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Sample records for cystinotic leukocytes affects

  1. 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.

  2. 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.

  3. Activation of the transcription factor EB rescues lysosomal abnormalities in cystinotic kidney cells.

    PubMed

    Rega, Laura R; Polishchuk, Elena; Montefusco, Sandro; Napolitano, Gennaro; Tozzi, Giulia; Zhang, Jinzhong; Bellomo, Francesco; Taranta, Anna; Pastore, Anna; Polishchuk, Roman; Piemonte, Fiorella; Medina, Diego L; Catz, Sergio D; Ballabio, Andrea; Emma, Francesco

    2016-04-01

    Nephropathic cystinosis is a rare autosomal recessive lysosomal storage disease characterized by accumulation of cystine into lysosomes secondary to mutations in the cystine lysosomal transporter, cystinosin. The defect initially causes proximal tubular dysfunction (Fanconi syndrome) which in time progresses to end-stage renal disease. Cystinotic patients treated with the cystine-depleting agent, cysteamine, have improved life expectancy, delayed progression to chronic renal failure, but persistence of Fanconi syndrome. Here, we have investigated the role of the transcription factor EB (TFEB), a master regulator of the autophagy-lysosomal pathway, in conditionally immortalized proximal tubular epithelial cells derived from the urine of a healthy volunteer or a cystinotic patient. Lack of cystinosin reduced TFEB expression and induced TFEB nuclear translocation. Stimulation of endogenous TFEB activity by genistein, or overexpression of exogenous TFEB lowered cystine levels within 24 hours in cystinotic cells. Overexpression of TFEB also stimulated delayed endocytic cargo processing within 24 hours. Rescue of other abnormalities of the lysosomal compartment was observed but required prolonged expression of TFEB. These abnormalities could not be corrected with cysteamine. Thus, these data show that the consequences of cystinosin deficiency are not restricted to cystine accumulation and support the role of TFEB as a therapeutic target for the treatment of lysosomal storage diseases, in particular of cystinosis. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  4. Cystinosin-LKG rescues cystine accumulation and decreases apoptosis rate in cystinotic proximal tubular epithelial cells.

    PubMed

    Taranta, Anna; Bellomo, Francesco; Petrini, Stefania; Polishchuk, Elena; De Leo, Ester; Rega, Laura Rita; Pastore, Anna; Polishchuk, Roman; De Matteis, Maria Antonietta; Emma, Francesco

    2017-01-01

    Nephropathic cystinosis is a lysosomal storage disease that is caused by mutations in the CTNS gene encoding a cystine/proton symporter cystinosin and an isoform cystinosin-LKG which is generated by an alternative splicing of exon 12. We have investigated the physiological role of the cystinosin-LKG that is widely expressed in epithelial tissues. We have analyzed the intracellular localization and the function of the cystinosin-LKG conjugated with DsRed (cystinosin-LKG-RFP) in Madin-Darby canine kidney cells (MDCK II) and in proximal tubular epithelial cells carrying a deletion of the CTNS gene (cystinotic PTEC), respectively. Cystinosin-LKG-RFP colocalized with markers of lysosomes, late endosomes and was also expressed on the apical surface of polarized MDCK II cells. Moreover, immune-electron microscopy images of MDCK II cells overexpressing cystinosin-LKG-RFP showed stacked lamellar membranes inside perinuclear lysosomal structures. To study the role of LKG-isoform, we have investigated cystine accumulation and apoptosis that have been described in cystinotic cells. Cystinosin-LKG decreased cystine levels by approximately 10-fold similarly to cystinosin-RFP. The levels of TNFα- and actinomycin D-inducted apoptosis dropped in cystinotic cells expressing LKG-isoform. This effect was also similar to the main isoform. Our results suggest that cystinosin-LKG and cystinosin move similar functional activities in cells.

  5. Photoperiod affects the expression of sex and species differences in leukocyte number and leukocyte trafficking in congeneric hamsters.

    PubMed

    Bilbo, S D; Dhabhar, F S; Viswanathan, K; Saul, A; Nelson, R J

    2003-11-01

    Sex differences in immune function are well documented. These sex differences may be modulated by social and environmental factors. Individuals of polygynous species generally exhibit more pronounced sex differences in immune parameters than individuals of monogamous species, often displaying an energetic trade-off between enhanced immunity and high mating success. During winter, animals contend with environmental conditions (e.g. low temperatures and decreased food availability) that evoke energetic-stress responses; many mammals restrict reproduction in response to photoperiod as part of an annual winter coping strategy. To test the hypothesis that extant sex and species differences in immune surveillance may be modulated by photoperiod, we examined leukocyte numbers in males and females of two closely related hamster species (Phodopus). As predicted, uniparental P. sungorus exhibited a robust sex difference, with total white blood cells, total lymphocytes, T cells, and B cells higher in females than males, during long days when reproduction occurs, but not during short days when reproduction usually stops. In contrast, biparental male and female P. campbelli exhibited comparable leukocyte numbers during both long and short days. To study sex differences in stress responses, we also examined immune cell trafficking in response to an acute (2 h) restraint stressor. During stressful challenges, it appears beneficial for immune cells to exit the blood and move to primary immune defense areas such as the skin, in preparation for potential injury or infection. Acute stress moved lymphocytes and monocytes out of the blood in all animals. Blood cortisol concentrations were increased in P. sungorus females compared to males at baseline (52%) and in response to restraint stress (38%), but only in long days. P. campbelli males and females exhibited comparable blood cortisol and stress responses during both long and short days. Our results suggest that interactions among

  6. Leukocyte adhesion deficiency-III is caused by mutations in KINDLIN3 affecting integrin activation

    PubMed Central

    Svensson, Lena; Howarth, Kimberley; McDowall, Alison; Patzak, Irene; Evans, Rachel; Ussar, Siegfried; Moser, Markus; Metin, Ayse; Fried, Mike; Tomlinson, Ian; Hogg, Nancy

    2009-01-01

    Integrins are the major adhesion receptors of leukocytes and platelets. β1 and β2 integrin function on leukocytes is crucial for a successful immune response and the platelet integrin αIIbβ3 initiates the process of blood clotting through binding fibrinogen1-3. Integrins on circulating cells bind poorly to their ligands but become active after ‘inside-out’ signaling through other membrane receptors4,5. Subjects with leukocyte adhesion deficiency-1 (LAD-I) do not express β2 integrins because of mutations in the gene specifying the β2 subunit, and they suffer recurrent bacterial infections6,7. Mutations affecting αIIbβ3 integrin cause the bleeding disorder termed Glanzmann’s thrombasthenia3. Subjects with LAD-III show symptoms of both LAD-I and Glanzmann’s thrombasthenia. Their hematopoietically-derived cells express β1, β2 and β3 integrins, but defective inside-out signaling causes immune deficiency and bleeding problems8. The LAD-III lesion has been attributed to a C→A mutation in the gene encoding calcium and diacylglycerol guanine nucleotide exchange factor (CALDAGGEF1; official symbol RASGRP2) specifying the CALDAG-GEF1 protein9, but we show that this change is not responsible for the LAD-III disorder. Instead, we identify mutations in the KINDLIN3 (official symbol FERMT3) gene specifying the KINDLIN-3 protein as the cause of LAD-III in Maltese and Turkish subjects. Two independent mutations result in decreased KINDLIN3 messenger RNA levels and loss of protein expression. Notably, transfection of the subjects’ lymphocytes with KINDLIN3 complementary DNA but not CALDAGGEF1 cDNA reverses the LAD-III defect, restoring integrin-mediated adhesion and migration. PMID:19234463

  7. 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.

  8. Stress differentially affects the systemic and leukocyte estrogen network in common carp.

    PubMed

    Szwejser, Ewa; Pijanowski, Lukasz; Maciuszek, Magdalena; Ptak, Anna; Wartalski, Kamil; Duda, Malgorzata; Segner, Helmut; Verburg-van Kemenade, B M Lidy; Chadzinska, Magdalena

    2017-09-01

    Both systemic and locally released steroid hormones, such as cortisol and estrogens, show immunomodulatory actions. This research gives evidence that circulating and leukocyte-derived estrogens can be involved in the regulation of the immune response in common carp, during homeostasis and upon restraining stress. It was found that stress reduced level of blood 17β-estradiol (E2) and down-regulated the gene expression of components of the "classical" estrogen system: the nuclear estrogen receptors and the aromatase CYP19, in the hypothalamus, the pituitary and in the ovaries. In contrast, higher gene expression of the nuclear estrogen receptors and cyp19a was found in the head kidney of stressed animals. Moreover, stress induced changes in the E2 level and in the estrogen sensitivity at local/leukocyte level. For the first time in fish, we showed the presence of physiologically relevant amounts of E2 and the substrates for its conversion (estrone - E1 and testosterone - T) in head kidney monocytes/macrophages and found that its production is modulated upon stress. Moreover, stress reduced the sensitivity of leukocytes towards estrogens, by down-regulation the expression of the erb and cyp19 genes in carp phagocytes. In contrast, era expression was up-regulated in the head kidney monocytes/macrophages and in PBLs derived from stressed animals. We hypothesize that, the increased expression of ERα, that was observed during stress, can be important for the regulation of leukocyte differentiation, maturation and migration. In conclusion, these results indicate that, in fish, the estrogen network can be actively involved in the regulation of the systemic and local stress response and the immune response. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Stem Cell Microvesicles Transfer Cystinosin to Human Cystinotic Cells and Reduce Cystine Accumulation In Vitro

    PubMed Central

    Taranta, Anna; Bellomo, Francesco; Emma, Francesco; Besouw, Martine; Levtchenko, Elena; Toelen, Jaan; van den Heuvel, Lambertus; Chu, LeeLee; Zhao, Jing; Young, Yoon Kow; Eliopoulos, Nicoletta; Goodyer, Paul

    2012-01-01

    Cystinosis is a rare disease caused by homozygous mutations of the CTNS gene, encoding a cystine efflux channel in the lysosomal membrane. In Ctns knockout mice, the pathologic intralysosomal accumulation of cystine that drives progressive organ damage can be reversed by infusion of wildtype bone marrow-derived stem cells, but the mechanism involved is unclear since the exogeneous stem cells are rarely integrated into renal tubules. Here we show that human mesenchymal stem cells, from amniotic fluid or bone marrow, reduce pathologic cystine accumulation in co-cultured CTNS mutant fibroblasts or proximal tubular cells from cystinosis patients. This paracrine effect is associated with release into the culture medium of stem cell microvesicles (100–400 nm diameter) containing wildtype cystinosin protein and CTNS mRNA. Isolated stem cell microvesicles reduce target cell cystine accumulation in a dose-dependent, Annexin V-sensitive manner. Microvesicles from stem cells expressing CTNSRed transfer tagged CTNS protein to the lysosome/endosome compartment of cystinotic fibroblasts. Our observations suggest that exogenous stem cells may reprogram the biology of mutant tissues by direct microvesicle transfer of membrane-associated wildtype molecules. PMID:22912749

  10. Oligonol Supplementation Affects Leukocyte and Immune Cell Counts after Heat Loading in Humans

    PubMed Central

    Lee, Jeong Beom; Shin, Young Oh

    2014-01-01

    Oligonol is a low-molecular-weight form of polyphenol and has antioxidant and anti-inflammatory activity, making it a potential promoter of immunity. This study investigates the effects of oligonol supplementation on leukocyte and immune cell counts after heat loading in 19 healthy male volunteers. The participants took a daily dose of 200 mg oligonol or a placebo for 1 week. After a 2-week washout period, the subjects were switched to the other study arm. After each supplement, half-body immersion into hot water was made, and blood was collected. Then, complete and differential blood counts were performed. Flow cytometry was used to enumerate and phenotype lymphocyte subsets. Serum concentrations of interleukin (IL)-1β and IL-6 in blood samples were analyzed. Lymphocyte subpopulation variables included counts of total T cells, B cells, and natural killer (NK) cells. Oligonol intake attenuated elevations in IL-1β (an 11.1-fold change vs. a 13.9-fold change immediately after heating; a 12.0-fold change vs. a 12.6-fold change 1h after heating) and IL-6 (an 8.6-fold change vs. a 9.9-fold change immediately after heating; a 9.1-fold change vs. a 10.5-fold change 1h after heating) immediately and 1 h after heating in comparison to those in the placebo group. Oligonol supplementation led to significantly higher numbers of leukocytes (a 30.0% change vs. a 21.5% change immediately after heating; a 13.5% change vs. a 3.5% change 1h after heating) and lymphocytes (a 47.3% change vs. a 39.3% change immediately after heating; a 19.08% change vs. a 2.1% change 1h after heating) relative to those in the placebo group. Oligonol intake led to larger increases in T cells, B cells, and NK cells at rest (p < 0.05, p < 0.05, and p < 0.001, respectively) and immediately after heating (p < 0.001) in comparison to those in the placebo group. In addition, levels of T cells (p < 0.001) and B cells (p < 0.001) were significantly higher 1 h after heating in comparison to those in the

  11. Heterotropic modulation of selectin affinity by allosteric antibodies affects leukocyte rolling

    PubMed Central

    Riese, Sebastian B; Kuehne, Christian; Tedder, Thomas F; Hallmann, Rupert; Hohenester, Erhard; Buscher, Konrad

    2013-01-01

    Selectins are a family of adhesion receptors designed for efficient leukocyte tethering to the endothelium under shear. As a key property to resist premature bond disruption, selectin adhesiveness is enhanced by tensile forces that promote the conversion of a bent into an extended conformation of the N-terminal lectin and EGF-like domains. Conformation-specific antibodies have been invaluable in deciphering the activation mechanism of integrins, but similar reagents are not available for selectins. Here we show that the anti-human L-selectin monoclonal antibodies DREG-55 and LAM1.5 but not DREG-56, -200 or LAM1.1 heterotropically modulate adhesion presumably by stabilizing the extended receptor conformation. Force-free affinity assays, flow chamber and microkinetic studies reveal a ligand-specific modulation of L-selectin affinity by DREG-55 mAb, resulting in a dramatic decrease of rolling velocity under flow. Furthermore, secondary tethering of polymorphonuclear cells was blocked by DREG-200 but significantly boosted by DREG-55 mAb. The results emphasize the need for a new classification for selectin antibodies and introduce the new concept of heterotropic modulation of receptor function. PMID:24431230

  12. Acrocentric Chromosomes in Cultured Leukocytes from Mothers of Children Affected With the G1- Trisomy Syndrome

    ERIC Educational Resources Information Center

    And Others; Cotton, James E.

    1973-01-01

    Analysis of venous blood samples from 24 mothers of G1-trisomy-affected (Down's Syndrome) children and 23 mothers of chromosomally normal children indicated that mothers of G1-trisomy-affected children had a greater than expected involvement of the G-chromosomes in associations of acrocentric satellited (chromosome configuration) chromosomes.…

  13. Leukocyte count affects expression of reference genes in canine whole blood samples

    PubMed Central

    2011-01-01

    Background The dog is frequently used as a model for hematologic human diseases. In this study the suitability of nine potential reference genes for quantitative RT-PCR studies in canine whole blood was investigated. Findings The expression of these genes was measured in whole blood samples of 263 individual dogs, representing 73 different breeds and a group of 40 mixed breed dogs, categorized into healthy dogs and dogs with internal and hematological diseases, and dogs that underwent a surgical procedure. GeNorm analysis revealed that a combination of 5 to 6 of the most stably expressed genes constituted a stable normalizing factor. Evaluation of the expression revealed different ranking of reference genes in Normfinder and GeNorm. The disease category and the white blood cell count significantly affected reference gene expression. Conclusions The discrepancy between the ranking of reference genes in this study by Normfinder and Genorm can be explained by differences between the experimental groups such as "disease category" and "WBC count". This stresses the importance of assessing the expression stability of potential reference genes for gene experiments in canine whole blood anew for each specific experimental condition. PMID:21303565

  14. 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.

  15. Poliovirus Receptor-Related 2: A Cholesterol-Responsive Gene Affecting Atherosclerosis Development by Modulating Leukocyte Migration.

    PubMed

    Rossignoli, Aránzazu; Shang, Ming-Mei; Gladh, Hanna; Moessinger, Christine; Foroughi Asl, Hassan; Talukdar, Husain Ahammad; Franzén, Oscar; Mueller, Steffen; Björkegren, Johan L M; Folestad, Erika; Skogsberg, Josefin

    2017-03-01

    Recently, poliovirus receptor-related 2 (Pvrl2) emerged as a top gene in a global gene expression study aiming to detect plasma cholesterol-responsive genes causally related to atherosclerosis regression in hypercholesterolemic mice. PVRL2 is an adherens junction protein implied to play a role in transendothelial migration of leukocytes, a key feature in atherosclerosis development. In this study, we investigated the effect of Pvrl2 deficiency on atherosclerosis development and transendothelial migration of leukocytes activity. APPROACH AND RESULTS: Pvrl2-deficient mice bred onto an atherosclerosis-prone background (Pvrl2(-/-)Ldlr(-/-)Apob(100/100)) had less atherosclerotic lesions and more stable plaques compared with littermate controls (Pvrl2(+/+)Ldlr(-/-)Apob(100/100)). Pvrl2(-/-)Ldlr(-/-)Apob(100/100) mice also showed a 49% decrease in transendothelial migration of leukocytes activity observed using the in vivo air pouch model. In accordance, augmented arterial wall expression of Pvrl2 during atherosclerosis progression coincided with an increased gene expression of migrating leukocytes into the vessel wall. Both in human and mice, gene and protein expression of PVRL2 was predominantly observed in the vascular endothelium according to the immunohistochemical and gene expression data. In addition, the cholesterol responsiveness of PVRL2 was also observed in humans. PVRL2 is a plasma cholesterol-responsive gene acting at endothelial sites of vascular inflammation that could potentially be a new therapeutic target for atherosclerosis prevention through its suggested transendothelial migration of leukocytes modulating activity. © 2017 American Heart Association, Inc.

  16. 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.

  17. Rifampin affects polymorphonuclear leukocyte interactions with bacterial and synthetic chemotaxins but not interactions with serum-derived chemotaxins.

    PubMed Central

    Gray, G D; Smith, C W; Hollers, J C; Chenoweth, D E; Fiegel, V D; Nelson, R D

    1983-01-01

    Three independent experimental approaches support the hypothesis that rifampin competes for receptors on polymorphonuclear leukocytes (PMLs) with small peptide chemoattractants, e.g., N-formylmethionylleucylphenylalanine (FMLP), but not with serum-derived chemoattractants (C5a). First, rifampin inhibited chemotaxis induced with FMLP but reversed the immobilization of PMLs that occurred at high FMLP concentrations. Second, rifampin competed with radiolabeled FMLP for binding sites on PMLs and displaced already-bound radiolabeled FMLP. Third, rifampin blocked and reversed the bipolar shape changes induced in PMLs by FMLP. These effects occurred at concentrations attained during rifampin therapy and were not due to rifampin toxicity. In contrast, no effect of rifampin was observed on serum-derived chemoattractants (C5a) in any of the three systems. The evidence suggests, therefore, that rifampin is a ligand for FMLP-type receptors on PMLs. PMID:6318656

  18. A short‐term extremely low frequency electromagnetic field exposure increases circulating leukocyte numbers and affects HPA‐axis signaling in mice

    PubMed Central

    de Kleijn, Stan; Ferwerda, Gerben; Wiese, Michelle; Trentelman, Jos; Cuppen, Jan; Kozicz, Tamas; de Jager, Linda; Hermans, Peter W. M.

