C in leukocyte retention in the RA synovium, in vitro and in situ adhesion assays and RA ST fibroblast transmigration assays were performed. Results JAM-C was highly expressed by RA ST lining cells, and its expression was increased in OA ST and RA ST endothelial cells compared with normal ST endothelial cells. JAM-C was also expressed on the surface of OA ST and RA ST fibroblasts. Furthermore, we demonstrated that myeloid U937 cell adhesion to both OA ST and RA ST fibroblasts and to RA ST was dependent on JAM-C. U937 cell migration through an RA ST fibroblast monolayer was enhanced in the presence of neutralizing antibodies against JAM-C. Conclusion Our results highlight the novel role of JAM-C in recruiting and retaining leukocytes in the RA synovium and suggest that targeting JAM-C may be important in combating inflammatory diseases such as RA. PMID:18821692

  19. Leukocyte myeloperoxidase deficiency and disseminated candidiasis: the role of myeloperoxidase in resistance to Candida infection

    PubMed Central

    Lehrer, Robert I.; Cline, Martin J.

    1969-01-01

    The neutrophils and monocytes of a patient with disseminated candidiasis were found to lack detectable levels of the lysosomal enzyme myeloperoxidase (MPO), although they had normal levels of other granule-associated enzymes. Leukocytes from one of the patient's sisters also lacked detectable MPO; leukocytes from his four sons contained approximately one-third of mean normal peroxidase levels. Neither the patient nor his affected relatives had experienced frequent or unusual bacterial infections. The phagocytic activity of the patient's MPO-deficient neutrophils was intact, and the cells displayed normal morphologic and metabolic responses to phagocytosis. In contrast to normal leukocytes which killed 30.5±7.3% of ingested Candida albicans in 1 hr, however, the patient's neutrophils killed virtually none. His leukocytes also failed to kill the strain of C. albicans recovered from his lesions, as well as other Candida species. These MPO-deficient neutrophils killed Serratia marcescens and Staphylococens aureus 502A at an abnormally slow rate, requiring 3-4 hr to achieve the bactericidal effect attained by normal leukocytes after 45 min. No other abnormalities in his cellular or humoral immune responses were demonstrated. These findings suggest that hereditary MPO deficiency is transmitted as an autosomal recessive characteristic, that the homozygous state conveys enhanced susceptibility to disseminated candidiasis, and that MPO is necessary for candidacidal activity in human neutrophils. Although lending support to the suggested bactericidal role of MPO in leukocytes, the data indicate that alternative bactericidal mechanisms, effective in the absence of MPO, are functionally dominant in the human neutrophil. Images PMID:5796360

  20. [The effect of bromantane on the erythro- and leukocytic profile of the peripheral blood in rats].

    PubMed

    Bugaeva, L I; Spasov, A A; Morozov, I S

    1999-01-01

    The specific effect of bromantan on blood tissue is demonstrated in rats. In chronic injection in a dose of 30 mg/kg bromantan raised the level of hemoglobin and leukocytes. In doses of 150 and 600 mg/kg it increased at first (3 months) and then reduced the level of erythrocytes, hemoglobin, and leukocytes. Reversible poikilocytosis, granulocytosis, and agranulocytosis were encountered. Hyperchromatosis and hypertrophy of the hepatocytes were found in the tissues of the liver and hemosiderosis was discovered in the spleen. It is suggested that the blood tissue is a "target" in the toxic effect of bromantan.

  1. A proposal for the definition of terms related to locomotion of leukocytes and other cells*

    PubMed Central

    1980-01-01

    There is currently much confusion of terms relating to locomotion of leukocytes and other cells. Standardized and precise use of terms is, however, indispensable for analysis of the basic mechanisms controlling such locomotion. The present proposal is intended to serve as a basis for a standardized system of reporting locomotor behaviour of leukocytes and other cells and their responses to environmental stimuli. Definitions of the terms random locomotion, directional locomotion, chemotaxis, chemokinesis, and intrinsic locomotor capacity are proposed. Examples are given to demonstrate the application of the terms in the experimental and clinical analysis of cell locomotion. PMID:6968255

  2. Scaling deterministic lateral displacement arrays for high throughput and dilution-free enrichment of leukocytes

    NASA Astrophysics Data System (ADS)

    Inglis, David W.; Lord, Megan; Nordon, Robert E.

    2011-05-01

    A disposable device for fractionation of blood into its components that is simple to operate and provides throughput of greater than 1 mL min-1 is highly sought after in medical diagnostics and therapies. This paper describes a device with parallel deterministic lateral displacement devices for enrichment of leukocytes from blood. We show capture of 98% and approximately ten-fold enrichment of leukocytes in whole blood. We demonstrate scaling up through the integration of six parallel devices to achieve a flow rate of 115 µL of undiluted blood per minute per atmosphere of applied pressure.

  3. Shorter leukocyte telomere length in patients at ultra high risk for psychosis.

    PubMed

    Maurya, Pawan Kumar; Rizzo, Lucas Bortolotto; Xavier, Gabriela; Tempaku, Priscila Farias; Zeni-Graiff, Maiara; Santoro, Marcos L; Mazzotti, Diego Robles; Zugman, André; Pan, Pedro; Noto, Cristiano; Maes, Michael; Asevedo, Elson; Mansur, Rodrigo B; Cunha, Graccielle R; Gadelha, Ary; Bressan, Rodrigo A; Belangero, Sintia Iole; Brietzke, Elisa

    2017-03-05

    Telomere length attrition has been demonstrated in schizophrenia but not in individuals in ultra high risk (UHR) for psychosis. The present study aimed to compare the leukocyte telomere length (TL) between patients at UHR for psychosis and healthy controls (HC). Twenty-two participants with UHR and 88 HC were enrolled in this study. Telomere lengths were determined using a multiplex qPCR assay. After adjustment for age, sex, ethnicity, and education, patients in UHR, compared with HC groups, had shorter telomere length (RR: 0.929, p=0.031). Shorter leukocyte telomere length in UHR could represent early signs of accelerated aging in this population.

  4. Indium-111-labeled autologous leukocyte scanning in gastrointestinal graft versus host disease (GVHD)

    SciTech Connect

    Saverymuttu, S.H.; Peters, A.M.; O'Brien, C.; Chadwick, V.S.; Lavender, J.P.; Goldman, J.M.; Gordon-Smith, E.C.; Hodgson, H.J.

    1986-08-01

    The technique of scanning with indium-111 autologous leukocytes has been used to assess gastrointestinal graft-versus-host disease (GVHD) following allogenic marrow transplantation. In patients with active disease, abdominal scans showed extensive abnormal localization in the bowel, while in those whose disease was quiescent after responding to treatment, scans showed localized ileocecal involvement. Rectal histology showed excellent agreement with scanning in the diagnosis of GVHD, but in three of six cases with active disease underestimated disease severity. Indium-111 leukocyte scanning is a useful noninvasive technique for the diagnosis and assessment of gut GVHD.

  5. INDEPENDENT ROLES OF MACROPHAGE MIGRATION INHIBITORY FACTOR AND ENDOGENOUS, BUT NOT EXOGENOUS GLUCOCORTICOIDS IN REGULATING LEUKOCYTE TRAFFICKING

    PubMed Central

    Gregory, Julia L.; Hall, Pam; Leech, Michelle; Morand, Eric F.; Hickey, Michael J.

    2011-01-01

    Objectives Macrophage migration inhibitory factor (MIF) promotes leukocyte recruitment and antagonizes the anti-inflammatory effects of glucocorticoids (GC). The aim of this study was to examine whether interaction between MIF and GC underlies the ability of MIF to promote leukocyte-endothelial cell interactions. Methods Intravital microscopy was used to assess leukocyte-endothelial cell interactions in wild-type and MIF−/− mice following treatment with LPS, the GC dexamethasone, and inhibition of endogenous GC using the GC receptor antagonist, RU486. Results Dexamethasone reduced LPS-induced leukocyte interactions in wild-type mice to levels similar to those observed in MIF−/− mice not treated with dexamethasone, whereas in MIF−/− mice, leukocyte interactions were not further inhibited by dexamethasone. RU486 increased LPS-induced leukocyte adhesion and emigration to a similar extent in both wild-type and MIF−/− mice, indicating that endogenous GC exert a similar inhibitory effect on leukocyte trafficking in wild-type and MIF−/− mice. Both MIF deficiency and RU486 treatment reduced VCAM-1 expression, while neither treatment modulated expression of ICAM-1 or chemokines CCL2, KC and MIP-2. Conclusions These results suggest that endogenous MIF and GC regulate leukocyte-endothelial cell interactions in vivo reciprocally but through predominantly independent mechanisms, and that the anti-inflammatory effect of MIF deficiency is comparable to that of exogenous GC. PMID:19905972

  6. Swine Leukocyte Antigen Diversity in Canadian Specific Pathogen-Free Yorkshire and Landrace Pigs

    PubMed Central

    Gao, Caixia; Quan, Jinqiang; Jiang, Xinjie; Li, Changwen; Lu, Xiaoye; Chen, Hongyan

    2017-01-01

    The highly polymorphic swine major histocompatibility complex (MHC), termed swine leukocyte antigen (SLA), is associated with different levels of immunologic responses to infectious diseases, vaccines, and transplantation. Pig breeds with known SLA haplotypes are important genetic resources for biomedical research. Canadian Yorkshire and Landrace pigs represent the current specific pathogen-free (SPF) breeding stock maintained in the isolation environment at the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. In this study, we identified 61 alleles at five polymorphic SLA loci (SLA-1, SLA-2, SLA-3, DRB1, and DQB1) representing 17 class I haplotypes and 11 class II haplotypes using reverse transcription-polymerase chain reaction (RT-PCR) sequence-based typing and PCR-sequence specific primers methods in 367 Canadian SPF Yorkshire and Landrace pigs. The official designation of the alleles has been assigned by the SLA Nomenclature Committee of the International Society for Animal Genetics and released in updated Immuno Polymorphism Database-MHC SLA sequence database [Release 2.0.0.3 (2016-11-03)]. The submissions confirmed some unassigned alleles and standardized nomenclatures of many previously unconfirmed alleles in the GenBank database. Three class I haplotypes, Hp-37.0, 63.0, and 73.0, appeared to be novel and have not previously been reported in other pig populations. One crossover within the class I region and two between class I and class II regions were observed, resulting in three new recombinant haplotypes. The presence of the duplicated SLA-1 locus was confirmed in three class I haplotypes Hp-28.0, Hp-35.0, and Hp-63.0. Furthermore, we also analyzed the functional diversities of 19 identified frequent SLA class I molecules in this study and confirmed the existence of four supertypes using the MHCcluster method. These results will be useful for studying the adaptive immune response and immunological phenotypic differences in

  7. Leukocyte Recruitment in Inflammation: Basic Concepts and New Mechanistic Insights Based on New Models and Microscopic Imaging Technologies

    PubMed Central

    Leick, Marion; Azcutia, Veronica; Newton, Gail; Luscinskas, Francis W.

    2014-01-01

    The immune cell system is a critical component of host defense. Recruitment of immune cells to sites of infection, immune reaction, or injury is complex and involves coordinated adhesive interactions between the leukocyte and the endothelial cell monolayer that lines blood vessels. This article will review basic mechanisms in the recruitment of leukocytes to tissues, and then selectively review new concepts that are emerging based on advances in live cell imaging microscopy and mouse strains. These emerging concepts are altering the conventional paradigms of inflammatory leukocyte recruitment established in the early 1990’s. Indeed, recent publications have identified previously unrecognized contributions from pericytes and interstitial leukocytes and their secreted products that guide leukocytes to their targets. It is also notable that investigators have begun to design organs on a chip. Recent reports indicate that this avenue of research holds much promise. PMID:24562377

  8. Polymorphonuclear leukocyte and monocyte activation induced by plasma from patients with heparin-induced thrombocytopenia in whole blood.

    PubMed

    Khairy, Mahnouch; Lasne, Dominique; Amelot, Aymeric; Crespin, Malvina; Rendu, Francine; Aiach, Martine; Bachelot-Loza, Christilla

    2004-12-01

    Heparin-induced thrombocytopenia (HIT), a severe complication of heparin therapy, results from platelet activation by heparin-dependent antibodies. Previously, we have shown that plasma from patients with HIT (HIT plasma) induces leukocyteplatelet aggregation in blood. In this report, we examined leukocyte activation by HIT plasma and the contribution of heparin and platelets to this activation, in whole blood. Degranulation of leukocytes from HIT patients was evaluated as a leukocyte activation marker. We showed that polymorphonuclear leukocytes (PMN) and monocytes were the leukocyte subpopulations involved in platelet-leukocyte aggregation induced by HIT plasma in healthy donor blood. PMN and monocyte activation, reflected by increased surface expression of the CD11b adhesion molecule, was induced by HIT plasma in a heparin-dependent manner. The CD11b increase induced by HIT plasma was observed on PMN only when they were associated with platelets. Moreover, the increased CD11b expression on monocytes and PMN correlated strongly with the degree of platelet adhesion to these cells. Degranulation of leukocytes from HIT patients and control subjects (non-HIT heparin-treated patients and healthy subjects) was evaluated in vivo by measuring the plasma myeloperoxidase concentration. HIT plasma contained higher myeloperoxidase concentrations than control plasma, suggesting leukocyte degranulation during HIT. In conclusion, this study provides the first evidence that PMN activation is induced by HIT plasma. HIT plasma induced PMN and monocyte activation in a heparin-dependent manner. In whole blood, platelet association with monocytes and PMN, and the activation of these leukocytes by HIT plasma were interrelated. Finally, leukocyte degranulation could be involved in HIT physiopathology.

  9. Role of β1 integrins and bacterial adhesins for Yop injection into leukocytes in Yersinia enterocolitica systemic mouse infection.

    PubMed

    Deuschle, Eva; Keller, Birgit; Siegfried, Alexandra; Manncke, Birgit; Spaeth, Tanja; Köberle, Martin; Drechsler-Hake, Doreen; Reber, Julia; Böttcher, Ralph T; Autenrieth, Stella E; Autenrieth, Ingo B; Bohn, Erwin; Schütz, Monika

    2016-02-01

    Injection of Yersinia outer proteins (Yops) into host cells by a type III secretion system is an important immune evasion mechanism of Yersinia enterocolitica (Ye). In this process Ye invasin (Inv) binds directly while Yersinia adhesin A (YadA) binds indirectly via extracellular matrix (ECM) proteins to β1 integrins on host cells. Although leukocytes turned out to be an important target of Yop injection by Ye, it was unclear which Ye adhesins and which leukocyte receptors are required for Yop injection. To explain this, we investigated the role of YadA, Inv and β1 integrins for Yop injection into leukocytes and their impact on the course of systemic Ye infection in mice. Ex vivo infection experiments revealed that adhesion of Ye via Inv or YadA is sufficient to promote Yop injection into leukocytes as revealed by a β-lactamase reporter assay. Serum factors inhibit YadA- but not Inv-mediated Yop injection into B and T cells, shifting YadA-mediated Yop injection in the direction of neutrophils and other myeloid cells. Systemic Ye mouse infection experiments demonstrated that YadA is essential for Ye virulence and Yop injection into leukocytes, while Inv is dispensable for virulence and plays only a transient and minor role for Yop injection in the early phase of infection. Ye infection of mice with β1 integrin-depleted leukocytes demonstrated that β1 integrins are dispensable for YadA-mediated Yop injection into leukocytes, but contribute to Inv-mediated Yop injection. Despite reduced Yop injection into leukocytes, β1 integrin-deficient mice exhibited an increased susceptibility for Ye infection, suggesting an important role of β1 integrins in immune defense against Ye. This study demonstrates that Yop injection into leukocytes by Ye is largely mediated by YadA exploiting, as yet unknown, leukocyte receptors.

  10. Donor exosomes rather than passenger leukocytes initiate alloreactive T cell responses after transplantation

    PubMed Central

    Marino, Jose; Babiker-Mohamed, Mohamed H.; Crosby-Bertorini, Patrick; Paster, Joshua T.; LeGuern, Christian; Germana, Sharon; Abdi, Reza; Uehara, Mayuko; Kim, James I.; Markmann, James F.; Tocco, Georges; Benichou, Gilles

    2016-01-01

    Transplantation of allogeneic organs and tissues represents a lifesaving procedure for a variety of patients affected with end-stage diseases. Although current immunosuppressive therapy prevents early acute rejection, it is associated with nephrotoxicity and increased risks for infection and neoplasia. This stresses the need for selective immune-based therapies relying on manipulation of lymphocyte recognition of donor antigens. The passenger leukocyte theory states that allograft rejection is initiated by recipient T cells recognizing donor major histocompatibility complex (MHC) molecules displayed on graft leukocytes migrating to the host’s lymphoid organs. We revisited this concept in mice transplanted with allogeneic skin, heart, or islet grafts using imaging flow cytometry. We observed no donor cells in the lymph nodes and spleen of skin-grafted mice, but we found high numbers of recipient cells displaying allogeneic MHC molecules (cross-dressed) acquired from donor microvesicles (exosomes). After heart or islet transplantation, we observed few donor leukocytes (100 per million) but large numbers of recipient cells cross-dressed with donor MHC (>90,000 per million). Last, we showed that purified allogeneic exosomes induced proinflammatory alloimmune responses by T cells in vitro and in vivo. Collectively, these results suggest that recipient antigen-presenting cells cross-dressed with donor MHC rather than passenger leukocytes trigger T cell responses after allotransplantation. PMID:27942611

  11. TRPC6 is the endothelial calcium channel that regulates leukocyte transendothelial migration during the inflammatory response

    PubMed Central

    Weber, Evan W.; Han, Fei; Tauseef, Mohammad; Birnbaumer, Lutz; Mehta, Dolly

    2015-01-01

    Leukocyte transendothelial migration (TEM) is a tightly regulated, multistep process that is critical to the inflammatory response. A transient increase in endothelial cytosolic free calcium ion concentration (↑[Ca2+]i) is required for TEM. However, the mechanism by which endothelial ↑[Ca2+]i regulates TEM and the channels mediating this ↑[Ca2+]i are unknown. Buffering ↑[Ca2+]i in endothelial cells does not affect leukocyte adhesion or locomotion but selectively blocks TEM, suggesting a role for ↑[Ca2+]i specifically for this step. Transient receptor potential canonical 6 (TRPC6), a Ca2+ channel expressed in endothelial cells, colocalizes with platelet/endothelial cell adhesion molecule-1 (PECAM) to surround leukocytes during TEM and clusters when endothelial PECAM is engaged. Expression of dominant-negative TRPC6 or shRNA knockdown in endothelial cells arrests neutrophils apically over the junction, similar to when PECAM is blocked. Selectively activating endothelial TRPC6 rescues TEM during an ongoing PECAM blockade, indicating that TRPC6 functions downstream of PECAM. Furthermore, endothelial TRPC6 is required for trafficking of lateral border recycling compartment membrane, which facilitates TEM. Finally, mice lacking TRPC6 in the nonmyeloid compartment (i.e., endothelium) exhibit a profound defect in neutrophil TEM with no effect on leukocyte trafficking. Our findings identify endothelial TRPC6 as the calcium channel mediating the ↑[Ca2+]i required for TEM at a step downstream of PECAM homophilic interactions. PMID:26392222

  12. Neuroendocrine modulation of the inflammatory response in common carp: adrenaline regulates leukocyte profile and activity.

    PubMed

    Kepka, M; Verburg-van Kemenade, B M L; Chadzinska, M

    2013-07-01

    Inflammatory responses have to be carefully controlled, as high concentrations and/or prolonged action of inflammation-related molecules (e.g. reactive oxygen species, nitric oxide and pro-inflammatory cytokines) can be detrimental to host tissue and organs. One of the potential regulators of the inflammatory process are stress mediators including adrenaline. In vivo effects of adrenaline were studied during zymosan-induced (Z) peritoneal inflammation in the common carp Cyprinus carpio L. Adrenaline injected together with zymosan (ZA) did not change the number of inflammatory leukocytes in the peritoneal cavity, however at 24h post-injection it significantly reduced the percentage of monocytes/macrophages. Moreover, compared to cells retrieved from fish treated with PBS or zymosan only, adrenaline increased the percentage of apoptotic leukocytes in the focus of inflammation. Furthermore, adrenaline significantly reduced the expression of chemokine CXCL8_L1 (a functional homolog of mammalian IL-8) and its receptors (CXCR1 and CXCR2), indicating changes in leukocyte recruitment after stress. We conclude that adrenaline may contribute to a coordinated reaction by influencing the inflammatory response via direct regulation of leukocyte migration and/or apoptosis.

  13. Molecular characterization of swine leukocyte antigen (SLA) class II genes in outbred pig populations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The highly polymorphic swine leukocyte antigen (SLA) genes are one of the most important determinants in swine immune, disease and vaccine responses. Thus, understanding how SLA gene polymorphism affects immunity, especially in outbred pig populations with a diverse genetic background, requires accu...

  14. MOLECULAR CHARACTERIZATION OF SWINE LEUKOCYTE ANTIGEN (SLA) CLASS I GENES IN OUTBRED PIG POPULATIONS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The highly polymorphic swine leukocyte antigen (SLA) genes are one of the most important determinants in swine immune responses to infectious diseases, vaccines, and in transplantation. Study of SLA influence requires accurate and effective typing methods. We developed a simple and rapid method to t...

  15. P38 and JNK have opposing effects on persistence of in vivo leukocyte migration in zebrafish

    PubMed Central

    Taylor, Harriet B; Liepe, Juliane; Barthen, Charlotte; Bugeon, Laurence; Huvet, Maxime; Kirk, Paul DW; Brown, Simon B; Lamb, Jonathan R; Stumpf, Michael PH; Dallman, Margaret J

    2013-01-01

    The recruitment and migration of macrophages and neutrophils is an important process during the early stages of the innate immune system in response to acute injury. Transgenic pu.1:EGFP zebrafish permit the acquisition of leukocyte migration trajectories during inflammation. Currently, these high-quality live-imaging data are mainly analysed using general statistics, for example, cell velocity. Here, we present a spatio-temporal analysis of the cell dynamics using transition matrices, which provide information of the type of cell migration. We find evidence that leukocytes exhibit types of migratory behaviour, which differ from previously described random walk processes. Dimethyl sulfoxide treatment decreased the level of persistence at early time points after wounding and ablated temporal dependencies observed in untreated embryos. We then use pharmacological inhibition of p38 and c-Jun N-terminal kinase mitogen-activated protein kinases to determine their effects on in vivo leukocyte migration patterns and discuss how they modify the characteristics of the cell migration process. In particular, we find that their respective inhibition leads to decreased and increased levels of persistent motion in leukocytes following wounding. This example shows the high level of information content, which can be gained from live-imaging data if appropriate statistical tools are used. PMID:23165607

  16. Rac-null leukocytes are associated with increased inflammation-mediated alveolar bone loss.

    PubMed

    Sima, Corneliu; Gastfreund, Shoshi; Sun, Chunxiang; Glogauer, Michael

    2014-02-01

    Periodontitis is characterized by altered host-biofilm interactions that result in irreversible inflammation-mediated alveolar bone loss. Genetic and epigenetic factors that predispose to ineffective control of biofilm composition and maintenance of tissue homeostasis are not fully understood. We elucidated how leukocytes affect the course of periodontitis in Rac-null mice. Mouse models of acute gingivitis and periodontitis were used to assess the early inflammatory response and patterns of chronicity leading to loss of alveolar bone due to inflammation in Rac-null mice. Leukocyte margination was differentially impaired in these mice during attachment in conditional Rac1-null (granulocyte/monocyte lineage) mice and during rolling and attachment in Rac2-null (all blood cells) mice. Inflammatory responses to subgingival ligatures, assessed by changes in peripheral blood differential leukocyte numbers, were altered in Rac-null compared with wild-type mice. In response to persistent subgingival ligature-mediated challenge, Rac-null mice had increased loss of alveolar bone with patterns of resorption characteristic of aggressive forms of periodontitis. These findings were partially explained by higher osteoclastic coverage of the bone-periodontal ligament interface in Rac-null compared with wild-type mice. In conclusion, this study demonstrates that leukocyte defects, such as decreased endothelial margination and tissue recruitment, are rate-limiting steps in the periodontal inflammatory process that lead to more aggressive forms of periodontitis.