    2016-01-01

    There is still uncertainty whether extremely low frequency electromagnetic fields (ELF‐EMF) can induce health effects like immunomodulation. Despite evidence obtained in vitro, an unambiguous association has not yet been established in vivo. Here, mice were exposed to ELF‐EMF for 1, 4, and 24 h/day in a short‐term (1 week) and long‐term (15 weeks) set‐up to investigate whole body effects on the level of stress regulation and immune response. ELF‐EMF signal contained multiple frequencies (20–5000 Hz) and a magnetic flux density of 10 μT. After exposure, blood was analyzed for leukocyte numbers (short‐term and long‐term) and adrenocorticotropic hormone concentration (short‐term only). Furthermore, in the short‐term experiment, stress‐related parameters, corticotropin‐releasing hormone, proopiomelanocortin (POMC) and CYP11A1 gene‐expression, respectively, were determined in the hypothalamic paraventricular nucleus, pituitary, and adrenal glands. In the short‐term but not long‐term experiment, leukocyte counts were significantly higher in the 24 h‐exposed group compared with controls, mainly represented by increased neutrophils and CD4 ± lymphocytes. POMC expression and plasma adrenocorticotropic hormone were significantly lower compared with unexposed control mice. In conclusion, short‐term ELF‐EMF exposure may affect hypothalamic‐pituitary‐adrenal axis activation in mice. Changes in stress hormone release may explain changes in circulating leukocyte numbers and composition. Bioelectromagnetics. 37:433–443, 2016. © 2016 The Authors. Bioelectromagnetics Published by Wiley Periodicals, Inc. PMID:27553635

  19. A short-term extremely low frequency electromagnetic field exposure increases circulating leukocyte numbers and affects HPA-axis signaling in mice.

    PubMed

    de Kleijn, Stan; Ferwerda, Gerben; Wiese, Michelle; Trentelman, Jos; Cuppen, Jan; Kozicz, Tamas; de Jager, Linda; Hermans, Peter W M; Verburg-van Kemenade, B M Lidy

    2016-10-01

    There is still uncertainty whether extremely low frequency electromagnetic fields (ELF-EMF) can induce health effects like immunomodulation. Despite evidence obtained in vitro, an unambiguous association has not yet been established in vivo. Here, mice were exposed to ELF-EMF for 1, 4, and 24 h/day in a short-term (1 week) and long-term (15 weeks) set-up to investigate whole body effects on the level of stress regulation and immune response. ELF-EMF signal contained multiple frequencies (20-5000 Hz) and a magnetic flux density of 10 μT. After exposure, blood was analyzed for leukocyte numbers (short-term and long-term) and adrenocorticotropic hormone concentration (short-term only). Furthermore, in the short-term experiment, stress-related parameters, corticotropin-releasing hormone, proopiomelanocortin (POMC) and CYP11A1 gene-expression, respectively, were determined in the hypothalamic paraventricular nucleus, pituitary, and adrenal glands. In the short-term but not long-term experiment, leukocyte counts were significantly higher in the 24 h-exposed group compared with controls, mainly represented by increased neutrophils and CD4 ± lymphocytes. POMC expression and plasma adrenocorticotropic hormone were significantly lower compared with unexposed control mice. In conclusion, short-term ELF-EMF exposure may affect hypothalamic-pituitary-adrenal axis activation in mice. Changes in stress hormone release may explain changes in circulating leukocyte numbers and composition. Bioelectromagnetics. 37:433-443, 2016. © 2016 The Authors. Bioelectromagnetics Published by Wiley Periodicals, Inc.

  20. 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

  1. Cryopreservation of Human Mucosal Leukocytes.

    PubMed

    Hughes, Sean M; 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

    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. 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. 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.

  2. Cysteamine treatment restores the in vitro ability to differentiate along the osteoblastic lineage of mesenchymal stromal cells isolated from bone marrow of a cystinotic patient.

    PubMed

    Conforti, Antonella; Taranta, Anna; Biagini, Simone; Starc, Nadia; Pitisci, Angela; Bellomo, Francesco; Cirillo, Valentina; Locatelli, Franco; Bernardo, Maria Ester; Emma, Francesco

    2015-05-07

    Cystinosis is a rare autosomal recessive disease caused by mutations of the CTNS gene, which encodes for a lysosomal cystine/H(+) symporter. In mice, inactivation of the CTNS gene causes intralysosomal cystine accumulation and progressive organ damage that can be reversed, at least in part, by infusion of mesenchymal stromal cells (MSCs). Little is known on the mesenchymal compartment of cystinotic patients. The aim of the study was to test the phenotypical and functional properties of cystinotic MSCs (Cys-MSCs) isolated from bone marrow (BM) aspirate of a patient with nephropathic cystinosis. Morphology, proliferative capacity (measured as population doublings), immunophenotype (by flow-cytometry) and immunomodulatory properties (as phytohemagglutinin-induced peripheral blood mononuclear cell proliferation) were analyzed. The osteogenic differentiation potential of Cys-MSCs was evaluated by histological staining (alkaline phosphatase activity, Alzarin Red and von Kossa staining) spectrophotometry and Quantitative Reverse Transcriptase Polymerase Chain Reaction for osteigenic markers in the presence and in the absence of cysteamine. Cys-MSCs were compared with those isolated and expanded ex vivo from three healthy donors (HD-MSCs). Despite a slightly lower proliferative capacity, Cys-MSCs displayed a characteristic spindle-shaped morphology and similar immunephenotype as HD-MSCs. Cys-MSCs and HD-MSCs prevented proliferation of PHA-stimulated allogeneic peripheral blood mononuclear cells to the same extent. After in vitro induction into osteoblasts, Cys-MSCs showed reduced alkaline phosphatase (ALP) activity, calcium depositions and expression of ALP and collagen type 1. When Cys-MSCs were treated in vitro with increasing doses of cysteamine (50-100-200 μM/L) during the differentiation assay, recovery of Cys-MSCs differentiation capacity into osteoblasts was observed. No difference in adipogenic differentiation was found between Cys-MSCs and HD-MSCs. Our results

  3. Blood storage affects the detection of cellular prion protein on peripheral blood leukocytes and circulating dendritic cells in part by promoting platelet satellitism.

    PubMed

    Glier, Hana; Holada, Karel

    2012-06-29

    Flow cytometry represents an attractive approach for developing currently unavailable screening tests for prion diseases. Several studies have reported significant differences in the binding of antibodies directed against cellular prion protein (PrP(C)) to blood cells of prion-infected subjects compared with healthy controls. However, flow cytometry data usually show large individual variations in detected PrP(C) levels in both infected and control groups, rendering the interpretation of individual patient data difficult. To determine how pre-analytical variables, such as the choice of anticoagulant, whether or not the blood was stored, and the storage temperature, affect the detection of PrP(C) in blood cells. Blood from healthy donors was collected in EDTA or citrate anticoagulant and processed either immediately or after storage overnight at room temperature or at 4°C. The expression of PrP(C) by T cells, B cells, NK cells, monocytes and circulating dendritic cells was evaluated using quantitative flow cytometry with the PrP(C) monoclonal antibodies AG4 and AH6. The anticoagulation of blood with citrate resulted in decreased levels of PrP(C) on monocytes but not the other cell types. The storage of blood prior to analysis led to a significant decrease in the levels of PrP(C) on the cells studied, although there were substantial differences between the cell populations. This decrease was more pronounced when using mAb AG4, which targets the N-terminal portion of the PrP(C) molecule, or following storage at room temperature. Moreover, we identified platelet satellitism on leukocytes, especially on monocytes and granulocytes, as an additional factor contributing to the heterogeneity of PrP(C) detection in stored blood. Our study demonstrates that the storage of blood prior to analysis greatly affects the detection of PrP(C) by flow cytometry. To limit the inclusion of storage-generated artifacts, we recommend the processing of blood samples immediately after their

  4. Hypothesis: Leukocyte Endogenous Mediator/Endogenous Pyrogen/Lymphocyte-Activating Factor Modulates the Development of Nonspecific and Specific Immunity and Affects Nutritional Status

    DTIC Science & Technology

    1982-04-01

    813-20. 55. 19. Bodel P. Colchicine stimulation of pyrogen produc- 36. Hubbard WJ. Hypothesis: alpha-2 macroglobulin- tion by human blood leukocytes. J...and repair systems. In: Po- 1970:131:727-43. wanda MC, Canonico PG, eds. Infection: the phys- 23. Bodel P. Studies on the mechanism of endogenous

  5. A motility test of leukocytes under agar.

    PubMed

    Goedemans, W T; de Jong, M M

    1985-01-01

    A migration test under agar for leukocytes was developed. Leukocytes moved quite a distance under anaerobic Blood Agar Base (blood agar), a Gibco product. Migration on stained and coloured plates was visualized by projection with a profile projector, making the use of a light microscope superfluous. A migration index was defined. Reproducibility was good enough to allow paired comparisons of leukocyte populations subjected to different treatments. Migration was the result of spontaneous and chemotactically directed migration. Cell-labelling complexes as 111In-oxinate and 111In-tropolonate--ligand concentration 3.5 micrograms/mL in the ultimate cell preparation--did not affect leukocyte migration. 111In-pyrithionate (mercapto pyridine-N-oxide) significantly impaired cell motility. The motility test described could be used as retrospective analysis in abscess localization studies using 111In labelled leukocytes.

  6. Genetic variability in swine leukocyte antigen class II and Toll-like receptors affects immune responses to vaccination for bacterial infections in pigs.

    PubMed

    Shinkai, H; Arakawa, A; Tanaka-Matsuda, M; Ide-Okumura, H; Terada, K; Chikyu, M; Kawarasaki, T; Ando, A; Uenishi, H

    2012-12-01

    The genes encoding swine leukocyte antigen (SLA) and Toll-like receptor (TLR) are highly polymorphic in pig populations, and likely have influences on infection and the effects of vaccination. We explored the associations of different genotypes of SLA class II and of the genes TLR1, TLR4, TLR5, and TLR6 with antibody responses after vaccination against Erysipelothrix rhusiopathiae (ER) and Actinobacillus pleuropneumoniae (APP) serotypes 1, 2, and 5 in 191 Duroc pigs maintained under specific pathogen-free conditions. We demonstrated close relationships between SLA class II and ER antibody response and between TLR genes other than TLR4 and APP antibody responses. Pigs with specific haplotypes in SLA class II or TLR5 showed decreased antibody response to ER vaccination or increased responses to APP2 and APP5 vaccination, respectively. It might be possible to breed for responsiveness to vaccination and to implement new vaccine development strategies unaffected by genetic backgrounds of pigs.

  7. Modeling Leukocyte-Leukocyte Non-Contact Interactions in a Lymph Node

    PubMed Central

    Gritti, Nicola; Caccia, Michele; Sironi, Laura; Collini, Maddalena; D'Alfonso, Laura; Granucci, Francesca; Zanoni, Ivan; Chirico, Giuseppe

    2013-01-01

    The interaction among leukocytes is at the basis of the innate and adaptive immune-response and it is largely ascribed to direct cell-cell contacts. However, the exchange of a number of chemical stimuli (chemokines) allows also non-contact interaction during the immunological response. We want here to evaluate the extent of the effect of the non-contact interactions on the observed leukocyte-leukocyte kinematics and their interaction duration. To this aim we adopt a simplified mean field description inspired by the Keller-Segel chemotaxis model, of which we report an analytical solution suited for slowly varying sources of chemokines. Since our focus is on the non-contact interactions, leukocyte-leukocyte contact interactions are simulated only by means of a space dependent friction coefficient of the cells. The analytical solution of the Keller-Segel model is then taken as the basis of numerical simulations of interactions between leukocytes and their duration. The mean field interaction force that we derive has a time-space separable form and depends on the chemotaxis sensitivity parameter as well as on the chemokines diffusion coefficient and their degradation rate. All these parameters affect the distribution of the interaction durations. We draw a successful qualitative comparison between simulated data and sets of experimental data for DC-NK cells interaction duration and other kinematic parameters. Remarkably, the predicted percentage of the leukocyte-leukocyte interactions falls in the experimental range and depends (≅25% increase) upon the chemotactic parameter indicating a non-negligible direct effect of the non-contact interaction on the leukocyte interactions. PMID:24204669

  8. Biomechanics of leukocyte rolling

    PubMed Central

    Sundd, Prithu; Pospieszalska, Maria K.; Cheung, Luthur Siu-Lun; Konstantopoulos, Konstantinos; Ley, Klaus

    2011-01-01

    Leukocyte rolling on endothelial cells and other P-selectin substrates is mediated by P-selectin binding to P-selectin glycoprotein ligand-1 expressed on the tips of leukocyte microvilli. Leukocyte rolling is a result of rapid, yet balanced formation and dissociation of selectin-ligand bonds in the presence of hydrodynamic shear forces. The hydrodynamic forces acting on the bonds may either increase (catch bonds) or decrease (slip-bonds) their lifetimes. The force-dependent ‘catch-slip’ bond kinetics are explained using the ‘two pathway model’ for bond dissociation. Both the ‘sliding-rebinding’ and the ‘allosteric’ mechanisms attribute ‘catch-slip’ bond behavior to the force-induced conformational changes in the lectin-EGF domain hinge of selectins. Below a threshold shear stress, selectins cannot mediate rolling. This ‘shear-threshold’ phenomenon is a consequence of shear-enhanced tethering and catch-bond enhanced rolling. Quantitative dynamic footprinting microscopy has revealed that leukocytes rolling at venular shear stresses (> 0.6 Pa) undergo cellular deformation (large footprint) and form long tethers. The hydrodynamic shear force and torque acting on the rolling cell are thought to be synergistically balanced by the forces acting on tethers and stressed microvilli, however, their relative contribution remains to be determined. Thus, improvement beyond the current understanding requires in silico models that can predict both cellular and microvillus deformation and experiments that allow measurement of forces acting on individual microvilli and tethers. PMID:21515934

  9. 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

  10. 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.

  11. Microparticle Profile and Procoagulant Activity of Fresh Frozen Plasma is Affected by Whole Blood-Leukocyte Depletion Rather Than 24-Hour Room Temperature-Hold

    PubMed Central

    Chan, Kasey Sze-Kei; Sparrow, Rosemary L

    2014-01-01

    BACKGROUND Microparticles (MPs) are small phospholipid-containing vesicles that have pro-coagulant properties. MPs are thought to contribute to the hemostatic potential of plasma. This study investigated the effects of WB-hold time and leukodepletion (LD) on the MP profile and hemostatic potential of fresh-frozen plasma (FFP). STUDY DESIGN AND METHODS WB units (n=12) from healthy donors were divided into two pairs and each pair was held at 20–24°C for 6 or 24 hours. At the designated hold-time, one unit from the pair was leukodepleted while the other unit was not leukodepleted. FFP was prepared by standard procedures, aliquoted and frozen. The MP content was determined by flow cytometry using an absolute count assay and specific labels for red cells (CD235a), platelets (CD41) and phosphatidylserine. The hemostatic potential was determined by thrombelastography (TEG) and coagulation factor assays. RESULTS Compared to non-LD FFP, LD-FFP had significantly lower numbers of MPs, particularly CD41+ MPs and phosphatidylserine-positive MPs (p<0.03). LD-FFP, compared to non-LD FFP, had a slower clot formation time (p=0.002), lower clot strength (p<0.001) and lower FVIII, FXII and fibrinogen levels (p<0.01). With longer WB hold-time, the TEG profile was unchanged, although FVIII levels were decreased as expected (p<0.01). On average FFP units met quality requirements. CONCLUSION LD of WB resulted in lower hemostatic potential of FFP in conjunction with depletion of MPs and coagulation factors. Longer WB hold-time did not significantly affect the hemostatic potential of FFP as measured by TEG. Acceptable hemostatic quality was maintained for all FFP processing conditions studied. PMID:24635475

  12. Evolution of cytokine responses: IL-1beta directly affects intracellular Ca2+ concentration of teleost fish leukocytes through a receptor-mediated mechanism.

    PubMed

    Benedetti, S; Randelli, E; Buonocore, F; Zou, J; Secombes, C J; Scapigliati, G

    2006-04-01

    In this work we studied the biological activities of recombinant IL-1beta from the teleosts sea bass (Dicentrarchus labrax) and rainbow trout (Oncorhynchus mykiss) by investigating the effects induced on intracellular Ca2+ concentrations ([Ca2+]i) of spleen leucocytes. Splenocytes were loaded with the Ca2+-permeant Fura-2AM, and then stimulated with rIL-1beta. The emitted fluorescence was read for 5 min at 1 min intervals on a dual excitation fluorescence fluorimeter. Results showed that rIL-1beta induced in both species a rise in [Ca2+]i, and a subsequent decrease until 5 min after stimulation. The stimulating effect was dose-dependent in both species reaching a plateau at 200 ng/ml of rIL-1beta, was abolished by heat-treatment of rIL-1beta, and affected in a dose-dependent fashion by treatment of leucocytes with trypsin. These features suggested a functional IL-1 receptor was involved in the binding. The observed rise in [Ca2+]i was not detected in human PBMC and was species-specific, since rIL-1beta from sea bass, trout, and human were unable to interfere each other in the assay. Moreover, incubation of splenocytes with rIL-1beta induced a rapid tyrosine phosphorylation of a 24 kDa polypeptide in both species. This work represents the first evidence of a direct effect on [Ca2+]i induced by IL-1beta and suggests that in the evolution of IL-1 activities, teleost fishes display a peculiar IL-1-associated behaviour that is lacking in mammals.

  13. Pulsatility of Parafoveal Capillary Leukocytes

    PubMed Central

    Martin, Joy A.; Roorda, Austin

    2009-01-01

    The use of adaptive optics (AO) in a confocal scanning laser ophthalmoscope (AOSLO) allows for long-term imaging of parafoveal capillary leukocyte movement and measurement of leukocyte velocity without contrast dyes. We applied the AOSLO to investigate the possible role of the cardiac cycle on capillary leukocyte velocity by directly measuring capillary leukocyte pulsatility. The parafoveal regions of 8 eight normal healthy subjects with clear ocular media were imaged with an AOSLO. All subjects were dilated and cyclopleged. The AOSLO field of view was either 1.4 × 1.5 degrees or 2.35 × 2.5 degrees, the imaging wavelength was 532 nm and the frame rate was 30 fps. A photoplethysmograph was used to record the subject’s pulse synchronously with each AOSLO video. Parafoveal capillary leukocyte velocities and pulsatility were determined for two or three capillaries per subject. Leukocyte velocity and pulsatility were determined for all eight subjects. The mean parafoveal capillary leukocyte velocity for all subjects was Vmean = 1.30 mm/sec (SD = +/− 0.40 mm/sec). There was a statistically significant difference between leukocyte velocities, Vmax and Vmin, over the pulse cycle for each subject (p<0.05). The mean pulsatility was Pmean= 0.45 (+/− 0.09). Parafoveal capillary leukocyte pulsatility can be directly and non-invasively measured without the use of contrast dyes using an AOSLO. A substantial amount of the variation found in leukocyte velocity is due to the pulsatility that is induced by the cardiac cycle. By controlling for the variation in leukocyte velocity caused by the cardiac cycle, we can better detect other changes in retinal leukocyte velocity induced by disease or pharmaceutical agents. PMID:18708051

  14. [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.

  15. 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.

  16. Interstitial leukocyte migration in vivo

    PubMed Central

    Lam, Pui-ying; Huttenlocher, Anna

    2013-01-01

    Rapid leukocyte motility is essential for immunity and host defense. There has been progress in understanding the molecular signals that regulate leukocyte motility both in vitro and in vivo. However, a gap remains in understanding how complex signals are prioritized to result in directed migration, which is critical for both adaptive and innate immune function. Here we focus on interstitial migration and how external cues are translated into intracellular signaling pathways that regulate leukocyte polarity, directional sensing and motility in three-dimensional spaces. PMID:23797028

  17. Inflammation, leukocytes and menstruation.

    PubMed

    Evans, Jemma; Salamonsen, Lois A

    2012-12-01

    Menstruation has many of the features of an inflammatory process. The complexity and sequence of inflammatory-type events leading to the final tissue breakdown and bleeding are slowly being unravelled. Progesterone has anti-inflammatory properties, and its rapidly declining levels (along with those of estrogen) in the late secretory phase of each non-conception cycle, initiates a sequence of interdependent events of an inflammatory nature involving local inter-cellular interactions within the endometrium. Intracellular responses to loss of progesterone (in decidualized stromal, vascular and epithelial cells) lead to decreased prostaglandin metabolism and loss of protection from reactive oxygen species (ROS). Increased ROS results in release of NFκB from suppression with activation of target gene transcription and increased synthesis of pro-inflammatory prostaglandins, cytokines, chemokines and matrix metalloproteinases (MMP). The resultant leukocyte recruitment, with changing phenotypes and activation, provide further degradative enzymes and MMP activators, which together with a hypoxic environment induced by prostaglandin actions, lead to the tissue breakdown and bleeding characteristic of menstruation. In parallel, at sites where shedding is complete, microenvironmentally-induced changes in phenotypes of neutrophils and macrophages from pro- to anti-inflammatory, in addition to induction of growth factors, contribute to the very rapid re-epithelialization and restoration of tissue integrity.