  17. Leukocytes in chemotactic-fragment-induced lung inflammation. Vascular emigration and alveolar surface migration.

    PubMed Central

    Shaw, J. O.

    1980-01-01

    Lung inflammation was induced in rabbits by intratracheal injections of chemotactic fragments obtained from zymosan-activated serum (CF-ZAS), and the route of vascular emigration and alveolar surface interaction of polymorphonuclear leukocytes (PMNs) and monocytes migrating into the lung was characterized by transmission (TEM) and scanning (SEM) electron-microscopic examination. Leukocytes migrated from capillaries and venules into the alveolar wall interstitium by adherence to the vascular endothelium and migration through the endothelial intracellular junction to attain a position between a reapposed endothelial cell junction and the vascular basement membrane. The cells then migrated into the interstitium through a narrow opening in the basement membrane. Leukocyte entrance into the alveolar space from the interstitium appeared to occur through small openings in the epithelial basement membrane at or near the Type I epithelial intercellular junction. Once in the alveolus, PMNs and macrophages demonstrated surface adherence and spreading along with evidence of migration, pseudopod extension, interalveolar pore transit, and retraction fiber formation. This study indicates the leukocyte influx into the alveolus in acute chemotactic-factor-induced inflammation is via a continuum of migrational activity, beginning at the pulmonary capillary endothelial surface and persisting on the alveolar epithelial surface. Images Figure 10 Figure 11 Figure 12 Figure 1 Figure 2 Figure 3 Figure 13 Figure 14 Figure 4 Figure 5 Figure 6 Figure 15 Figure 7 Figure 8 Figure 16 Figure 9 PMID:7435538

  18. A New Machine Classification Method Applied to Human Peripheral Blood Leukocytes.

    ERIC Educational Resources Information Center

    Rorvig, Mark E.; And Others

    1993-01-01

    Discusses pattern classification of images by computer and describes the Two Domain Method in which expert knowledge is acquired using multidimensional scaling of judgments of dissimilarities and linear mapping. An application of the Two Domain Method that tested its power to discriminate two patterns of human blood leukocyte distribution is…

  19. Phylogenetic analysis of the swine leukocyte antigen - 2 gene for Korean native pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to investigate genetic distances of the SLA-2 gene, to characterize SLA-2 alleles, and to provide basic genetic information of Korean pigs. The swine leukocyte antigen - 2 (SLA-2) gene in the MHC classical region was cloned with spleen tissues from Korean native pigs ...

  20. No Evidence of Human Leukocyte Antigen Gene Association With Rheumatic Fever Among Children in Samoa.

    PubMed

    Erdem, Guliz; Seifried, Steven E

    2015-03-01

    Human leukocyte antigens (HLAs) have been implicated in rheumatic fever pathogenesis. This pilot whole genome association study compares genotypes of Samoan children with rheumatic fever to unaffected siblings and unrelated healthy controls. No risk-related genotypes were associated with HLA genes. Thirteen Regions of Interest were identified as candidates for further study.

  1. Longer leukocyte telomeres are associated with ultra-endurance exercise independent of cardiovascular risk factors.

    PubMed

    Denham, Joshua; Nelson, Christopher P; O'Brien, Brendan J; Nankervis, Scott A; Denniff, Matthew; Harvey, Jack T; Marques, Francine Z; Codd, Veryan; Zukowska-Szczechowska, Ewa; Samani, Nilesh J; Tomaszewski, Maciej; Charchar, Fadi J

    2013-01-01

    Telomere length is recognized as a marker of biological age, and shorter mean leukocyte telomere length is associated with increased risk of cardiovascular disease. It is unclear whether repeated exposure to ultra-endurance aerobic exercise is beneficial or detrimental in the long-term and whether it attenuates biological aging. We quantified 67 ultra-marathon runners' and 56 apparently healthy males' leukocyte telomere length (T/S ratio) using real-time quantitative PCR. The ultra-marathon runners had 11% longer telomeres (T/S ratio) than controls (ultra-marathon runners: T/S ratio = 3.5±0.68, controls: T/S ratio = 3.1±0.41; β = 0.40, SE = 0.10, P = 1.4×10(-4)) in age-adjusted analysis. The difference remained statistically significant after adjustment for cardiovascular risk factors (P = 2.2×10(-4)). The magnitude of this association translates into 16.2±0.26 years difference in biological age and approximately 324-648bp difference in leukocyte telomere length between ultra-marathon runners and healthy controls. Neither traditional cardiovascular risk factors nor markers of inflammation/adhesion molecules explained the difference in leukocyte telomere length between ultra-marathon runners and controls. Taken together these data suggest that regular engagement in ultra-endurance aerobic exercise attenuates cellular aging.

  2. Extracellular adenosine triphosphate activates calcium mobilization in human phagocytic leukocytes and neutrophil/monocyte progenitor cells.

    PubMed Central

    Cowen, D S; Lazarus, H M; Shurin, S B; Stoll, S E; Dubyak, G R

    1989-01-01

    We have examined the ability of extracellular ATP to elicit intracellular Ca2+ mobilization in a broad range of human leukocytes at particular stages of hematopoietic differentiation. The average cytosolic [Ca2+] in various leukocyte populations was measured in Fura 2-loaded cell suspensions while the cytosolic [Ca2+] in individual, Indo 1-loaded leukocytes was assayed by flow cytometric methods. Utilizing normal blood- and marrow-derived cells, human leukemic cell lines, and mononuclear cell fractions derived from the blood of patients with various leukemias, we have found that ATP-induced Ca2+ mobilization appears restricted to leukocytes of neutrophil/monocyte ontogeny. Significant ATP-induced increases in cytosolic [Ca2+] were observed in neutrophils, monocytes, and myeloid progenitor cells as immature as myeloblasts, but not in lymphocytes. Extensive characterization of the ATP-induced changes in [Ca2+] observed in the HL-60 promyelocytic cell line have indicated these Ca2+-mobilizing effects of ATP can be correlated with an activation of inositol phospholipid breakdown via the occupation of P2-purinergic receptors Significantly, of the various agonists (FMLP, platelet-activating factor, LTB4, and ATP) which elicit equivalent and maximal Ca2+ mobilization in mature neutrophils and monocytes, ATP was the most efficacious stimulant of Ca2+ mobilization in immature neutrophil/monocyte precursors. Thus, expression of putative P2-purinergic receptors for ATP appears to precede expression of other receptor types known to activate the inositol phospholipid signaling cascades in terminally differentiated phagocytes. PMID:2708526

  3. Harvesting the noncirculating pool of polymorphonuclear leukocytes in rats by hetastarch exchange transfusion (HET): yield and functional assessment

    SciTech Connect

    Williams, J.H. Jr.; Moser, K.M.; Ulich, T.; Cairo, M.S.

    1987-11-01

    Isolation of polymorphonuclear leukocytes (PMN) provides an opportunity to study PMN activity in vitro and to label PMN for study of in vivo kinetics. However, simple phlebotomy (SP) of a small animal frequently yields too few PMN for in vitro handling, while PMN harvested from an induced-peritonitis may not accurately reflect PMN in a less stimulated state. We report a novel method of harvesting PMN from the circulation of rats, using hetastarch exchange transfusion (HET), which is both time and animal sparing. HET harvested 8-fold more PMN than SP. In vitro cell function was examined with assays of adherence, chemotaxis, bacterial killing, and superoxide generation. No significant (p less than 0.05) difference was found between PMN obtained by HET and pooled-PMN obtained by SP. In vivo function was examined following labeling with indium 111-oxine. The kinetics pattern described suggested normal migratory activity when compared to previous reports. The data demonstrate that rats possess a relatively large, noncirculating pool of PMN which is readily accessible by HET.

  4. Degeneration and atrophy of the thymus of lethally irradiated dogs, rescued by transfusion of cryopreserved autologous blood leukocytes

    SciTech Connect

    Calvo, W.; Fliedner, T.M.; Herbst, E.W.; Huegl, E.B.; Boedey, B.

    1987-12-01

    Dogs exposed to a fatal radiation dose of 12 Gy were rescued by transfusion of autologous blood leukocytes. A severe acute and long-lasting damage to the thymus was observed. The acute damage, as observed on the tenth day, consisted of a marked reduction in the number of lymphocytes, degeneration of Hassall's bodies, and hemorrhage. Long-term effects, observed several months after irradiation, were partial to total atrophy of the thymus. Regeneration, when it occurred, was limited to a few small isolated areas in which lymphopoiesis was supported by epithelial reticular cells. In contrast, the lymph nodes of all dogs had abundant cortical lymphopoiesis. The abundant hemopoiesis present in the marrow from the tenth day after irradiation until the end of the observation period should have provided sufficient circulating precursor cells to seed the thymus and regenerate the organ to the same extent as that observed in the other blood-forming organs. The impairment of lymphopoietic regeneration in the thymus seems to be due, therefore, to damage caused by irradiation on the specific stroma of the organ, which is not able to support such activity.

  5. Interactions of TANGO and leukocyte integrin CD11c/CD18 regulate the migration of human monocytes.

    PubMed

    Arndt, Stephanie; Melle, Christian; Mondal, Krishna; Klein, Gerd; von Eggeling, Ferdinand; Bosserhoff, Anja-Katrin

    2007-12-01

    The TANGO gene was originally identified as a new member of the MIA gene family. It codes for a protein of yet unknown function. TANGO revealed a very broad expression pattern in contrast to the highly restricted expression pattern determined for the other family members. The only cells lacking TANGO expression are cells of the hematopoietic system. One of the major differences between mature hematopoietic cells and other tissue cells is the lack of adhesion until these cells leave the bloodstream. In this study, we observed that TANGO expression was induced after adhesion of human monocytic cells to substrate. To understand the mechanism of TANGO function during monocyte adhesion we isolated interacting proteins and found an interaction between TANGO and the leukocyte-specific integrin CD11c. In functional assays, we observed reduced attachment of human monocytic cells to fibrinogen, ICAM-1 and to human microvascular endothelial cells (HMECs) after stimulation with recombinant TANGO protein. Additionally, the migrating capacity of premonocytic cells through fibrinogen or HMECs was increased after stimulation of these cells with recombinant TANGO. Therefore, we suggest that TANGO reduced the attachment to fibrinogen or other cell adhesion molecules. As TANGO does not compete for CD11c ligand binding directly, we hypothesize TANGO function by modulation of integrin activity. Taken together, the results from this study present TANGO as a novel ligand for CD11c, regulating migratory processes of hematopoietic cells.

  6. Purification of the active C5a receptor from human polymorphonuclear leukocytes as a receptor - G sub i complex

    SciTech Connect

    Rollins, T.E.; Siciliano, S.; Kobayashi, S.; Cianciarulo, D.N.; Bonilla-Argudo, V.; Collier, K.; Springer, M.S. )

    1991-02-01

    The authors have isolated, in an active state, the C5a receptor from human polymorphonuclear leukocytes. The purification was achieved in a single step using a C5a affinity column in which the C5a molecule was coupled to the resin through its N terminus. The purified receptor, like the crude solubilized molecule, exhibited a single class of high-affinity binding sites with a K{sub d} of 30 pM. Further, the binding of C5a retained its sensitivity to guanine nucleotides, implying that the purified receptor contained a guanine nucleotide-binding protein (G protein). SDS/PAGE revealed the presence of three polypeptides with molecular masses of 42, 40, and 36 kDa, which were determined to be the C5a-binding subunit and the {alpha} and {beta} subunits of G{sub i}, respectively. The 36- and 40-kDa polypeptides were identified by immunoblotting and by the ability of pertussis toxin to ADP-ribosylate the 40-kDa molecule. These results confirm their earlier hypothesis that the receptor exists as a complex with a G protein in the presence or absence of C5a. The tight coupling between the receptor and G protein should make possible the identification of the G protein(s) involved in the transduction pathways used by C5a to produce its many biological effects.

  7. Primary Amine Oxidase of Escherichia coli Is a Metabolic Enzyme that Can Use a Human Leukocyte Molecule as a Substrate

    PubMed Central

    Maksimow, Mikael; Elima, Kati; Yegutkin, Gennady G.; Skurnik, Mikael; Dobrindt, Ulrich; Siitonen, Anja; McPherson, Michael J.

    2015-01-01

    Escherichia coli amine oxidase (ECAO), encoded by the tynA gene, catalyzes the oxidative deamination of aromatic amines into aldehydes through a well-established mechanism, but its exact biological role is unknown. We investigated the role of ECAO by screening environmental and human isolates for tynA and characterizing a tynA-deletion strain using microarray analysis and biochemical studies. The presence of tynA did not correlate with pathogenicity. In tynA+ Escherichia coli strains, ECAO enabled bacterial growth in phenylethylamine, and the resultant H2O2 was released into the growth medium. Some aminoglycoside antibiotics inhibited the enzymatic activity of ECAO, which could affect the growth of tynA+ bacteria. Our results suggest that tynA is a reserve gene used under stringent environmental conditions in which ECAO may, due to its production of H2O2, provide a growth advantage over other bacteria that are unable to manage high levels of this oxidant. In addition, ECAO, which resembles the human homolog hAOC3, is able to process an unknown substrate on human leukocytes. PMID:26556595

  8. Functional role of gap junctions in cytokine-induced leukocyte adhesion to endothelium in vivo

    PubMed Central

    Véliz, Loreto P.; González, Francisco G.; Duling, Brian R.; Sáez, Juan C.; Boric, Mauricio P.

    2008-01-01

    To assess the hypothesis that gap junctions (GJs) participate on leukocyte-endothelium interactions in the inflammatory response, we compared leukocyte adhesion and transmigration elicited by cytokine stimulation in the presence or absence of GJ blockers in the hamster cheek pouch and also in the cremaster muscle of wild-type (WT) and endothelium-specific connexin 43 (Cx43) null mice (Cx43e−/−). In the cheek pouch, topical tumor necrosis factor-α (TNF-α; 150 ng/ml, 15 min) caused a sustained increment in the number of leukocytes adhered to venular endothelium (LAV) and located at perivenular regions (LPV). Superfusion with the GJ blockers 18-α-glycyrrhetinic acid (AGA; 75 μM) or 18-β-glycyrrhetinic acid (50 μM) abolished the TNF-α-induced increase in LAV and LPV; carbenoxolone (75 μM) or oleamide (100 μM) reduced LAV by 50 and 75%, respectively, and LPV to a lesser extent. None of these GJ blockers modified venular diameter, blood flow, or leukocyte rolling. In contrast, glycyrrhizin (75 μM), a non-GJ blocker analog of AGA, was devoid of effect. Interestingly, when AGA was removed 90 min after TNF-α stimulation, LAV started to rise at a similar rate as in control. Conversely, application of AGA 90 min after TNF-α reduced the number of previously adhered cells. In WT mice, intrascrotal injection of TNF-α (0.5 μg/0.3 ml) increased LAV (fourfold) and LPV (threefold) compared with saline-injected controls. In contrast to the observations in WT animals, TNF-α stimulation did not increase LAV or LPV in Cx43e−/− mice. These results demonstrate an important role for GJ communication in leukocyte adhesion and transmigration during acute inflammation in vivo and further suggest that endothelial Cx43 is key in these processes. PMID:18599597

  9. Oxidative DNA Damage to Sperm Cells and Peripheral Blood Leukocytes in Infertile Men

    PubMed Central

    Taken, Kerem; Alp, Hamit Hakan; Eryilmaz, Recep; Donmez, Muhammet Irfan; Demir, Murat; Gunes, Mustafa; Aslan, Rahmi; Sekeroglu, Mehmet Ramazan

    2016-01-01

    Background Oxidative DNA damage is associated with male infertility. The aim of this study was to evaluate the oxidative DNA damage of sperm cells and blood leukocytes and to determine the levels of MDA and NO levels in seminal and blood plasma of idiopathic infertile men. Material/Methods The study enrolled 52 patients, including 30 infertile and 22 fertile men. MDA, NO, and 8-OHdG/106dG were estimated using spectrophotometry and high-pressure liquid chromatography (HPLC)-based methods in seminal and blood plasma. The association with the sperm parameters was assessed, particularly sperm counts and motility. Results The mean sperm concentration and sperm motility of the fertile men were significantly higher than that of the infertile men. The mean MDA and NO concentration in the seminal and blood samples of the infertile men were higher than that of fertile men. Also, the mean numbers of sperm cells and leukocytes 8-OHdG/106dG of the infertile men were significantly higher than that of fertile men (p=0.04 and p<0.001, respectively). Sperm motility and sperm count were negatively correlated with leukocyte and sperm cell 8-OHdG/106dG ratio. However, progressive motility was significantly negatively correlated with sperm cell and leukocyte 8-OHdG/106dG ratio (R=−0.357, p=0.026; R=−0.388, p=0.024, respectively). Conclusions Oxidative stress is an important factor in male infertility. Therefore, biochemical detection of 8-OHdG/106dG in sperm cells and blood leukocytes may be an additional tool in the diagnosis of male infertility. PMID:27837200

  10. Red blood cell and leukocyte alloimmunization in patients awaiting kidney transplantation

    PubMed Central

    da Silva, Silvia Fernandes Ribeiro; Ferreira, Gláucia Maria; da Silva, Sonia Leite; Alves, Tânia Maria de Oliveira; Ribeiro, Ilana Farias; Ribeiro, Thyciana Rodrigues; Cavalcante, Maria do Carmo Serpa

    2013-01-01

    Objective To determine the rates of red blood cell and leukocyte alloimmunization in patients with chronic kidney disease awaiting kidney transplantation. Methods In this cross-sectional and prospective study, the serum of 393 chronic kidney disease patients on a transplant waiting list in Ceará, Northeastern Brazil were tested for red cell and leukocyte antibodies. In addition, demographic, clinical and laboratory data were collected. Results The average age in the sample of 393 patients was 34.1 ± 14 years. Slightly more than half (208; 52.9%) were male. The average numbers of transfusions and gestations were 3.1 ± 3.3 and 1.6 ± 6, respectively. One third (33.6%) were alloimmunized: 78% with leukocyte antibodies, 9.1% with red cell antibodies and 12.9% with both. Red cell antibodies were detected in 29 cases (7.4%), 17 of whom were women, who had received more transfusions than the males (p-value < 0.0001). The most frequently detected red cell antibodies belonged to the Rh (24.1%) and Kell (13.8%) blood group systems. Leukocyte antibodies were detected in 30.5% of cases, 83 of whom were women, who had received more transfusions than the males (p-value < 0.0001) and were more reactive to panel reactive antibodies (p-value < 0.0001). The mean alloreactivity to panel reactive antibodies was 47.7 ± 31.2%. Conclusion Chronic kidney disease patients on the transplant waiting list in Ceará, Brazil, display high rates of red cell (7.4%) and leukocyte (30.5%) alloimmunization. In this sample, alloimmunization was significantly associated with the number of transfusions and gender. PMID:23904808

  11. Determination of an unrelated donor pool size for human leukocyte antigen-matched platelets in Brazil

    PubMed Central

    Bub, Carolina Bonet; Torres, Margareth Afonso; Moraes, Maria Elisa; Hamerschlak, Nelson; Kutner, José Mauro

    2015-01-01

    Background Successful transfusion of platelet refractory patients is a challenge. Many potential donors are needed to sustain human leukocyte antigen matched-platelet transfusion programs because of the different types of antigens and the constant needs of these patients. For a highly mixed population such as the Brazilian population, the pool size required to provide adequate platelet support is unknown. Methods A mathematical model was created to estimate the appropriate size of an unrelated donor pool to provide human leukocyte antigen-compatible platelet support for a Brazilian population. A group of 154 hematologic human leukocyte antigen-typed patients was used as the potential patient population and a database of 65,500 human leukocyte antigen-typed bone marrow registered donors was used as the donor population. Platelet compatibility was based on the grading system of Duquesnoy. Results Using the mathematical model, a pool containing 31,940, 1710 and 321 donors would be necessary to match more than 80% of the patients with at least five completely compatible (no cross-reactive group), partial compatible (one cross-reactive group) or less compatible (two cross-reactive group) donors, respectively. Conclusion The phenotypic diversity of the Brazilian population has probably made it more difficulty to find completely compatible donors. However, this heterogeneity seems to have facilitated finding donors when cross-reactive groups are accepted as proposed by the grading system of Duquesnoy. The results of this study may help to establish unrelated human leukocyte antigen-compatible platelet transfusions, a procedure not routinely performed in most Brazilian transfusion services. PMID:26969768

  12. Association between Leukocyte Mitochondrial DNA Copy Number and Regular Exercise in Postmenopausal Women

    PubMed Central

    Chang, Yu Kyung; Kim, Da Eun; Cho, Soo Hyun

    2016-01-01

    Background Previous studies suggest that habitual exercise can improve skeletal mitochondrial function; however, to date, the association between exercise and mitochondrial function in peripheral leukocytes has not been reported. The aim of this study was to evaluate the relationship between regular exercise and mitochondrial function by measuring leukocyte mitochondrial DNA (mtDNA) copy number in postmenopausal women. Methods This cross-sectional study included 144 relatively healthy, non-diabetic, non-smoking, postmenopausal women. Clinical parameters, including anthropometric measurements and cardio-metabolic parameters, were assessed. Regular exercise was defined as at least 150 minutes per week of moderate-intensity activity, or an equivalent combination of moderate and vigorous-intensity activity, over a duration of at least 6 months. Leukocyte mtDNA copy numbers were measured using real-time polymerase chain reaction assays, and these were normalized to the β-globin copy number to give the relative mtDNA copy number. Results The mtDNA copy number of peripheral leukocytes was significantly greater in the exercise group (1.33±0.02) than in the no exercise group (1.05±0.02, P<0.01). Stepwise multiple regression analysis showed that regular exercise was independently associated with mtDNA copy number (β=0.25, P<0.01) after adjusting for the variables age, body mass index, waist-to-hip ratio, systolic and diastolic blood pressure, homeostasis model assessment of insulin resistance value, and levels of high-density lipoprotein cholesterol, triglycerides, and homocysteine. Conclusion Regular exercise is associated with greater leukocyte mtDNA copy number in postmenopausal women. PMID:27900071

  13. Increased expression of telomere-regulating genes in endurance athletes with long leukocyte telomeres.

    PubMed

    Denham, Joshua; O'Brien, Brendan J; Prestes, Priscilla R; Brown, Nicholas J; Charchar, Fadi J

    2016-01-15

    Leukocyte telomeres shorten with age, and excessive shortening is associated with age-related cardiometabolic diseases. Exercise training may prevent disease through telomere length maintenance although the optimal amount of exercise that attenuates telomere attrition is unknown. Furthermore, the underlying molecular mechanisms responsible for the enhanced telomere maintenance observed in endurance athletes is poorly understood. We quantified the leukocyte telomere length and analyzed the expression of telomere-regulating genes in endurance athletes and healthy controls (both n = 61), using quantitative PCR. We found endurance athletes have significantly longer (7.1%, 208-416 nt) leukocyte telomeres and upregulated TERT (2.0-fold) and TPP1 (1.3-fold) mRNA expression compared with controls in age-adjusted analysis. The telomere length and telomere-regulating gene expression differences were no longer statistically significant after adjustment for resting heart rate and relative V̇O(2 max) (all P > 0.05). Resting heart rate emerged as an independent predictor of leukocyte telomere length and TERT and TPP1 mRNA expression in stepwise regression models. To gauge whether volume of exercise was associated with leukocyte telomere length, we divided subjects into running and cycling tertiles (distance covered per week) and found individuals in the middle and highest tertiles had longer telomeres than individuals in the lowest tertile. These data emphasize the importance of cardiorespiratory fitness and exercise training in the prevention of biological aging. They also support the concept that moderate amounts of exercise training protects against biological aging, while higher amounts may not elicit additional benefits.