  18. Elemental composition of leukocyte subfractions

    NASA Astrophysics Data System (ADS)

    Admans, L. L.; Spyrou, N. M.

    2003-01-01

    The aim of this preliminary investigation was to determine the elemental concentration of various subfractions of leukocytes in a normal subject. Little work has been published on the elemental composition of these subfractions. First, a reliable technique for separation of these subfractions had to be established so that it could be applied to the determination of elemental concentrations in leukocyte subfractions from patients undergoing heart bypass surgery. Instrumental neutron activation analysis (INAA) utilising short irradiation and counting was the technique employed. Various washing media were examined during the separation of the leukocyte subfractions, for contamination of these small samples of peripheral blood mononuclear cells (PBMC) and polymorphonuclearcytes (PMN). Early results showed Mg and Se were present in these subfractions. Possibilities for further work are also discussed.

  19. Intracellular Penetration and Activity of Gemifloxacin in Human Polymorphonuclear Leukocytes

    PubMed Central

    García, Isabel; Pascual, Alvaro; Ballesta, Sofía; Joyanes, Providencia; Perea, Evelio J.

    2000-01-01

    The intracellular penetration and activity of gemifloxacin in human polymorphonuclear leukocytes (PMN) were evaluated. Gemifloxacin reached intracellular concentrations eight times higher than extracellular concentrations. The uptake was rapid, reversible, and nonsaturable and was affected by environmental temperature, cell viability, and membrane stimuli. At therapeutic extracellular concentrations, gemifloxacin showed intracellular activity against Staphylococcus aureus. PMID:11036051

  20. 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.

  1. Selective Harvesting of Marginating-hepatic Leukocytes.

    PubMed

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

    2016-07-21

    Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

  2. Regulation of leukocyte-endothelium interaction and leukocyte transendothelial migration by intercellular adhesion molecule 1-fibrinogen recognition.

    PubMed

    Languino, L R; Duperray, A; Joganic, K J; Fornaro, M; Thornton, G B; Altieri, D C

    1995-02-28

    Although primarily recognized for its role in hemostasis, fibrinogen is also required for competent inflammatory reactions in vivo. It is now shown that fibrinogen promotes adhesion to and migration across an endothelial monolayer of terminally differentiated myelomonocytic cells. This process does not require chemotactic/haptotactic gradients or cytokine stimulation of the endothelium and is specific for the association of fibrinogen with intercellular adhesion molecule 1 (ICAM-1) on endothelium. Among other adhesive plasma proteins, fibronectin fails to increase the binding of leukocytes to endothelium, or transendothelial migration, whereas vitronectin promotes the binding but not the migration. The fibrinogen-mediated leukocyte adhesion and transendothelial migration could be inhibited by a peptide from the fibrinogen gamma-chain sequence N117NQKIVNL-KEKVAQLEA133, which blocks the binding of fibrinogen to ICAM-1. This interaction could also be inhibited by new anti-ICAM-1 monoclonal antibodies that did not affect the ICAM-1-CD11a/CD18 recognition, thus suggesting that the fibrinogen binding site on ICAM-1 may be structurally distinct from regions previously implicated in leukocyte-endothelium interaction. Therefore, binding of fibrinogen to vascular cell receptors is sufficient to initiate (i) increased leukocyte adhesion to endothelium and (ii) leukocyte transendothelial migration. These two processes are the earliest events of immune inflammatory responses and may also contribute to atherosclerosis.

  3. 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.

  4. 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

  5. Role of cell adhesion molecules in leukocyte recruitment in the liver and gut

    PubMed Central

    Ala, A; Dhillon, AP; Hodgson, HJ

    2003-01-01

    This article reviews the evidence that adhesion molecules are critical in leukocyte recirculation and pathogenesis of diseases affecting the closely related tissues of the liver and gut, which offer novel opportunities for treatment. PMID:12694483

  6. Leukocyte integrins: role in leukocyte recruitment and as therapeutic targets in inflammatory disease.

    PubMed

    Mitroulis, Ioannis; Alexaki, Vasileia I; Kourtzelis, Ioannis; Ziogas, Athanassios; Hajishengallis, George; Chavakis, Triantafyllos

    2015-03-01

    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 signaling 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. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. 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

  8. Periovulatory leukocyte infiltration in the rat ovary.

    PubMed

    Oakley, Oliver R; Kim, HeyYoung; El-Amouri, Ismail; Lin, Po-Ching Patrick; Cho, Jongki; Bani-Ahmad, Mohammad; Ko, Chemyong

    2010-09-01

    Ovulation is preceded by intraovarian inflammatory reactions that occur in response to the preovulatory gonadotropin surge. As a main inflammatory event, leukocytes infiltrate the ovary and release proteolytic enzymes that degrade the extracellular matrix weakening the follicular wall, a required step for follicle rupture. This study aimed to quantitatively measure the infiltrating leukocytes, determine their cell types, and localize infiltration sites in the periovulatory rat ovary. Cycling adult and gonadotropin-stimulated immature rats were used as animal models. Ovaries were collected at five different stages of estrous cycle in the adult rats (diestrus, 1700 h; proestrus, 1500 h; proestrus, 2400 h; estrus, 0600 h; and metestrus, 1700 h) and at five different time points after superovulation induction in the immature rats (pregnant mare's serum gonadotrophin, 0 h; pregnant mare's serum gonadotrophin, 48 h; human chorionic gonadotropin, 6 h; human chorionic gonadotropin, 12 h; and human chorionic gonadotropin, 24 h). The ovaries were either dissociated into a single cell suspension for flow cytometric analysis or fixed for immunohistochemical localization of the leukocytes. Similar numbers of leukocytes were seen throughout the estrous cycle (approximately 500,000/ovary), except proestrus 2400 when 2-fold higher numbers of leukocytes were found (approximately 1.1 million/ovary). A similar trend of periovulatory rise of leukocyte numbers was seen in the superovulation-induced immature rat model, recapitulating a dramatic increase in leukocyte numbers upon gonadotropin stimulation. Both macrophage/granulocytes and lymphocytes were among the infiltrating leukocytes and were localized in the theca and interstitial tissues, where platelet-endothelial cell adhesion molecule-1 and intercellular adhesion molecule-1 may play roles in the transmigration of leukocytes, because their expressions correlates spatiotemporally with the infiltrating leukocytes. In addition, a

  9. 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

  10. 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.

  11. Binding of CD40L to Mac-1’s I-domain involves the EQLKKSKTL motif and mediates leukocyte recruitment and atherosclerosis – but does not affect immunity and thrombosis in mice

    PubMed Central

    Wolf, Dennis; Hohmann, Jan-David; Wiedemann, Ansgar; Bledzka, Kamila; Blankenbach, Hermann; Marchini, Timoteo; Gutte, Katharina; Zeschky, Katharina; Bassler, Nicole; Hoppe, Natalie; Rodriguez, Alexandra Ortiz; Herr, Nadine; Hilgendorf, Ingo; Stachon, Peter; Willecke, Florian; Dürschmied, Daniel; von zur Mühlen, Constantin; Soloviev, Dmitry A.; Zhang, Li; Bode, Christoph; Plow, Edward F.; Libby, Peter; Peter, Karlheinz; Zirlik, Andreas

    2012-01-01

    Rationale CD40L figures prominently in chronic inflammatory diseases such as atherosclerosis. However, since CD40L potently regulates immune function and haemostasis by interaction with CD40 receptor and the platelet integrin GPIIb/IIIa, its global inhibition compromises host defense and generated thromboembolic complications in clinical trials. We recently reported that CD40L mediates atherogenesis independently of CD40 and proposed Mac-1 as an alternate receptor. Objective Here, we molecularly characterized the CD40L-Mac-1 interaction and tested whether its selective inhibition by a small peptide modulates inflammation and atherogenesis in vivo. Methods and Results CD40L concentration-dependently bound to Mac-1 I-domain in solid phase binding assays, and a high affinity interaction was revealed by surface-plasmon-resonance analysis. We identified the motif EQLKKSKTL, an exposed loop between the α1 helix and the β-sheet B, on Mac-1 as binding site for CD40L. A linear peptide mimicking this sequence, M7, specifically inhibited the interaction of CD40L and Mac-1. cM7, a cyclisized version optimized for in vivo use, decreased peritoneal inflammation and inflammatory cell recruitment in vivo. Finally, LDLr-/- mice treated with intraperitoneal injections of cM7 developed smaller, less inflamed atherosclerotic lesions featuring characteristics of stability. However, cM7 did not interfere with CD40L-CD40 binding in vitro and CD40L-GPIIb/IIIa-mediated thrombus formation in vivo. Conclusions We present the novel finding that CD40L binds to the EQLKKSKTL motif on Mac-1 mediating leukocyte recruitment and atherogenesis. Specific inhibition of CD40L-Mac-1 binding may represent an attractive anti-inflammatory treatment strategy for atherosclerosis and other inflammatory conditions, potentially avoiding the unwanted immunologic and thrombotic effects of global inhibition of CD40L. PMID:21998326

  12. Evaluation of methylglyoxal toxicity in human erythrocytes, leukocytes and platelets.

    PubMed

    Prestes, Alessandro de Souza; Dos Santos, Matheus Mülling; Ecker, Assis; Zanini, Daniela; Schetinger, Maria Rosa Chitolina; Rosemberg, Denis Broock; da Rocha, João Batista Teixeira; Barbosa, Nilda Vargas

    2017-05-01

    Methylglyoxal (MG) is a reactive dicarbonyl metabolite originated mainly from glucose degradation pathway that plays an important role in the pathogenesis of diabetes mellitus (DM). Reactions of MG with biological macromolecules (proteins, DNA and lipids) can induce cytotoxicity and apoptosis. Here, human erythrocytes, leukocytes and platelets were acutely exposed to MG at concentration ranging from 0.025 to 10 mM. Afterwards, hemolysis and osmotic fragility in erythrocytes, DNA damage and cell viability in leukocytes, and the activity of purinergic ecto-nucleotidases in platelets were evaluated. The levels of glycated products from leukocytes and free amino groups from erythrocytes and platelets were also measured. MG caused fragility of membrane, hemolysis and depletion of amino groups in erythrocytes. DNA damage, loss of cell viability and increased levels of glycated products were observed in leukocytes. In platelets, MG inhibited the activity of enzymes NTPDase, 5'-nucleotidase and adenosine deaminase (ADA) without affecting the levels of free amino groups. Our findings provide insights for understanding the mechanisms involved in MG acute toxicity towards distinct blood cells.

  13. Effects of new peritoneal dialysis solutions on leukocyte recruitment in the rat peritoneal membrane.

    PubMed

    Mortier, Siska; Faict, Dirk; Gericke, Marion; Lameire, Norbert; De Vriese, An

    2005-01-01

    Peritonitis remains a principal cause of dropout in peritoneal dialysis (PD). The physiological host response to a peritoneal infection involves a rise in numbers of circulating leukocytes to the peritoneal cavity. We evaluated the effects of (1) conventional peritoneal dialysis fluid (PDF), (2) bicarbonate-based PDF, low in glucose degradation products, and (3) non-glucose PDF on peritoneal leukocyte recruitment in response to an inflammatory stimulus using intravital microscopy. The visceral peritoneum was exposed to EBSS, conventional lactate-buffered and bicarbonate/lactate-buffered glucose-based PDF and three lactate-buffered non-glucose PDF-icodextrin, amino acid-based PDF and amino acid/glycerol-based PDF. The number of rolling, adhering and extravasated leukocytes and leukocyte rolling velocity was assessed at different time intervals after stimulation with lipopolysaccharide (LPS). Exposure to LPS dissolved in EBSS dramatically increased the number of rolling, adhering and extravasated leukocytes and decreased leukocyte rolling velocity. Conventional PDF completely abolished LPS-induced leukocyte recruitment. Bicarbonate/lactate-buffered PDF only minimally affected the process of leukocyte recruitment, whereas icodextrin PDF resulted in partial inhibition of the immune response. The amino acid-based and the amino acid/glycerol-based PDF inhibited leukocyte recruitment to a similar extent as conventional PDF. Bicarbonate/lactate-buffered PDF has superior biocompatibility towards peritoneal host defense, in spite of its high glucose concentrations. Lactate-buffered non-glucose containing PDF has substantial inhibitory effects on leukocyte recruitment, indicating that the bioincompatibility of high lactate concentrations and/or low pH may not be underestimated. Copyright 2005 S. Karger AG, Basel.

  14. Meisoindigo, but not its core chemical structure indirubin, inhibits zebrafish interstitial leukocyte chemotactic migration.

    PubMed

    Ye, Baixin; Xiong, Xiaoxing; Deng, Xu; Gu, Lijuan; Wang, Qiongyu; Zeng, Zhi; Gao, Xiang; Gao, Qingping; Wang, Yueying

    2017-12-01

    Inflammatory disease is a big threat to human health. Leukocyte chemotactic migration is required for efficient inflammatory response. Inhibition of leukocyte chemotactic migration to the inflammatory site has been shown to provide therapeutic targets for treating inflammatory diseases. Our study was designed to discover effective and safe compounds that can inhibit leukocyte chemotactic migration, thus providing possible novel therapeutic strategy for treating inflammatory diseases. In this study, we used transgenic zebrafish model (Tg:zlyz-EGFP line) to visualize the process of leukocyte chemotactic migration. Then, we used this model to screen the hit compound and evaluate its biological activity on leukocyte chemotactic migration. Furthermore, western blot analysis was performed to evaluate the effect of the hit compound on the AKT or ERK-mediated pathway, which plays an important role in leukocyte chemotactic migration. In this study, using zebrafish-based chemical screening, we identified that the hit compound meisoindigo (25 μM, 50 μM, 75 μM) can significantly inhibit zebrafish leukocyte chemotactic migration in a dose-dependent manner (p = 0.01, p = 0.0006, p < 0.0001). Also, we found that meisoindigo did not affect the process of leukocyte reverse migration (p = 0.43). Furthermore, our results unexpectedly showed that indirubin, the core structure of meisoindigo, had no significant effect on zebrafish leukocyte chemotactic migration (p = 0.6001). Additionally, our results revealed that meisoindigo exerts no effect on the Akt or Erk-mediated signalling pathway. Our results suggest that meisoindigo, but not indirubin, is effective for inhibiting leukocyte chemotactic migration, thus providing a potential therapeutic agent for treating inflammatory diseases.

  15. Detection and characterization of carrier-mediated cationic amino acid transport in lysosomes of normal and cystinotic human fibroblasts. Role in therapeutic cystine removal

    SciTech Connect

    Pisoni, R.L.; Thoene, J.G.; Christensen, H.N.

    1985-04-25

    The discovery of a trans-stimulation property associated with lysine exodus from lysosomes of human fibroblasts has enabled us to characterize a system mediating the transport of cationic amino acids across the lysosomal membrane of human fibroblasts. The cationic amino acids arginine, lysine, ornithine, diaminobutyrate, histidine, 2-aminoethylcysteine, and the mixed disulfide of cysteine and cysteamine all caused trans-stimulation of the exodus of radiolabeled lysine from the lysosomal fraction of human fibroblasts at pH 6.5. In contrast, neutral and acidic amino acids did not affect the rate of lysine exodus. Trans-stimulation of lysine exodus was observed over the pH range from 5.5 to 7.6, was specific for the L-isomer of the cationic amino acid, and was intolerant to methylation of the alpha-amino group of the amino acid. The lysosomotropic amine, chloroquine, greatly retarded lysine exodus, whereas the presence of sodium ion was without effect. The specificity and lack of Na+ dependence of this lysosomal transport system is similar to that of System y+ present on the plasma membrane of human fibroblasts. An important mechanism by which cysteamine treatment of cystinosis allows cystine escape from lysosomes may be the ability of the mixed disulfide of cysteine and cysteamine formed by sulfhydryl-disulfide exchange to migrate by this newly discovered system mediating cationic amino acid transport.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. Acute Fluoxetine Treatment Induces Slow Rolling of Leukocytes on Endothelium in Mice

    PubMed Central

    Herr, Nadine; Mauler, Maximilian; Witsch, Thilo; Stallmann, Daniela; Schmitt, Stefanie; Mezger, Julius; Bode, Christoph; Duerschmied, Daniel

    2014-01-01

    Objective Activated platelets release serotonin at sites of inflammation where it acts as inflammatory mediator and enhances recruitment of neutrophils. Chronic treatment with selective serotonin reuptake inhibitors (SSRI) depletes the serotonin storage pool in platelets, leading to reduced leukocyte recruitment in murine experiments. Here, we examined the direct and acute effects of SSRI on leukocyte recruitment in murine peritonitis. Methods C57Bl/6 and Tph1−/− (Tryptophan hydroxylase1) mice underwent acute treatment with the SSRI fluoxetine or vehicle. Serotonin concentrations were measured by ELISA. Leukocyte rolling and adhesion on endothelium was analyzed by intravital microscopy in mesentery venules with and without lipopolysaccharide challenge. Leukocyte extravasation in sterile peritonitis was measured by flow cytometry of abdominal lavage fluid. Results Plasma serotonin levels were elevated 2 hours after fluoxetine treatment (0.70±0.1 µg/ml versus 0.27±0.1, p = 0.03, n = 14), while serum serotonin did not change. Without further stimulation, acute fluoxetine treatment increased the number of rolling leukocytes (63±8 versus 165±17/0.04 mm2min−1) and decreased their velocity (61±6 versus 28±1 µm/s, both p<0.0001, n = 10). In Tph1−/− mice leukocyte rolling was not significantly influenced by acute fluoxetine treatment. Stimulation with lipopolysaccharide decreased rolling velocity and induced leukocyte adhesion, which was enhanced after fluoxetine pretreatment (27±3 versus 36±2/0.04 mm2, p = 0.008, n = 10). Leukocyte extravasation in sterile peritonitis, however, was not affected by acute fluoxetine treatment. Conclusions Acute fluoxetine treatment increased plasma serotonin concentrations and promoted leukocyte-endothelial interactions in-vivo, suggesting that serotonin is a promoter of acute inflammation. E-selectin was upregulated on endothelial cells in the presence of serotonin, possibly explaining the observed

  1. Leukocyte nucleus segmentation and nucleus lobe counting.

    PubMed

    Chan, Yung-Kuan; Tsai, Meng-Hsiun; Huang, Der-Chen; Zheng, Zong-Han; Hung, Kun-Ding

    2010-11-12

    Leukocytes play an important role in the human immune system. The family of leukocytes is comprised of lymphocytes, monocytes, eosinophils, basophils, and neutrophils. Any infection or acute stress may increase or decrease the number of leukocytes. An increased percentage of neutrophils may be caused by an acute infection, while an increased percentage of lymphocytes can be caused by a chronic bacterial infection. It is important to realize an abnormal variation in the leukocytes. The five types of leukocytes can be distinguished by their cytoplasmic granules, staining properties of the granules, size of cell, the proportion of the nuclear to the cytoplasmic material, and the type of nucleolar lobes. The number of lobes increased when leukemia, chronic nephritis, liver disease, cancer, sepsis, and vitamin B12 or folate deficiency occurred. Clinical neutrophil hypersegmentation has been widely used as an indicator of B12 or folate deficiency.Biomedical technologists can currently recognize abnormal leukocytes using human eyes. However, the quality and efficiency of diagnosis may be compromised due to the limitations of the biomedical technologists' eyesight, strength, and medical knowledge. Therefore, the development of an automatic leukocyte recognition system is feasible and necessary. It is essential to extract the leukocyte region from a blood smear image in order to develop an automatic leukocyte recognition system. The number of lobes increased when leukemia, chronic nephritis, liver disease, cancer, sepsis, and vitamin B12 or folate deficiency occurred. Clinical neutrophil hypersegmentation has been widely used as an indicator of B12 or folate deficiency. The purpose of this paper is to contribute an automatic leukocyte nuclei image segmentation method for such recognition technology. The other goal of this paper is to develop the method of counting the number of lobes in a cell nucleus. The experimental results demonstrated impressive segmentation accuracy

  2. 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

  3. 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.