  14. Leukocyte Subset Changes in Response to a 164-km Road Cycle Ride in a Hot Environment

    PubMed Central

    LUK, HUI-YING; MCKENZIE, AMY L.; DUPLANTY, ANTHONY A.; BUDNAR, RONALD G.; LEVITT, DANIELLE; FERNANDEZ, ALEX; LEE, ELAINE C.; ARMSTRONG, LAWRENCE E.; VINGREN, JAKOB L.

    2016-01-01

    The purpose of this observational study was to determine the circulating leukocyte subset response to completing the 2013 Hotter’N Hell Hundred recreational 164-km road cycle event in a hot and humid environmental condition. Twenty-eight men and four women were included in this study. Whole blood samples were obtained 1–2 hours before (PRE) and immediately after (POST) the event. Electronic sizing/sorting and cytometry were used to determine complete blood counts (CBC) including neutrophil, monocyte, and lymphocyte subsets. The concentration of circulating total leukocytes (103·μL−1) increased 134% from PRE to POST with the greatest increase in neutrophils (319%, p<0.0001). Circulating monocytes (including macrophages) increased 24% (p=0.004) and circulating lymphocytes including B and T cells increased 53% (p<0.0001). No association was observed between rolling time or relative intensity and leukocyte subset. Completing the Hotter n′ Hell Hundred (HHH), a 100 mile recreational cycling race in extreme (hot and humid) environmental conditions, induces a substantial increase in total leukocytes in circulation. The contribution of increases in specific immune cell subsets is not equal, with neutrophils increasing to greater than 4-fold starting values from PRE to POST race. It is likely that exercise in stressful environmental conditions affects the complement of circulating immune cells, although activational state and characterization of specific leukocyte subsets remains unclear. The observed increase in circulating cell sub-populations suggests that the circulating immune surveillance system may be acutely affected by exercise in hot and humid conditions. PMID:27293505

  15. Interaction of complement and leukocytes in severe acute pancreatitis: potential for therapeutic intervention.

    PubMed

    Hartwig, Werner; Klafs, Martina; Kirschfink, Michael; Hackert, Thilo; Schneider, Lutz; Gebhard, Martha-Maria; Büchler, Markus W; Werner, Jens

    2006-11-01

    In acute pancreatitis, local as well as systemic organ complications are mediated by the activation of various inflammatory cascades. The role of complement in this setting is unclear. The aim of the present study was to determine the level of complement activation in experimental pancreatitis, to evaluate the interaction of complement and leukocyte-endothelium activation, and to assess the effects of complement inhibition by soluble complement receptor 1 (sCR1) in this setting. Necrotizing pancreatitis was induced in Wistar rats by the combination of intravenous cerulein and retrograde infusion of glycodeoxycholic acid into the biliopancreatic duct; edematous pancreatitis was induced by intravenous cerulein only. In control animals, a sham operation (midline laparotomy) was performed. Complement activation, leukocyte sequestration, and pancreatic as well as pulmonary injury were assessed in the presence/absence of sCR1. Increased levels of C3a were found in necrotizing but not in edematous pancreatitis. When complement activation in necrotizing pancreatitis was blocked by sCR1, levels of C3a and total hemolytic activity (CH50) were decreased. Leukocyte-endothelial interaction, as assessed by intravital microscopy, and pancreatic as well as pulmonary organ injury (wet-to-dry weight ratio, MPO activity, and histology) were ameliorated by sCR1. As a result of the present study, necrotizing but not edematous pancreatitis is characterized by significant and early complement activation. Based on the interaction of complement and leukocytes, complement inhibition by sCR1 may be a valuable option in the treatment of leukocyte-associated organ injury in severe pancreatitis.

  16. Inhibitory Effect of Serotonin Antagonist on Leukocyte-Endothelial Interactions In Vivo and In Vitro

    PubMed Central

    Kataoka, Hiroshi; Ariyama, Yuno; Deushi, Michiyo; Osaka, Mizuko; Nitta, Kosaku; Yoshida, Masayuki

    2016-01-01

    Background Although 5-HT2A serotonergic antagonists have been used to treat vascular disease in patients with diabetes mellitus or obesity, their effects on leukocyte-endothelial interactions have not been fully investigated. In this study, we assessed the effects of sarpogrelate hydrochloride (SRPO), a 5-HT2A receptor inverse agonist, on leukocyte-endothelial cell interactions in obesity both in vivo and in vitro. Methods and Findings In the in vivo experiment, C57BL/6 mice were fed a high-fat high-fructose diet (HFFD), comprising 20% fat and 30% fructose, with or without intraperitoneal injection of 5 mg/kg/day SRPO for 4 weeks. The body weight, visceral fat weight, and serum monocyte chemoattractant protein-1 levels in the mice increased significantly with the HFFD, but these effects were prevented by chronic injections of SRPO. Intravital microscopy of the femoral artery detected significant leukocyte-endothelial interactions after treatment with HFFD, but these leukocyte-endothelial interactions were reduced in the mice injected with SRPO. In the in vitro experiment, pre-incubation of activated human umbilical vein endothelial cells (HUVECs) with platelet-rich plasma (PRP) induced THP-1 cell adhesion under physiological flow conditions, but the adhesion was reduced by pretreatment of PRP with SRPO. A fluorescent immunobinding assay showed that PRP induced significant upregulation of E-selectin in HUVECs, but this upregulation was reduced by pretreatment of PRP with SRPO. In other in vitro conditions, pre-incubation of THP-1 cells with phorbol 12-myristate 13-acetate increased the adhesion of THP-1 cells to activated HUVECs under rotational conditions, but this adhesion was reduced by pretreatment with SRPO. Western blotting analysis showed that protein kinase C α activation in THP-1 cells was inhibited by SRPO. Conclusion Our findings indicated that SRPO inhibits vascular inflammation in obesity via inactivation of platelets and leukocytes, and improvement of

  17. Roscovitine blocks leukocyte extravasation by inhibition of cyclin-dependent kinases 5 and 9

    PubMed Central

    Berberich, Nina; Uhl, Bernd; Joore, Jos; Schmerwitz, Ulrike K; Mayer, Bettina A; Reichel, Christoph A; Krombach, Fritz; Zahler, Stefan; Vollmar, Angelika M; Fürst, Robert

    2011-01-01

    BACKGROUND AND PURPOSE Roscovitine, a cyclin-dependent kinase (CDK) inhibitor that induces tumour cell death, is under evaluation as an anti-cancer drug. By triggering leukocyte apoptosis, roscovitine can also enhance the resolution of inflammation. Beyond death-inducing properties, we tested whether roscovitine affects leukocyte-endothelial cell interaction, a vital step in the onset of inflammation. EXPERIMENTAL APPROACH Leukocyte-endothelial cell interactions were evaluated in venules of mouse cremaster muscle, using intravital microscopy. In primary human endothelial cells, we studied the influence of roscovitine on adhesion molecules and on the nuclear factor-κB (NF-κB) pathway. A cellular kinome array, in vitro CDK profiling and RNAi methods were used to identify targets of roscovitine. KEY RESULTS In vivo, roscovitine attenuated the tumour necrosis factor-α (TNF-α)-induced leukocyte adherence to and transmigration through, the endothelium. In vitro, roscovitine strongly inhibited TNF-α-evoked expression of endothelial adhesion molecules (E-selectin, intercellular cell adhesion molecule, vascular cell adhesion molecule). Roscovitine blocked NF-κB-dependent gene transcription, but not the NF-κB activation cascade [inhibitor of κB (IκB) kinase activity, IκB-α degradation, p65 translocation]. Using a cellular kinome array and an in vitro CDK panel, we found that roscovitine inhibited protein kinase A, ribosomal S6 kinase and CDKs 2, 5, 7 and 9. Experiments using kinase inhibitors and siRNA showed that the decreased endothelial activation was due solely to blockade of CDK5 and CDK9 by roscovitine. CONCLUSIONS AND IMPLICATIONS Our study highlights a novel mode of action for roscovitine, preventing endothelial activation and leukocyte-endothelial cell interaction by inhibition of CDK5 and 9. This might expand its usage as a promising anti-inflammatory compound. PMID:21391976

  18. Candida albicans stimulates cytokine production and leukocyte adhesion molecule expression by endothelial cells.

    PubMed Central

    Filler, S G; Pfunder, A S; Spellberg, B J; Spellberg, J P; Edwards, J E

    1996-01-01

    Endothelial cells have the potential to influence significantly the host immune response to blood-borne microbial pathogens, such as Candida albicans. We investigated the ability (of this organism to stimulate endothelial cell responses relevant to host defense in vitro. Infection with C. albicans induced endothelial cells to express mRNAs encoding E-selectin, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, interleukin 6, interleukin 8, monocyte chemoattractant protein 1, and inducible cyclooxygenase (cox2). All three leukocyte adhesion molecule proteins were expressed on the surfaces of the endothelial cells after 8 h of exposure to C. albicans. An increase in secretion of all three cytokines was found after 12 h of infection. Cytochalasin D inhibited accumulation of the endothelial cell cytokine and leukocyte adhesion molecule mRNAs in response to C. albicans, suggesting that endothelial cell phagocytosis of the organism is required to induce this response. Live Candida tropicalis, Candida glabrata, a nongerminating strain of C. albicans, and killed C. albicans did not stimulate the expression of any of the cytokine or leukocyte adhesion molecule mRNAs. These findings indicate that a factor associated with live, germinating C. albicans is required for induction of endothelial cell mRNA expression. Furthermore, since endothelial cells phagocytize killed C. albicans, phagocytosis is likely necessary but not sufficient for this organism to stimulate mRNA accumulation. In conclusion, the secretion of proinflammatory cytokines and expression of leukocyte adhesion molecules by endothelial cells in response to C. albicans could enhance the host defense against this organism by contributing to the recruitment of activated leukocytes to sites of intravascular infection. PMID:8698486

  19. SRC protein tyrosine kinase, c-Jun N-terminal kinase (JNK), and NF-kappaBp65 signaling in commercial and wild-type turkey leukocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Studies comparing signaling in wild-type turkey (WT) leukocytes and commercial turkey (CT) leukocytes found that the activity of protein tyrosine kinases (PTK) and MAP kinases, ERK 1/2 and p38, were significantly higher in WT leukocytes compared to CT lines upon exposure to both SE and OPSE on days...

  20. Channel surface patterning of alternating biomimetic protein combinations for enhanced microfluidic tumor cell isolation.

    PubMed

    Launiere, Cari; Gaskill, Marissa; Czaplewski, Gregory; Myung, Ja Hye; Hong, Seungpyo; Eddington, David T

    2012-05-01

    Here, we report a new method for multicomponent protein patterning in a microchannel and also a technique for improving immunoaffinity-based circulating tumor cell (CTC) capture by patterning regions of alternating adhesive proteins using the new method. The first of two proteins, antiepithelial cell adhesion molecule (anti-EpCAM), provides the specificity for CTC capture. The second, E-selectin, increases CTC capture under shear. Patterning regions with and without E-selectin allows captured leukocytes, which also bind E-selectin and are unwanted impurities in CTC isolation, to roll a short distance and detach from the capture surface. This reduces leukocyte capture by up to 82%. The patterning is combined with a leukocyte elution step in which a calcium chelating buffer effectively deactivates E-selectin so that leukocytes may be rinsed away 60% more efficiently than with a buffer containing calcium. The alternating patterning of this biomimetic protein combination, used in conjunction with the elution step, reduces capture of leukocytes while maintaining a high tumor cell capture efficiency that is up to 1.9 times higher than the tumor cell capture efficiency of a surface with only anti-EpCAM. The new patterning technique described here does not require mask alignment and can be used to spatially control the immobilization of any two proteins or protein mixtures inside a sealed microfluidic channel.

  1. Cytokine-mediated induction of endothelial adhesion molecule and histocompatibility leukocyte antigen expression by cytomegalovirus-activated T cells.

    PubMed Central

    Waldman, W. J.; Knight, D. A.

    1996-01-01

    Cytomegalovirus (CMV) has been associated with allograft rejection and transplantation-associated arteriosclerosis. CMV infects endothelium, the interface between allograft tissue and the host immune system; however, mechanisms by which such interaction might exacerbate the rejection process remain unresolved. Here we test the hypothesis that host immune activity, triggered by CMV-infected graft endothelial cells (ECs), can result in the production of cytokines capable of enhancing the alloimmunogenicity of nearby uninfected endothelia. To model these phenomena in vitro, confluent monolayers of ECs derived from human umbilical vein or adult gonadal vein were incubated 5 days beneath trans-well culture inserts containing CMV-seropositive or CMV-seronegative donor-derived CD3+ or CD4+ T cells alone or in combination with CMV-infected or uninfected allogeneic ECs. The extent of T cell proliferation was determined by [3H]thymidine labeling of trans-well contents after transfer to microtiter plates. Endothelial responses to soluble factors elaborated by CMV-activated T cells were determined by immunohistochemical staining and immunofluorescence flow cytometric analysis of underlying EC monolayers. Results of experiments with CMV-seropositive donor-derived CD4+ T cells demonstrated enhancement of ICAM-1 and histocompatibility leukocyte antigen class I, as well as induction of histocompatibility leukocyte antigen DR on ECs incubated beneath T cell/EC/CMV trans-well co-cultures. Total (CD3+) T cells co-cultured with EC/CMV induced VCAM-1 as well. Furthermore, [3H]thymidine incorporation by these T cells indicated a strong proliferative response. Endothelial responses to T cells alone or in combination with uninfected ECs were minimal, and T cells cultured under these conditions showed little proliferative activity. Similarly, little or no endothelial responses were apparent in monolayers beneath trans-wells containing T cells isolated from CMV-seronegative individuals

  2. The role of leukocytes from L-PRP/L-PRF in wound healing and immune defense: new perspectives.

    PubMed

    Bielecki, Tomasz; Dohan Ehrenfest, David M; Everts, Peter A; Wiczkowski, Andrzej

    2012-06-01

    Platelet concentrates for topical use are innovative tools of regenerative medicine and their effects in various therapeutical situations are hotly debated. Unfortunately, this field of research mainly focused on the platelet growth factors, and the fibrin architecture and the leukocyte content of these products are too often neglected. In the four families of platelet concentrates, 2 families contain significant concentrations of leukocytes: L-PRP (Leukocyte- and Platelet-Rich Plasma) and L-PRF (Leukocyte- and Platelet-Rich Fibrin). The presence of leukocytes has a great impact on the biology of these products, not only because of their immune and antibacterial properties, but also because they are turntables of the wound healing process and the local factor regulation. In this article, the various kinds of leukocytes present in a platelet concentrate are described (particularly the various populations of granulocytes and lymphocytes), and we insist on the large diversity of factors and pathways that these cells can use to defend the wound site against infections and to regulate the healing process. Finally, the impact of these cells in the healing properties of the L-PRP and L-PRF is also discussed: if antimicrobial properties were already pointed out, effects in the regulation of cell proliferation and differentiation were also hypothesized. Leukocytes are key actors of many platelet concentrates, and a better understanding of their effects is an important issue for the development of these technologies.

  3. Real-time analysis of integrin-dependent transendothelial migration and integrin-independent interstitial motility of leukocytes.

    PubMed

    Shulman, Ziv; Alon, Ronen

    2012-01-01

    The role of integrins in leukocyte migration across endothelial barriers is widely accepted. In contrast, the contribution of integrins to interstitial motility of leukocytes is still elusive. Chemokine binding to G-protein-coupled receptors expressed on the surface of leukocytes plays key roles in both of these processes by directly activating integrin conformations favorable for ligand binding and integrin microclustering. Chemokines can also serve as weak adhesive ligands and potent inducers of actin cytoskeleton remodeling. Real-time assays utilizing live imaging microscopy have been implemented to dissect these versatile roles of chemokines in different leukocyte migration processes. Here, we review several in vitro assays useful for exploring the contribution of chemokine signals and shear forces to integrin activation and function during various stages of leukocyte transendothelial migration. In addition, we describe a new assay that assesses the contribution of chemokines to integrin-independent interstitial leukocyte motility. These assays can also follow the outcome of specific genetic or biochemical manipulations of either the leukocyte or the endothelial barrier on distinct migratory steps. Following fixation, subcellular changes in the distribution of integrin subsets and of specific integrin-associated adaptors can be further dissected by immunofluorescence tools and by ultrastructural electron microscopic analysis.

  4. Isolation and characterization of human defensin cDNA clones

    SciTech Connect

    Daher, K.A.; Lehrer, R.I.; Ganz, T.; Kronenberg, M. )

    1988-10-01

    Four clones that encode defensins, a group of microbicidal and cytotoxic peptides made by neutrophils, were isolated from an HL-60 human promyelocytic leukemia cDNA library. Analysis of these clones indicated that the defensins are made as precursor proteins, which must be cleaved to yield the mature peptides. Defensin mRNA was detected in normal bone marrow cells, but not in normal peripheral blood leukocytes. Defensin transcripts were also found in the peripheral leukocytes of some leukemia patients and in some lung and intestine tissues. Defensin mRNA content was augmented by treatment of HL-60 cells with dimethyl sulfoxide. These results define important aspects of the mechanism of synthesis and the tissue-specific expression of a major group of neutrophil granule proteins.

  5. A high-salt diet enhances leukocyte adhesion in association with kidney injury in young dahl salt-sensitive rats.

    PubMed

    Takahashi, Hidenori; Nakagawa, Suguru; Wu, Yaqiong; Kawabata, Yukari; Numabe, Atsushi; Yanagi, Yasuo; Tamaki, Yasuhiro; Uehara, Yoshio; Araie, Makoto

    2017-03-16

    Salt-sensitive hypertension is associated with severe organ damage. Generating oxygen radicals is an integral component of salt-induced kidney damage, and activated leukocytes are important in oxygen radical biosynthesis. We hypothesized that a high-salt diet causes the upregulation of immune-related mechanisms, thereby contributing to the susceptibility of Dahl salt-sensitive rats to hypertensive kidney damage. For verifying the hypothesis, we investigated leukocytes adhering to retinal vessels when Dahl salt-sensitive rats were challenged with a high-salt (8% NaCl) diet using acridine orange fluoroscopy and a scanning laser ophthalmoscope. The high-salt diet increased leukocyte adhesion after 3 days and was associated with a significant increase in mRNA biosynthesis of monocyte chemotactic protein-1 and intercellular adhesion molecule-1 (ICAM-1) -related molecules in the kidney. Losartan treatment did not affect increased leukocyte adhesion during the early, pre-hypertensive phase of high salt loading; however, losartan attenuated the adhesion of leukocytes during the hypertensive stage. Moreover, the inhibition of leukocyte adhesion in the pre-hypertensive stage by anti-CD18 antibodies decreased tethering of leukocytes and was associated with the attenuation of functional and morphological kidney damage without affecting blood pressure elevation. In conclusion, a high-salt challenge rapidly increased leukocyte adhesion through the over-expression of ICAM-1. Increased leukocyte adhesion in the pre-hypertensive stage is responsible for subsequent kidney damage in Dahl salt-sensitive rats. Immune system involvement may be a key component that initiates kidney damage in a genetic model of salt-induced hypertension.Hypertension Research advance online publication, 16 March 2017; doi:10.1038/hr.2017.31.

  6. Visualization of a prosthetic vascular graft due to platelet contamination during /sup 111/Indium-labeled leukocyte scintigraphy

    SciTech Connect

    Oates, E.; Ramberg, K.

    1988-09-01

    A prosthetic axillo-femoral bypass graft was visualized during /sup 111/In-labeled leukocyte scintigraphy in a patient referred for possible abdominal abscess. The presence of significant cardiac blood-pool activity raised the possibility that this uptake was due to deposition of contaminating labeled platelets rather than labeled leukocytes. An analysis of a small sample of the patient's blood confirmed that the circulating activity was due to labeled platelets. Increased activity along prosthetic vascular grafts in patients undergoing /sup 111/In-labeled leukocyte scintigraphy may be due to adherent platelet, and not indicative of infection.

  7. Apoptosis of peripheral blood leukocytes from rabbits infected with non-haemagglutinating strains of rabbit haemorrhagic disease virus (RHDV).

    PubMed

    Niedźwiedzka-Rystwej, Paulina; Deptuła, Wiesław

    2012-09-15

    The report demonstrates that the induction of apoptosis in peripheral blood granulocytes and lymphocytes of rabbits infected with three non-haemagglutinating RHDV strains (English Rainham, German Frankfurt, and Spanish Asturias) is a crucial determinant of the pathogenesis of rabbit haemorrhagic disease. Apoptosis was measured by flow cytometric detection of caspase activity. These studies demonstrated that the investigated RHDV (rabbit haemorrhagic disease virus) viral strains affected leukocyte apoptosis to varying degrees. Enhanced leukocyte apoptosis was detected between 4 and 36 h after infection and was more pronounced in lymphocytes than in granulocytes. The data presented here thus provide a preliminary understanding of the kinetics of apoptosis in leukocytes of rabbits infected with RHDV.

  8. The value of indium 111 leukocyte scanning in the evaluation of painful or infected total knee arthroplasties

    SciTech Connect

    Rand, J.A.; Brown, M.L. )

    1990-10-01

    Evaluation of painful total knee arthroplasties (TKAs) for infection can be difficult. Indium 111 ({sup 111}In) leukocyte bone scanning provides a minimally invasive technique for evaluation of possible infection. Thirty-eight patients with a painful TKA who had surgical exploration after {sup 111}In leukocyte scanning were reviewed. The scan had an accuracy of 84%, a sensitivity of 83%, and a specificity of 85%. The {sup 111}In leukocyte scans must be interpreted in conjunction with the clinical evaluation of the patient because they are less accurate for study of TKAs than of total hip arthroplasties.

  9. Lipids and leukocytes in newborn umbilical vein blood, birth weight and maternal body mass index.

    PubMed

    Brittos, T; de Souza, W B; Anschau, F; Pellanda, L

    2016-12-01

    Maternal obesity during pregnancy may influence fetal development and possibly predispose offspring to cardiovascular disease. The aim of the present study was to evaluate the relationship between maternal pre-pregnancy body mass index (BMI) and weight gain during pregnancy, and newborn birth weight, with lipid profile, high-sensitivity C-reactive protein (hs-CRP) and leukocyte in newborns. We performed a cross-sectional study of 245 mothers and their children. Blood was collected from the umbilical vein and assayed for lipid profile, hs-CRP and leukocyte count. Newborns average weight was 3241 g, total cholesterol 53.9 mg/dl, high-density lipoprotein cholesterol (HDL-c) 21.9 mg/dl, low-density lipoprotein cholesterol (LDL-c) 26.2 mg/dl, triglyceride 29.5 mg/dl and leukocytes 13,777/mm3. There was a direct correlation of pre-pregnancy BMI of overweight mothers with total cholesterol (r=0.220, P=0.037) and LDL-c (r=0.268, P=0.011) of newborns. Total cholesterol, LDL-c and HDL-c were higher in pre-term newborns (66.3±19.7, 35.9±14.6 and 25.2±7.7 mg/dl, respectively) that in full-term (52.4±13.1, 25.0±8.7 and 21.5±6.0 mg/dl), with P=0.001, 0.001 and 0.003, respectively. Leukocyte counts were higher in full-term newborns (14,268±3982/mm3) compared with pre-term (9792±2836/mm3, P<0.0001). There was a direct correlation between birth weight and leukocyte counts of newborns (r=0.282, P<0.0001). These results suggest the possible interaction of maternal weight and fetal growth with lipid metabolism and leukocyte count in the newborn, which may be linked to programming of the immune system.

  10. Promotion of DNA strand breaks in cocultured mononuclear leukocytes by protein kinase C-dependent prooxidative interactions of benoxaprofen, human polymorphonuclear leukocytes, and ultraviolet radiation

    SciTech Connect

    Schwalb, G.; Beyers, A.D.; Anderson, R.; Nel, A.E.