  4. Interactions between stably rolling leukocytes in vivo

    NASA Astrophysics Data System (ADS)

    King, Michael R.; Ruscio, Aimee D.; Kim, Michael B.; Sarelius, Ingrid H.

    2005-03-01

    We have characterized the two-dimensional spatial dependence of the hydrodynamic interactions between two adhesively rolling leukocytes in a live venule in the mouse cremaster muscle. Two rolling leukocytes were observed to slow each other down when rolling together in close proximity due to mutual sheltering from the external blood flow in the vessel lumen. A previous study of leukocyte rolling interactions using carbohydrate-coated beads in a parallel-plate flow chamber and a detailed computer model of adhesion in a multicellular environment is in qualitative agreement with the current in vivo results.

  5. 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.

  6. 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.

  7. Chromate transport in human leukocytes.

    PubMed

    Lilien, D L; Spivak, J L; Goldman, I D

    1970-08-01

    Chromium is a trace metal of importance in human physiology and, in addition, as 51-chromate, has been extensively used as a label in the study of blood cell pool sizes and intravascular kinetics. The transport characteristics of 51-chromate were investigated in normal human leukocytes. Chromate uptake is unidirectional over a 1 hr incubation with extracellular chromate concentrations up to 200 mumoles/liter. Under these conditions, intracellular 51-chromium is in a form in which it is nonexchangeable. Influx is temperature sensitive with a Q(10) of approximately 2 and may be energy dependent since a variety of metabolic poisons strongly inhibit uptake. The unidirectional influx of chromate follows Michaelis-Menten kinetics; the maximum velocity is 52 mmumoles/g dry weight of cells per min and the chromate concentration at which influx velocity is half maximal is 87 mumoles/liter. This transport mechanism is highly specific for chromate; other divalent tetrahedral anions only slightly inhibit influx at concentrations up to 10 times that of chromate. Metavanadate, however, competitively inhibits chromate influx at equimolar concentrations. Exposure of cells to unlabeled chromate leads to inhibition of subsequent influx of 51-chromate. It is suggested that this is due to a primary inhibitory effect of chromate on cellular energy metabolism.

  8. Chromate transport in human leukocytes

    PubMed Central

    Lilien, David L.; Spivak, Jerry L.; Goldman, I. David

    1970-01-01

    Chromium is a trace metal of importance in human physiology and, in addition, as 51-chromate, has been extensively used as a label in the study of blood cell pool sizes and intravascular kinetics. The transport characteristics of 51-chromate were investigated in normal human leukocytes. Chromate uptake is unidirectional over a 1 hr incubation with extracellular chromate concentrations up to 200 μmoles/liter. Under these conditions, intracellular 51-chromium is in a form in which it is nonexchangeable. Influx is temperature sensitive with a Q10 of approximately 2 and may be energy dependent since a variety of metabolic poisons strongly inhibit uptake. The unidirectional influx of chromate follows Michaelis-Menten kinetics; the maximum velocity is 52 mμmoles/g dry weight of cells per min and the chromate concentration at which influx velocity is half maximal is 87 μmoles/liter. This transport mechanism is highly specific for chromate; other divalent tetrahedral anions only slightly inhibit influx at concentrations up to 10 times that of chromate. Metavanadate, however, competitively inhibits chromate influx at equimolar concentrations. Exposure of cells to unlabeled chromate leads to inhibition of subsequent influx of 51-chromate. It is suggested that this is due to a primary inhibitory effect of chromate on cellular energy metabolism. PMID:5431664

  9. 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

  10. The multiple faces of leukocyte interstitial migration

    PubMed Central

    Lämmermann, Tim; Germain, Ronald N.

    2014-01-01

    Spatiotemporal control of leukocyte dynamics within tissues is critical for successful innate and adaptive immune responses. Homeostatic trafficking and coordinated infiltration into and within sites of inflammation and infection rely on signaling in response to extracellular cues that in turn controls a variety of intracellular protein networks regulating leukocyte motility, migration, chemotaxis, positioning, and cell–cell interaction. In contrast to mesenchymal cells, leukocytes migrate in an amoeboid fashion by rapid cycles of actin polymerization and actomyosin contraction, and their migration in tissues is generally referred to as low adhesive and nonproteolytic. The interplay of actin network expansion, contraction, and adhesion shapes the exact mode of amoeboid migration, and in this review, we explore how leukocyte subsets potentially harness the same basic biomechanical mechanisms in a cell-type-specific manner. Most of our detailed understanding of these processes derives from in vitro migration studies in three-dimensional gels and confined spaces that mimic geometrical aspects of physiological tissues. We summarize these in vitro results and then critically compare them to data from intravital imaging of leukocyte interstitial migration in mouse tissues. We outline the technical challenges of obtaining conclusive mechanistic results from intravital studies, discuss leukocyte migration strategies in vivo, and present examples of mode switching during physiological interstitial migration. These findings are also placed in the context of leukocyte migration defects in primary immunodeficiencies. This overview of both in vitro and in vivo studies highlights recent progress in understanding the molecular and biophysical mechanisms that shape robust leukocyte migration responses in physiologically complex and heterogeneous environments. PMID:24573488

  11. 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

  12. Leukocyte Trafficking to the Small Intestine and Colon

    PubMed Central

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

    2016-01-01

    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. PMID:26551552

  13. 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.

  14. 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.

  15. Leukocyte activation by triglyceride-rich lipoproteins.

    PubMed

    Alipour, Arash; van Oostrom, Antonie J H H M; Izraeljan, Alisa; Verseyden, Caroline; Collins, Jennifer M; Frayn, Keith N; Plokker, Thijs W M; Elte, Jan Willem F; Castro Cabezas, Manuel

    2008-04-01

    Postprandial lipemia has been linked to atherosclerosis and inflammation. Because leukocyte activation is obligatory for atherogenesis, leukocyte activation by triglyceride-rich lipoproteins (TRLs) was investigated. The expression of CD11b and CD66b after incubation with glucose and native and artificial TRLs (NTRL and ATRL) in vivo and in vitro was evaluated by flowcytometry. Oral fat loading tests showed an increased expression of CD11b on monocytes and neutrophils and CD66b on neutrophils. In 11 volunteers, postprandial leukocytes became enriched with meal-derived fatty acids ([1-(13)C]16:0) suggesting uptake of exogenous fat. ApoB binding on leukocytes measured by flowcytometry in 65 subjects was highest on neutrophils and monocytes suggesting adherence of apoB-containing lipoproteins. Physiological concentrations of TRLs showed 62% increased neutrophil CD11b and a dose-dependent increased monocyte CD11b up to 84% in vitro. Incubations with lipid emulsions in the hypertriglyceridemic range showed a 5-fold increased monocyte CD11b expression, which was higher than the positive control (fMLP), and a dose-dependent 2- to 3-fold increased neutrophil CD11b and CD66b. The oxidative scavenger DMTU decreased the neutrophil CD66b expression by 36%. Acute hypertriglyceridemia is a leukocyte activator most likely by direct interaction between TRLs and leukocytes and uptake of fatty acids. TG-mediated leukocyte activation is an alternative proinflammatory and proatherogenic mechanism of hypertriglyceridemia in part associated to the generation of oxidative stress.

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. High leukocyte mtDNA content contributes to poor prognosis through ROS-mediated immunosuppression in hepatocellular carcinoma patients

    PubMed Central

    Zhou, Feng; Zhou, Xingchun; Chen, Yibing; Guo, Xu; Li, Jibin; Huang, Qichao; Yang, Yefa; Lyu, Zhuomin; Zhang, Hongxin; Xing, Jinliang

    2016-01-01

    Compelling epidemiological evidences indicate a significant association between leukocyte mitochondrial DNA (mtDNA) content and incidence risk of several malignancies, including hepatocellular carcinoma (HCC). However, whether leukocyte mtDNA content affect prognosis of HCC patients and underlying mechanism has never been explored. In our study, leukocyte mtDNA content was measured in 618 HCC patients and its prognostic value was analyzed. Moreover, we detected the immunophenotypes of peripheral blood mononuclear cells (PBMCs) and plasma concentrations of several cytokines in 40 HCC patients and assessed the modulating effects of mtDNA content on immunosuppression in cell models. Our results showed that HCC patients with high leukocyte mtDNA content exhibited a significantly worse recurrence-free survival (RFS) and overall survival (OS) than those with low leukocyte mtDNA content. Leukocyte mtDNA content and TNM stage exhibited a notable joint effect in prognosis prediction. Furthermore, we found that patients with high leukocyte mtDNA content exhibited a higher frequency of CD4+CD25+FOXP3+ regulatory T (Treg) cells and lower frequency of NK cells in PBMCs and had higher TGF-β1 and lower TNF-α and IFN-γ plasma concentration when compared with those with low leukocyte mtDNA content, which suggests an immunosuppressive status. High leukocyte mtDNA content significantly enhanced the ROS-mediated secretion of TGF-β1, which accounted for higher Treg and lower NK frequency in PBMCs. In a conclusion, our study for the first time demonstrates that leukocyte mtDNA content is an independent prognostic marker complementing TNM stage and associated with an ROS-mediated immunosuppressive phenotype in HCC patients. PMID:26985767

  1. 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.

  2. Bactericidal mechanisms of human breast milk leukocytes.

    PubMed Central

    Johnson, D F; France, G L; Marmer, D J; Steele, R W

    1980-01-01

    The functional capacity of human breast milk phagocytes was evaluated with both bactericidal and biochemical assays. Acridine orange was used as a vital stain for bacteria to directly visualize phagocytosis and killing. Bactericidal capabilities were further examined by colony count and chemiluminescent methods. Cytocentrifuged specimens stained for myeloperoxidase exhibited enzyme activity in breast milk leukocytes equal to that of peripheral neutrophils. A radioisotopic assay of hexose monophosphate shunt activity demonstrated metabolic activity in breast milk leukocytes greater than that in peripheral blood neutrophils. However, the chemiluminescent response of breast cells was negligible, apparently the result of quenching secondary to fat present in the milk; preincubation of human blood leukocytes with the fatty layer of breast milk produced similar inhibition in the chemiluminescence assay. By most parameters breast milk phagocytes are at least equal to blood neutrophils. PMID:6249738

  3. 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.

  4. Leukocytic Promotion of Prostate Cellular Proliferation

    PubMed Central

    McDowell, Kristy L.; Begley, Lesa A.; Mor-Vaknin, Nirit; Markovitz, David M.; Macoska, Jill A.

    2011-01-01

    BACKGROUND Histological evidence of pervasive inflammatory infiltrate has been noted in both benign prostatic hyperplasia/hypertrophy (BPH) and prostate cancer (PCa). Cytokines known to attract particular leukocyte subsets are secreted from prostatic stroma consequent to aging and also from malignant prostate epithelium. Therefore, we hypothesized that leukocytes associated with either acute or chronic inflammation attracted to the prostate consequent to aging or tumorigenesis may promote the abnormal cellular proliferation associated with BPH and PCa. METHODS An in vitro system designed to mimic the human prostatic microenvironment incorporating prostatic stroma (primary and immortalized prostate stromal fibroblasts), epithelium (N15C6, BPH-1, LNCaP, and PC3 cells), and inflammatory infiltrate (HL-60 cells, HH, and Molt-3 T-lymphocytes) was developed. Modified Boyden chamber assays were used to test the ability of prostate stromal and epithelial cells to attract leukocytes and to test the effect of leukocytes on prostate cellular proliferation. Antibody arrays were used to identify leukocyte-secreted cytokines mediating prostate cellular proliferation. RESULTS Leukocytic cells migrated towards both prostate stromal and epithelial cells. CD4+ T-lymphocytes promoted the proliferation of both transformed and non-transformed prostate epithelial cell lines tested, whereas CD8+ T-lymphocytes as well as dHL-60M macrophagic and dHL-60N neutrophilic cells selectively promoted the proliferation of PCa cells. CONCLUSIONS The results of these studies show that inflammatory cells can be attracted to the prostate tissue microenvironment and can selectively promote the proliferation of non-transformed or transformed prostate epithelial cells, and are consistent with differential role(s) for inflammatory infiltrate in the etiologies of benign and malignant proliferative disease in the prostate. PMID:19866464

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. Molecular characterization of swine leukocyte antigen (SLA) class II genes in outbred pig populations

    USDA-ARS?s Scientific Manuscript database

    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...

  10. In vivo temporal and spatial profile of leukocyte adhesion and migration after experimental traumatic brain injury in mice

    PubMed Central

    2013-01-01

    Background Leukocytes are believed to be involved in delayed cell death following traumatic brain injury (TBI). However, data demonstrating that blood-borne inflammatory cells are present in the injured brain prior to the onset of secondary brain damage have been inconclusive. We therefore investigated both the interaction between leukocytes and the cerebrovascular endothelium using in vivo imaging and the accumulation of leukocytes in the penumbra following experimentally induced TBI. Methods Experimental TBI was induced in C57/Bl6 mice (n = 42) using the controlled cortical impact (CCI) injury model, and leukocyte-endothelium interactions (LEI) were quantified using both intravital fluorescence microscopy (IVM) of superficial vessels and 2-photon microscopy of cortical vessels for up to 14 h post-CCI. In a separate experimental group, leukocyte accumulation and secondary lesion expansion were analyzed in mice that were sacrificed 15 min, 2, 6, 12, 24, or 48 h after CCI (n = 48). Finally, leukocyte adhesion was blocked with anti-CD18 antibodies, and the effects on LEI and secondary lesion expansion were determined 16 (n = 12) and 24 h (n = 21), respectively, following TBI. Results One hour after TBI leukocytes and leukocyte-platelet aggregates started to roll on the endothelium of pial venules, whereas no significant LEI were observed in pial arterioles or in sham-operated mice. With a delay of >4 h, leukocytes and aggregates did also firmly adhere to the venular endothelium. In deep cortical vessels (250 μm) LEIs were much less pronounced. Transmigration of leukocytes into the brain parenchyma only became significant after the tissue became necrotic. Treatment with anti-CD18 antibodies reduced adhesion by 65%; however, this treatment had no effect on secondary lesion expansion. Conclusions LEI occurred primarily in pial venules, whereas little or no LEI occurred in arterioles or deep cortical vessels. Inhibiting LEI did not affect secondary lesion

  11. 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

  12. Gravity sedimentation of leukocytes is partially independent from erythrocyte sedimentation.

    PubMed

    Bogar, L L; Tarsoly, P P

    2006-01-01

    Leukocyte function tests are suitable for monitoring the severity of chronic inflammatory and acute infectious diseases. The tests usually require time consuming leukocyte separation techniques while the original character of leukocytes can substantially alter. In contrast, we noted that gravity sedimentation properties of leukocytes is simple to measure and it also reflects non-specific inflammatory reactions of leukocytes. Our novel test is named leukocyte antisedimentation rate (LAR) which is measured by leukocyte counting in the upper (U) and lower (L) half of the sedimentation blood column after one-hour gravity sedimentation of the whole blood. The formula LAR=100.(U-L)/(U+L) is used to calculate the percentage of leukocytes crosses the middle line of sedimentation blood column upward during one-hour sedimentation (normal range<15%, inter-assay coefficient of variation<5%). In this study we found that in vitro pre-treatment of septic patients' blood samples with protamine, lidocaine and prednisolone decreased leukocyte antisedimentation rate in a concentration dependent manner without effecting erythrocyte sedimentation rate. Leukocyte adherence was measured by the retention rate of leukocytes in a nylon fibre column. There was a significant positive correlation between leukocyte antisedimentation rate and leukocyte adherence (p<0.01), hematocrit (p<0.05), erythrocyte sedimentation rate (p<0.05) when blood samples of 35 healthy individuals were analysed. We concluded that leukocyte antisedimentation rate in septic patients is significantly elevated comparing to healthy controls and as a bedside test it can reflect leukocyte involvement in infections. In vitro protamine, lidocaine and prednisolone pre-treatment of septic patients' blood samples indicates that leukocyte antisedimentation process is partially independent from the ongoing erythrocyte sedimentation.

  13. Endothelial Src kinase regulates membrane recycling from the lateral border recycling compartment during leukocyte transendothelial migration.

    PubMed

    Dasgupta, Bidisha; Muller, William A

    2008-12-01

    When leukocytes cross endothelial cells during the inflammatory response, membrane from the recently described lateral border recycling compartment (LBRC) is selectively targeted around diapedesing leukocytes. This "targeted recycling" is critical for leukocyte transendothelial migration. Blocking homophilic PECAM interactions between leukocytes and endothelial cells blocks targeted recycling from the LBRC and blocks diapedesis. However, the cellular signaling pathways that trigger targeted recycling are not known. We show that targeted recycling from the LBRC is dependent on Src kinase. The selective Src kinase inhibitor PP2 blocked targeted recycling and blocked diapedesis by over 70%. However, Src kinase inhibition did not affect the structure or normal constitutive recycling of membrane from the LBRC in the absence of leukocytes. PECAM, a Src kinase substrate, traffics between the LBRC and the endothelial surface at the cell border. However, virtually all of the PECAM in the cell that was phosphorylated on tyrosine residues was found in the LBRC. These findings demonstrate that Src kinase activity is critical for the targeted recycling of membrane from the LBRC to the site of transendothelial migration and that the PECAM in the LBRC is qualitatively different from the PECAM on the surface of endothelial cells.

  14. 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.

  15. 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.

  16. 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.

  17. Positive indium-111 leukocyte scan in Nocardia brain abscess

    SciTech Connect

    Bauman, J.M.; Osenbach, R.; Hartshorne, M.F.; Youngblood, L.; Crooks, L.; Landry, A.J.; Cawthon, M.A.

    1986-01-01

    We report a case of clinically unsuspected nocardia brain abscess detected by /sup 111/In-labeled autologous leukocytes. Clinical and computed tomographic findings supported the diagnosis of primary or metastatic tumor and the patient was treated with dexamethasone for 30 days prior to the leukocyte scan. Labeled leukocytes may provide a sensitive discriminator for brain abscess despite previous therapy with steroids.

  18. Differential sensitivity of human leukocyte subpopulations to ultraviolet light

    SciTech Connect

    Spielberg, H.; June, C.; Cereb, N.; Nystrom-Rosander, C.; Deeg, H.J.

    1989-02-01

    Ultraviolet (UV) light, in addition to its effect on DNA, can cause cell-membrane alterations and modification of cell surface antigens, and interfere with interactions of different cell populations, antigen presentation, and cytokine release. The mechanisms are still poorly understood. However, current data indicate that UV irradiation profoundly affects donor/host interactions in transplantation models by altering the immunogenicity of donor tissue and the immune response of the recipient. In the present study we used intracellular Ca{sup 2+} (Ca{sup 2+}i) mobilization as a parameter of how various cell populations responded to UV light. These data show that UV treatment profoundly affects leukocytes. There is a preferential inhibition of lymphocytes compared to hemopoietic precursor cells and of CD{sup 8+} compared to CD{sup 8{minus}} negative T-cells.

  19. HDAC inhibitors: modulating leukocyte differentiation, survival, proliferation and inflammation.

    PubMed

    Sweet, Matthew J; Shakespear, Melanie R; Kamal, Nabilah A; Fairlie, David P

    2012-01-01

    Therapeutic effects of histone deacetylase (HDAC) inhibitors in cancer models were first linked to their ability to cause growth arrest and apoptosis of tumor cells. It is now clear that these agents also have pleiotropic effects on angiogenesis and the immune system, and some of these properties are likely to contribute to their anti-cancer activities. It is also emerging that inhibitors of specific HDACs affect the differentiation, survival and/or proliferation of distinct immune cell populations. This is true for innate immune cells such as macrophages, as well as cells of the acquired immune system, for example, T-regulatory cells. These effects may contribute to therapeutic profiles in some autoimmune and chronic inflammatory disease models. Here, we review our current understanding of how classical HDACs (HDACs 1-11) and their inhibitors impact on differentiation, survival and proliferation of distinct leukocyte populations, as well as the likely relevance of these effects to autoimmune and inflammatory disease processes. The ability of HDAC inhibitors to modulate leukocyte survival may have implications for the rationale of developing selective inhibitors as anti-inflammatory drugs.

  20. Pentoxifylline modulation of plasma membrane functions in human polymorphonuclear leukocytes.

    PubMed Central

    Hand, W L; Butera, M L; King-Thompson, N L; Hand, D L

    1989-01-01

    Pentoxifylline is known to have major effects on cell membrane function in mammalian cells, including human leukocytes. The protective effects of this agent in animal models of infection and inflammation may be due to alterations in phagocyte (neutrophil and macrophage) function. However, the exact mechanism of action of pentoxifylline is unknown. In this study, we evaluated the effect of the drug on several membrane-associated activities in human polymorphonuclear neutrophils and investigated possible mechanisms for the observed changes in neutrophil function. Pentoxifylline inhibited ingestion of microbial particles (Staphylococcus aureus and zymosan); decreased superoxide generation activated by zymosan, formyl-methionyl-leucyl-phenylalanine, and concanavalin A (but not phorbol myristate acetate); and decreased uptake (transport) of adenosine stimulated by formyl-methionyl-leucyl-phenylalanine and zymosan. In contrast, pentoxifylline actually increased clindamycin uptake in zymosan-stimulated polymorphonuclear neutrophils. However, pentoxifylline had no effect on uptake of adenosine or clindamycin in unstimulated neutrophils. In comparison with known inhibitors of nucleoside transport (nitrobenzylthioinosine and dipyridamole), the results suggested that pentoxifylline does not bind to membrane nucleoside transport receptors. At concentrations which inhibit neutrophil function, pentoxifylline activity is not mediated through external membrane nucleoside regulatory sites. Thus, pentoxifylline affects the activation signal chain at a point beyond the membrane receptors. Whatever its precise mechanism of action, pentoxifylline has a striking modulatory effect on cell membrane-associated responses in stimulated leukocytes and may prove useful for control of injurious inflammatory states. PMID:2553608

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. Leukocyte set points in metabolic disease.

    PubMed

    Odegaard, Justin I; Chawla, Ajay

    2012-01-01

    Vertebrate tissues comprise precise admixtures of parenchymal and hematopoietic cells, whose interactions are vital to proper tissue function. By regulating this interaction, vertebrates are able to mitigate environmental stress and coordinate dramatic physiologic adaptations. For instance, under conditions of chronic nutrient excess, leukocyte recruitment and activation increase in an effort to decrease excess nutrient storage and alleviate adipocyte stress. While basal equilibria may be reestablished upon normalization of nutrient intake, a new set point characterized by insulin resistance and chronic inflammation is established if the stress persists. Consequently, although this response is adaptive in settings of acute overfeeding and infection, it has catastrophic health consequences in the modern context of obesity. Understanding how leukocyte set points (numbers and activation status) are established, maintained, and regulated in tissues is, thus, critical to our understanding of, and intervention in, chronic metabolic diseases, such as obesity and diabetes.

  9. 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.

  10. 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.

  11. 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

  12. [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.

  13. The effect of Intravenous Immunoglobulin (IVIG) on \\textit{ex vivo} activation of human leukocytes.

    PubMed

    Basyreva, Liliya Yu; Brodsky, Ilya B; Gusev, Alexander A; Zhapparova, Olga N; Mikhalchik, Elena V; Gusev, Sergey A; Shor, Dana Ben-Ami; Dahan, Shani; Blank, Miri; Shoenfeld, Yehuda

    2016-01-01

    Intravenous immunoglobulin (IVIG) has been widely used to treat various conditions, including inflammatory and autoimmune diseases. IVIG has been shown to have a direct influence on neutrophils, eosinophils and lymphocytes. However, many aspects IVIG's effect on neutrophils activation still remain unknown. To evaluate the effect of IVIG on PMA-induced activation of neutrophils, with and without priming with TNF-α, in a series of in vitro experiments performed on whole blood. Our data coincided with well-known literature indicating that the effect of phorbol 12-myristate 13-acetate (PMA) on human leukocytes includes activation of neutrophils, monocytes and eosinophils, increase of chemiluminescence (CL) and induction of netosis, resulting in assembly of traps. In presence of IVIG (10 mg/mL), CL was reduced by 25% in response to PMA compared to PMA-induced leukocyte activation without IVIG. Decreasing IVIG concentration to 1 mg/mL and below did not inhibit PMA-induced activation of CL.PMA-induced activation after TNF-α priming resulted in approximately 50% increase of amplitude of CL response to PMA. Moreover, maximum CL was reached by minute 5, which was more rapid than in the absence of TNF-α-priming (in this case maximum CL was reached by minute 15).The IVIG concentrations did not affect morphological changes of leukocytes after sequential addition of TNF-α and PMA. IVIG had no effect on leukocyte content and on PMA-induced CL of primed leukocytes.Addition of IVIG under TNF-α priming significantly increased the number of traps in the smears in response to PMA activation. Of note, such an increase in the number of traps was depended on the IVIG concentration in plasma. In conclusion, we suggest that IVIG is able to reduce the degradation of traps under priming with TNF-α. Moreover, IVIG might switch the activation of primed leukocytes to netosis.

  14. 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

  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. 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.

  17. 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.

  18. A computational study of leukocyte adhesion and its effect on flow pattern in microvessels.

    PubMed

    Pappu, Vijay; Doddi, Sai K; Bagchi, Prosenjit

    2008-09-21

    Three-dimensional computational modeling and simulation are presented on the adhesive rolling of deformable leukocytes over a P-selectin coated surface in parabolic shear flow in microchannels. The computational model is based on the immersed boundary method for cell deformation and Monte Carlo simulation for receptor/ligand interaction. The simulations are continued for at least 1s of leukocyte rolling during which the instantaneous quantities such as cell deformation index, cell/substrate contact area, and fluid drag remain statistically stationary. The characteristic 'stop-and-go' motion of rolling leukocytes, and the 'tear-drop' shape of adherent leukocytes as observed in experiments are reproduced by the simulations. We first consider the role of cell deformation and cell concentration on rolling characteristics. We observe that compliant cells roll slower and more stably than rigid cells. Our simulations agree with previous in vivo observation that the hydrodynamic interactions between nearby leukocytes affect cell rolling, and that the rolling velocity decreases inversely with the separation distance, irrespective of cell deformability. We also find that cell deformation decreases, and the cells roll more stably with reduced velocity fluctuation, as the cell concentration is increased. However, the effect of nearby cells on the rolling characteristics is found to be more significant for rigid cells than compliant cells. We then address the effect of cell deformability and rolling velocity on the flow resistance due to, and the fluid drag on, adherent leukocytes. While several earlier computational works have addressed this problem, two key features of leukocyte adhesion, such as cell deformation and rolling, were often neglected. Our results suggest that neglecting cell deformability and rolling velocity may significantly overpredict the flow resistance and drag force. Increasing the cell concentration is shown to increase the flow resistance and reduce the

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. [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.

  4. 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.

  5. 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.

  6. Coronin 1 is dispensable for leukocyte recruitment and liver injury in concanavalin A-induced hepatitis.

    PubMed

    Siegmund, Kerstin; Lee, Woo-Yong; Tchang, Vincent S; Stiess, Michael; Terracciano, Luigi; Kubes, Paul; Pieters, Jean

    2013-06-01

    Coronin 1, a member of the evolutionary conserved coronin protein family, is highly expressed in all leukocytes. In mice and human, genetic inactivation of coronin 1 results in immuno-deficiencies that are linked to a strong reduction of naïve T cell numbers in peripheral organs, while memory/effector T cells, B cells, monocytes and neutrophils are less or not at all affected. Whether or not coronin 1 is important for leukocyte functions such as migration and phagocytosis has been a matter of debate. The current work addresses coronin 1-dependent leukocyte function by analyzing the response of coronin 1-deficient mice in a model of concanavalin A (Con A)-induced liver injury. Histological evaluation and determination of serum liver enzyme levels showed that coronin 1-deficient mice develop signs of acute hepatitis similar to Con A-treated wild type mice despite a reduced activation of T cells in the absence of coronin 1. Furthermore, analysis by intravital microscopy following Con A stimulation revealed that Gr-1+ neutrophils and CD4+ T cell adhesion in the post-sinusoidal venules increased in wild type as well as in coronin 1-deficient mice. These results suggest that coronin 1, while important for naïve T cell survival, is dispensable for other leukocyte function under inflammatory conditions in vivo. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. 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.

  8. Leukocyte integrin Mac-1 regulates thrombosis via interaction with platelet GPIbα

    PubMed Central

    Wang, Yunmei; Gao, Huiyun; Shi, Can; Erhardt, Paul W.; Pavlovsky, Alexander; A. Soloviev, Dmitry; Bledzka, Kamila; Ustinov, Valentin; Zhu, Liang; Qin, Jun; Munday, Adam D.; Lopez, Jose; Plow, Edward; Simon, Daniel I.

    2017-01-01

    Inflammation and thrombosis occur together in many diseases. The leukocyte integrin Mac-1 (also known as integrin αMβ2, or CD11b/CD18) is crucial for leukocyte recruitment to the endothelium, and Mac-1 engagement of platelet GPIbα is required for injury responses in diverse disease models. However, the role of Mac-1 in thrombosis is undefined. Here we report that mice with Mac-1 deficiency (Mac-1−/−) or mutation of the Mac-1-binding site for GPIbα have delayed thrombosis after carotid artery and cremaster microvascular injury without affecting parameters of haemostasis. Adoptive wild-type leukocyte transfer rescues the thrombosis defect in Mac-1−/− mice, and Mac-1-dependent regulation of the transcription factor Foxp1 contributes to thrombosis as evidenced by delayed thrombosis in mice with monocyte-/macrophage-specific overexpression of Foxp1. Antibody and small-molecule targeting of Mac-1:GPIbα inhibits thrombosis. Our data identify a new pathway of thrombosis involving leukocyte Mac-1 and platelet GPIbα, and suggest that targeting this interaction has anti-thrombotic therapeutic potential with reduced bleeding risk. PMID:28555620

  9. Leukocyte integrin Mac-1 regulates thrombosis via interaction with platelet GPIbα.

    PubMed

    Wang, Yunmei; Gao, Huiyun; Shi, Can; Erhardt, Paul W; Pavlovsky, Alexander; A Soloviev, Dmitry; Bledzka, Kamila; Ustinov, Valentin; Zhu, Liang; Qin, Jun; Munday, Adam D; Lopez, Jose; Plow, Edward; Simon, Daniel I

    2017-05-30

    Inflammation and thrombosis occur together in many diseases. The leukocyte integrin Mac-1 (also known as integrin αMβ2, or CD11b/CD18) is crucial for leukocyte recruitment to the endothelium, and Mac-1 engagement of platelet GPIbα is required for injury responses in diverse disease models. However, the role of Mac-1 in thrombosis is undefined. Here we report that mice with Mac-1 deficiency (Mac-1(-/-)) or mutation of the Mac-1-binding site for GPIbα have delayed thrombosis after carotid artery and cremaster microvascular injury without affecting parameters of haemostasis. Adoptive wild-type leukocyte transfer rescues the thrombosis defect in Mac-1(-/-) mice, and Mac-1-dependent regulation of the transcription factor Foxp1 contributes to thrombosis as evidenced by delayed thrombosis in mice with monocyte-/macrophage-specific overexpression of Foxp1. Antibody and small-molecule targeting of Mac-1:GPIbα inhibits thrombosis. Our data identify a new pathway of thrombosis involving leukocyte Mac-1 and platelet GPIbα, and suggest that targeting this interaction has anti-thrombotic therapeutic potential with reduced bleeding risk.

  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. Effects of cadmium on metallothionein levels in human peripheral blood leukocytes: a comparison with zinc.

    PubMed

    Yurkow, E J; DeCoste, C J

    1999-11-12

    Metallothioneins (MT) are low-molecular-weight, cysteine-rich proteins that are induced in response to a variety of chemical stresses and therefore can be used to assess human exposure to environmental agents. In the current study, flow cytometry was used to characterize the basal and cadmium-induced expression of MT in the three major leukocyte populations of human peripheral blood. In the analysis, monocytes were the most sensitive leukocytes to this toxic metal, with significant increases in cellular MT levels being detected at concentrations of cadmium as low as 0.1 microM (24 h). The lymphocyte population also exhibited pronounced treatment-associated elevations in cellular MT, while the granulocyte population was found to be nonresponsive. Although both CdCl2 (3 microM) and ZnCl2 (50 microM) induced MT expression in monocytes to a similar degree and did not affect the expression of this protein in granulocytes, cadmium but not zinc treatment induced dramatic increases in MT levels of lymphocytes. Our results indicate that cellular MT protein levels, as determined by this flow cytometric method, may be used to characterize the differential responsiveness of the major human leukocyte subpopulations to transitional metals. It is evident from the current work that the responsiveness of all peripheral blood leukocyte populations should be analyzed in exposure assessment studies.

  13. Changes in peripheral blood leukocyte populations in pigs with naturally occurring exudative epidermitis.

    PubMed

    Nofrarías, M; Pujols, J; Segalés, J; Gibert, X; Majó, N

    2006-10-01

    The objective of the study was to analyze changes in peripheral blood leukocyte subsets in cases of naturally occurring exudative epidermitis (EE) in pigs. Five of ten piglets developed the chronic clinical form of EE 2-5 days after weaning (PW). Blood samples were obtained at 7, 14 and 21 days from both normal and clinically affected piglets for routine haematology and for the determination of CD45, CD21, CD4, CD8 and gammadeltaTCR cell markers by flow cytometry. When compared with clinically normal piglets EE affected pigs showed significantly decreased values of monocytes at 14 and 21 days PW, and increased numbers of neutrophils and leukocytes at 21 days PW. The EE affected pigs also had an early significant CD4(+) and CD8(high+) T lymphocyte proliferative response at 7 days PW. However affected pigs had a significantly reduced number of B (CD21(+)) and gammadeltaTCR(+) T lymphocytes in blood at 21 days PW. Although all values remained within the normal range, the significant differences in some peripheral blood leukocyte subsets between the two groups of piglets suggest that the generalised cutaneous infection with Staphylococcus hyicus is severe enough to induce a systemic inflammatory and immune responses.

  14. Leukocyte involvement in renal reperfusion-induced liver damage.

    PubMed

    Khastar, Hossein; Kadkhodaee, Mehri; Sadeghipour, Hamid Reza; Seifi, Behjat; Hadjati, Jamshid; Delavari, Fatemeh; Soleimani, Manoocher

    2011-01-01

    Renal ischemia-reperfusion (IR) induces organ damage in remote organs. The aim of this study was to assess the role of leukocytes in the induction of liver damage after renal IR injury. Inbred mice were subjected to either sham operation or bilateral renal IR injury (60 min ischemia followed by 3 h reperfusion). Mice were then anesthetized for collection of leukocytes by heart puncture. Isolated leukocytes were transferred to two other groups: intact recipient mice that received leukocytes from IR mice and intact recipient mice that received leukocytes from sham-operated control mice. After 24 h, recipient mice were anesthetized and samples were collected. Alanine aminotransferase, aspartate aminotransferase, and hepatic malondialdehyde increased significantly, and hepatic glutathione decreased significantly in intact recipient mice that received leukocytes from IR mice in comparison with intact recipient mice that received leukocytes from sham-operated control mice. Loss of normal liver architecture, cytoplasmic vacuolization, and focal infiltration of leukocytes were seen. These results suggest that leukocytes are one of the possible factors that contribute to liver damage after renal IR injury and this damage is partly due to the induction of oxidative stress.

  15. 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.

  16. 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.

  17. 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

  18. Computerized detection of leukocytes in microscopic leukorrhea images.

    PubMed

    Zhang, Jing; Zhong, Ya; Wang, Xiangzhou; Ni, Guangming; Du, Xiaohui; Liu, Juanxiu; Liu, Lin; Liu, Yong

    2017-09-01

    Detection of leukocytes is critical for the routine leukorrhea exam, which is widely used in gynecological examinations. An elevated vaginal leukocyte count in women with bacterial vaginosis is a strong predictor of vaginal or cervical infections. In the routine leukorrhea exam, the counting of leukocytes is primarily performed by manual techniques. However, the viewing and counting of leukocytes from multiple high-power viewing fields on a glass slide under a microscope leads to subjectivity, low efficiency, and low accuracy. To date, many biological cells in stool, blood, and breast cancer have been studied to realize computerized detection; however, the detection of leukocytes in microscopic leukorrhea images has not been studied. Thus, there is an increasing need for computerized detection of leukocytes. There are two key processes in the computerized detection of leukocytes in digital image processing. One is segmentation; the other is intelligent classification. In this paper, we propose a combined ensemble to detect leukocytes in the microscopic leukorrhea image. After image segmentation and selecting likely leukocyte subimages, we obtain the leukocyte candidates. Then, for intelligent classification, we adopt two methods: feature extraction and classification by a support vector machine (SVM); applying a modified convolutional neural network (CNN) to the larger subimages. If different methods classify a candidate in the same category, the process is finished. If not, the outputs of the methods are provided to a classifier to further classify the candidate. After acquiring leukocyte candidates, we attempted three methods to perform classification. The first approach using features and SVM achieved 88% sensitivity, 97% specificity, and 92.5% accuracy. The second method using CNN achieved 95% sensitivity, 84% specificity, and 89.5% accuracy. Then, in the combination approach, we achieved 92% sensitivity, 95% specificity, and 93.5% accuracy. Finally, the images

  19. Roscovitine blocks leukocyte extravasation by inhibition of cyclin-dependent kinases 5 and 9.

    PubMed

    Berberich, Nina; Uhl, Bernd; Joore, Jos; Schmerwitz, Ulrike K; Mayer, Bettina A; Reichel, Christoph A; Krombach, Fritz; Zahler, Stefan; Vollmar, Angelika M; Fürst, Robert

    2011-07-01

    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. 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. 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. 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. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  20. 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

  1. Stress and skin leukocyte trafficking as a dual-stage process.

    PubMed

    Neeman, Elad; Shaashua, Lee; Benish, Marganit; Page, Gayle G; Zmora, Oded; Ben-Eliyahu, Shamgar

    2012-02-01

    Stress responses are known to modulate leukocyte trafficking. In the skin, stress was reported both to enhance and reduce skin immunity, and the chronicity of stress exposure was suggested as a key determining factor. We here propose a dual-stage hypothesis, suggesting that stress, of any duration, reduces skin immunity during its course, while its cessation is potentially followed by a period of enhanced skin immunity. To start testing this hypothesis, rats were subcutaneously implanted with sterile surgical sponges for four-hours, during or after exposure to one of several acute stress paradigms, or to a chronic stress paradigm. Our findings, in both males and females, indicate that numbers of sponge-infiltrating leukocytes, and their specific subsets, were reduced during acute or chronic stress, and increased after stress cessation. Studying potential mediating mechanisms of the reduction in leukocyte numbers during acute stress, we found that neither adrenalectomy nor the administration of beta-adrenergic or glucocorticoid antagonists prevented this reduction. Additionally, administration of corticosterone or epinephrine to adrenalectomized rats did not impact skin leukocyte numbers, whereas, in the blood, these treatments did affect numbers of leukocytes and their specific subsets, as was also reported previously. Overall, our findings support the proposed dual-stage hypothesis, which can be evolutionally rationalized and accounts for most of the apparent inconsistencies in the literature regarding stress and skin immunity. Other aspects of the hypothesis should be tested, also using additional methodologies, and its predictions may bear clinical significance in treatment of skin disorders related to hyper- or hypo-immune function. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Niacin decreases leukocyte myeloperoxidase: mechanistic role of redox agents and Src/p38MAP kinase.