    1988-06-01

    At concentrations of 5 micrograms/ml and greater the nonsteroidal antiinflammatory drug benoxaprofen caused dose-related activation of lucigenin-enhanced chemiluminescence in human polymorphonuclear leukocytes (PMNL). Benoxaprofen-mediated activation of lucigenin-enhanced chemiluminescence by PMNL was increased by UV radiation and was particularly sensitive to inhibition by the selective protein kinase C inhibitor H-7. To identify the molecular mechanism of the prooxidative activity of benoxaprofen, the effects of the nonsteroidal antiinflammatory drug on the activity of purified protein kinase C in a cell-free system were investigated. Benoxaprofen caused a dose-related activation of protein kinase C by interaction with the binding site for the physiological activator phosphatidylserine, but could not replace diacylglycerol. When autologous mononuclear leukocytes (MNL) were cocultured with PMNL and benoxaprofen in combination, but not individually, the frequency of DNA strand breaks in MNL was markedly increased. UV radiation significantly potentiated damage to DNA mediated by benoxaprofen and PMNL. Inclusion of superoxide dismutase, H-7, and, to a much lesser extent, catalase during exposure of MNL to benoxaprofen-activated PMNL prevented oxidant damage to DNA. These results clearly demonstrate that potentially carcinogenic prooxidative interactions, which are unlikely to be detected by conventional assays of mutagenicity, may occur between phagocytes, UV radiation, and certain pharmacological agents.

  11. Comparative Genome Analysis of Three Eukaryotic Parasites with Differing Abilities To Transform Leukocytes Reveals Key Mediators of Theileria-Induced Leukocyte Transformation

    PubMed Central

    Hayashida, Kyoko; Hara, Yuichiro; Abe, Takashi; Yamasaki, Chisato; Toyoda, Atsushi; Kosuge, Takehide; Suzuki, Yutaka; Sato, Yoshiharu; Kawashima, Shuichi; Katayama, Toshiaki; Wakaguri, Hiroyuki; Inoue, Noboru; Homma, Keiichi; Tada-Umezaki, Masahito; Yagi, Yukio; Fujii, Yasuyuki; Habara, Takuya; Kanehisa, Minoru; Watanabe, Hidemi; Ito, Kimihito; Gojobori, Takashi; Sugawara, Hideaki; Imanishi, Tadashi; Weir, William; Gardner, Malcolm; Pain, Arnab; Shiels, Brian; Hattori, Masahira; Nene, Vishvanath; Sugimoto, Chihiro

    2012-01-01

    ABSTRACT We sequenced the genome of Theileria orientalis, a tick-borne apicomplexan protozoan parasite of cattle. The focus of this study was a comparative genome analysis of T. orientalis relative to other highly pathogenic Theileria species, T. parva and T. annulata. T. parva and T. annulata induce transformation of infected cells of lymphocyte or macrophage/monocyte lineages; in contrast, T. orientalis does not induce uncontrolled proliferation of infected leukocytes and multiplies predominantly within infected erythrocytes. While synteny across homologous chromosomes of the three Theileria species was found to be well conserved overall, subtelomeric structures were found to differ substantially, as T. orientalis lacks the large tandemly arrayed subtelomere-encoded variable secreted protein-encoding gene family. Moreover, expansion of particular gene families by gene duplication was found in the genomes of the two transforming Theileria species, most notably, the TashAT/TpHN and Tar/Tpr gene families. Gene families that are present only in T. parva and T. annulata and not in T. orientalis, Babesia bovis, or Plasmodium were also identified. Identification of differences between the genome sequences of Theileria species with different abilities to transform and immortalize bovine leukocytes will provide insight into proteins and mechanisms that have evolved to induce and regulate this process. The T. orientalis genome database is available at http://totdb.czc.hokudai.ac.jp/. PMID:22951932

  12. Experimental bacterial pneumonia in rabbits: polymorphonuclear leukocyte margination and sequestration in rabbit lungs and quantitation and kinetics of /sup 51/Cr-labeled polymorphonuclear leukocytes in E. coli-induced lung lesions

    SciTech Connect

    Cybulsky, M.I.; Movat, H.Z.

    1982-12-01

    A relationship between the circulating and marginal polymorphonuclear leukocyte (PMN) pools was documented using /sup 51/Cr-labeled leukocytes as a marker. /sup 51/Cr-leukocytes marginating in the lungs were found to decrease following a first-order exponential decline, while /sup 51/Cr radioactivity accumulated in the liver and the spleen. Intravenously administered endotoxin caused a rapid selective disappearance of PMNs from the circulation. The percentage of infused /sup 51/Cr cells disappearing was equal to the percentage of disappearance of host cells. The PMNs were found to sequester in the lungs, with peak sequestration of labeled cells occurring 5 min after an endotoxin challenge. Over the next 25 min the /sup 51/Cr radioactivity in the lungs declined. Large numbers of PMNs, probably newly derived from the bone marrow, were observed histologically to be sequestered in the lung vasculature 90 min after an endotoxin dose, while the early sequestration of circulating leukocytes could not be assessed histologically. Pulmonary inflammatory lesions were induced selectively with Escherichia coli in the left lower lobes of rabbits, leaving the right lower lobes as intrinsic controls. PMN-accumulation into the lesions was quantitated using /sup 51/Cr-labeled blood leukocytes. With the aid of /sup 125/I-labeled E. coli, a logarithmic dose-response relationship was found between the number of E. coli and of PMNs. Over a 6-hr period circulating PMNs were found to accumulate in a lesion in the left lower lobe, whereas in the control right lower lobe, leukocyte radioactivity declined. These findings were confirmed with the aid of lavages of the right and left lungs. Two peaks of PMN-accumulation were found by studying leukocyte kinetics: a larger peak between 0 and 6 hr and a smaller peak 18-24 hr after instillation of the microorganisms. Histologic studies confirmed the accumulation of leukocytes, and by 3 weeks showed a complete resolution of the lesions.

  13. Two-dimensional electrophoretic analysis of human leukocyte proteins from patients with rheumatoid arthritis

    SciTech Connect

    Willard, K.E.; Thorsrud, A.K.; Munthe, E.; Jellum, E.

    1982-04-01

    Human leukocyte proteins from more than 150 patients with rheumatoid arthritis, together with age- and sex-matched controls, were analyzed by use of the ISO-DALT technique of two-dimensional polyacrylamide gel electrophoresis. Patients with ankylosing spondylitis, polymyalgia rheumatica, psoriatic arthritis, calcium tendinitis, post-infectious arthritis, and asymmetrical seronegative arthritis were also included as positive controls. Synthesis of several proteins, referred to by number as members of the Rheuma set, is shown to increase in the leukocyte preparations from patients with classical rheumatoid arthritis. Several of these proteins are specific to monocytes or granulocytes; others are of unknown cellular origin, but appear to be unique to rheumatoid arthritis. The Rheuma proteins appear to be indicators of disease activity, because their increased synthesis can be correlated with sedimentation rate and other clinical indices of rheumatoid disease activity.

  14. Cellular Barriers after Extravasation: Leukocyte Interactions with Polarized Epithelia in the Inflamed Tissue

    PubMed Central

    Reglero-Real, Natalia; García-Weber, Diego; Millán, Jaime

    2016-01-01

    During the inflammatory response, immune cells egress from the circulation and follow a chemotactic and haptotactic gradient within the tissue, interacting with matrix components in the stroma and with parenchymal cells, which guide them towards the sites of inflammation. Polarized epithelial cells compartmentalize tissue cavities and are often exposed to inflammatory challenges such as toxics or infections in non-lymphoid tissues. Apicobasal polarity is critical to the specialized functions of these epithelia. Indeed, a common feature of epithelial dysfunction is the loss of polarity. Here we review evidence showing that apicobasal polarity regulates the inflammatory response: various polarized epithelia asymmetrically secrete chemotactic mediators and polarize adhesion receptors that dictate the route of leukocyte migration within the parenchyma. We also discuss recent findings showing that the loss of apicobasal polarity increases leukocyte adhesion to epithelial cells and the consequences that this could have for the inflammatory response towards damaged, infected or transformed epithelial cells. PMID:26941485

  15. Synthetic nanoparticles functionalized with biomimetic leukocyte membranes possess cell-like functions

    NASA Astrophysics Data System (ADS)

    Parodi, Alessandro; Quattrocchi, Nicoletta; van de Ven, Anne L.; Chiappini, Ciro; Evangelopoulos, Michael; Martinez, Jonathan O.; Brown, Brandon S.; Khaled, Sm Z.; Yazdi, Iman K.; Enzo, Maria Vittoria; Isenhart, Lucas; Ferrari, Mauro; Tasciotti, Ennio

    2013-01-01

    The therapeutic efficacy of systemic drug-delivery vehicles depends on their ability to evade the immune system, cross the biological barriers of the body and localize at target tissues. White blood cells of the immune system--known as leukocytes--possess all of these properties and exert their targeting ability through cellular membrane interactions. Here, we show that nanoporous silicon particles can successfully perform all these actions when they are coated with cellular membranes purified from leukocytes. These hybrid particles, called leukolike vectors, can avoid being cleared by the immune system. Furthermore, they can communicate with endothelial cells through receptor-ligand interactions, and transport and release a payload across an inflamed reconstructed endothelium. Moreover, leukolike vectors retained their functions when injected in vivo, showing enhanced circulation time and improved accumulation in a tumour.

  16. Phagocytosis of bovine blood and milk polymorphonuclear leukocytes after ozone gas administration in vitro.

    PubMed

    Ducusin, Rio John T; Nishimura, Masakazu; Sarashina, Takao; Uzuka, Yuji; Tanabe, Shigeyuki; Otani, Masayuki

    2003-04-01

    To determine the effects of ozone on the phagocytosis of bovine polymorphonuclear leukocytes (PMNs), ozone gas was administered in vitro on the blood and milk of healthy lactating cows, cows with acute mastitis, and cows with milk fever. In the blood of healthy dairy cattle, although there was no significant effect of ozone gas on the viability of the leukocytes, phagocytosis of PMNs significantly decreased. In contrast, ozone gas administration in vitro significantly increased phagocytosis of PMNs from the blood of cows with acute mastitis and milk fever, and from mastitic milk. These findings showed that ozone administration in vitro has positive and negative effects on bovine PMN phagocytosis, depending on the health status of the animal.

  17. [Gamma interferon induced in human leukocytes by phytohemagglutinin: its production and biological characteristics].

    PubMed

    Danielescu, G; Maniu, H; Georgescu, T; Cajal, N

    1988-01-01

    Human gamma type interferon (IFN) preparations were obtained through phytohemagglutinin stimulation of leukocytes from the peripheral blood. Biological value of these preparations varied between 160 u and 800 u/ml, depending on leukocyte incubation medium, culture system and inductor conservation. The rising of the antiviral activity through association between gamma (3 u) and alpha (27 u) interferons was revealed by the virus quantity reduction (in this case the vesicular stomatitis virus was used) during a 24-hour multiplication cycle. The protection ensured by the mixture of the two types of interferon was about ten times higher than the additive effect of the two preparations. Study of the antiproliferative activity of a gamma interferon preparation was conducted on two human cell lines of tumoral origin (T-10 from a glioblastoma, and HEp-2) and revealed the difficulties to quantify precisely this property of the crude gamma interferon preparations.

  18. Intracytoplasmic inclusions in circulating leukocytes from an eastern box turtle (Terrapene carolina carolina) with iridoviral infection.

    PubMed

    Allender, Matthew C; Fry, Michael M; Irizarry, Armando R; Craig, Linden; Johnson, April J; Jones, Michael

    2006-07-01

    A free-ranging adult female eastern box turtle (Terrapene carolina carolina) was presented to the University of Tennessee in October 2003 because of suspected trauma and blindness. Physical examination revealed lethargy, clear ocular and nasal discharges, and white oral and laryngeal plaques. Intracytoplasmic inclusions within heterophils and large mononuclear leukocytes were observed on routine blood smear examination. Postmortem findings included necrosis of epithelial and parenchymal cells with intracytoplasmic inclusions. Ultrastructurally, the leukocyte inclusions consisted of variably electron-dense granular material and viral particles consistent with the Iridoviridae family of viruses. The virus shared 100% sequence identity to a 420-base pair sequence of frog virus 3 (family Iridoviridae, genus Ranavirus) as determined by polymerase chain reaction and gene sequencing targeting a portion of the Ranavirus major capsid protein gene.

  19. Detection of microparticles of leukocytic origin in the peripheral blood in normal pregnancy and preeclampsia.

    PubMed

    Mikhailova, V A; Ovchinnikova, O M; Zainulina, M S; Sokolov, D I; Sel'kov, S A

    2014-10-01

    Microparticles are microvesicles forming during cell activation and as a result of apoptotic cell death. Normal pregnancy is associated with apoptosis induction in active immune system cells, present in the decidual tissue. Preeclampsia is associated with activation of the peripheral blood leukocytes and more intense apoptosis of the trophoblast cells. As a result, the number of microparticles in the peripheral blood is changing in normal gestation and in preeclampsia. The content of the leukocytic microparticles in the peripheral blood is evaluated in normal pregnancy and preeclampsia. The content of neutrophilic and monocytic microparticles is higher than normally in preeclampsia, this indicating activation of these cells. The number of microparticles formed by NK cells is low in preeclampsia, which can reflect the incompetence of immunological tolerance mechanisms under these conditions.

  20. Biology and structure of leukocyte β 2 integrins and their role in inflammation

    PubMed Central

    Arnaout, M. Amin

    2016-01-01

    Integrins comprise a large family of αβ heterodimeric cell adhesion receptors that are expressed on all cells except red blood cells and that play essential roles in the regulation of cell growth and function. The leukocyte integrins, which include members of the β 1, β 2, β 3, and β 7 integrin family, are critical for innate and adaptive immune responses but also can contribute to many inflammatory and autoimmune diseases when dysregulated. This review focuses on the β 2 integrins, the principal integrins expressed on leukocytes. We review their discovery and role in host defense, the structural basis for their ligand recognition and activation, and their potential as therapeutic targets. PMID:27781085

  1. Polyvalent lactose-quantum dot conjugate for fluorescent labeling of live leukocytes.

    PubMed

    Yu, Min; Yang, Yang; Han, Rongcheng; Zheng, Qiang; Wang, Lijun; Hong, Yuankai; Li, Zhongjun; Sha, Yinlin

    2010-06-01

    Oligosaccharides play crucial roles in many biorecognition processes by the so-called "cluster glycosidic effect". We here report a facile synthesis of lactose-CdSeS/ZnS quantum dot conjugate (Lac-QDs) by use of 1-thiol-beta-D-lactose via ligand exchange, which exhibits significantly high affinity and specificity to leukocytes in contrast to the monovalent lactose. Structural analyses indicate that there are about 132 lactosyl molecules assembled on single QDs and the hydrodynamic diameter is small, close to 8.2 nm. Further, Lac-QDs display good fluorescence and physicochemical stability in physiological conditions, as well as extremely low cytotoxicity. These properties facilitate the use of Lac-QDs in fluorescent labeling of live leukocytes.

  2. Effects of Himatanthus lancifolius on human leukocyte chemotaxis and their adhesion to integrins.

    PubMed

    Nardin, Jeanine Marie; de Souza, Wesley Maurício; Lopes, Juliano Ferreira; Florão, Angela; de Moraes Santos, Cid Aimbiré; Weffort-Santos, Almeriane Maria

    2008-08-01

    The aim of this work was to investigate the anti-inflammatory activities of the uleine-rich fraction extracted from the barks of Himatanthus lancifolius (Muell. Arg.) Woodson (Apocynaceae). To achieve this, we focused on its in vitro effects on some steps of the inflammatory response using peripheral human leukocytes. The results presented herein show that the uleine-rich fraction significantly inhibits the migration of casein-induced granulocytes and their adhesion to fibronectin and vitronectin, along with mononuclear cells, by down-regulating the expression of alpha 4beta1 and alpha5beta1 integrins. The data suggest that H. LANCIFOLIUS has the potential of interferring with leukocyte trafficking through its uleine-rich fraction, emphasizing its usefulness in inflammatory conditions. DEXA:dexamethasone disodium phosphate FN:fibronectin PMN:polymorphonuclear URF:uleine-rich fraction VN:vitronectin.

  3. Competitive evaluation of data mining algorithms for use in classification of leukocyte subtypes with Raman microspectroscopy.

    PubMed

    Maguire, A; Vega-Carrascal, I; Bryant, J; White, L; Howe, O; Lyng, F M; Meade, A D

    2015-04-07

    Raman microspectroscopy has been investigated for some time for use in label-free cell sorting devices. These approaches require coupling of the Raman spectrometer to complex data mining algorithms for identification of cellular subtypes such as the leukocyte subpopulations of lymphocytes and monocytes. In this study, three distinct multivariate classification approaches, (PCA-LDA, SVMs and Random Forests) are developed and tested on their ability to classify the cellular subtype in extracted peripheral blood mononuclear cells (T-cell lymphocytes from myeloid cells), and are evaluated in terms of their respective classification performance. A strategy for optimisation of each of the classification algorithm is presented with emphasis on reduction of model complexity in each of the algorithms. The relative classification performance and performance characteristics are highlighted, overall suggesting the radial basis function SVM as a robust option for classification of leukocytes with Raman microspectroscopy.

  4. How accurate are leukocyte indices and C-reactive protein for diagnosis of neonatal sepsis?

    PubMed Central

    da Silva, Orlando; Ohlsson, Arne

    1998-01-01

    Early diagnosis of neonatal sepsis is often difficult to make. Treatment on the basis of clinical suspicion and risk factors may result in overtreatment. A previous review of the usefulness of C-reactive protein and leukocyte indices concluded that these test results should be interpreted with caution. The present paper reviews and, when appropriate, revises, in light of new information, the conclusions reached in the previous systematic review of the topic. PMID:20401235

  5. The internal architecture of leukocyte lipid body organelles captured by three-dimensional electron microscopy tomography.

    PubMed

    Melo, Rossana C N; Paganoti, Guillherme F; Dvorak, Ann M; Weller, Peter F

    2013-01-01

    Lipid bodies (LBs), also known as lipid droplets, are complex organelles of all eukaryotic cells linked to a variety of biological functions as well as to the development of human diseases. In cells from the immune system, such as eosinophils, neutrophils and macrophages, LBs are rapidly formed in the cytoplasm in response to inflammatory and infectious diseases and are sites of synthesis of eicosanoid lipid mediators. However, little is known about the structural organization of these organelles. It is unclear whether leukocyte LBs contain a hydrophobic core of neutral lipids as found in lipid droplets from adipocytes and how diverse proteins, including enzymes involved in eicosanoid formation, incorporate into LBs. Here, leukocyte LB ultrastructure was studied in detail by conventional transmission electron microscopy (TEM), immunogold EM and electron tomography. By careful analysis of the two-dimensional ultrastructure of LBs from human blood eosinophils under different conditions, we identified membranous structures within LBs in both resting and activated cells. Cyclooxygenase, a membrane inserted protein that catalyzes the first step in prostaglandin synthesis, was localized throughout the internum of LBs. We used fully automated dual-axis electron tomography to study the three-dimensional architecture of LBs in high resolution. By tracking 4 nm-thick serial digital sections we found that leukocyte LBs enclose an intricate system of membranes within their "cores". After computational reconstruction, we showed that these membranes are organized as a network of tubules which resemble the endoplasmic reticulum (ER). Our findings explain how membrane-bound proteins interact and are spatially arranged within LB "cores" and support a model for LB formation by incorporating cytoplasmic membranes of the ER, instead of the conventional view that LBs emerge from the ER leaflets. This is important to understand the functional capabilities of leukocyte LBs in health and

  6. Prenatal Arsenic Exposure and DNA Methylation in Maternal and Umbilical Cord Blood Leukocytes

    PubMed Central

    Baccarelli, Andrea; Hoffman, Elaine; Tarantini, Letizia; Quamruzzaman, Quazi; Rahman, Mahmuder; Mahiuddin, Golam; Mostofa, Golam; Hsueh, Yu-Mei; Wright, Robert O.; Christiani, David C.

    2012-01-01

    Background: Arsenic is an epigenetic toxicant and could influence fetal developmental programming. Objectives: We evaluated the association between arsenic exposure and DNA methylation in maternal and umbilical cord leukocytes. Methods: Drinking-water and urine samples were collected when women were at ≤ 28 weeks gestation; the samples were analyzed for arsenic using inductively coupled plasma mass spectrometry. DNA methylation at CpG sites in p16 (n = 7) and p53 (n = 4), and in LINE-1 and Alu repetitive elements (3 CpG sites in each), was quantified using pyrosequencing in 113 pairs of maternal and umbilical blood samples. We used general linear models to evaluate the relationship between DNA methylation and tertiles of arsenic exposure. Results: Mean (± SD) drinking-water arsenic concentration was 14.8 ± 36.2 μg/L (range: < 1–230 μg/L). Methylation in LINE-1 increased by 1.36% [95% confidence interval (CI): 0.52, 2.21%] and 1.08% (95% CI: 0.07, 2.10%) in umbilical cord and maternal leukocytes, respectively, in association with the highest versus lowest tertile of total urinary arsenic per gram creatinine. Arsenic exposure was also associated with higher methylation of some of the tested CpG sites in the promoter region of p16 in umbilical cord and maternal leukocytes. No associations were observed for Alu or p53 methylation. Conclusions: Exposure to higher levels of arsenic was positively associated with DNA methylation in LINE-1 repeated elements, and to a lesser degree at CpG sites within the promoter region of the tumor suppressor gene p16. Associations were observed in both maternal and fetal leukocytes. Future research is needed to confirm these results and determine if these small increases in methylation are associated with any health effects. PMID:22466225

  7. Reduction of Leukocyte Counts by Hydroxyurea Improves Cardiac Function in Rats with Acute Myocardial Infarction.

    PubMed

    Zhu, Guiyue; Yao, Yucai; Pan, Lingyun; Zhu, Wei; Yan, Suhua

    2015-12-17

    BACKGROUND This study aimed to decrease leukocytes counts by hydroxyurea (Hu) in an acute myocardial infarction (AMI) rat model and examine its effect on the inflammatory response of myocardial infarction and cardiac functions. MATERIAL AND METHODS AMI was successfully caused in 36 rats, and 12 control rats received sham operation. Rats in the AMI group were then randomly divided into Hu and vehicle group with 18 rats each. Rats in the Hu AMI group received Hu (200 mg/kg) intragastrically while vehicle AMI group received saline. Leukocytes counts, cardiac functions, myocardial tissue morphology, and levels of soluble intercellular adhesion molecule-1 (sICAM), P-selectin and platelet activating factor (PAF) were measured and compared among the three groups four weeks after AMI induction. RESULTS Leukocytes, neutrophils, and leukomonocyte counts in vehicle AMI rats were significantly higher than that of the normal control group (p<0.05). However, Hu treatment decreased their counts significantly (p<0.05). sICAM, P-selectin, and PAF level in vehicle AMI group were significantly higher than those of the normal group, and their level was also decreased by Hu treatment (p<0.05). Echocardiography analysis showed that Hu treatment increased left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) compared to that of vehicle AMI group (p<0.05). Histopathological examination showed that Hu significantly reduced the swelling of the heart muscle fiber in necrotic foci and the number of inflammatory cells infiltrated into myocardial interstitium compared to vehicle AMI group. CONCLUSIONS Decrease leukocytes counts by Hu significantly reduced inflammatory reaction and improved cardiac functions in AMI rats.

  8. False-positive indium-111 labeled leukocyte scintigram in a patient with a painful hip prosthesis

    SciTech Connect

    Feldman, N.; Makler, P.T. Jr.; Alavi, A.

    1986-01-01

    A Tronzo hip prosthesis is designed to elicit an inflammatory reaction in order to promote prosthesis stability. A three-phased bone scan and Ga-67 imaging in conjunction with physical examination and laboratory findings failed to demonstrate evidence for osteomyelitis in a patient with a painful hip prosthesis, in whom images obtained with In-111-labeled leukocytes were positive. This observation demonstrated that the interpretation of the latter technique in demonstrating inflammation can cause a false impression of an infectious process.