    PubMed

    Ganji, Shobha H; Kamanna, Vaijinath S; Kashyap, Moti L

    2014-08-01

    Leukocyte myeloperoxidase (MPO) is a major player in the pathogenesis of various chronic diseases including atherosclerosis. This study proposes the novel concept that niacin, through reactive oxygen species (ROS)-mediated signaling, decreases neutrophil MPO release and its activity, protects apolipoprotein-AI (apo-AI) modification and improves HDL function. Human blood leukocytes and leukocytic cell line HL-60 cells were treated with niacin, and stimulated with phorbol myristate acetate (PMA). Cellular and released MPO activity in the medium was measured by assessing chlorination of MPO-specific substrate. MPO protein release in the medium and apo-AI degradation was measured by Western blot analysis. Monocyte adhesion to human aortic primary endothelial cells was measured to assess biological function of HDL/apo-AI. PMA significantly increased leukocyte MPO activity in both intracellular extract and medium. Niacin (0.25-0.5 mM) decreased PMA-induced MPO activity (cellular and released in the media). Niacin also decreased MPO protein mass in the medium without affecting its mRNA expression. Increased NADPH oxidase and ROS production by PMA were also significantly inhibited by niacin. Studies with specific inhibitors suggest that ROS-dependent Src and p38MAP kinase mediate decreased MPO activity by niacin. Niacin blocked apo-AI degradation, and apo-AI from niacin treated cells decreased monocyte adhesion to aortic endothelial cells. These findings identify niacin as a potent inhibitor of leukocyte MPO release and MPO-mediated formation of dysfunctional HDL. Niacin and niacin-related chemical entities may form important therapeutic agents for MPO-mediated inflammatory diseases. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  3. 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.

  4. Relationship of Stress, Leukocyte Functions and Acute Ulcerative Gingivitis.

    DTIC Science & Technology

    1982-10-22

    IONAL 1jjl (I Y AN[)AAU> I,)(, 4 •. . . . .i -AD (Report Number 3 , Lf) RELATIONSHIP OF STRESS, LEUKOCYTE FUNCTION AND ACUTE ULCERATIVE GINGIVITIS...AIk £It. KEY WORDS (C~mntm. a reers old. A *1acoa and Identit by block number) Acute Necrotic Ulcerative Gingivitis (ANUG)), Stress 4 Leukocyte

  5. 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.

  6. Mixed wife-husband leukocyte migration inhibition test after normal pregnancy.

    PubMed

    Halbrecht, I; Komlos, L; Ben-Efraim, S

    1979-01-01

    The leukocyte migration inhibition test was performed in mixed wife-husband leukocyte suspensions in 11 cases of normal pregancy. Migration of leukocytes was significantly inhibited in the presence of paternal, as compared to maternal serum.

  7. 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

  8. 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.

  9. A novel method to analyze leukocyte rolling behavior in vivo

    PubMed Central

    Dunne, Jessica L.; Goobic, Adam P.; Acton, Scott T.

    2004-01-01

    Leukocyte endothelial cell interaction is a fundamentally important process in many disease states. Current methods to analyze such interactions include the parallel-plate flow chamber and intravital microscopy. Here, we present an improvement of the traditional intravital microscopy that allows leukocyte-endothelial cell interaction to be studied from the time the leukocyte makes its initial contact with the endothelium until it adheres to or detaches from the endothelium. The leukocyte is tracked throughout the venular tree with the aid of a motorized stage and the rolling and adhesive behavior is measured off-line. Because this method can involve human error, methods to automate the tracking procedure have been developed. This novel tracking method allows for a more detailed examination of leukocyte-endothelial cell interactions. PMID:15346173

  10. A major allogenic leukocyte antigen in the agnathan hagfish.

    PubMed

    Takaba, Hiroyuki; Imai, Takeshi; Miki, Shoji; Morishita, Yasuyuki; Miyashita, Akihiro; Ishikawa, Naoko; Nishizumi, Hirofumi; Sakano, Hitoshi

    2013-01-01

    All vertebrates, from jawless fish to mammals, possess adaptive immune systems that can detect and inactivate non-self-antigens through a vast repertoire of antigen receptors. Unlike jawed vertebrates, the hagfish utilizes variable lymphocyte receptors (VLRs) that are unrelated to immunoglobulin molecules but are diversified by copy-choice gene conversion mechanism. Here, we report that hagfish VLRs react with allogenic leukocyte antigens but not with self-antigens. We found that a highly polymorphic membrane protein, NICIR3, is recognized by VLRs as an allogenic leukocyte antigen (ALA). In a serological cross-reactivity test, a close correlation was observed between the amino acid differences in the protein sequences and the VLR cross-reactivities. This leukocyte antigen was predominantly expressed in phagocytic leukocytes, where it was associated with phagocytosed protein antigens. These findings suggest that a polymorphic leukocyte antigen, NICIR3/ALA, plays a pivotal role in jawless vertebrate adaptive immunity.

  11. A major allogenic leukocyte antigen in the agnathan hagfish

    PubMed Central

    Takaba, Hiroyuki; Imai, Takeshi; Miki, Shoji; Morishita, Yasuyuki; Miyashita, Akihiro; Ishikawa, Naoko; Nishizumi, Hirofumi; Sakano, Hitoshi

    2013-01-01

    All vertebrates, from jawless fish to mammals, possess adaptive immune systems that can detect and inactivate non-self-antigens through a vast repertoire of antigen receptors. Unlike jawed vertebrates, the hagfish utilizes variable lymphocyte receptors (VLRs) that are unrelated to immunoglobulin molecules but are diversified by copy-choice gene conversion mechanism. Here, we report that hagfish VLRs react with allogenic leukocyte antigens but not with self-antigens. We found that a highly polymorphic membrane protein, NICIR3, is recognized by VLRs as an allogenic leukocyte antigen (ALA). In a serological cross-reactivity test, a close correlation was observed between the amino acid differences in the protein sequences and the VLR cross-reactivities. This leukocyte antigen was predominantly expressed in phagocytic leukocytes, where it was associated with phagocytosed protein antigens. These findings suggest that a polymorphic leukocyte antigen, NICIR3/ALA, plays a pivotal role in jawless vertebrate adaptive immunity. PMID:23612706

  12. 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

  13. Oxidation of glucosamine by human polymorphonuclear leukocytes.

    PubMed

    Swendsen, C L; DeChatelet, L R

    1981-03-01

    When exposed to a phagocytic stimulus (opsonized zymosan), human polymorphonuclear leukocytes (PMNs) produced 14CO2 from [1-14C]glucosamine at a rate 10-25% of that produced from glucose under the same conditions. The production of CO2 from glucosamine by intact PMNs was inhibited by glucose and dependent upon activation of the hexosemonophosphate shunt (HMPS). However, the metabolic pathways for the oxidation of glucose and glucosamine by PMNs are not identical. This is suggested by the fact that glucose-6-phosphate dehydrogenase, the initiating enzyme for the HMPS, did not utilize glucosamine-6-phosphate as a substrate. In addition, glucosamine was not oxidized by sonically disrupted PMNs whereas oxidation of glucose by the same preparation was increased sevenfold over intact cells. Taken together, the data suggest that PMNs oxidize glucosamine by converting it to a compound compatible with the enzymes of the HMPS. This conversion requires intact PMNs and/or an as yet unidentified cofactor.

  14. Leukocyte Count and Intracerebral Hemorrhage Expansion.

    PubMed

    Morotti, Andrea; Phuah, Chia-Ling; Anderson, Christopher D; Jessel, Michael J; Schwab, Kristin; Ayres, Alison M; Pezzini, Alessandro; Padovani, Alessandro; Gurol, M Edip; Viswanathan, Anand; Greenberg, Steven M; Goldstein, Joshua N; Rosand, Jonathan

    2016-06-01

    Acute leukocytosis is a well-established response to intracerebral hemorrhage (ICH). Leukocytes, because of their interaction with platelets and coagulation factors, may in turn play a role in hemostasis. We investigated whether admission leukocytosis was associated with reduced bleeding after acute ICH. Consecutive patients with primary ICH were prospectively collected from 1994 to 2015 and retrospectively analyzed. We included subjects with a follow-up computed tomographic scan available and automated complete white blood cell count performed within 48 hours from onset. Baseline and follow-up hematoma volumes were calculated with semiautomated software, and hematoma expansion was defined as volume increase >30% or 6 mL. The association between white blood cell count and ICH expansion was investigated with multivariate logistic regression. A total of 1302 subjects met eligibility criteria (median age, 75 years; 55.8% men), of whom 207 (15.9%) experienced hematoma expansion. Higher leukocyte count on admission was associated with reduced risk of hematoma expansion (odds ratio for 1000 cells increase, 0.91; 95% confidence interval, 0.86-0.96; P=0.001). The risk of hematoma expansion was inversely associated with neutrophil count (odds ratio, 0.90; 95% confidence interval, 0.85-0.96; P=0.001) and directly associated with monocyte count (odds ratio, 2.71; 95% confidence interval, 1.08-6.83; P=0.034). There was no association between lymphocyte count and ICH expansion (odds ratio, 0.96; 95% confidence interval, 0.79-1.17; P=0.718). Higher admission white blood cell count is associated with lower risk of hematoma expansion. This highlights a potential role of the inflammatory response in modulating the coagulation cascade after acute ICH. © 2016 American Heart Association, Inc.

  15. 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

  16. Flow cytometry based profiling of leukocytes: A new method for diagnosis of tropical theileriosis in crossbred cattle

    PubMed Central

    Jagtap, Ramesh B.; Gupta, Amit; Chaphalkar, Sushama R.

    2015-01-01

    Aim: In India, dairy industries are important for the livelihood of small scale farmers and dairy owners. Tropical theileriosis, mostly affecting dairy cattle and buffaloes is a major threat to dairy and related industries. Tropical theileriosis is caused by Theileria annulata, a hemoprotozoan parasite transmitted by Ixodid ticks of Hyalomma spp. In the present study, we examined the clinical signs, hematological parameters and flow cytometric profile of whole blood in 30 theileriosis affected crossbred cattle. The aim of our study is to analyze, in comparison with clinical and hematological diagnosis, whether flow cytometry based profiling of leukocytes could be used as better, quick and alternative method for diagnosis and screening of bovine tropical theileriosis in crossbred cattle. Materials and Methods: In this study, we screened parasites in 30 peripheral blood samples from clinical cases of theileriosis by Giemsa’s staining technique in crossbred cattle. Hematological analysis was done to estimate hemoglobin (Hb) content, total red blood cell (RBC) count, total leukocyte count and differential leukocyte count. Further, flow cytometric analysis of whole blood was carried out to study leukocytes profile in affected cattle. Results: Microscopic examination of stained blood films revealed the presence of piroplasms in erythrocytes and schizonts in lymphocytes. Hematological examination revealed significant (p<0.05) decrease of Hb percent (Hb %), reduced total RBC and total leukocytes, lymphocytosis, eosinopenia, and neutropenia compared to that of apparently healthy cattle. Flow cytometric profiling of leukocytes revealed the severe effect on shape, size, and granularity of leukocytes, marked decrease in granulocytes and 3-5 fold increase in lymphocytes count compared to clinically healthy cattle. Thus, in both methods, namely conventional and flow cytometric analysis, marked lymphocytosis and decrease in other blood cell counts were observed compared to

  17. Electrophoretic detection of protein p53 in human leukocytes

    SciTech Connect

    Paponov, V.D.; Kupsik, E.G.; Shcheglova, E.G.; Yarullin, N.N.

    1986-01-01

    The authors have found an acid-soluble protein with mol. wt. of about 53 kD in peripheral blood leukocytes of persons with Down's syndrome. It was present in different quantities in all 20 patients tested, but was virtually not discovered in 12 healthy blood donors. This paper determines the possible identity of this protein with protein p53 from mouse ascites carcinoma by comparing their electrophoretic mobilities, because the accuracy of electrophoretic determination of the molecular weight of proteins is not sufficient to identify them. The paper also describes experiments to detect a protein with electrophoretic mobility identical with that of a protein in the leukocytes of patients with Down's syndrome in leukocytes of patients with leukemia. To discover if protein p53 is involved in cell proliferation, the protein composition of leukocytes from healthy blood donors, cultured in the presence and absence of phytohemagglutinin (PHA), was compared. Increased incorporation of H 3-thymidine by leukocytes of patients with Down's syndrome is explained by the presence of a population of immature leukocytes actively synthesizing DNA in the peripheral blood of these patients, and this can also explain the presence of protein p53 in the leukocytes of these patients.

  18. Bacterial lipopolysaccharide induction of leukocyte-derived corticotropin and endorphins.

    PubMed Central

    Harbour-McMenamin, D; Smith, E M; Blalock, J E

    1985-01-01

    Previous reports have shown that there is an endogenous opioid component associated with pathophysiological responses to endotoxin. It has been shown that these responses are alleviated by naloxone, a specific opiate antagonist. Results of another study have indicated that leukocytes may mediate some of those responses since leukocyte depletion alleviated the effects of lipopolysaccharide. In view of the above reports as well as the finding that leukocytes produce immunoreactive (ir-) endorphins and corticotropin (ACTH) when stimulated with Newcastle disease virus or ACTH-releasing factor, we postulated that leukocytes may serve as an extrapituitary source of endorphins produced in response to bacterial endotoxin. To test this hypothesis, human peripheral blood leukocytes as well as mouse spleen cells were cultured in vitro with Escherichia coli lipopolysaccharide for 48 h. The lipopolysaccharide (i.e., endotoxin) was shown to induce de novo synthesis of ir-ACTH and ir-endorphins. The leukocyte-derived ir-ACTH had a molecular weight of approximately 2,900 and demonstrated a bioactivity similar to that of pituitary-derived ACTH. The lymphocyte-derived ir-endorphin comigrated with alpha- and gamma-endorphin at approximately 1,800 daltons and was shown to bind to brain opiate receptors. These findings imply that leukocyte-derived endorphins may be involved in the pathophysiological response to endotoxin. PMID:2987131

  19. 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

  20. Fibronectin connecting segment-1 peptide inhibits pathogenic leukocyte trafficking and inflammatory demyelination in experimental models of chronic inflammatory demyelinating polyradiculoneuropathy.

    PubMed

    Dong, Chaoling; Greathouse, Kelsey M; Beacham, Rebecca L; Palladino, Steven P; Helton, E Scott; Ubogu, Eroboghene E

    2017-06-01

    The molecular determinants of pathogenic leukocyte migration across the blood-nerve barrier (BNB) in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) are unknown. Specific disease modifying therapies for CIDP are also lacking. Fibronectin connecting segment-1 (FNCS1), an alternatively spliced fibronectin variant expressed by microvascular endothelial cells at sites of inflammation in vitro and in situ, is a counterligand for leukocyte α4 integrin (also known as CD49d) implicated in pathogenic leukocyte trafficking in multiple sclerosis and inflammatory bowel disease. We sought to determine the role of FNCS1 in CIDP patient leukocyte trafficking across the BNB in vitro and in severe chronic demyelinating neuritis in vivo using a representative spontaneous murine CIDP model. Peripheral blood mononuclear leukocytes from 7 untreated CIDP patients were independently infused into a cytokine-treated, flow-dependent in vitro BNB model system. Time-lapse digital video microscopy was performed to visualize and quantify leukocyte trafficking, comparing FNCS1 peptide blockade to relevant controls. Fifty 24-week old female B7-2 deficient non-obese diabetic mice with spontaneous autoimmune peripheral polyneuropathy (SAPP) were treated daily with 2mg/kg FNCS1 peptide for 5days via intraperitoneal injection with appropriate controls. Neurobehavioral measures of disease severity, motor nerve electrophysiology assessments and histopathological quantification of inflammation and morphometric assessment of demyelination were performed to determine in vivo efficacy. The biological relevance of FNCS1 and CD49d in CIDP was evaluated by immunohistochemical detection in affected patient sural nerve biopsies. 25μM FNCS1 peptide maximally inhibited CIDP leukocyte trafficking at the human BNB in vitro. FNCS1 peptide treatment resulted in significant improvements in disease severity, motor electrophysiological parameters of demyelination and histological measures of

  1. 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.

  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. 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.

  4. 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.

  5. Association between Snoring and Leukocyte Telomere Length.

    PubMed

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

    2016-04-01

    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. 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. 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. Our findings suggest that snoring may influence telomere attrition independent of sleep apnea. © 2016 Associated Professional Sleep Societies, LLC.

  6. 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

  7. 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.

  8. 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

  9. Leukocyte adhesion deficiency: a case report and review.

    PubMed

    Nagendran, J; Prakash, Chandra; Anandakrishna, Latha; Gaviappa, Dhananjaya; Ganesh, Dhanu

    2012-01-01

    Leukocyte adhesion deficiency (LAD) is a rare inherited primary immunodeficiency disorder characterized by the presence of a defect of phagocytic function resulting from a lack of leukocyte cell surface expression of β₂ integrin molecules (CD11 and CD18) that are essential for leukocyte adhesion to endothelial cells and chemotaxis. A small number of patients with LAD-1 have a milder defect, with residual expression of CD18. These patients tend to survive beyond infancy; they manifest progressive severe periodontitis, leading to partial or total premature loss of the primary and permanent dentitions. Close cooperation with pediatricians and immunologists is often the key to successful management of pediatric patients with LAD. The purpose of this report was to present the case of a 5-year-old boy with moderate leukocyte adhesion deficiency-1 and severe periodontitis, cellulitis and illustrate the need for periodic oral checkups to avoid the progression of oral diseases and prevent premature tooth loss.

  10. 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.

  11. 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.

  12. Genetics Home Reference: leukocyte adhesion deficiency type 1

    MedlinePlus

    ... navigation Home Page Search Home Health Conditions Genes Chromosomes & mtDNA Resources Help Me Understand Genetics Share: Email ... with leukocyte adhesion deficiency type 1 develop serious bacterial and fungal infections. One of the first signs ...

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. Navigating the leukocyte signaling maze guided by Ariadne's thread.

    PubMed

    Altman, Amnon; Koretzky, Gary A; Tsoukas, Constantine D

    2009-11-01

    Ariadne is the legendary Minoan goddess of the Labyrinth. The term 'Ariadne's thread' is used to describe the understanding of complex issues. Immunologists attending the 5th Leukocyte Signal Transduction Workshop discussed the Ariadne's thread woven about intracellular signaling pathways.

  18. 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.

  19. 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.

  20. 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

  1. Isolation of Leukocytes from the Human Maternal-fetal Interface

    PubMed Central

    Xu, Yi; Plazyo, Olesya; Romero, Roberto; Hassan, Sonia S.; Gomez-Lopez, Nardhy

    2015-01-01

    Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface. PMID:26067211

  2. Astrocyte-shed extracellular vesicles regulate the peripheral leukocyte response to inflammatory brain lesions.

    PubMed

    Dickens, Alex M; Tovar-Y-Romo, Luis B; Yoo, Seung-Wan; Trout, Amanda L; Bae, Mihyun; Kanmogne, Marlene; Megra, Bezawit; Williams, Dionna W; Witwer, Kennith W; Gacias, Mar; Tabatadze, Nino; Cole, Robert N; Casaccia, Patrizia; Berman, Joan W; Anthony, Daniel C; Haughey, Norman J

    2017-04-04

    Brain injury induces a peripheral acute cytokine response that directs the transmigration of leukocytes into the brain. Because this brain-to-peripheral immune communication affects patient recovery, understanding its regulation is important. Using a mouse model of inflammatory brain injury, we set out to find a soluble mediator for this phenomenon. We found that extracellular vesicles (EVs) shed from astrocytes in response to intracerebral injection of interleukin-1β (IL-1β) rapidly entered into peripheral circulation and promoted the transmigration of leukocytes through modulation of the peripheral acute cytokine response. Bioinformatic analysis of the protein and microRNA cargo of EVs identified peroxisome proliferator-activated receptor α (PPARα) as a primary molecular target of astrocyte-shed EVs. We confirmed in mice that astrocytic EVs promoted the transmigration of leukocytes into the brain by inhibiting PPARα, resulting in the increase of nuclear factor κB (NF-κB) activity that triggered the production of cytokines in liver. These findings expand our understanding of the mechanisms regulating communication between the brain and peripheral immune system and identify astrocytic EVs as a molecular regulator of the immunological response to inflammatory brain damage. Copyright © 2017, American Association for the Advancement of Science.