  9. Impaired Leukocyte Trafficking and Skin Inflammatory Responses in Hamsters Lacking a Functional Circadian System

    PubMed Central

    Prendergast, Brian J.; Cable, Erin J.; Patel, Priyesh N.; Pyter, Leah M.; Onishi, Kenneth G.; Stevenson, Tyler J.; Ruby, Norman F.; Bradley, Sean P.

    2013-01-01

    The immune system is under strong circadian control, and circadian desynchrony is a risk factor for metabolic disorders, inflammatory responses and cancer. Signaling pathways that maintain circadian rhythms (CRs) in immune function in vivo, and the mechanisms by which circadian desynchrony impairs immune function, remain to be fully-identified. These experiments tested the hypothesis that the hypothalamic circadian pacemaker in the suprachiasmatic nucleus (SCN) drives CRs in the immune system, using a non-invasive model of SCN circadian arrhythmia. Robust CRs in blood leukocyte trafficking, with a peak during the early light phase (ZT4) and nadir in the early dark phase (ZT18), were absent in arrhythmic hamsters, as were CRs in spleen clock gene (per1, bmal1) expression, indicating that a functional pacemaker in the SCN is required for the generation of CRs in leukocyte trafficking and for driving peripheral clocks in secondary lymphoid organs. Pinealectomy was without effect on CRs in leukocyte trafficking, but abolished CRs in spleen clock gene expression, indicating that nocturnal melatonin secretion is necessary for communicating circadian time information to the spleen. CRs in trafficking of antigen presenting cells (CD11c+ dendritic cells) in the skin were abolished, and antigen-specific delayed-type hypersensitivity skin inflammatory responses were markedly impaired in arrhythmic hamsters. The SCN drives robust CRs in leukocyte trafficking and lymphoid clock gene expression; the latter of which is not expressed in the absence of melatonin. Robust entrainment of the circadian pacemaker provides a signal critical to diurnal rhythms in immunosurveilliance and optimal memory T-cell dependent immune responses. PMID:23474187

  10. ErbB3 mRNA leukocyte levels as a biomarker for major depressive disorder

    PubMed Central

    2012-01-01

    Background In recent years, the identification of peripheral biomarkers that are associated with psychiatric diseases, such as Major Depressive Disorder (MDD), has become relevant because these biomarkers may improve the efficiency of the differential diagnosis process and indicate targets for new antidepressant drugs. Two recent candidate genes, ErbB3 and Fgfr1, are growth factors whose mRNA levels have been found to be altered in the leukocytes of patients that are affected by bipolar disorder in a depressive state. On this basis, the aim of the study was to determine if ErbB3 and Fgfr1 mRNA levels could be a biomarkers of MDD. Methods We measured by Real Time PCR ErbB3 and Fgfr1 mRNA expression levels in leukocytes of MDD patients compared with controls. Successively, to assess whether ErbB3 mRNA levels were influenced by previous antidepressant treatment we stratified our patients sample in two cohorts, comparing drug-naive versus drug-free patients. Moreover, we evaluated the levels of the transcript in MDD patients after 12 weeks of antidepressant treatment, and in prefrontal cortex of rats stressed and treated with an antidepressant drug of the same class. Results These results showed that ErbB3 but not Fgfr1 mRNA levels were reduced in leukocytes of MDD patients compared to healthy subjects. Furthermore, ErbB3 levels were not affected by antidepressant treatment in either human or animal models Conclusions Our data suggest that ErbB3 might be considered as a biomarker for MDD and that its deficit may underlie the pathopsysiology of the disease and is not a consequence of treatment. Moreover the study supports the usefulness of leukocytes as a peripheral system for identifying biomarkers in psychiatric diseases. PMID:22989054

  11. Effects of radiotherapy and chemotherapy on angiogenesis and leukocyte infiltration in rectal cancer

    SciTech Connect

    Baeten, Coen . E-mail: C.Baeten@surgery.azm.nl; Castermans, Karolien; Lammering, Guido; Hillen, Femke; Wouters, Bradly G.; Hillen, Harry; Griffioen, Arjan W.; Baeten, Cornelius G.M.I.

    2006-11-15

    Background: We and others have shown that angiogenesis and leukocyte infiltration are important prognostic factors in rectal cancer. However, little is known about its possible changes in response to radiotherapy (RTX), which is frequently given to rectal tumors as a neoadjuvant treatment to improve the prognosis. We therefore investigated the biologic effects of RTX on these parameters using fresh-frozen biopsy samples of tumor and normal mucosa tissue before and after RTX. Methods: Biopsy samples were taken from a total of 34 patients before and after either a short course or long course of RTX combined with chemotherapy. The following parameters were analyzed by immunohistochemistry, flow cytometry, or quantitative real-time polymerase chain reaction: Microvessel density, leukocyte infiltration, proliferating epithelial and tumor cells, proliferating endothelial cells, adhesion molecule expression on endothelial cells, and the angiogenic mRNA profile. Results: The tumor biopsy samples taken after RTX treatment demonstrated a significant decrease in microvessel density and the number of proliferating tumor cells and proliferating endothelial cells (p < 0.001). In contrast, the leukocyte infiltration, the levels of basic fibroblast growth factor in carcinoma tissue, and the adhesion molecule expression on endothelial cells in normal as well as carcinoma tissue increased significantly (p < 0.05). Conclusion: Our data show that together with an overall decrease in tumor cell and endothelial cell proliferation, RTX results in an increase in the expression of adhesion molecules that stimulate leukocyte infiltration. This suggests the possibility that, in addition to its direct cytotoxic effect, radiation may also stimulate an immunologic tumor response that could contribute to the documented improvement in local tumor control and distal failure rate of rectal cancers.

  12. Inhalation of ultrafine particles alters blood leukocyte expression of adhesion molecules in humans.

    PubMed

    Frampton, Mark W; Stewart, Judith C; Oberdörster, Günter; Morrow, Paul E; Chalupa, David; Pietropaoli, Anthony P; Frasier, Lauren M; Speers, Donna M; Cox, Christopher; Huang, Li-Shan; Utell, Mark J

    2006-01-01

    Ultrafine particles (UFPs; aerodynamic diameter < 100 nm) may contribute to the respiratory and cardiovascular morbidity and mortality associated with particulate air pollution. We tested the hypothesis that inhalation of carbon UFPs has vascular effects in healthy and asthmatic subjects, detectable as alterations in blood leukocyte expression of adhesion molecules. Healthy subjects inhaled filtered air and freshly generated elemental carbon particles (count median diameter approximately 25nm, geometric standard deviation approximately 1.6), for 2 hr, in three separate protocols: 10 microg/m3 at rest, 10 and 25 microg/m3 with exercise, and 50 microg/m3 with exercise. In a fourth protocol, subjects with asthma inhaled air and 10 microg/m3 UFPs with exercise. Peripheral venous blood was obtained before and at intervals after exposure, and leukocyte expression of surface markers was quantitated using multiparameter flow cytometry. In healthy subjects, particle exposure with exercise reduced expression of adhesion molecules CD54 and CD18 on monocytes and CD18 and CD49d on granulocytes. There were also concentration-related reductions in blood monocytes, basophils, and eosinophils and increased lymphocyte expression of the activation marker CD25. In subjects with asthma, exposure with exercise to 10 microg/m3 UFPs reduced expression of CD11b on monocytes and eosinophils and CD54 on granulocytes. Particle exposure also reduced the percentage of CD4+ T cells, basophils, and eosinophils. Inhalation of elemental carbon UFPs alters peripheral blood leukocyte distribution and expression of adhesion molecules, in a pattern consistent with increased retention of leukocytes in the pulmonary vascular bed.

  13. COMPARISON OF TOTAL LEUKOCYTE QUANTIFICATION METHODS IN FREE-LIVING GALAPAGOS TORTOISES (CHELONOIDIS SPP.).

    PubMed

    Sheldon, Julie D; Stacy, Nicole I; Blake, Stephen; Cabrera, Fredy; Deem, Sharon L

    2016-03-01

    Reptile hematologic data provide important health information for conservation efforts of vulnerable wildlife species such as the Galapagos tortoise (Chelonoidis spp.). Given the reported discrepancies between manual leukocyte counts for nonmammalian species, two manual leukocyte quantification methods, the Natt and Herrick's (NH) and the Eopette (EO), were compared to white blood cell (WBC) estimates from blood films of 42 free-living, clinically healthy, adult female Galapagos tortoises. To investigate the effects of delay in sample processing, estimated WBC counts and leukocyte differentials were compared for blood films prepared at time of collection under field conditions (T0) to blood films prepared from samples that were stored for 18-23 hr at 4°C in the laboratory (T1). Passing-Bablok regression analysis revealed no constant or proportional error between the NH and WBC estimates (T0 and T1) with slopes of 1.1 and 0.9, respectively. However both constant and proportional errors were present between EO and WBC estimates (T0 and T1) with slopes of 3.1 and 2.7, respectively. Bland Altman plots also showed agreement between the NH and WBC estimates where the points fell within the confidence-interval limit lines and were evenly distributed about the mean. In contrast, the EO and WBC estimate comparisons showed numerous points above the upper limit line, especially at higher concentrations. WBC estimates obtained from T0 and T1 films were in agreement, whereas heterophil and monocyte percentages based on differentials were not. Cell morphology and preservation were superior in T0 blood films because thrombocytes exhibited swelling after storage, becoming difficult to differentiate from lymphocytes. In this study, the highest quality and most reliable hematologic data in Galapagos tortoises were obtained by combining immediate blood film preparation with the NH leukocyte quantification method and a confirmatory WBC estimate from the blood film.

  14. A Killer Immunoglobulin - Like Receptor Gene - Content Haplotype and A Cognate Human Leukocyte Antigen Ligand are Associated with Autism

    PubMed Central

    Torres, Anthony; Westover, Jonna; Benson, Michael; Johnson, Randall; Dykes, Annelise

    2016-01-01

    The killing activity of natural killer cells is largely regulated by the binding of class I human leukocyte antigen cognate ligands to killer cell immunoglobulin - like receptor proteins. The killer cell immunoglobulin - like receptor gene - complex contains genes that activate and others that inhibit the killing state of natural killer cells depending on the binding of specific human leukocyte antigen cognate ligands. It has been suggested in previous publications that activating human leukocyte antigen/killer - cell immunoglobulin - like receptor complexes are increased in people with autism. We present data, which suggests that an activating cB01/tA01 killer cell immunoglobulin - like receptor gene - content haplotype and the cognate ligand human leukocyte antigen - C1k that activates this haplotype is significantly increased in autism. This is an important observation suggesting that the interaction between two proteins encoded on different chromosomes increases natural killer cell killing in autism. PMID:27853655

  15. Aloe-emodin affects the levels of cytokines and functions of leukocytes from Sprague-Dawley rats.

    PubMed

    Yu, Chun-Shu; Yu, Fu-Shun; Chan, Jack Kai-Sheng; Li, Te-Mao; Lin, Song-Shei; Chen, Ssu-Ching; Hsia, Te-Chun; Chang, Yung-Hsien; Chung, Jing-Gung

    2006-01-01

    Aloe-emodin has shown anti-neoplastic activity against some human cancer cell lines. This study aimed to explore the effects of aloe-emodin on the phagocytosis of macrophages, the activity of natural killer (NK) cells and the expression of cytokines in leukocytes from Sprague-Dawley rats. Leukocytes were collected, placed into culture plates and the functions of macrophages and NK cells and the percentage of viable cells were determined by flow cytometric analysis. Incubation of leukocytes with various concentrations of aloe-emodin caused a dose-dependent decrease of viable cells, a decrease of phagocytosis by macrophages, and a decrease of the activity of NK cells. Evaluation of cytokines in leukocytes by ELISA indicated that aloe-emodin increased the levels of interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. The results were also confirmed by PCR assay for the mRNA expression of the examined cytokines.

  16. Increased production of plasminogen activator inhibitor in vitro by pleural leukocytes from rats intratracheally instilled with crocidolite asbestos

    SciTech Connect

    Xiao Yang Li; Brown, G.M.; Donaldson, K. ); Lamb, D. )

    1991-08-01

    The authors have previously reported that normal pleural leukocytes secrete a urokinase-type plasminogen activator inhibitor (PAI) in culture. In view of the pathogenic effects of asbestos on the pleura, in particular pleural fibrosis, they have extended these observation to crocidolite asbestos-exposed rats. Pleural leukocytes from rats exposed to crocidolite asbestos were found to secrete more PAI in culture than controls. The activity of PAI in pleural leukocyte-conditioned medium increased in a dose-dependent manner in relation to the quantity of asbestos injected into the lung. However, with increasing time post asbestos instillation, there was no significant change in the secretion of PAI by pleural leukocytes in culture compared with earlier time points of crocidolite-exposed rats. Plasminogen activator was not detectable in the conditioned medium at any time point. The data derived from this study may help to elucidate the pathogenesis of some pleural disorders caused by exposure to fibrous dusts in the lungs.

  17. A Killer Immunoglobulin - Like Receptor Gene - Content Haplotype and A Cognate Human Leukocyte Antigen Ligand are Associated with Autism.

    PubMed

    Torres, Anthony; Westover, Jonna; Benson, Michael; Johnson, Randall; Dykes, Annelise

    2016-04-01

    The killing activity of natural killer cells is largely regulated by the binding of class I human leukocyte antigen cognate ligands to killer cell immunoglobulin - like receptor proteins. The killer cell immunoglobulin - like receptor gene - complex contains genes that activate and others that inhibit the killing state of natural killer cells depending on the binding of specific human leukocyte antigen cognate ligands. It has been suggested in previous publications that activating human leukocyte antigen/killer - cell immunoglobulin - like receptor complexes are increased in people with autism. We present data, which suggests that an activating cB01/tA01 killer cell immunoglobulin - like receptor gene - content haplotype and the cognate ligand human leukocyte antigen - C1k that activates this haplotype is significantly increased in autism. This is an important observation suggesting that the interaction between two proteins encoded on different chromosomes increases natural killer cell killing in autism.

  18. Effect of cigarette smoke extract on the polymorphonuclear leukocytes chemiluminescence: influence of a filter containing glutathione.

    PubMed

    Zappacosta, B; Persichilli, S; Minucci, A; Fasanella, S; Scribano, D; Giardina, B; De Sole, P

    2005-01-01

    Cigarette smoking is known to be a risk factor for several chronic and neoplastic diseases. Many compounds formed by cigarette burning, ranging from particulate materials to water solutes and gaseous extracts, are considered to be noxious agents, and many biochemical and molecular mechanisms have been proposed for the toxic effects of cigarette smoke. The oral cavity and the upper respiratory tract represent the first contact areas for smoke compounds; even a single cigarette can produce marked effects on some components of the oral cavity, either chemical compounds, such as glutathione and enzymes, or cellular elements, such as polymorphonuclear leukocytes. Several studies suggest a protective role of glutathione against the noxious effects of tobacco smoke; the sulphydril groups of glutathione, in fact, could react with some smoke products, such as unsaturated aldehydes, leading to the formation of harmless intermediate compounds and simultaneously preventing the inactivation of metabolically essential molecules, such as some enzymes. In this paper we analyse the effect of a filter containing glutathione on the respiratory burst of polymorphonuclear leukocytes exposed to aqueous extract of cigarette smoke, measuring their chemiluminescence activity. The results of this paper indicate that the GSH-containing filter has a likely protective effect against the inhibition of cigarette smoke extract on polymorphonuclear leukocyte activity.

  19. Inhibition of bacterial and leukocyte adhesion under shear stress conditions by material surface chemistry.

    PubMed

    Patel, Jasmine D; Ebert, Michael; Stokes, Ken; Ward, Robert; Anderson, James M

    2003-01-01

    Biomaterial-centered infections, initiated by bacterial adhesion, persist due to a compromised host immune response. Altering implant materials with surface modifying endgroups (SMEs) may enhance their biocompatibility by reducing bacterial and inflammatory cell adhesion. A rotating disc model, which generates shear stress within physiological ranges, was used to characterize adhesion of leukocytes and Staphylococcus epidermidis on polycarbonate-urethanes and polyetherurethanes modified with SMEs (polyethylene oxide, fluorocarbon and dimethylsiloxane) under dynamic flow conditions. Bacterial adhesion in the absence of serum was found to be mediated by shear stress and surface chemistry, with reduced adhesion exhibited on materials modified with polydimethylsiloxane and polyethylene oxide SMEs. In contrast, bacterial adhesion was enhanced on materials modified with fluorocarbon SMEs. In the presence of serum, bacterial adhesion was primarily neither material nor shear dependent. However, bacterial adhesion in serum was significantly reduced to < or = 10% compared to adhesion in serum-free media. Leukocyte adhesion in serum exhibited a shear dependency with increased adhesion occurring in regions exposed to lower shear-stress levels of < or = 7 dyne/cm2. Additionally, polydimethylsiloxane and polyethylene oxide SMEs reduced leukocyte adhesion on polyether-urethanes. In conclusion, these results suggest that surface chemistry and shear stress can mediate bacterial and cellular adhesion. Furthermore, materials modified with polyethylene oxide SMEs are capable of inhibiting bacterial adhesion, consequently minimizing the probability of biomaterial-centered infections.

  20. Effects of Tapinanthus globiferus and Zanthoxylum zanthoxyloides extracts on human leukocytes in vitro

    PubMed Central

    Ogunbolude, Yetunde; Ibrahim, Mohammad; Elekofehinti, Olusola Olalekan; Adeniran, Adekunle; Abolaji, Amos Olalekan; Rocha, João Batista Teixeira; Kamdem, Jean Paul

    2014-01-01

    Objective: This study aimed at investigating the genotoxicity and cytotoxicity effect of Tapinanthus globiferus and Zanthoxylum zanthoxyloides to human leukocytes. In addition, the reductive potential and the chemical composition of the two plant extracts were also determined. Materials and Methods: Human leukocytes were obtained from healthy volunteer donors. The genotoxicity and cytotoxicity of T. globiferus and Z. zanthoxyloides were assessed using the comet assay and trypan blue exclusion, respectively. The antioxidant activity of the plant extracts was evaluated by the reducing power assay. Furthermore, high-performance liquid chromatography-diode array detector was used to characterize and quantify the constituents of these plants. Results: T. globiferus (10-150 µg/mL) was neither genotoxic nor cytotoxic at the concentrations tested, suggesting that it can be consumed safely at relatively high concentrations. However, Z. zanthoxyloides showed cytoxicity and genotoxicity to human leukocytes at the highest concentration tested (150 µg/mL). In addition, the total reducing power of T. globiferus was found higher than Z. zanthoxyloides in potassium ferricyanide reduction. Both plants extract contained flavonoids (rutin and quercetin) and phenolic acids (chlorogenic and caffeic). Conclusion: The results obtained support the fact that some caution should be paid regarding the dosage and the frequency of use of Z. zanthoxyloides extract. PMID:26401368

  1. Quantitation of complement and leukocyte binding to a parasitic helminth species.

    PubMed

    Giacomin, Paul R; Wang, Hui; Gordon, David L; Dent, Lindsay A

    2004-06-01

    Methods used to quantify complement deposition and cell adherence to parasitic helminths usually involve subjective visual comparisons of immunofluorescence or time-consuming manual counting of bound cells. Such targets are relatively large and, generally, few individual organisms can be analysed. More objective and efficient radiometric assays are available, but these also have considerable disadvantages. We have developed an improved immunofluorescence-based method for quantitation of complement deposition on viable third-stage larvae of the nematode Nippostrongylus brasiliensis (L3). A similar strategy was also applied to measuring leukocyte adherence to the parasite. Fluorescein isothiocyanate (FITC)-conjugated antibodies were used to detect complement on serum-treated larvae. The adherence of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled mouse leukocytes to larvae was investigated using the same basic approach. Images of fluorescent larvae or fluorescent cells attached to larvae were generated with a Bio-Rad Molecular Imager FX and fluorescence intensity was quantified. Hundreds of larvae can be analysed simultaneously in multiple samples, and these strategies allow rapid and sensitive quantitation that is directly proportional to the amount of protein or the number of leukocytes added to cultures. These techniques may also be applicable to other large objects, organisms or biological surfaces.

  2. Label-free in vivo imaging of human leukocytes using two-photon excited endogenous fluorescence

    NASA Astrophysics Data System (ADS)

    Zeng, Yan; Yan, Bo; Sun, Qiqi; Teh, Seng Khoon; Zhang, Wei; Wen, Zilong; Qu, Jianan Y.

    2013-04-01

    We demonstrate that two-photon excited endogenous fluorescence enables label-free morphological and functional imaging of various human blood cells. Specifically, we achieved distinctive morphological contrast to visualize morphology of important leukocytes, such as polymorphonuclear structure of granulocyte and mononuclear feature of agranulocyte, through the employment of the reduced nicotinamide adenine dinucleotide (NADH) fluorescence signals. In addition, NADH fluorescence images clearly reveal the morphological transformation process of neutrophils during disease-causing bacterial infection. Our findings also show that time-resolved NADH fluorescence can be potentially used for functional imaging of the phagocytosis of pathogens by leukocytes (neutrophils) in vivo. In particular, we found that free-to-bound NADH ratios measured in infected neutrophils increased significantly, which is consistent with a previous study that the energy consumed in the phagocytosis of neutrophils is mainly generated through the glycolysis pathway that leads to the accumulation of free NADH. Future work will focus on further developing and applying label-free imaging technology to investigate leukocyte-related diseases and disorders.

  3. TNF{alpha} release from peripheral blood leukocytes depends on a CRM1-mediated nuclear export

    SciTech Connect

    Miskolci, Veronika; Ghosh, Chandra C.; Rollins, Janet; Romero, Carlos; Vu, Hai-Yen; Robinson, Staci; Davidson, Dennis; Vancurova, Ivana . E-mail: vancuroi@stjohns.edu

    2006-12-15

    Tumor necrosis factor-{alpha} (TNF{alpha}) is a potent pro-inflammatory cytokine that plays a major role in the pathogenesis of acute and chronic inflammatory disorders such as septic shock and arthritis, respectively. Leukocytes stimulated with inflammatory signals such as lipopolysaccharide (LPS) are the predominant producers of TNF{alpha}, and thus control of TNF{alpha} release from stimulated leukocytes represents a potential therapeutic target. Here, we report that leptomycin B (LMB), a specific inhibitor of CRM1-dependent nuclear protein export, inhibits TNF{alpha} release from LPS-stimulated human peripheral blood neutrophils and mononuclear cells. In addition, immunofluorescence confocal microscopy and immunoblotting analysis indicate that TNF{alpha} is localized in the nucleus of human neutrophils and mononuclear cells. This study demonstrates that the cellular release of TNF{alpha} from stimulated leukocytes is mediated by the CRM1-dependent nuclear export mechanism. Inhibition of CRM1-dependent cellular release of TNF{alpha} could thus provide a novel therapeutic approach for disorders involving excessive TNF{alpha} release.

  4. Leukocyte telomere length in Hispanic schizophrenia patients under treatment with olanzapine.

    PubMed

    Monroy-Jaramillo, Nancy; Rodríguez-Agudelo, Yaneth; Aviña-Cervantes, Luis Carlos; Roberts, David L; Velligan, Dawn I; Walss-Bass, Consuelo

    2017-02-10

    Different lines of evidence indicate that patients with schizophrenia (SZ) exhibit accelerated aging. Leukocyte telomere length (TL), an aging marker, is associated with age-related and chronic pathologies, including schizophrenia. We analyzed leukocyte TL in 170 SZ patients of Hispanic ancestry grouped based on antipsychotic treatment, compared to 126 matched controls. The group under treatment with atypical antipsychotics was further subdivided according to the risk of medication to cause metabolic syndrome (MetS). Our results show significant erosion in the TL of SZ patients under treatment with the atypical antipsychotics clozapine and olanzapine, which cause high-risk for MetS, compared to healthy controls and patients under treatment with medium and low-risk antipsychotics. However, when the analysis was done separately for clozapine and olanzapine, a significant difference remained only for olanzapine. These findings suggest that atypical antipsychotics that cause high-risk for MetS, particularly olanzapine, may modulate leukocyte TL in SZ patients. Future research is required to elucidate if in fact atypical antipsychotics are involved in TL maintenance in SZ subjects and the mechanism by which this occurs.

  5. Preconditioning-induced CXCL12 upregulation minimizes leukocyte infiltration after stroke in ischemia-tolerant mice.

    PubMed

    Selvaraj, Uma Maheswari; Ortega, Sterling B; Hu, Ruilong; Gilchrist, Robert; Kong, Xiangmei; Partin, Alexander; Plautz, Erik J; Klein, Robyn S; Gidday, Jeffrey M; Stowe, Ann M

    2017-03-01

    Repetitive hypoxic preconditioning creates long-lasting, endogenous protection in a mouse model of stroke, characterized by reductions in leukocyte-endothelial adherence, inflammation, and infarct volumes. The constitutively expressed chemokine CXCL12 can be upregulated by hypoxia and limits leukocyte entry into brain parenchyma during central nervous system inflammatory autoimmune disease. We therefore hypothesized that the sustained tolerance to stroke induced by repetitive hypoxic preconditioning is mediated, in part, by long-term CXCL12 upregulation at the blood-brain barrier (BBB). In male Swiss Webster mice, repetitive hypoxic preconditioning elevated cortical CXCL12 protein levels, and the number of cortical CXCL12+ microvessels, for at least two weeks after the last hypoxic exposure. Repetitive hypoxic preconditioning-treated mice maintained more CXCL12-positive vessels than untreated controls following transient focal stroke, despite cortical decreases in CXCL12 mRNA and protein. Continuous administration of the CXCL12 receptor (CXCR4) antagonist AMD3100 for two weeks following repetitive hypoxic preconditioning countered the increase in CXCL12-positive microvessels, both prior to and following stroke. AMD3100 blocked the protective post-stroke reductions in leukocyte diapedesis, including macrophages and NK cells, and blocked the protective effect of repetitive hypoxic preconditioning on lesion volume, but had no effect on blood-brain barrier dysfunction. These data suggest that CXCL12 upregulation prior to stroke onset, and its actions following stroke, contribute to the endogenous, anti-inflammatory phenotype induced by repetitive hypoxic preconditioning.

  6. TNFα promotes CAR-dependent migration of leukocytes across epithelial monolayers

    PubMed Central

    Morton, Penny E.; Hicks, Alexander; Ortiz-Zapater, Elena; Raghavan, Swetavalli; Pike, Rosemary; Noble, Alistair; Woodfin, Abigail; Jenkins, Gisli; Rayner, Emma; Santis, George; Parsons, Maddy

    2016-01-01

    Trans-epithelial migration (TEpM) of leukocytes during inflammation requires engagement with receptors expressed on the basolateral surface of the epithelium. One such receptor is Coxsackie and Adenovirus Receptor (CAR) that binds to Junctional Adhesion Molecule-like (JAM-L) expressed on leukocytes. Here we provide the first evidence that efficient TEpM of monocyte-derived THP-1 cells requires and is controlled by phosphorylation of CAR. We show that TNFα acts in a paracrine manner on epithelial cells via a TNFR1-PI3K-PKCδ pathway leading to CAR phosphorylation and subsequent transmigration across cell junctions. Moreover, we show that CAR is hyper-phosphorylated in vivo in acute and chronic lung inflammation models and this response is required to facilitate immune cell recruitment. This represents a novel mechanism of feedback between leukocytes and epithelial cells during TEpM and may be important in controlling responses to pro-inflammatory cytokines in pathological settings. PMID:27193388

  7. Human Eosinophil Leukocytes Express Protein Disulfide Isomerase in Secretory Granules and Vesicles: Ultrastructural Studies.

    PubMed

    Dias, Felipe F; Amaral, Kátia B; Carmo, Lívia A S; Shamri, Revital; Dvorak, Ann M; Weller, Peter F; Melo, Rossana C N

    2014-06-01

    Protein disulfide isomerase (PDI) has fundamental roles in the oxidative folding of proteins in the endoplasmic reticulum (ER) of eukaryotic cells. The study of this molecule has been attracting considerable attention due to its association with other cell functions and human diseases. In leukocytes, such as neutrophils, PDI is involved with cell adhesion, signaling and inflammation. However, the expression of PDI in other leukocytes, such as eosinophils, important cells in inflammatory, allergic and immunomodulatory responses, remains to be defined. Here we used different approaches to investigate PDI expression within human eosinophils. Western blotting and flow cytometry demonstrated high PDI expression in both unstimulated and CCL11/eotaxin-1-stimulated eosinophils, with similar levels in both conditions. By using an immunogold electron microscopy technique that combines better epitope preservation and secondary Fab-fragments of antibodies linked to 1.4-nm gold particles for optimal access to microdomains, we identified different intracellular sites for PDI. In addition to predictable strong PDI labeling at the nuclear envelope, other unanticipated sites, such as secretory granules, lipid bodies and vesicles, including large transport vesicles (eosinophil sombrero vesicles), were also labeled. Thus, we provide the first identification of PDI in human eosinophils, suggesting that this molecule may have additional/specific functions in these leukocytes.

  8. Dielectric properties of human leukocyte subpopulations determined by electrorotation as a cell separation criterion.

    PubMed

    Yang, J; Huang, Y; Wang, X; Wang, X B; Becker, F F; Gascoyne, P R

    1999-06-01

    The separation and purification of human blood cell subpopulations is an essential step in many biomedical applications. New dielectrophoretic fractionation methods have great potential for cell discrimination and manipulation, both for microscale diagnostic applications and for much larger scale clinical problems. To discover whether human leukocyte subpopulations might be separable by such methods, the dielectric characteristics of the four main leukocyte subpopulations, namely, B- and T-lymphocytes, monocytes, and granulocytes, were measured by electrorotation over the frequency range 1 kHz to 120 MHz. The subpopulations were derived from human peripheral blood by magnetically activated cell sorting (MACS) and sheep erythrocyte rosetting methods, and the quality of cell fractions was checked by flow cytometry. Mean specific membrane capacitance values were calculated from the electrorotation data as 10.5 (+/- 3.1), 12.6 (+/- 3.5), 15.3 (+/- 4.3), and 11.0 (+/- 3.2) mF/m2 for T- and B-lymphocytes, monocytes, and granulocytes, respectively, according to a single-shell dielectric model. In agreement with earlier findings, these values correlated with the richness of the surface morphologies of the different cell types, as revealed by scanning electron microscopy (SEM). The data reveal that dielectrophoretic cell sorters should have the ability to discriminate between, and to separate, leukocyte subpopulations under appropriate conditions.

  9. Leukocyte transepithelial migration in lung induced by DMSA functionalized magnetic nanoparticles.

    PubMed

    Azevedo, Ricardo Bentes; Valois, Caroline Rodrigues Alves; Chaves, Sacha Braun; Silva, Jaqueline Rodrigues; Garcia, Monica Pereira

    2011-01-01

    Magnetic nanoparticles surface-covered with meso-2,3-dimercaptosuccinic acid (MNPs-DMSA) constitute a promising approach for tissue- and cell-targeted delivery of therapeutic drugs in the lung. However, they can also induce a transient transendothelial migration of leukocytes in the organ as a side effect after endovenous administration of MNPs-DMSA. We demonstrated that monocytes/macrophages constitute the main subpopulation of leukocytes involved in this process. Our recent research found that MNPs-DMSA up-regulated the mRNA expression of E-, L- and P-selectin and macrophage-1 antigen, and increased concentration of tumor necrosis factor-α in lung, in a time dependent manner. The critical relevance of the β2 integrin-dependent pathway in leukocyte transmigration elicited by MNPs-DMSA was demonstrated by use of knockout mice. Our work characterizes mechanisms of the pro-inflammatory effects of MNPs-DMSA in the lung, and identifies β2 integrin-targeted interventions as promising strategies to reduce pulmonary side effects of MNPs-DMSA during biomedical applications. In addition, MNPs-DMSA could be used as modulators of lung immune response.

  10. Use of computed tomography and radiolabeled leukocytes in a cat with pancreatitis.

    PubMed

    Head, Laurie L; Daniel, Gregory B; Becker, Timothy J; Lidbetter, David A

    2005-01-01

    The normal feline pancreas has been evaluated using radiolabeled leukocytes (99mTc-HMPAO) and computed tomography. The purpose of this report is to describe a clinical case where both modalities were utilized to assess the inflamed feline pancreas. A nine year old female cat presented with anorexia, depression and some vomiting. Blood values were unremarkable. Radiographs and ultrasound were suggestive of pancreatitis. The cat's leukocytes were separated and labeled according to an established protocol. Whole body images were acquired immediately, at 5 and 30 min, and at 1, 2, 4, and 17 hours post injection. Approximately 48 h later, the animal was anesthetized and computed tomography of the abdomen was preformed both pre and post contrast. Surgical biopsies were taken. The distribution of the WBCs was similar to that documented in normal animals, however, at 2 h there was faint uptake seen in the region of the pancreas. This uptake became more intense at 4 h and persisted at 17 h. Computed tomography showed irregular margination of the pancreas, it was larger than normal and inhomogeneous. Contrast enhancement was inhomogeneous and its peak enhancement was not reached until 10 min post injection; normal feline pancreas enhances homogeneously and peaks immediately. Histopathology confirmed pancreatitis with lymphocytic, plasmacytic, neutrophilic and eosinophilic inflammation and fibrosis. Radiolabeled leukocytes can be used to document pancreatic inflammation and this is best seen 4 h after injection. Computed tomography allows superior visualization of the pancreas. Both the appearance and contrast enhancement pattern of the inflamed pancreas differ from normal.

  11. Molecular cloning and characterization of plastin, a human leukocyte protein expressed in transformed human fibroblasts.

    PubMed Central

    Lin, C S; Aebersold, R H; Kent, S B; Varma, M; Leavitt, J

    1988-01-01

    The phosphoprotein plastin was originally identified as an abundant transformation-induced polypeptide of chemically transformed neoplastic human fibroblasts. This abundant protein is normally expressed only in leukocytes, suggesting that it may play a role in hemopoietic cell differentiation. Protein microsequencing of plastin purified from leukemic T lymphocytes by high-resolution two-dimensional gel electrophoresis produced eight internal oligopeptide sequences. An oligodeoxynucleotide probe corresponding to one of the oligopeptides was used to clone cDNAs from transformed human fibroblasts that encoded the seven other oligopeptides predicted for human plastin. Sequencing and characterization of two cloned cDNAs revealed the existence of two distinct, but closely related, isoforms of plastin--l-plastin, which is expressed in leukocytes and transformed fibroblasts, and t-plastin, which is expressed in normal cells of solid tissues and transformed fibroblasts. The leukocyte isoform l-plastin is expressed in a diverse variety of human tumor cell lines, suggesting that it may be involved in the neoplastic process of some solid human tumors. Images PMID:3211125

  12. Modulation of leukocyte infiltration and phenotype in microporous tissue engineering scaffolds via vector induced IL-10 expression.

    PubMed

    Gower, R Michael; Boehler, Ryan M; Azarin, Samira M; Ricci, Christine F; Leonard, Joshua N; Shea, Lonnie D

    2014-02-01

    Biomaterial scaffolds are central to many tissue engineering strategies as they create a space for tissue growth and provide a support for cell adhesion and migration. However, biomaterial implantation results in unavoidable injury resulting in an inflammatory response, which can impair integration with the host and tissue regeneration. Toward the goal of reducing inflammation, we investigated the hypothesis that a lentiviral gene therapy-based approach to localized and sustained IL-10 expression at a scaffold could modulate the number, relative proportions, and cytokine production of infiltrating leukocyte populations. Flow cytometry was used to quantify infiltration of six leukocyte populations for 21 days following implantation of PLG scaffolds into intraperitoneal fat. Leukocytes with innate immune functions (i.e., macrophages, dendritic cells, neutrophils) were most prevalent at early time points, while T lymphocytes became prevalent by day 14. Reporter gene delivery indicated that transgene expression persisted at the scaffold for up to 28 days and macrophages were the most common leukocyte transduced, while transduced dendritic cells expressed the greatest levels of transgene. IL-10 delivery decreased leukocyte infiltration by 50% relative to controls, increased macrophage IL-10 expression, and decreased macrophage, dendritic cell, and CD4 T cell IFN-γ expression. Thus, IL-10 gene delivery significantly decreased inflammation following scaffold implant into the intraperitoneal fat, in part by modulating cytokine expression of infiltrating leukocytes.

  13. Comparative evaluation of leukocyte- and platelet-rich plasma and pure platelet-rich plasma for cartilage regeneration

    PubMed Central

    Xu, Zhengliang; Yin, Wenjing; Zhang, Yuelei; Qi, Xin; Chen, Yixuan; Xie, Xuetao; Zhang, Changqing

    2017-01-01

    Platelet-rich plasma (PRP) has gained growing popularity in the treatment of articular cartilage lesions in the last decade. However, the potential harmful effects of leukocytes in PRP on cartilage regeneration have seldom been studied in vitro, and not at all in vivo yet. The objective of the present study is to compare the effects of leukocyte- and platelet-rich plasma (L-PRP) and pure platelet-rich plasma (P-PRP) on cartilage repair and NF-κB pathway, in order to explore the mechanism underlying the function of leukocytes in PRP in cartilage regeneration. The constituent analysis showed that P-PRP had significantly lower concentrations of leukocytes and pro-inflammatory cytokines compared with L-PRP. In addition, cell proliferation and differentiation assays indicated P-PRP promoted growth and chondrogenesis of rabbit bone marrow mesenchymal stem cells (rBMSC) significantly compared with L-PRP. Despite similarity in macroscopic appearance, the implantation of P-PRP combining rBMSC in vivo yielded better cartilage repair results than the L-PRP group based on histological examination. Importantly, the therapeutic effects of PRP on cartilage regeneration could be enhanced by removing leukocytes to avoid the activation of the NF-κB pathway. Thus, PRP without concentrated leukocytes may be more suitable for the treatment of articular cartilage lesions. PMID:28265109

  14. A novel algorithm based on visual saliency attention for localization and segmentation in rapidly-stained leukocyte images.

    PubMed

    Zheng, Xin; Wang, Yong; Wang, Guoyou; Chen, Zhong

    2014-01-01

    In this paper, we propose a fast hierarchical framework of leukocyte localization and segmentation in rapidly-stained leukocyte images (RSLI) with complex backgrounds and varying illumination. The proposed framework contains two main steps. First, a nucleus saliency model based on average absolute difference is built, which locates each leukocyte precisely while effectively removes dyeing impurities and erythrocyte fragments. Secondly, two different schemes are presented for segmenting the nuclei and cytoplasm respectively. As for nuclei segmentation, to solve the overlap problem between leukocytes, we extract the nucleus lobes first and further group them. The lobes extraction is realized by the histogram-based contrast map and watershed segmentation, taking into account the saliency and similarity of nucleus color. Meanwhile, as for cytoplasm segmentation, to extract the blurry contour of the cytoplasm under instable illumination, we propose a cytoplasm enhancement based on tri-modal histogram specification, which specifically improves the contrast of cytoplasm while maintaining others. Then, the contour of cytoplasm is quickly obtained by extraction based on parameter-controlled adaptive attention window. Furthermore, the contour is corrected by concave points matching in order to solve the overlap between leukocytes and impurities. The experiments show the effectiveness of the proposed nucleus saliency model, which achieves average localization accuracy with F1-measure greater than 95%. In addition, the comparison of single leukocyte segmentation accuracy and running time has demonstrated that the proposed segmentation scheme outperforms the former approaches in RSLI.

  15. Complement (C5)-derived chemotactic activity accounts for accumulation of polymorphonuclear leukocytes in cerebrospinal fluid of rabbits with pneumococcal meningitis.

    PubMed Central

    Ernst, J D; Hartiala, K T; Goldstein, I M; Sande, M A

    1984-01-01

    Experiments were performed to identify the chemoattractant for polymorphonuclear leukocytes that appears in the cerebrospinal fluid of rabbits with experimental pneumococcal meningitis. Meningitis was induced in anesthetized New Zealand white rabbits by injecting 10(4) cells of stationary-phase Streptococcus pneumoniae type III intracisternally. Before bacteria were injected, cerebrospinal fluid contained neither polymorphonuclear leukocytes nor chemotactic activity. Significant chemotactic activity for rabbit polymorphonuclear leukocytes was detected 12 h after inoculation with bacteria and was maximal after 18 to 20 h. Chemotactic activity appeared in cerebrospinal fluid while concentrations of pneumococci and total protein were increasing but before there was any accumulation of polymorphonuclear leukocytes. The chemotactic activity in cerebrospinal fluid was heat stable (56 degrees C for 30 min), eluted from Sephadex G-75 with a profile identical to that of the chemotactic activity in zymosan-activated rabbit serum, and was inhibited by treatment with antibodies to native human C5. In addition, preincubation of polymorphonuclear leukocytes with partially purified rabbit C5a selectively inhibited their subsequent chemotactic responses to cerebrospinal fluid. These data indicate that complement (C5)-derived chemotactic activity appears in cerebrospinal fluid during the course of experimental pneumococcal meningitis in rabbits and suggest that this activity accounts for the accumulation of polymorphonuclear leukocytes observed in this infection. PMID:6480117

  16. The Rho-guanine nucleotide exchange factor Trio controls leukocyte transendothelial migration by promoting docking structure formation.

    PubMed

    van Rijssel, Jos; Kroon, Jeffrey; Hoogenboezem, Mark; van Alphen, Floris P J; de Jong, Renske J; Kostadinova, Elena; Geerts, Dirk; Hordijk, Peter L; van Buul, Jaap D

    2012-08-01

    Leukocyte transendothelial migration involves the active participation of the endothelium through the formation of apical membrane protrusions that embrace adherent leukocytes, termed docking structures. Using live-cell imaging, we find that prior to transmigration, endothelial docking structures form around 80% of all neutrophils. Previously we showed that endothelial RhoG and SGEF control leukocyte transmigration. In this study, our data reveal that both full-length Trio and the first DH-PH (TrioD1) domain of Trio, which can activate Rac1 and RhoG, interact with ICAM-1 and are recruited to leukocyte adhesion sites. Moreover, upon clustering of ICAM-1, the Rho-guanine nucleotide exchange factor Trio activates Rac1, prior to activating RhoG, in a filamin-dependent manner. We further show that docking structure formation is initiated by ICAM-1 clustering into ring-like structures, which is followed by apical membrane protrusion. Interestingly, we find that Rac1 is required for ICAM-1 clustering, whereas RhoG controls membrane protrusion formation. Finally, silencing endothelial Trio expression or reducing TrioD1 activity without affecting SGEF impairs both docking structure formation and leukocyte transmigration. We conclude that Trio promotes leukocyte transendothelial migration by inducing endothelial docking structure formation in a filamin-dependent manner through the activation of Rac1 and RhoG.

  17. The effect of ibuprofen on accumulation of /sup 111/In-labeled platelets and leukocytes in experimental myocardial infarction

    SciTech Connect

    Romson, J.L.; Hook, B.G.; Rigot, V.H.; Schork, M.A.; Swanson, D.P.; Lucchesi, B.R.

    1982-11-01

    To assess the ability of ibuprofen to influence the extent of platelet aggregation and leukocyte infiltration during acute myocardial infarction, autologous indium-111 (/sup 111/In)-labeled platelets or leukocytes were injected before 60 minutes of left circumflex coronary artery (LCx) occlusion, followed by 24 hours of reperfusion in the canine heart. Myocardial infarct size, as a percent of the area at risk, was reduced in the ibuprofen-treated group (12.5 mg/kg i.v. every 4 hours beginning 30 minutes before LCx occlusion) by 40%, from 48 +/- 4% in control animals to 29 +/- 4% in ibuprofen-treated dogs (p . 0.005). Quantification of the platelet-associated /sup 111/In radioactivity in irreversibly injured myocardium indicated that ibuprofen did not alter the accumulation of platelets in infarcted myocardium. In contrast, leukocyte accumulation in infarcted tissue was reduced significantly. In tissue samples with 0.41-0.60 gram infarct, the infarcted/normal ratio of leukocyte radioactivity was 12 +/- 2 in control dogs and 4 +/- 1 in ibuprofen-treated dogs, which represents a 67% reduction in leukocyte accumulation in ibuprofen-treated compared with control dogs. Similar reductions were found in other gram-infarct-weight categories. Although both platelets and leukocytes accumulate in infarcted canine myocardium, ibuprofen may exert its beneficial effect on ischemic myocardium by suppressing the inflammatory response associated with myocardial ischemia and infarction.

  18. The effect of ibuprofen on accumulation of indium-111-labeled platelets and leukocytes in experimental myocardial infarction

    SciTech Connect

    Romson, J.L.; Hook, B.G.; Rigot, V.H.; Schark, M.A.; Swanson, D.P.; Lucchesi, B.R.

    1982-11-01

    To assess the ability of ibuprofen to influence the extent of platelet aggregation and leukocyte infiltration during acute myocardial infarction, autologous indium-111 (/sup 111/In)-labeled platelets or leukocytes were injected before 60 minutes of left circumflex coronary artery (LCx) occlusion, followed by 24 hours of reperfusion in the canine heart. Myocardial infarct size, as a percent of the area at risk, was reduced in the ibuprofen-treated group (12.5 mg/kg i.v. every 4 hours beginning 30 minutes before LCx occulsion) by 40%, from 48 +/- 4% in control animals to 29 +/- 4% in ibuprofen-treated dogs (p=0.005). Quantification of the platelet-associated /sup 111/In radioactivity in irreversibly injured myocardium indicated that ibuprofen did not alter the accumulation of platelets in infarcted myocardium. In contrast, leukocyte accumulation in infarcted tissue was reduced significantly. In tissue samples with 0.41-0.60 gram infarct, the infarcted/normal ratio of leukocyte radioactivity was 12 +/- 2 in control dogs and 4 +/- 1 in ibuprofen-treated dogs, which represents a 67% reduction in leukocyte accumulation in ibuprofen-treated compared with control dogs. Similar reductions were found in other gram-infarct-weight categories. Although both platelets and leukocytes acumulate in infarcted canine myocardium, ibuprofen may exert its beneficial effect on ischemic myocardium by suppressing the inflammatory response associated with myocardial ischemia and infarction.