  3. Injustice at work and leukocyte glucocorticoid sensitivity: findings from a cross-sectional study.

    PubMed

    Herr, Raphael M; Loerbroks, Adrian; van Vianen, Annelies E M; Hoffmann, Kristina; Fischer, Joachim E; Bosch, Jos A

    2015-06-01

    Organizational justice refers to perceived fairness at the workplace. Low organizational justice has been identified as a major source of distress and a predictor of poor health. Impaired regulation of immunological and inflammatory pathways may, in part, underlie these health effects. The present study investigated the association of organizational justice with leukocyte glucocorticoid sensitivity in vivo. Organizational justice was assessed among 541 male factory workers (mean [standard deviation] age = 46 [9] years) by questionnaire. Cortisol release was measured at three time points before blood collection and summed as the area under the curve. Blood was used to assess leukocyte (white blood cell [WBC] count) subsets (neutrophils [%WBC], lymphocytes [%WBC], and the neutrophil/lymphocyte ratio). Glucocorticoid sensitivity was operationalized as the correlation between cortisol release and these hematologic parameters. Associations were adjusted for demographics, work characteristics, and life-style variables. A dose-response relationship between organizational justice and glucocorticoid sensitivity was found. Cortisol and hematologic parameters showed the expected significant association among individuals reporting high (all β values ≥ |.26|; all p values ≤.001) or medium organizational justice (all β values ≥ |.15|; all p values ≤.050), but not among those reporting low organizational justice (all β values ≤ |.04|; all p values > .10). These regression slopes differed significantly between organizational justice groups (p values for interaction < .050). Low justice at work is associated with an impaired ability of endogenous cortisol to regulate leukocyte distribution in vivo. These findings identify a novel biological pathway by which organizational justice may affect health.

  4. In vitro effect of cadmium and copper on separated blood leukocytes of Dicentrarchus labrax.

    PubMed

    Vazzana, Mirella; Celi, Monica; Tramati, Cecilia; Ferrantelli, Vincenzo; Arizza, Vincenzo; Parrinello, Nicolò

    2014-04-01

    The immunotoxic effects of heavy metals on blood leukocytes of sea bass (Dicentrarchus labrax) were examined. The cells, separated by a discontinuous Percoll-gradients, were exposed in vitro to various sublethal concentrations of cadmium and copper (10(-7)M, 10(-5)M, and 10(-3)M) and their immunotoxic effect was then evaluated by measuring neutral red uptake, MTT assay, DNA fragmentation and Hsp70 gene expression. First of all, we demonstrated that the cells treated in vitro could incorporate Cd and Cu. A relationship between heavy metal exposure and dose-time-dependent alterations in responses of leukocytes from blood was found for both metals, but copper was more immunotoxic than cadmium in all assays performed. A significant reduction in the cells׳ ability to uptake neutral red and viability by MTT assay was recorded, indicating that both cadmium and copper could change the membrane permeability, inducing cellular apoptosis when the concentration of metals reached 10(-3)M. The apoptotic effect may also explain the high level of cytotoxicity found when the leukocytes were exposed to higher concentration of metals. These results demonstrated that toxic effect of copper and cadmium affect on the mechanisms of cell-mediated immunity reducing the immune defences of the organism. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Expression of toll-like receptor genes in leukocytes of patients with sudden sensorineural hearing loss.

    PubMed

    Yang, Chao-Hui; Hwang, Chung-Feng; Yang, Ming-Yu; Lin, Pai-Mei; Chuang, Jiin-Haur

    2015-12-01

    Sudden sensorineural hearing loss (SNNHL) is a disease entity that could be caused by multiple etiologies in which the innate immunity status of the patients might be involved. The aim of this study is to investigate the expression of Toll-like receptor (TLR) genes in peripheral blood leukocytes of SNNHL patients. Basic research. We examined the expression of six TLR genes in the peripheral blood leukocytes of SNNHL patients and normal controls using real-time quantitative reverse transcriptase-polymerase chain reaction. We found significantly higher expression of TLR2, TLR3, TLR4, TLR7, TLR8, and TLR9 genes in SNNHL patients as compared with normal controls (P < 0.05). Higher expression of the TLR2 gene was found in patients with profound hearing loss compared with those with less severe hearing loss (P < 0.05). The result was validated by the positively stained leukocytes for TLR2 protein in SNNHL patients using the immunocytochemical study. In addition, the percentage of CD14(+) monocytes expressing TLR2 in SNNHL patients was higher than in normal controls assessed by flow cytometry and significantly correlated with the hearing thresholds of the affected ear (P < 0.05). Our study implies a role for TLRs in SNNHL. The expression of TLR2 in particular correlates with the severity of the disease. N/A. © 2015 The American Laryngological, Rhinological and Otological Society, Inc.

  6. [Screening with angiographic images prior to (99m)Tc-HMPAO labelled leukocyte scintigraphy in the diagnosis of periprosthetic infection].

    PubMed

    Granados, U; Fuster, D; Soriano, A; García, S; Bori, G; Martínez, J C; Mayoral, M; Perlaza, P; Tomás, X; Pons, F

    2015-01-01

    To evaluate the impact of the angioscintigrapy of the three phase bone scan as screening method to rule out infection of the hip and knee prosthesis prior to performing the (99m)Tc-HMPAO leukocyte scintigraphy. A total of 120 (70 women, 50 men; mean age 71±11years) with clinical suspicion of hip (n=63) or knee (n=57) infection of the prosthesis and clinical suspicion of infection were evaluated prospectively. All patients underwent three-phase bone scan (angioscintigraphy, vascular and bone phase) and (99m)Tc-HMPAO-labelled white blood cell scintigraphy. Final diagnosis of infection was made by microbiological documentation or clinical follow-up for at least 12months. Eighteen out of 120 patients were diagnosed of infection of hip prosthesis (n=10) or knee prosthesis (n=8). The angioscintigraphy was positive in 15/18 infected cases and in 21/102 of the non-infected cases with a sensitivity of 83%, specificity of 79% and negative predictive value of 97%. Sensitivity and specificity of (99m)Tc-HMPAO leukocyte scintigraphy were 72% and 95%, respectively. If the leukocyte labeled scintigraphies had been used exclusively for patients with positive angioscintigraphy, this would have saved up to 70% of the (99m)Tc-HMPAO leukocyte scintigraphies performed. There were no cases of infection with positive labeled leukocyte scintigraphy and negative angioscintigraphy. Angioscintigraphy (blood flow phase of bone scan) is a useful technique for screening for hip and knee joint prosthesis infection, significantly reducing the need for (99m)Tc-HMPAO leukocyte scintigraphy without affecting the sensitivity of the technique. Copyright © 2014 Elsevier España, S.L.U. and SEMNIM. All rights reserved.

  7. 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.

  8. 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.

  9. Platelet-leukocyte mixed conjugates in patients with atrial fibrillation.

    PubMed

    Alberti, Silvia; Angeloni, Giulia; Tamburrelli, Chiara; Pampuch, Agnieszka; Izzi, Benedetta; Messano, Loredana; Parisi, Quintino; Santamaria, Matteo; Donati, Maria Benedetta; de Gaetano, Giovanni; Cerletti, Chiara

    2009-06-01

    Although platelets may contribute to the inflammatory component in atrial fibrillation (AF), the impact of platelet-leukocyte mixed conjugates has not yet been determined. Seventeen patients with persistent AF (8/9 m/f; mean age 68.1 +/- 2.5 years), not on anticoagulant therapy, were recruited and compared to 34 healthy controls with normal sinus rhythm (16/18 m/f; mean age 60.8 +/- 1.2 years). Platelet-leukocyte mixed conjugates, platelet P-selectin and leukocyte activation markers (CD11b, myeloperoxidase) were measured by flow-cytometry in whole blood both in basal condition and after in vitro ADP/collagen challenge. Plasma D-dimer and soluble P-selectin were also measured. Statistical analyses were performed by Mann-Whitney or Wilcoxon U test for intergroup differences. In AF patients platelet count, as well as platelet P-selectin expression and percent platelet-leukocyte conjugates were all significantly lower both in basal condition and upon activation with ADP/collagen. In contrast, both soluble P-selectin and D-dimer were significantly higher than in controls; white blood cell count and leukocyte activation markers were unchanged. In conclusion, the formation of platelet-leukocyte mixed conjugates was unexpectedly reduced in AF, possibly due to less reactive platelets as a consequence of previous in vivo activation by ongoing formation of trace amounts of thrombin.

  10. 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

    the endothelial cells are important in affecting the apparent reduction of toxic oxygen products derived from polymorphonuclear leukocytes attached to their surface. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:3030114

  11. 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

  12. 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.

  13. 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

  14. Expression of nitric oxide synthases in leukocytes in nasal polyps.

    PubMed

    Yoshimura, Tsuyoshi; Moon, Tae Chul; St Laurent, Chris D; Puttagunta, Lakshmi; Chung, Kerri; Wright, Erin; Yoshikawa, Mamoru; Moriyama, Hiroshi; Befus, A Dean

    2012-03-01

    Nitric oxide (NO) has various roles in airway physiology and pathophysiology. Monitoring exhaled NO levels is increasingly common to measure airways inflammation and inhaled NO studied for its therapeutic value in premature infants and adult respiratory distress syndrome. NO is produced by 3 isoforms of NO synthase (NOS1, 2, 3), and each can play distinct and perhaps overlapping roles in the airways. However, the distribution, regulation, and functions of NOS in various cells in the upper airways, particularly in leukocytes, are incompletely understood. To characterize the expression of NOS isoforms in leukocytes in normal middle turbinate tissues (MT) and in inflammatory nasal tissue (nasal polyps, NP). Normal MT tissue was collected from surgical specimens that were to be discarded. The NP samples were from surgical tissue archives of 15 patients with chronic rhinosinusitis. Isoforms of NOS in cells were identified by double immunostaining using NOS isoform-specific and leukocyte-specific (mast cell, eosinophil, macrophage, neutrophil, or T cell) antibodies. The proportion of total cells below the epithelium that were positive for each isoform of NOS was higher in NP than in MT. Each isoform of NOS was found in all leukocyte populations studied, and there were significant differences in the percentage of leukocytes expressing NOS isoforms between MT and NP. All isoforms of NOS are expressed in leukocytes in MT and NP, and their expression varies among leukocyte types. Our data provide a basis to investigate the regulation, cell distribution, and distinct functions of NOS isoforms in normal and inflamed nasal tissues. Copyright © 2012 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  15. [Leukocyte mobility in modulation of activity of the cell signalling system].

    PubMed

    Luĭk, A I; Mogilevich, S E; Radchenko, I V; Kondrashova, L N

    1993-01-01

    The mobility of the rat polymorphonuclear leukocytes (PMNL) has been studied. It was shown, that it is greatly determined by the balance of adenylate cyclase (AdC) and Ca-polyphosphoinositide (Ca-PPI) cell signalling systems. Various compounds whose action on the activity of the signalling systems was previously connected with the membrane receptors, proved to be capable to affect the activity of submembrane elements of these systems. It is concluded that multiple areas of bioregulators fixation within the limits of the signal cascades are available.

  16. The effect of sample storage on the Peroxidation of Leukocytes Index Ratio (PLIR) measure

    PubMed Central

    Peluso, Ilaria; Manafikhi, Husseen; Altieri, Fabio; Zanza, Christian; Palmery, Maura

    2014-01-01

    Delays in processing are frequent because of problems associated with transporting the samples to the laboratory. Thus, we aimed to evaluate the effect of sample storage on the Peroxidation of Leukocytes Index Ratio (PLIR). Differences between PLIR values of lymphocytes (PLIR-L), monocytes (PLIR-M) and granulocytes (PLIR-G) were observed in fresh samples. Sample storage affected the evaluation of PLIR. In particular, PLIR-L was lower in stored samples compared to fresh samples. In conclusion, our results suggest that fresh samples are recommended for assessing the PLIR. PMID:25290094

  17. Porphyromonas gingivalis virulence factors involved in subversion of leukocytes and microbial dysbiosis.

    PubMed

    Zenobia, Camille; Hajishengallis, George

    2015-01-01

    The oral bacterium Porphyromonas gingivalis has special nutrient requirements due to its asaccharolytic nature subsisting on small peptides cleaved from host proteins. Using proteases and other virulence factors, P. gingivalis thrives as a component of a polymicrobial community in nutritionally favorable inflammatory environments. In this regard, P. gingivalis has a number of strategies that subvert the host immune response in ways that promote its colonization and facilitate the outgrowth of the surrounding microbial community. The focus of this review is to discuss at the molecular level how P. gingivalis subverts leukocytes to create a favorable environment for a select community of bacteria that, in turn, adversely affects the periodontal tissues.

  18. [Effects of the glucoprotein component of musk on functions of rat polymorphonuclear leukocytes activated by LTB4 in vitro].

    PubMed

    Wang, W; Bai, J; Cheng, G; Zhu, X

    1998-04-01

    To investigate the effects of musk-1, a glucoprotein component isolated from the water extract of musk, on some functions of rat polymorphonuclear leukocytes activated by LTB4, an in vitro incubation system with rat polymorphonuclear leukocytes was used. The superoxide anion production was determined by cytochrome C reduction, and the beta-glucuronidase and lysozyme release was quantitated by enzyme reactions in which phenolphthaleinglucuronic acid and micrococcus lysodeikticus were used as the substrates. In comparison with the control, musk-1 at final concentrations of 1 microgram/ml-100 micrograms/ml can increase the superoxide anion production by 28.7%-202.1% and decrease the beta-glucuronidase and lysozyme release by 3%-46% and 6%-32% respectively in rat polymorphonuclear leukocytes. It is concluded that musk-1 can significantly affect the functions of rat polymorphonuclear leukocytes activated by LTB4. One of the mechanisms of this anti-inflammatory action of musk may consist in the inhibition of lysosomal enzyme release.

  19. Contour Detection of Leukocyte Cell Nucleus Using Morphological Image

    NASA Astrophysics Data System (ADS)

    Supriyanti, R.; Satrio, G. P.; Ramadhani, Y.; Siswandari, W.

    2017-04-01

    Leukocytes are blood cells that do not contain color pigments. Leukocyte function to the tool body’s defenses. Abnormal forms of leukocytes can be a sign of serious diseases such example is leukemia. Most laboratories still use cell morphology examination to assist the diagnosis of illness associated with white blood cells such example is leukemia because of limited resources, both infrastructure, and human resources as happens in developing nations, such as Indonesia. This examination is less expensive and quicker process. However, morphological review requires the expertise of a specialist clinical pathology were limited. This process is sometimes less valid cause in some cases trying to differentiate morphology blast cells into the type of myoblasts, lymphoblast, monoblast, or erythroblast thus potentially misdiagnosis. The goal of this research is to develop a detection device types of blood cells automatically as lower-priced, easy to use and accurate so that the tool can be distributed across all units in existing health services throughout Indonesia and in particular for remote areas. However, because the variables used in the identification of abnormal leukocytes are very complex, in this paper, we emphasize on the contour detection of leukocyte cell nucleus using the morphological image. The results show that this method is promising for further development.

  20. Endothelial-Leukocyte Interaction in Severe Malaria: Beyond the Brain

    PubMed Central

    Souza, Mariana C.; Padua, Tatiana A.; Henriques, Maria G.

    2015-01-01

    Malaria is the most important parasitic disease worldwide, accounting for 1 million deaths each year. Severe malaria is a systemic illness characterized by dysfunction of brain tissue and of one or more peripheral organs as lungs and kidney. The most severe and most studied form of malaria is associated with cerebral complications due to capillary congestion and the adhesion of infected erythrocytes, platelets, and leukocytes to brain vasculature. Thus, leukocyte rolling and adhesion in the brain vascular bed during severe malaria is singular and distinct from other models of inflammation. The leukocyte/endothelium interaction and neutrophil accumulation are also observed in the lungs. However, lung interactions differ from brain interactions, likely due to differences in the blood-brain barrier and blood-air barrier tight junction composition of the brain and lung endothelium. Here, we review the importance of endothelial dysfunction and the mechanism of leukocyte/endothelium interaction during severe malaria. Furthermore, we hypothesize a possible use of adjunctive therapies to antimalarial drugs that target the interaction between the leukocytes and the endothelium. PMID:26491221

  1. Endothelial-Leukocyte Interaction in Severe Malaria: Beyond the Brain.

    PubMed

    Souza, Mariana C; Padua, Tatiana A; Henriques, Maria G

    2015-01-01

    Malaria is the most important parasitic disease worldwide, accounting for 1 million deaths each year. Severe malaria is a systemic illness characterized by dysfunction of brain tissue and of one or more peripheral organs as lungs and kidney. The most severe and most studied form of malaria is associated with cerebral complications due to capillary congestion and the adhesion of infected erythrocytes, platelets, and leukocytes to brain vasculature. Thus, leukocyte rolling and adhesion in the brain vascular bed during severe malaria is singular and distinct from other models of inflammation. The leukocyte/endothelium interaction and neutrophil accumulation are also observed in the lungs. However, lung interactions differ from brain interactions, likely due to differences in the blood-brain barrier and blood-air barrier tight junction composition of the brain and lung endothelium. Here, we review the importance of endothelial dysfunction and the mechanism of leukocyte/endothelium interaction during severe malaria. Furthermore, we hypothesize a possible use of adjunctive therapies to antimalarial drugs that target the interaction between the leukocytes and the endothelium.

  2. Preferential Adhesion of Leukocytes Near Bifurcations is Endothelium Independent

    PubMed Central

    Tousi, Nazanin; Wang, Bin; Pant, Kapil; Kiani, Mohammad F.; Prabhakarpandian, Balabhaskar

    2010-01-01

    Leukocyte-endothelial interactions play central roles in many pathological conditions. However, the mechanisms responsible for non-uniform spatial distribution of adhering leukocytes to endothelial cells in microvascular networks in vivo are not clear. We used a combination of in vitro and in vivo methodologies for understanding of this complex phenomenon. A mouse cremaster muscle model was used to study the spatial distribution of leukocyte-endothelial cell interaction in vivo. A PDMS based synthetic microvascular network (SMN) device was used to study interactions of functionalized microspheres using a receptor-ligand system in a (endothelial) cell-free environment for the in vitro studies. Our in vivo and in vitro findings indicate that both leukocytes in vivo and microspheres in vitro preferentially adhere near bifurcation within 1-2 vessel diameters from the bifurcation. This adhesion pattern was found to be independent of the diameter of the vessels. These findings support our hypothesis that the fluidic patterns near bifurcations/junctions, and not the presence or absence of endothelial cells and/or receptors on their surfaces, may be the driving force behind the preferential adhesion patterns of leukocytes near bifurcations. PMID:20624406

  3. 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

  4. 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

  5. Leukocyte emigration in the early stages of laminitis.

    PubMed

    Black, Samuel J; Lunn, D Paul; Yin, Cailing; Hwang, Misako; Lenz, Stephen D; Belknap, James K

    2006-01-15

    The mechanisms that initiate the pathophysiologic changes in the digital laminae in equine laminitis are poorly understood. Due to the fact that (1) the horse at risk of laminitis has many similarities clinically to the human sepsis patient and (2) our recent finding of marked laminar proinflammatory cytokine expression at the developmental time point of the black walnut extract (BWE) model of laminitis, we tested the possibility that, similar to organ damage in human sepsis, leukocyte emigration is an early event in laminitis. Using immunoperoxidase methods with an anti-equine CD13 monoclonal antibody that recognizes neutrophils and monocytes, we discovered that, whereas the dermal microvasculature of the skin commonly has a marginal pool of leukocytes, the normal laminar dermal microvasculature has minimal to no perivascular leukocytes. However, increases in leukocyte numbers occurred around the dermal vasculature of both the laminae and the skin in the majority of BWE-treated horses in the developmental stage and at the onset of clinical signs of lameness in the BWE model. These findings indicate that, similar to organ failure in human sepsis, leukocyte emigration is likely to play a significant role in initiating numerous pathophysiologic mechanisms that lead to the development of laminitis.