  19. Rescue from acute neuroinflammation by pharmacological chemokine-mediated deviation of leukocytes

    PubMed Central

    2012-01-01

    Background Neutrophil influx is an important sign of hyperacute neuroinflammation, whereas the entry of activated lymphocytes into the brain parenchyma is a hallmark of chronic inflammatory processes, as observed in multiple sclerosis (MS) and its animal models of experimental autoimmune encephalomyelitis (EAE). Clinically approved or experimental therapies for neuroinflammation act by blocking leukocyte penetration of the blood brain barrier. However, in view of unsatisfactory results and severe side effects, complementary therapies are needed. We have examined the effect of chlorite-oxidized oxyamylose (COAM), a potent antiviral polycarboxylic acid on EAE. Methods EAE was induced in SJL/J mice by immunization with spinal cord homogenate (SCH) or in IFN-γ-deficient BALB/c (KO) mice with myelin oligodendrocyte glycoprotein peptide (MOG35-55). Mice were treated intraperitoneally (i.p.) with COAM or saline at different time points after immunization. Clinical disease and histopathology were compared between both groups. IFN expression was analyzed in COAM-treated MEF cell cultures and in sera and peritoneal fluids of COAM-treated animals by quantitative PCR, ELISA and a bioassay on L929 cells. Populations of immune cell subsets in the periphery and the central nervous system (CNS) were quantified at different stages of disease development by flow cytometry and differential cell count analysis. Expression levels of selected chemokine genes in the CNS were determined by quantitative PCR. Results We discovered that COAM (2 mg i.p. per mouse on days 0 and 7) protects significantly against hyperacute SCH-induced EAE in SJL/J mice and MOG35-55-induced EAE in IFN-γ KO mice. COAM deviated leukocyte trafficking from the CNS into the periphery. In the CNS, COAM reduced four-fold the expression levels of the neutrophil CXC chemokines KC/CXCL1 and MIP-2/CXCL2. Whereas the effects of COAM on circulating blood and splenic leukocytes were limited, significant alterations were

  20. In vitro leukocyte response of three-spined sticklebacks (Gasterosteus aculeatus) to helminth parasite antigens.

    PubMed

    Franke, Frederik; Rahn, Anna K; Dittmar, Janine; Erin, Noémie; Rieger, Jennifer K; Haase, David; Samonte-Padilla, Irene E; Lange, Joseph; Jakobsen, Per J; Hermida, Miguel; Fernández, Carlos; Kurtz, Joachim; Bakker, Theo C M; Reusch, Thorsten B H; Kalbe, Martin; Scharsack, Jörn P

    2014-01-01

    Helminth parasites of teleost fish have evolved strategies to evade and manipulate the immune responses of their hosts. Responsiveness of fish host immunity to helminth antigens may therefore vary depending on the degree of host-parasite counter-adaptation. Generalist parasites, infective for a number of host species, might be unable to adapt optimally to the immune system of a certain host species, while specialist parasites might display high levels of adaptation to a particular host species. The degree of adaptations may further differ between sympatric and allopatric host-parasite combinations. Here, we test these hypotheses by in vitro exposure of head kidney leukocytes from three-spined sticklebacks (Gasterosteus aculeatus) to antigens from parasites with a broad fish host range (Diplostomum pseudospathaceum, Triaenophorus nodulosus), a specific fish parasite of cyprinids (Ligula intestinalis) and parasites highly specific only to a single fish species as second intermediate host (Schistocephalus pungitii, which does not infect G. aculeatus, and Schistocephalus solidus, infecting G. aculeatus). In vitro responses of stickleback leukocytes to S. solidus antigens from six European populations, with S. solidus prevalence from <1% to 66% were tested in a fully crossed experimental design. Leukocyte cultures were analysed by means of flow cytometry and a chemiluminescence assay to quantify respiratory burst activity. We detected decreasing magnitudes of in vitro responses to antigens from generalist to specialist parasites and among specialists, from parasites that do not infect G. aculeatus to a G. aculeatus-infecting species. Generalist parasites seem to maintain their ability to infect different host species at the costs of relatively higher immunogenicity compared to specialist parasites. In a comparison of sympatric and allopatric combinations of stickleback leukocytes and antigens from S. solidus, magnitudes of in vitro responses were dependent on

  1. Long Non-Coding RNA and Alternative Splicing Modulations in Parkinson's Leukocytes Identified by RNA Sequencing

    PubMed Central

    Soreq, Lilach; Guffanti, Alessandro; Salomonis, Nathan; Simchovitz, Alon; Israel, Zvi; Bergman, Hagai; Soreq, Hermona

    2014-01-01

    The continuously prolonged human lifespan is accompanied by increase in neurodegenerative diseases incidence, calling for the development of inexpensive blood-based diagnostics. Analyzing blood cell transcripts by RNA-Seq is a robust means to identify novel biomarkers that rapidly becomes a commonplace. However, there is lack of tools to discover novel exons, junctions and splicing events and to precisely and sensitively assess differential splicing through RNA-Seq data analysis and across RNA-Seq platforms. Here, we present a new and comprehensive computational workflow for whole-transcriptome RNA-Seq analysis, using an updated version of the software AltAnalyze, to identify both known and novel high-confidence alternative splicing events, and to integrate them with both protein-domains and microRNA binding annotations. We applied the novel workflow on RNA-Seq data from Parkinson's disease (PD) patients' leukocytes pre- and post- Deep Brain Stimulation (DBS) treatment and compared to healthy controls. Disease-mediated changes included decreased usage of alternative promoters and N-termini, 5′-end variations and mutually-exclusive exons. The PD regulated FUS and HNRNP A/B included prion-like domains regulated regions. We also present here a workflow to identify and analyze long non-coding RNAs (lncRNAs) via RNA-Seq data. We identified reduced lncRNA expression and selective PD-induced changes in 13 of over 6,000 detected leukocyte lncRNAs, four of which were inversely altered post-DBS. These included the U1 spliceosomal lncRNA and RP11-462G22.1, each entailing sequence complementarity to numerous microRNAs. Analysis of RNA-Seq from PD and unaffected controls brains revealed over 7,000 brain-expressed lncRNAs, of which 3,495 were co-expressed in the leukocytes including U1, which showed both leukocyte and brain increases. Furthermore, qRT-PCR validations confirmed these co-increases in PD leukocytes and two brain regions, the amygdala and substantia

  2. Effects of Different Peep Levels on Mesenteric Leukocyte-Endothelial Interactions in Rats During Mechanical Ventilation

    PubMed Central

    Aikawa, Priscila; Farsky, Sandra Helena Poliselli; de Oliveira, Maria Aparecida; Pazetti, Rogério; Mauad, Thaís; Sannomiya, Paulina; Nakagawa, Naomi Kondo

    2009-01-01

    INTRODUCTION: Mechanical ventilation with positive end expiratory pressure (PEEP) improves oxygenation and treats acute pulmonary failure. However, increased intrathoracic pressure may cause regional blood flow alterations that may contribute to mesenteric ischemia and gastrointestinal failure. We investigated the effects of different PEEP levels on mesenteric leukocyte-endothelial interactions. METHODS: Forty-four male Wistar rats were initially anesthetized (Pentobarbital I.P. 50mg/kg) and randomly assigned to one of the following groups: 1) NAIVE (only anesthesia; n=9), 2) PEEP 0 (PEEP of 0 cmH2O, n=13), 3) PEEP 5 (PEEP of 5 cmH2O, n=12), and 4) PEEP 10 (PEEP of 10 cmH2O, n=13). Positive end expiratory pressure groups were tracheostomized and mechanically ventilated with a tidal volume of 10 mL/kg, respiratory rate of 70 rpm, and inspired oxygen fraction of 1. Animals were maintained under isoflurane anesthesia. After two hours, laparotomy was performed, and leukocyte-endothelial interactions were evaluated by intravital microscopy. RESULTS: No significant changes were observed in mean arterial blood pressure among groups during the study. Tracheal peak pressure was smaller in PEEP 5 compared with PEEP 0 and PEEP 10 groups (11, 15, and 16 cmH2O, respectively; p<0.05). After two hours of MV, there were no differences among NAIVE, PEEP 0 and PEEP 5 groups in the number of rollers (118±9,127±14 and 147±26 cells/10minutes, respectively), adherent leukocytes (3±1,3±1 and 4±2 cells/100μm venule length, respectively), and migrated leukocytes (2±1,2±1 and 2±1 cells/5,000μm2, respectively) at the mesentery. However, the PEEP 10 group exhibited an increase in the number of rolling, adherent and migrated leukocytes (188±15 cells / 10 min, 8±1 cells / 100 μm and 12±1 cells / 5,000 μm2, respectively; p<0.05). CONCLUSIONS: High intrathoracic pressure was harmful to mesenteric microcirculation in the experimental model of rats with normal lungs and stable

  3. Expression of selected proteins of the extrinsic and intrinsic pathways of apoptosis in human leukocytes exposed to N-nitrosodimethylamine.

    PubMed

    Iwaniuk, A; Jabłońska, E; Jabłoński, J; Ratajczak-Wrona, W; Garley, M

    2015-06-01

    N-nitrosodimethylamine (NDMA) is a xenobiotic widespread in human environment capable of regulating the lifespan of immune cells. In this study, we examined the roles of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/death receptor 5 (DR5) complex and the Fas molecule in the induction of the extrinsic apoptosis pathway in human neutrophils (polymorphonuclear neutrophils (PMNs)) and peripheral blood mononuclear cells (PBMCs) exposed to NDMA. Also we assessed these proteins ability to trigger the intrinsic apoptosis pathway in those cells. For this purpose, we examined the expression of Fas-associated protein with death domain, truncated Bid (tBid) proteins, and apoptogenic factors such as apoptosis-inducing factor, Smac/Diablo, Omi/HtrA2, and caspase-3 as an indication of accomplished apoptosis phenomenon. PMNs and PBMCs were isolated from whole blood by density gradient centrifugation using Polymorphrep. Apoptotic cells were assessed with flow cytometry using a ready-made kit. The expression of proapoptotic molecules was investigated by Western blot analysis of PMNs and PBMCs treated with NDMA and/or rhTRAIL. The obtained results confirm the proapoptotic effects of NDMA on the examined human leukocytes and indicate an active participation of the TRAIL/DR5 complex and Fas protein in the process of apoptosis. Moreover, the research revealed distinct mechanisms of intrinsic apoptosis pathway activation between PMNs and PBMCs exposed to NDMA, as confirmed by the different levels of tBid, Smac/Diablo, Omi/HtrA2, and caspase-3 expression in those cells.

  4. Sampling From the Proteome to the Human Leukocyte Antigen-DR (HLA-DR) Ligandome Proceeds Via High Specificity*

    PubMed Central

    Mommen, Geert P. M.; Marino, Fabio; Meiring, Hugo D.; Poelen, Martien C. M.; van Gaans-van den Brink, Jacqueline A. M.; Mohammed, Shabaz; Heck, Albert J. R.; van Els, Cécile A. C. M.

    2016-01-01

    Comprehensive analysis of the complex nature of the Human Leukocyte Antigen (HLA) class II ligandome is of utmost importance to understand the basis for CD4+ T cell mediated immunity and tolerance. Here, we implemented important improvements in the analysis of the repertoire of HLA-DR-presented peptides, using hybrid mass spectrometry-based peptide fragmentation techniques on a ligandome sample isolated from matured human monocyte-derived dendritic cells (DC). The reported data set constitutes nearly 14 thousand unique high-confident peptides, i.e. the largest single inventory of human DC derived HLA-DR ligands to date. From a technical viewpoint the most prominent finding is that no single peptide fragmentation technique could elucidate the majority of HLA-DR ligands, because of the wide range of physical chemical properties displayed by the HLA-DR ligandome. Our in-depth profiling allowed us to reveal a strikingly poor correlation between the source proteins identified in the HLA class II ligandome and the DC cellular proteome. Important selective sieving from the sampled proteome to the ligandome was evidenced by specificity in the sequences of the core regions both at their N- and C- termini, hence not only reflecting binding motifs but also dominant protease activity associated to the endolysosomal compartments. Moreover, we demonstrate that the HLA-DR ligandome reflects a surface representation of cell-compartments specific for biological events linked to the maturation of monocytes into antigen presenting cells. Our results present new perspectives into the complex nature of the HLA class II system and will aid future immunological studies in characterizing the full breadth of potential CD4+ T cell epitopes relevant in health and disease. PMID:26764012

  5. PSGL-1 on Leukocytes is a Critical Component of the Host Immune Response against Invasive Pneumococcal Disease

    PubMed Central

    Ramos-Sevillano, Elisa; Urzainqui, Ana; de Andrés, Belén; González-Tajuelo, Rafael; Domenech, Mirian; González-Camacho, Fernando; Sánchez-Madrid, Francisco; Brown, Jeremy S.; García, Ernesto; Yuste, Jose

    2016-01-01

    Bacterial uptake by phagocytic cells is a vital event in the clearance of invading pathogens such as Streptococcus pneumoniae. A major role of the P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes against invasive pneumococcal disease is described in this study. Phagocytosis experiments using different serotypes demonstrated that PSGL-1 is involved in the recognition, uptake and killing of S. pneumoniae. Co-localization of several clinical isolates of S. pneumoniae with PSGL-1 was demonstrated, observing a rapid and active phagocytosis in the presence of PSGL-1. Furthermore, the pneumococcal capsular polysaccharide and the main autolysin of the bacterium ―the amidase LytA― were identified as bacterial ligands for PSGL-1. Experimental models of pneumococcal disease including invasive pneumonia and systemic infection showed that bacterial levels were markedly increased in the blood of PSGL-1−/− mice. During pneumonia, PSGL-1 controls the severity of pneumococcal dissemination from the lung to the bloodstream. In systemic infection, a major role of PSGL-1 in host defense is to clear the bacteria in the systemic circulation controlling bacterial replication. These results confirmed the importance of this receptor in the recognition and clearance of S. pneumoniae during invasive pneumococcal disease. Histological and cellular analysis demonstrated that PSGL-1−/− mice have increased levels of T cells migrating to the lung than the corresponding wild-type mice. In contrast, during systemic infection, PSGL-1−/− mice had increased numbers of neutrophils and macrophages in blood, but were less effective controlling the infection process due to the lack of this functional receptor. Overall, this study demonstrates that PSGL-1 is a novel receptor for S. pneumoniae that contributes to protection against invasive pneumococcal disease. PMID:26975045

  6. The Dynamics of the Human Leukocyte Antigen Head Domain Modulates Its Recognition by the T-Cell Receptor.

    PubMed

    García-Guerrero, Estefanía; Pérez-Simón, José Antonio; Sánchez-Abarca, Luis Ignacio; Díaz-Moreno, Irene; De la Rosa, Miguel A; Díaz-Quintana, Antonio

    2016-01-01

    Generating the immune response requires the discrimination of peptides presented by the human leukocyte antigen complex (HLA) through the T-cell receptor (TCR). However, how a single amino acid substitution in the antigen bonded to HLA affects the response of T cells remains uncertain. Hence, we used molecular dynamics computations to analyze the molecular interactions between peptides, HLA and TCR. We compared immunologically reactive complexes with non-reactive and weakly reactive complexes. MD trajectories were produced to simulate the behavior of isolated components of the various p-HLA-TCR complexes. Analysis of the fluctuations showed that p-HLA binding barely restrains TCR motions, and mainly affects the CDR3 loops. Conversely, inactive p-HLA complexes displayed significant drop in their dynamics when compared with its free versus ternary forms (p-HLA-TCR). In agreement, the free non-reactive p-HLA complexes showed a lower amount of salt bridges than the responsive ones. This resulted in differences between the electrostatic potentials of reactive and inactive p-HLA species and larger vibrational entropies in non-elicitor complexes. Analysis of the ternary p-HLA-TCR complexes also revealed a larger number of salt bridges in the responsive complexes. To summarize, our computations indicate that the affinity of each p-HLA complex towards TCR is intimately linked to both, the dynamics of its free species and its ability to form specific intermolecular salt-bridges in the ternary complexes. Of outstanding interest is the emerging concept of antigen reactivity involving its interplay with the HLA head sidechain dynamics by rearranging its salt-bridges.

  7. Magnesium-dependent adenosine triphosphatase as a marker enzyme for the plasma membrane of human polymorphonuclear leukocytes.

    PubMed

    Harlan, J; DeChatelet, L R; Iverson, D B; McCall, C E

    1977-02-01

    The adenosine triphosphatase (ATPase) activities of human polymorphonuclear leukocytes (PMNL) were studied with an assay that monitored the release of 32P-labeled inorganic pyrophosphate (32P1) from gamma-[32P]adenosine 5'-triphosphate (ATP). In cell homogenates, (Na+ + K+)-sensitive, ouabain-inhibitable ATPase comprised an insignificant fraction of the total ATPase activity. Additions of p-nitrophenyl phosphate and beta-glycerophosphate (substrates for nonspecific acid and alkaline phosphatases) and of tartrate (inhibitor of acid phosphatase) gave no indication of inhibition. This suggested that the assay was relatively specific for ATP hydrolysis. The activity was found to have a pH optimum of 8.7 and a Km for ATP of 0.6 mM. There was an absolute requirement for Mg2+, with other divalent cations substituting less efficiently. When the Mg2+-dependent ATPase activity of intact cells was compared with that in homogenized cells, no significant difference was observed. The activity in intact cells was linear with respect to incubation time up to at least l0 min. Trypan blue staining and lactate dehydrogenase assays revealed that greater than 92% of the PMNL remained intact and viable during the assay. No soluble ATPase was released from the cells under assay conditions. In following the distribution of gamma[32P]ATP and 32P2 counts became cell associated. Since the experimental evidence supports the observation that PMNL remain intact and viable and that ATP does not penetrate the cell under assay conditions, it is proposed that greater than 90% of the Mg2+-dependent ATPase of the human PMNL is associated with a plasma membrnae enzyme. This would qualify the enzyme for the role of a plasma membrane marker for future fractionation and isolation attempts.

  8. Sampling From the Proteome to the Human Leukocyte Antigen-DR (HLA-DR) Ligandome Proceeds Via High Specificity.

    PubMed

    Mommen, Geert P M; Marino, Fabio; Meiring, Hugo D; Poelen, Martien C M; van Gaans-van den Brink, Jacqueline A M; Mohammed, Shabaz; Heck, Albert J R; van Els, Cécile A C M

    2016-04-01

    Comprehensive analysis of the complex nature of the Human Leukocyte Antigen (HLA) class II ligandome is of utmost importance to understand the basis for CD4(+)T cell mediated immunity and tolerance. Here, we implemented important improvements in the analysis of the repertoire of HLA-DR-presented peptides, using hybrid mass spectrometry-based peptide fragmentation techniques on a ligandome sample isolated from matured human monocyte-derived dendritic cells (DC). The reported data set constitutes nearly 14 thousand unique high-confident peptides,i.e.the largest single inventory of human DC derived HLA-DR ligands to date. From a technical viewpoint the most prominent finding is that no single peptide fragmentation technique could elucidate the majority of HLA-DR ligands, because of the wide range of physical chemical properties displayed by the HLA-DR ligandome. Our in-depth profiling allowed us to reveal a strikingly poor correlation between the source proteins identified in the HLA class II ligandome and the DC cellular proteome. Important selective sieving from the sampled proteome to the ligandome was evidenced by specificity in the sequences of the core regions both at their N- and C- termini, hence not only reflecting binding motifs but also dominant protease activity associated to the endolysosomal compartments. Moreover, we demonstrate that the HLA-DR ligandome reflects a surface representation of cell-compartments specific for biological events linked to the maturation of monocytes into antigen presenting cells. Our results present new perspectives into the complex nature of the HLA class II system and will aid future immunological studies in characterizing the full breadth of potential CD4(+)T cell epitopes relevant in health and disease.

  9. The Dynamics of the Human Leukocyte Antigen Head Domain Modulates Its Recognition by the T-Cell Receptor

    PubMed Central

    García-Guerrero, Estefanía; Pérez-Simón, José Antonio; Sánchez-Abarca, Luis Ignacio; Díaz-Moreno, Irene; De la Rosa, Miguel A.; Díaz-Quintana, Antonio

    2016-01-01

    Generating the immune response requires the discrimination of peptides presented by the human leukocyte antigen complex (HLA) through the T-cell receptor (TCR). However, how a single amino acid substitution in the antigen bonded to HLA affects the response of T cells remains uncertain. Hence, we used molecular dynamics computations to analyze the molecular interactions between peptides, HLA and TCR. We compared immunologically reactive complexes with non-reactive and weakly reactive complexes. MD trajectories were produced to simulate the behavior of isolated components of the various p-HLA-TCR complexes. Analysis of the fluctuations showed that p-HLA binding barely restrains TCR motions, and mainly affects the CDR3 loops. Conversely, inactive p-HLA complexes displayed significant drop in their dynamics when compared with its free versus ternary forms (p-HLA-TCR). In agreement, the free non-reactive p-HLA complexes showed a lower amount of salt bridges than the responsive ones. This resulted in differences between the electrostatic potentials of reactive and inactive p-HLA species and larger vibrational entropies in non-elicitor complexes. Analysis of the ternary p-HLA-TCR complexes also revealed a larger number of salt bridges in the responsive complexes. To summarize, our computations indicate that the affinity of each p-HLA complex towards TCR is intimately linked to both, the dynamics of its free species and its ability to form specific intermolecular salt-bridges in the ternary complexes. Of outstanding interest is the emerging concept of antigen reactivity involving its interplay with the HLA head sidechain dynamics by rearranging its salt-bridges. PMID:27124285

  10. Chemical, biochemical, pharmacokinetic, and biological properties of L-680,833: a potent, orally active monocyclic beta-lactam inhibitor of human polymorphonuclear leukocyte elastase.

    PubMed Central

    Doherty, J B; Shah, S K; Finke, P E; Dorn, C P; Hagmann, W K; Hale, J J; Kissinger, A L; Thompson, K R; Brause, K; Chandler, G O

    1993-01-01

    A series of potent and highly selective time-dependent monocyclic beta-lactam inhibitors of human polymorphonuclear leukocyte elastase (PMNE, EC 3.4.21.37) is described. The intrinsic potency of these compounds, as exemplified by L-680,833 (k(inactivation)/K(i) of 622,000 M-1.s-1), is reflected at the cellular level where it inhibits generation of the specific N-terminal cleavage product A alpha-(1-21) from the A alpha chain of fibrinogen by enzyme released from isolated polymorphonuclear leukocytes stimulated with fMet-Leu-Phe with an IC50 of 0.06 microM. The inhibitory activity of L-680,833 is also apparent in whole blood stimulated with A23187, where it inhibits formation of A alpha-(1-21) and PMNE-alpha 1-proteinase inhibitor complex formation with IC50 values of 9 microM. Pharmacokinetic studies indicate that after oral dosing L-680,833 is bioavailable in rats and rhesus monkeys. This oral bioavailability is reflected by the inhibition (i) of tissue damage elicited in hamster lungs by intratracheal instillation of human PMNE and (ii) enzyme released from human PMN stimulated after their transfer into the pleural cavity of mice. The properties of L-680,833 allow it to effectively supplement the activity of natural inhibitors of PMNE in vivo, suggesting that this type of low-molecular-weight synthetic inhibitor could have therapeutic value in diseases where PMNE damages tissue. PMID:8378355

  11. A new approach to determining the rates of recruitment of circulating leukocytes into tissues: Application to the measurement of leukocyte recruitment into atherosclerotic lesions

    PubMed Central

    Steinberg, Daniel; Khoo, John C.; Glass, Christopher K.; Palinski, Wulf; Almazan, Felicidad

    1997-01-01

    Recruitment of circulating monocytes into the artery wall is an important feature of early atherogenesis. In vitro studies have identified a number of adhesion molecules and chemokines that may control this process but very little work has been done to evaluate their relative importance in vivo, in part because there have been no methods available of sufficient sensitivity and reliability. This paper proposes a new approach in which advantage is taken of naturally occurring or transgenically induced mutations to “mark” donor cells and to follow their fate in recipient animals using highly sensitive PCR methods. The feasibility of the approach is demonstrated by preliminary studies of monocyte recruitment into atherosclerotic lesions. However, the method should in principle be applicable to the study of any of the circulating leukocytes and their rate of entry into any tissue or tissues of interest. PMID:9108101

  12. Killing of gram-negative bacteria by polymorphonuclear leukocytes: role of an O2-independent bactericidal system.

    PubMed

    Weiss, J; Victor, M; Stendhal, O; Elsbach, P

    1982-04-01

    Previous studies have suggested that a cationic bactericidal/permeability-increasing protein (BPI) present in both rabbit and human polymorphonuclear leukocytes is the principal O2-independent bactericidal agent of these cells toward several strains of Escherichia coli and Salmonella typhimurium (1978. J. Biol. Chem. 253: 2664--2672; 1979. J. Biol. Chem. 254: 11000--11009). To further evaluate the possible role of this protein in the killing of gram-negative bacteria by polymorphonuclear leukocytes, we have measured the bactericidal activity of intact rabbit peritoneal exudate leukocytes under aerobic or anaerobic conditions and of intact human leukocytes from a patient with chronic granulomatous disease. Anaerobic conditions were created by flushing the cells under a nitrogen stream. Effective removal of oxygen was demonstrated by the inability of nitrogen-flushed leukocytes to mount a respiratory burst (measured as increased conversion of 1-[14C]glucose leads to 14CO2 or by superoxide production) during bacterial ingestion. At a bacteria/leukocyte ratio of 10:1, killing of gram-positive, BPI-resistant, Staphylococcus epidermidis is markedly impaired in the absence of oxygen (76.4 +/- 3.3% killing in room air, 29.2 +/- 8.2% killing in nitrogen). Essentially all increased bacterial survival is intracellular. In contrast, both a nonopsonized rough strain (MR-10) and an opsonized smooth strain (MS) of S. typhimurium 395 are killed equally well in room air and nitrogen. A maximum of 70--80 MR-10 and 30--40 MS are killed per leukocyte either in the presence or absence of oxygen. There is no intracellular bacterial survival in either condition indicating that intracellular O2-independent bactericidal system(s) of rabbit polymorphonuclear leukocytes can at least match the leukocyte's ingestive capacity. Whole homogenates and crude acid extracts manifest similar bactericidal capacity toward S. typhimurium 395. This activity can be accounted for by the BPI content of these

  13. Killing of gram-negative bacteria by polymorphonuclear leukocytes: role of an O2-independent bactericidal system.

    PubMed Central

    Weiss, J; Victor, M; Stendhal, O; Elsbach, P

    1982-01-01

    Previous studies have suggested that a cationic bactericidal/permeability-increasing protein (BPI) present in both rabbit and human polymorphonuclear leukocytes is the principal O2-independent bactericidal agent of these cells toward several strains of Escherichia coli and Salmonella typhimurium (1978. J. Biol. Chem. 253: 2664--2672; 1979. J. Biol. Chem. 254: 11000--11009). To further evaluate the possible role of this protein in the killing of gram-negative bacteria by polymorphonuclear leukocytes, we have measured the bactericidal activity of intact rabbit peritoneal exudate leukocytes under aerobic or anaerobic conditions and of intact human leukocytes from a patient with chronic granulomatous disease. Anaerobic conditions were created by flushing the cells under a nitrogen stream. Effective removal of oxygen was demonstrated by the inability of nitrogen-flushed leukocytes to mount a respiratory burst (measured as increased conversion of 1-[14C]glucose leads to 14CO2 or by superoxide production) during bacterial ingestion. At a bacteria/leukocyte ratio of 10:1, killing of gram-positive, BPI-resistant, Staphylococcus epidermidis is markedly impaired in the absence of oxygen (76.4 +/- 3.3% killing in room air, 29.2 +/- 8.2% killing in nitrogen). Essentially all increased bacterial survival is intracellular. In contrast, both a nonopsonized rough strain (MR-10) and an opsonized smooth strain (MS) of S. typhimurium 395 are killed equally well in room air and nitrogen. A maximum of 70--80 MR-10 and 30--40 MS are killed per leukocyte either in the presence or absence of oxygen. There is no intracellular bacterial survival in either condition indicating that intracellular O2-independent bactericidal system(s) of rabbit polymorphonuclear leukocytes can at least match the leukocyte's ingestive capacity. Whole homogenates and crude acid extracts manifest similar bactericidal capacity toward S. typhimurium 395. This activity can be accounted for by the BPI content of these

  14. Pre-adjuvant chemotherapy leukocyte count may predict the outcome for advanced gastric cancer after radical resection.

    PubMed

    Pei, Dong; Zhu, Fang; Chen, Xiaofeng; Qian, Jing; He, Shaohua; Qian, Yingying; Shen, Hua; Liu, Yiqian; Xu, Jiali; Shu, Yongqian

    2014-03-01

    Gastric cancer (GC) has a high morbidity worldwide each year especially in China and advanced GC is well known with poor prognosis, for which surgical resection combine adjuvant chemotherapy is the optimal choice for therapy. Leukocyte is an important index during the treatment for its influence on drugs' dosage and tolerance. Therefore, peripheral blood leukocyte and its subsets during adjuvant chemotherapy may have great clinical value for predicting prognostic. In this retrospective study, we showed the distribution of white blood cell and its subsets in the baseline period before adjuvant chemotherapy in 399 patients who underwent radical resection for advanced GC from January 1, 2008 to August 31, 2012. We investigated the relationship between leukocyte count and overall survival (OS) as well as disease-free survival (DFS). In these patients, females were more likely to have less white blood cells after operation (P=0.016). Patients with pre-chemotherapy leukocyte count less than 4×10(9)/L got worse DFS (P=0.028) and OS (P=0.016). In multivariate analysis, tumor size ≥ 6cm (P=0.033), TNM stage IV (P=0.024), vascular or nerval invasion (P=0.005) and leukocyte count less than 4.0×10(9)/L (P=0.019) was associated with poor DFS. TNM stage IV (P=0.008), vascular or nerval invasion (P=0.001) and lower leukocyte count (P=0.045) were independent risk factors for poor OS. Taken together, our findings suggest that pre-adjuvant chemotherapy peripheral blood leukocyte count correlates with clinical outcome of patients with advanced GC after radical resection.

  15. Comparison of changes over time in leukocyte counts in Yucatan minipigs irradiated with simulated solar particle event-like radiation

    NASA Astrophysics Data System (ADS)

    Sanzari, Jenine K.; Steven Wan, X.; Muehlmatt, Amy; Lin, Liyong; Kennedy, Ann R.

    2015-01-01

    During a major solar particle event (SPE), astronauts in space are at risk of exposure to an increased dose of proton radiation. The whole body distribution of the absorbed SPE proton dose is inhomogeneous, and such an inhomogeneous SPE proton dose can be simulated by electron radiation. Using Yucatan minipigs as an animal model, we compared the time courses of leukocyte count changes after exposure to proton simulated SPE (pSPE) radiation or electron simulated SPE (eSPE) radiation. The results demonstrated that the time required after irradiation to reach the lowest leukocyte counts was generally comparable between the pSPE and eSPE radiation exposures. However, the leukocyte count often recovered faster after electron irradiation compared to proton irradiation at the corresponding doses. In addition, the radiation dose required to achieve comparable magnitudes of leukocyte count decrease was higher in the eSPE animals than for the pSPE animals. In conclusion, based on the magnitude of the decrease and the time required to reach the lowest leukocyte counts after irradiation, the pSPE radiation was more effective than the eSPE radiation in reducing the peripheral leukocyte counts. Lymphocytes appeared to be the most sensitive type of leukocytes in response to either type of SPE radiation. It is particularly noteworthy that following exposure to pSPE radiation at the skin doses >5 Gy, the neutrophils do not recover from the radiation damage at times up to 30 days, and the neutrophils have not recovered to their baseline levels even at 90 days post-irradiation. These results suggest a marked difference in the ability of the neutrophils to recover from pSPE radiation compared with the results observed for eSPE radiation.

  16. Resident peritoneal leukocytes are important sources of MMP-9 during zymosan peritonitis: superior contribution of macrophages over mast cells.

    PubMed

    Kolaczkowska, Elzbieta; Lelito, Monika; Kozakiewicz, Elzbieta; van Rooijen, Nico; Plytycz, Barbara; Arnold, Bernd

    2007-11-15

    Metalloproteinase 9 (MMP-9) is crucial for normal neutrophil infiltration into zymosan-inflamed peritoneum. During the course of zymosan peritonitis MMP-9 is produced in a biphasic-manner as its presence is detectable as early as 30 min post zymosan and then between 2 and 8 h of inflammation. As inflammatory leukocytes were shown to produce MMP-9 we asked if also resident leukocytes, mast cells and macrophages, contribute to its production. And furthermore, if their contribution is limited only to the early phase of inflammation or extends to the later stages. For this purpose some mice were depleted of either resident macrophages or functional mast cells and expression of MMP-9 in peritoneal leukocytes and its release to the exudate were monitored. It turned out that depletion of peritoneal macrophages decreased both MMP-9 content in the leukocytes and its release to the inflammatory exudate at 30 min and 6h of peritonitis. The functional depletion of mast cells also caused a significant decrease in the production/release of MMP-9 that was especially apparent at the early time point (30 min). Moreover, the study shows concomitant kinetics of MMP-9 expression in leukocytes and its release to the exudatory fluid. The findings indicate that resident tissue leukocytes, and among them especially macrophages, constitute an important source of MMP-9 during acute peritoneal inflammation. Overall, the study shows that resident tissue leukocytes, mostly macrophages, constitute an important cellular source(s) of inflammation-related factors and should be regarded as possible targets of anti-inflammatory treatment.

  17. Comparison of changes over time in leukocyte counts in Yucatan minipigs irradiated with simulated solar particle event-like radiation

    PubMed Central

    Sanzari, Jenine K.; Wan, X. Steven; Muehlmatt, Amy; Lin, Liyong; Kennedy, Ann R.

    2015-01-01

    During a major solar particle event (SPE), astronauts in space are at risk of exposure to an increased dose of proton radiation. The whole body distribution of the absorbed SPE proton dose is inhomogeneous, and such an inhomogeneous SPE proton dose can be simulated by electron radiation. Using Yucatan minipigs as an animal model, we compared the time courses of leukocyte count changes after exposure to proton simulated SPE (pSPE) radiation or electron simulated SPE (eSPE) radiation. The results demonstrated that the time required after irradiation to reach the lowest leukocyte counts was generally comparable between the pSPE and eSPE radiation exposures. However, the leukocyte count often recovered faster after electron irradiation compared to proton irradiation at the corresponding doses. In addition, the radiation dose required to achieve comparable magnitudes of leukocyte count decrease was higher in the eSPE animals than for the pSPE animals. In conclusion, based on the magnitude of the decrease and the time required to reach the lowest leukocyte counts after irradiation, the pSPE radiation was more effective than the eSPE radiation in reducing the peripheral leukocyte counts. Lymphocytes appeared to be the most sensitive type of leukocytes in response to either type of SPE radiation. It is particularly noteworthy that following exposure to pSPE radiation at the skin doses >5 Gy, the neutrophils do not recover from the radiation damage at times up to 30 days, and the neutrophils have not recovered to their baseline levels even at 90 days post-irradiation. These results suggest a marked difference in the ability of the neutrophils to recover from pSPE radiation compared the results observed for eSPE radiation. PMID:25774341

  18. Involvement of seminal leukocytes, reactive oxygen species, and sperm mitochondrial membrane potential in the DNA damage of the human spermatozoa.

    PubMed

    Lobascio, A M; De Felici, M; Anibaldi, M; Greco, P; Minasi, M G; Greco, E

    2015-03-01

    Measurement of reactive oxygen species (ROS) producing leukocytes in semen has been a standard component of the semen analysis, but its true significance remains still unknown. In this study, we have correlated the number of seminal leukocytes to various semen parameters. We found a negative correlation between the leukocyte number and sperm concentration (rs  = -0.22; p = 0.01) and motility (rs  = -0.20; p = 0.02). In contrast, a positive correlation between the number of leukocytes and both seminal ROS (rs  = 0.70, p < 0.001; n = 125) and the number of spermatozoa with DNA fragmentation (rs  = 0.43, p = 0.032; n = 25) was found. However, only a trend of positive correlation between ROS and the number of spermatozoa with TUNEL-detected DNA fragmentation was observed. Moreover, this latter was not correlated with loss of sperm mitochondrial membrane potential (MMP) (10% vs 35%, rs  = 0.25, p = 0.08; n = 50). Overall these results indicate that the presence of high number of leukocytes in the ejaculate negatively affects key semen parameters, as sperm concentration and motility, associated with infertility conditions. Moreover, they suggest that leukocytes are the major source of the seminal ROS and cause of sperm DNA fragmentation. However, the absence of a clear correlation between ROS and sperm DNA fragmentation, and spermatozoa with damaged DNA and MMP loss, suggest that ROS produced by leukocytes might be not the only cause of DNA damage in spermatozoa and that intrinsic mitochondrial-dependent apoptotic pathways might not have a major impact on sperm DNA fragmentation.

  19. CD31/PECAM-1 is a ligand for alpha v beta 3 integrin involved in adhesion of leukocytes to endothelium

    PubMed Central

    1995-01-01

    To protect the body efficiently from infectious organisms, leukocytes circulate as nonadherent cells in the blood and lymph, and migrate as adherent cells into tissues. Circulating leukocytes in the blood have first to adhere to and then to cross the endothelial lining. CD31/PECAM- 1 is an adhesion molecule expressed by vascular endothelial cells, platelets, monocytes, neutrophils, and naive T lymphocytes. It is a transmembrane glycoprotein of the immunoglobulin gene superfamily (IgSF), with six Ig-like homology units mediating leukocyte-endothelial interactions. The adhesive interactions mediated by CD31 are complex and include homophilic (CD31-CD31) or heterophilic (CD31-X) contacts. Soluble, recombinant forms of CD31 allowed us to study the heterophilic interactions in leukocyte adhesion assays. We show that the adhesion molecule alpha v beta 3 integrin is a ligand for CD31. The leukocytes revealed adhesion mediated by the second Ig-like domain of CD31, and this binding was inhibited by alpha v beta 3 integrin-specific antibodies. Moreover alpha v beta 3 was precipitated by recombinant CD31 from cell lysates. These data establish a third IgSF-integrin pair of adhesion molecules, CD31-alpha v beta 3 in addition to VCAM-1, MadCAM-1/alpha 4 integrins, and ICAM/beta 2 integrins, which are major components mediating leukocyte-endothelial adhesion. Identification of a further versatile adhesion pair broadens our current understanding of leukocyte-endothelial interactions and may provide the basis for the treatment of inflammatory disorders and metastasis formation. PMID:7542249

  20. Indium-111-labeled leukocyte and technetium-99m-sulfur colloid uptake by a malignant fibrous histiocytoma: Phagocytosis by tumor cells

    SciTech Connect

    Palestro, C.J.; Klein, M.; Kim, C.K.; Swyer, A.J.; Goldsmith, S.J. )

    1990-09-01

    Indium-111-labeled leukocyte imaging, performed on a patient with a calcified mass in the right thigh, demonstrated labeled leukocyte accumulation in this mass. Technetium-99m-sulfur colloid imaging was performed to differentiate labeled leukocyte uptake in heterotopic bone marrow from uptake in a focus of infection. Leukocyte and sulfur colloid images were virtually identical, and the study was interpreted as without evidence of infection. Excision of the mass revealed an angiomatoid malignant fibrous histiocytoma with metaplastic bone formation. While no marrow elements were present in either the tumor or the metaplastic bone, phagocytosis of leukocytes by tumor cells was identified. Phagocytosis of leukocytes by tumor cells may be another cause of white cell accumulation in uninfected neoplasms.

  1. Kinetics of staphylococcal opsonization, attachment, ingestion and killing by human polymorphonuclear leukocytes: a quantitative assay using [3H]thymidine labeled bacteria.

    PubMed

    Verhoef, J; Peterson, P K; Quie, P G

    1977-01-01

    A method has been developed for studying quantitatively the separate processes of bacterial opsonization, phagocytosis, and killing by human polymorphonuclear leukocytes using [3H]thymidine labeled Staphylococcus aureus. Phagocytosis is determined by assaying for leukocytes-associated radioactivity after differential centrifugation and washing the leukocytes. Opsonization is studied by incubating bacteria with an opsonic source for varying durations and then adding leukocytes. By treatment of samples with the muralytic enzyme, lysostaphin, the attachment and ingestion phases of phagocytosis can be separated. Sampling for colony forming units after disruption of the leukocytes permits the measurement of bacterial killing. Using this method, differences in the kinetics of staphylococcal opsonization by normal and C2 deficient sera were defined, opsonic influences on the attachment and ingestion phases of pH agocytosis were delineated, and the influences of different opsonins and leukocyte populations on killing were determined.

  2. Structure elucidation and antioxidant activity of (-)-isosilandrin isolated from Silybum marianum L.

    PubMed

    Samu, Zsuzsanna; Nyiredy, Szabolcs; Baitz-Gács, Eszter; Varga, Zsuzsa; Kurtán, Tibor; Dinya, Zoltán; Antus, Sándor

    2004-11-01

    A regioisomer of the known flavanolignan (-)-silandrin (3a), named (-)-isosilandrin (8a), was isolated from the fruits of a white-flowered variant of Silybum marianum L. populated in Hungary. Its structure was established both by spectroscopic methods and total synthesis, and its absolute configuration was determined by means of circular dichroism. This compound showed stronger inhibitory activity on the superoxide anion (O2*-) release by human polymorphonuclear leukocytes (PMNL) than (+)-silybin (1a,b).

  3. An anti-platelet-endothelial cell adhesion molecule-1 antibody inhibits leukocyte extravasation from mesenteric microvessels in vivo by blocking the passage through the basement membrane

    PubMed Central

    1996-01-01

    Platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31) plays an active role in the process of leukocyte migration through cultured endothelial cells in vitro and anti-PECAM-1 antibodies (Abs) inhibit accumulation of leukocytes into sites of inflammation in vivo. Despite the latter, it is still not clear at which stage of leukocyte emigration in vivo PECAM-1 is involved. To address this point directly, we studied the effect of an anti-PECAM-1 Ab, recognizing rat PECAM-1, on leukocyte responses within rat mesenteric microvessels using intravital microscopy. In mesenteric preparations activated by interleukin (IL)-1 beta, the anti-PECAM-1 Ab had no significant effect on the rolling or adhesion of leukocytes, but inhibited their migration into the surrounding extravascular tissue in a dose-dependent manner. Although in some vessel segments these leukocytes had come to a halt within the vascular lumen, often the leukocytes appeared to be trapped within the vessel wall. Analysis of these sections by electron microscopy revealed that the leukocytes had passed through endothelial cell junctions but not the basement membrane. In contrast to the effect of the Ab in mesenteric preparations treated with IL-1 beta, leukocyte extravasation induced by topical or intraperitoneal administration of the chemotactic peptide formyl-methionyl-leucyl-phenylalanine was not inhibited by the anti-PECAM-1 Ab. These results directly demonstrate a role for PECAM-1 in leukocyte extravasation in vivo and indicate that this involvement is selective for leukocyte extravasation elicited by certain inflammatory mediators. Further, our findings provide the first in vivo indication that PECAM-1 may have an important role in triggering the passage of leukocytes through the perivascular basement membrane. PMID:8691137

  4. Inflammatory Leukocyte Phenotypes Correlate with Disease Progression in Idiopathic Pulmonary Fibrosis

    PubMed Central

    Moore, Bethany B.; Fry, Chris; Zhou, Yueren; Murray, Susan; Han, MeiLan K.; Martinez, Fernando J.; Flaherty, Kevin R.

    2014-01-01

    Idiopathic pulmonary fibrosis (IPF) is characterized by progressive deposition of extracellular matrix, worsening dyspnea, and eventual mortality. Pathogenesis of IPF is poorly understood and the role inflammation and activated leukocytes play in the disease process is controversial. Previous studies demonstrated that activated leukocyte subsets characterize IPF patients. We sought to validate this observation in a well-defined cohort of 35 IPF patients and to correlate the observed leukocyte phenotypes with robust parameters of disease progression. We demonstrate that in univariate and multivariate analyses, increases in the CD14hi, CD16hi subset of monocytes measured at baseline correlated with disease progression, with a threshold value >0.5% of the total peripheral blood mononuclear cells being a significant predictor for worse outcome. In addition, several T cell subsets, including CD25 expressing CD4 cells, and CXCR3 expressing CD4 and CD8 subsets correlated with disease progression when found in increased percentages in the peripheral blood of IPF patients when sampled at baseline. Somewhat surprising in comparison to previous literature, the CD4 T cells did not appear to have lost expression of the co-stimulatory molecule, CD28, but the CD8 T cells did. Taken together, these results are consistent with the presence of an inflammatory process in IPF patients who eventually progress. However, when longitudinal measurements of these same markers were examined, there was significant heterogeneity of expression and these biomarkers did not necessarily remain elevated in IPF patients with progressive disease. We interpret this heterogeneity to suggest that IPF patients experience episodic inflammatory events that once triggered, may lead to disease progression. This longitudinal heterogeneity in biomarker analyses may explain why such markers are not consistently measured in all IPF cohorts. PMID:25580363

  5. An investigation of the immunotoxicity of oil sands processed water and leachates in trout leukocytes.

    PubMed

    Gagné, F; Bruneau, A; Turcotte, P; Gagnon, C; Lacaze, E

    2017-03-14

    Increased oil sands (OS) mining activity has raised concerns about impacts on aquatic organisms. This study sought to examine the effects of single representative compounds from OS (benzo(a)pyrene, naphthalene), a mixture of naphthenic acids (NAs), OS-processed water (OSPW) and OS leachate (OSL) extracts on rainbow trout leukocytes. Primary cultures of trout leukocytes were exposed to increasing concentrations of benzo(a)pyrene, naphthalene, NAs, OSPW and OSL for 48h at 18°C. Immunocompetence was followed by measuring changes in lymphocyte and macrophage viability and phagocytosis. Changes in the expression of 10 transcripts were also followed: interleukin 1, 2 and 6 (Il-1, Il-2 and Il-6), calreticulin (CRT), caspase 9 (Cas9), aryl hydrocarbon receptor (AhR), cyclooxygenase-2 (COX2), glutathione S-transferase (GST), catalase (CAT) and p53 tumor suppressor. The results revealed that exposure to OSPW extracts decreased the capacity of macrophages to engulf three beads or more, while the other compounds generally increased phagocytosis activity. Lymphocyte apoptosis was increased by all compounds and mixtures except naphthalene. Both OSPW and OSL induced apoptosis in macrophages. At the gene expression level, Cas9, CRT, Il-1 (inhibition) and Il-2 were specifically influenced by OSPW, while CAT, p53, COX2 and Il-1 (induction) transcripts were specifically expressed by OSL. Leukocyte exposure to OSPW produced characteristic changes in immunocompetence and genes involved in proinflammatory, apoptosis and protein damage (CRT) pathways which could not be explained by OSL, benzo(a)pyrene, naphthalene and NA mixture.

  6. Lower Methylation of the ANGPTL2 Gene in Leukocytes from Post-Acute Coronary Syndrome Patients

    PubMed Central

    Nguyen, Albert; Mamarbachi, Maya; Turcot, Valérie; Lessard, Samuel; Yu, Carol; Luo, Xiaoyan; Lalongé, Julie; Hayami, Doug; Gayda, Mathieu; Juneau, Martin; Thorin-Trescases, Nathalie; Lettre, Guillaume; Nigam, Anil; Thorin, Eric

    2016-01-01

    DNA methylation is believed to regulate gene expression during adulthood in response to the constant changes in environment. The methylome is therefore proposed to be a biomarker of health through age. ANGPTL2 is a circulating pro-inflammatory protein that increases with age and prematurely in patients with coronary artery diseases; integrating the methylation pattern of the promoter may help differentiate age- vs. disease-related change in its expression. We believe that in a pro-inflammatory environment, ANGPTL2 is differentially methylated, regulating ANGPTL2 expression. To test this hypothesis we investigated the changes in promoter methylation of ANGPTL2 gene in leukocytes from patients suffering from post-acute coronary syndrome (ACS). DNA was extracted from circulating leukocytes of post-ACS patients with cardiovascular risk factors and from healthy young and age-matched controls. Methylation sites (CpGs) found in the ANGPTL2 gene were targeted for specific DNA methylation quantification. The functionality of ANGPTL2 methylation was assessed by an in vitro luciferase assay. In post-ACS patients, C-reactive protein and ANGPTL2 circulating levels increased significantly when compared to healthy controls. Decreased methylation of specific CpGs were found in the promoter of ANGPTL2 and allowed to discriminate age vs. disease associated methylation. In vitro DNA methylation of specific CpG lead to inhibition of ANGPTL2 promoter activity. Reduced leukocyte DNA methylation in the promoter region of ANGPTL2 is associated with the pro-inflammatory environment that characterizes patients with post-ACS differently from age-matched healthy controls. Methylation of different CpGs in ANGPTL2 gene may prove to be a reliable biomarker of coronary disease. PMID:27101308

  7. Alterations in leukocyte transcriptional control pathway activity associated with major depressive disorder and antidepressant treatment.

    PubMed

    Mellon, S H; Wolkowitz, O M; Schonemann, M D; Epel, E S; Rosser, R; Burke, H B; Mahan, L; Reus, V I; Stamatiou, D; Liew, C-C; Cole, S W

    2016-05-24

    Major depressive disorder (MDD) is associated with a significantly elevated risk of developing serious medical illnesses such as cardiovascular disease, immune impairments, infection, dementia and premature death. Previous work has demonstrated immune dysregulat