  6. Flow cytometric analysis of glyoxalase-1 expression in human leukocytes.

    PubMed

    Skapare, Elina; Riekstina, Una; Liepinsh, Edgars; Konrade, Ilze; Makrecka, Marina; Maurina, Baiba; Dambrova, Maija

    2011-03-01

    Altered glyoxalase-1 (GLO-1) activity and expression is associated with the development of late diabetic complications, malignancy and oxidative stress- and aging-related diseases. In the present study, we developed a flow cytometry method for GLO-1 detection in human leukocytes isolated from peripheral blood samples to investigate GLO-1 expression in leukocyte subsets from type 1 and 2 diabetes mellitus patients (n = 11) and healthy subjects (n = 8). The flow cytometry analysis of GLO-1 in leukocytes showed that expression index of GLO-1-positive cells was slightly increased in mononuclear leukocytes from diabetic patients. This result correlated with the increase in GLO-1 activity in the whole blood samples of type 2 diabetes patients. In conclusion, the present study demonstrates that flow cytometry is suitable for the detection of the GLO-1 enzyme in human leukocytes and that this method could be used to investigate the fast adaptation of the glyoxalase system related to the pathogenesis of late complications of diabetes mellitus and other glycation stress-related disorders. Copyright © 2011 John Wiley & Sons, Ltd.

  7. Systemic leukocyte alterations are associated with invasive uterine cervical cancer.

    PubMed

    Tavares-Murta, Beatriz M; Mendonça, Maria A O; Duarte, Natália L; da Silva, Juliana A; Mutão, Taciana S; Garcia, Cristiana B; Murta, Eddie F C

    2010-10-01

    The aim of the study was to evaluate blood leukocyte counts in patients with uterine cervical neoplasia. Patients treated at a university hospital were reviewed retrospectively. Disease progression was monitored, beginning in 1990 to 2002, for at least 5 years. Blood count parameters included absolute leukocyte, neutrophil and lymphocyte counts, leukocytosis (white blood cells > 10³/μL), neutrophilia (neutrophils ≥ 70% of leukocytes), lymphopenia (lymphocytes ≤ 15% of leukocytes), and the neutrophil-lymphocyte ratio (NLR), categorized as less than 5 or 5 or greater. A total of 315 patients were enrolled: 182 (57.8%) with preinvasive neoplasia (cervical intraepithelial neoplasia [CIN] group), 95 (30.1%) with stages I to II (early group), and 38 patients (12.1%) with stages III to IV neoplasia (advanced group). Neutrophil and lymphocyte counts were elevated and reduced, respectively, at advanced stages compared with the CIN group (P < 0.05). Leukocytosis, neutrophilia, lymphopenia, and an NLR of 5 or greater were more frequent at advanced stages compared with the CIN and early-stage groups (P < 0.05). Moreover, neutrophilia was also significantly more frequent at early stage compared with the CIN group. The advanced group with neutrophilia had increased frequency of recidivism and metastasis than patients in the CIN group with neutrophilia (P < 0.05). Patients with advanced cervical cancer had significantly higher frequency of leukocyte alterations, although they may occur apart from the preinvasive stages. Overall, neutrophilia was the best indicator of cancer invasiveness.

  8. The role of leukocytes in the pathogenesis of fibrin deposition in bovine acute lung injury.

    PubMed Central

    Car, B. D.; Suyemoto, M. M.; Neilsen, N. R.; Slauson, D. O.

    1991-01-01

    The peculiarly fibrinous nature of bovine acute lung injury due to infection with Pasteurella haemolytica A1 suggests an imbalance between leukocyte-directed procoagulant and profibrinolytic influences in the inflamed bovine lung. Calves with experimental pneumonia produced by intratracheal inoculation with P. haemolytica A1 developed acute locally extensive cranioventral fibrinopurulent bronchopneumonia. Pulmonary alveolar macrophages (PAM) recovered by segmental lavage from affected lung lobes were 30 times more procoagulant than PAM obtained from unaffected lung lobes and 37-fold more procoagulant than PAM from control calf lungs. Unlike the enhancement of procoagulant activity, profibrinolytic activity (plasminogen activator amidolysis) of total lung leukocytes (PAM and plasminogen activator neutrophils [PMN]) was decreased 23 times in cells obtained from affected lung lobes and also was decreased four times in cells obtained from unaffected lobes of infected animals. This marked imbalance in cellular procoagulant and fibrinolytic activity probably contributes significantly to enhanced fibrin deposition and retarded fibrin removal. In addition, PAM from inflamed lungs were strongly positive for bovine tissue factor antigen as demonstrated by immunocytochemistry. Intensely tissue factor-positive PAM enmeshed in fibrinocellular exudates and positive alveolar walls were situated such that they were likely to have, in concert, initiated extrinsic activation of coagulation in the acutely inflamed lung. These data collectively suggest that enhanced PAM-directed procoagulant activity and diminished PAM- and PMN-directed profibrinolytic activity represent important modifications of local leukocyte function in bovine acute lung injury that are central to the pathogenesis of lesion development with extensive fibrin deposition and retarded fibrin removal. Images Figure 2 Figure 3 PMID:2024707

  9. Treatment of Obstructive Sleep Apnea Alters Cancer-associated Transcriptional Signatures in Circulating Leukocytes

    PubMed Central

    Gharib, Sina A.; Seiger, Ashley N.; Hayes, Amanda L.; Mehra, Reena; Patel, Sanjay R.

    2014-01-01

    Rationale: Obstructive sleep apnea (OSA) has been associated with a number of chronic disorders that may improve with effective therapy. However, the molecular pathways affected by continuous positive airway pressure (CPAP) treatment are largely unknown. We sought to assess the system-wide consequences of CPAP therapy by transcriptionally profiling peripheral blood leukocytes (PBLs). Methods: Subjects in whom severe OSA was diagnosed were treated with CPAP, and whole-genome expression measurement of PBLs was performed at baseline and following therapy. We used gene set enrichment analysis (GSEA) to identify pathways that were differentially enriched. Network analysis was then applied to highlight key drivers of processes influenced by CPAP. Results: Eighteen subjects with significant OSA underwent CPAP therapy and microarray analysis of their PBLs. Treatment with CPAP improved apnea-hypopnea index (AHI), daytime sleepiness, and blood pressure, but did not affect anthropometric measures. GSEA revealed a number of enriched gene sets, many of which were involved in neoplastic processes and displayed downregulated expression patterns in response to CPAP. Network analysis identified several densely connected genes that are important modulators of cancer and tumor growth. Conclusions: Effective therapy of OSA with CPAP is associated with alterations in circulating leukocyte gene expression. Functional enrichment and network analyses highlighted transcriptional suppression in cancer-related pathways, suggesting potentially novel mechanisms linking OSA with neoplastic signatures. Citation: Gharib SA; Seiger AN; Hayes AL; Mehra R; Patel SR. Treatment of obstructive sleep apnea alters cancer-associated transcriptional signatures in circulating leukocytes. SLEEP 2014;37(4):709-714. PMID:24688164

  10. Platelet subpopulation bearing leukocyte specific antigen and tissue factor.

    PubMed

    Gabbasov, Z A; Saburova, O S; Antonova, O A; Golubeva, N V; Khaspekova, S G; Shustova, O N; Zyuryaev, I T; Ruda, M Ya; Mazurov, A V

    2016-11-01

    Platelets bearing leukocyte antigen CD45 were identified in the blood of patients with myocardial infarction (MI) and healthy donors by flow cytofluorimetry. Part of these platelets contained tissue factor (TF)-primary initiator of blood clotting. The number of CD45(+) and CD45(+)/TF(+) platelets in MI patients at the first day was comparable with their level in healthy donors, but was increased at 8-12 days after MI onset. At that time in some patients the amount of CD45(+) and CD45(+)/TF(+) platelets reached 5-6 and 2-3% of their total number. It is assumed that CD45(+)/TF(+) platelets could be formed as a result of platelet interaction with leukocytes or leukocyte produced membrane microparticles.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. Diagnosis of brain abscesses with indium-111-labeled leukocytes

    SciTech Connect

    Rehncrona, S.; Brismar, J.; Holtas, S.

    1985-01-01

    Sixteen patients with intracerebral mass lesions where computed tomography (CT) was not fully conclusive with respect to the differential diagnosis between brain tumor and abscess were examined with leukocyte brain scintigraphy (LBS). Autologous leukocytes were labeled with indium-111 oxinate and were reinjected intravenously; registration with a gamma camera was performed after 24 and 48 hours. In 10 of 11 patients with the final diagnosis of a brain tumor, no accumulation of radiolabeled leukocytes could be detected in the brain. In 4 of 5 patients with the final diagnosis of brain abscess, scintigraphy showed a pronounced increase of focal activity corresponding to the lesion demonstrated with CT. The reasons for the one false-positive and the one false-negative result are discussed, and it is concluded that LBS (a) can be used to detect intracranial infection and (b) may be a useful diagnostic tool for distinguishing between brain abscess and brain tumor.

  16. Occult Purulent Pericarditis Detected by Indium-111 Leukocyte Imaging*

    PubMed Central

    Greenberg, Mark L.; Niebulski, Harvey I. J; Uretsky, Barry F.; Salerni, Rosemarie; Klein, Herbert A.; Forstate, William J.; Starzl, Thomas E.

    2011-01-01

    Leukocyte imaging with indium-111 is-a relatively new technique which, to this point in time, has been discussed almost exclusively in the radiologic literature. Although this procedure has been used mainly to detect intra-abdominal infection, the thorax is routinely imaged along with the abdomen, and therefore detection of cardiac disease may be feasible. This case report is of a young woman after liver transplantation who developed occult purulent pericarditis initially detected by a leukocyte scan with indium-111. This case demonstrates that striking pericardial uptake on a whole-body indium-111 leukocyte scan can occur with purulent pericarditis, and it reemphasizes how insidiously purulent pericarditis may present in an immunosuppressed patient. PMID:6370622

  17. 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.

  18. Intravascular staining for discrimination of vascular and tissue leukocytes.

    PubMed

    Anderson, Kristin G; Mayer-Barber, Katrin; Sung, Heungsup; Beura, Lalit; James, Britnie R; Taylor, Justin J; Qunaj, Lindor; Griffith, Thomas S; Vezys, Vaiva; Barber, Daniel L; Masopust, David

    2014-01-01

    Characterization of the cellular participants in tissue immune responses is crucial to understanding infection, cancer, autoimmunity, allergy, graft rejection and other immunological processes. Previous reports indicate that leukocytes in lung vasculature fail to be completely removed by perfusion. Several studies suggest that intravascular staining may discriminate between tissue-localized and blood-borne cells in the mouse lung. Here we outline a protocol for the validation and use of intravascular staining to define innate and adaptive immune cells in mice. We demonstrate application of this protocol to leukocyte analyses in many tissues and we describe its use in the contexts of lymphocytic choriomeningitis virus and Mycobacterium tuberculosis infections or solid tumors. Intravascular staining and organ isolation usually takes 5-30 min per mouse, with additional time required for any subsequent leukocyte isolation, staining and analysis. In summary, this simple protocol should help enable interpretable analyses of tissue immune responses.

  19. Intravascular staining for discrimination of vascular and tissue leukocytes

    PubMed Central

    Anderson, Kristin G; Mayer-Barber, Katrin; Sung, Heungsup; Beura, Lalit; James, Britnie R; Taylor, Justin J; Qunaj, Lindor; Griffith, Thomas S; Vezys, Vaiva; Barber, Daniel L; Masopust, David

    2015-01-01

    Characterization of the cellular participants in tissue immune responses is crucial to understanding infection, cancer, autoimmunity, allergy, graft rejection and other immunological processes. previous reports indicate that leukocytes in lung vasculature fail to be completely removed by perfusion. several studies suggest that intravascular staining may discriminate between tissue-localized and blood-borne cells in the mouse lung. Here we outline a protocol for the validation and use of intravascular staining to define innate and adaptive immune cells in mice. We demonstrate application of this protocol to leukocyte analyses in many tissues and we describe its use in the contexts of lymphocytic choriomeningitis virus and Mycobacterium tuberculosis infections or solid tumors. Intravascular staining and organ isolation usually takes 5–30 min per mouse, with additional time required for any subsequent leukocyte isolation, staining and analysis. In summary, this simple protocol should help enable interpretable analyses of tissue immune responses. PMID:24385150

  20. Elevated platelet and leukocyte response to oral bacteria in periodontitis.

    PubMed

    Nicu, E A; Van der Velden, U; Nieuwland, R; Everts, V; Loos, B G

    2009-01-01

    Periodontitis is associated with an increased risk for cardiovascular diseases (CVD), but the underlying mechanisms are poorly understood. Recently, we showed that platelets from periodontitis patients are more activated than those from controls. Given the regularly occurring bacteremic episodes in periodontitis patients, we hypothesized that platelets and/or leukocytes from periodontitis patients are more sensitive to stimulation by oral bacteria, in particular the known periodontal pathogens, than platelets from control subjects. Three-color flow cytometry analysis was performed to quantify activation of platelets (P-selectin, PAC-1, CD63) and leukocytes (CD11b) in whole blood from patients with periodontitis (n = 19) and controls (n = 18), with and without stimulation by oral bacteria. Phagocytosis was assessed by using green-fluorescent protein (GFP)-expressing Aggregatibacter actinomycetemcomitans (Aa). Neutrophils and monocytes were activated by all species of oral bacteria tested, but no differences were observed between patients and controls. In response to several species of oral bacteria, platelets from periodontitis patients showed, compared with controls, increased exposure of P-selectin (P = 0.027) and increased formation of platelet-monocyte complexes (P = 0.040). Platelet-leukocyte complexes bound and/or phagocytosed more GFP-Aa than platelet-free leukocytes (for neutrophils and monocytes, in both patients and controls, P < 0.001). In periodontitis, increased platelet response to oral bacteria is paralleled by increased formation of platelet-leukocyte complexes with elevated capacity for bacterial clearance. We speculate that activated platelets and leukocytes might contribute to increased atherothrombotic activity.

  1. Upregulation of Leukocytic Syncytin-1 in Acute Myeloid Leukemia Patients.

    PubMed

    Sun, Yi; Zhu, Hongyan; Song, Jianxin; Jiang, Yaxian; Ouyang, Hongmei; Huang, Rongzhong; Zhang, Guiqian; Fan, Xin; Tao, Rui; Jiang, Jie; Niu, Hua

    2016-07-09

    BACKGROUND Syncytin-1, a cell membrane-localizing fusogen, is abnormally expressed in several cancers, including endometrial cancer, breast cancer, and leukemia. Although abnormal syncytin-1 expression has been detected in two-thirds of leukemia blood samples, its expression profile in acute leukemia patients has not yet been analyzed. MATERIAL AND METHODS Bone marrow samples from 50 acute myelogenous leukemia (AML) cases and 14 B-cell acute lymphocytic leukemia (B-cell ALL) patients were subjected to flow cytometry to assess leukocyte type distributions and leukocytic syncytin-1 surface expression. RT-PCR was applied to assess leukocytic syncytin-1 mRNA expression. Statistical analysis was applied to compare syncytin-1 expression between AML and B-cell ALL patients across blasts, granulocytes, lymphocytes, and monocytes as well as to determine clinical factors statistically associated with changes in syncytin-1 expression. RESULTS The leukocyte type distributions of the AML and B-cell ALL cohorts highly overlapped, with an observable difference in blast distribution between the 2 cohorts. The AML cohort displayed significantly greater syncytin-1 surface and mRNA expression (p<0.05). Syncytin-1 surface and mRNA expression was significantly increased across all 4 leukocyte types (p<0.05). The percentage of syncytin-1-expressing blasts was significantly greater in AML patients (p<0.05), with blasts showing the largest fold-change in syncytin-1 expression (p<0.05). M5, M5a, and M5b AML patients displayed significantly higher syncytin-1 surface expression relative to all other AML French-American-British (FAB) classifications (p<0.05). CONCLUSIONS These findings suggest leukocytic syncytin-1 expression may play a role in the development and/or maintenance of the AML phenotype and the acute monocytic leukemia phenotype in particular.

  2. 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

  3. In Vivo Quantitation of Local Anesthetic Suppression of Leukocyte Adherence

    PubMed Central

    Giddon, D. B.; Lindhe, J.

    1972-01-01

    Using intravital microscopy, topically applied amide-type local anesthetics suppressed the adherence of leukocytes to the venular endothelium within surgical defects in the hamster cheek pouch. The response was reversible with physiologic saline and was localized to venules within the defect. Quantitation in terms of the percent of initially adhering leukocytes remaining in place on the venule wall at each minute following application of lidocaine and physiologic saline, respectively, revealed the suppression to be reliably related to the concentration, viz: 20.0 >10.0 >5.0 >0.0 mg ml of commercially available Xylocaine® (lidocaine) HCl. ImagesFig 1Fig 1 PMID:5049429

  4. 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

  5. Endothelial cells proactively form microvilli-like membrane projections upon intercellular adhesion molecule 1 engagement of leukocyte LFA-1.

    PubMed

    Carman, Christopher V; Jun, Chang-Duk; Salas, Azucena; Springer, Timothy A

    2003-12-01

    Specific leukocyte/endothelial interactions are critical for immunity and inflammation, yet the molecular details of this interaction interface remain poorly understood. Thus, we investigated, with confocal microscopy, the distribution dynamics of the central adhesion molecules ICAM-1 and LFA-1 in this context. Monolayers of activated HUVECs stained with fluorescent anti-ICAM-1 Fabs or Chinese hamster ovary-K1 cells expressing ICAM-1-green fluorescent protein were allowed to bind LFA-1-bearing monocytes, neutrophils, or K562 LFA-1 transfectants. ICAM-1 was rapidly relocalized to newly formed microvilli-like membrane projections in response to binding LFA-1 on leukocytes. These ICAM-1-enriched projections encircled the leukocytes extending up their sides and clustered LFA-1 underneath into linear tracks. Projections formed independently of VCAM-1/very late Ag 4 interactions, shear, and proactive contributions from the LFA-1-bearing cells. In the ICAM-1-bearing endothelial cells, projections were enriched in actin but not microtubules, required intracellular calcium, and intact microfilament and microtubule cytoskeletons and were independent of Rho/Rho kinase signaling. Disruption of these projections with cytochalasin D, colchicine, or BAPTA-AM had no affect on firm adhesion. These data show that in response to LFA-1 engagement the endothelium proactively forms an ICAM-1-enriched cup-like structure that surrounds adherent leukocytes but is not important for firm adhesion. This finding leaves open a possible role in leukocyte transendothelial migration, which would be consistent with the geometry and kinetics of formation of the cup-like structure.

  6. Treatment of obstructive sleep apnea alters cancer-associated transcriptional signatures in circulating leukocytes.

    PubMed

    Gharib, Sina A; Seiger, Ashley N; Hayes, Amanda L; Mehra, Reena; Patel, Sanjay R

    2014-04-01

    Obstructive sleep apnea (OSA) has been associated with a number of chronic disorders that may improve with effective therapy. However, the molecular pathways affected by continuous positive airway pressure (CPAP) treatment are largely unknown. We sought to assess the system-wide consequences of CPAP therapy by transcriptionally profiling peripheral blood leukocytes (PBLs). Subjects in whom severe OSA was diagnosed were treated with CPAP, and whole-genome expression measurement of PBLs was performed at baseline and following therapy. We used gene set enrichment analysis (GSEA) to identify pathways that were differentially enriched. Network analysis was then applied to highlight key drivers of processes influenced by CPAP. Eighteen subjects with significant OSA underwent CPAP therapy and microarray analysis of their PBLs. Treatment with CPAP improved apnea-hypopnea index (AHI), daytime sleepiness, and blood pressure, but did not affect anthropometric measures. GSEA revealed a number of enriched gene sets, many of which were involved in neoplastic processes and displayed downregulated expression patterns in response to CPAP. Network analysis identified several densely connected genes that are important modulators of cancer and tumor growth. Effective therapy of OSA with CPAP is associated with alterations in circulating leukocyte gene expression. Functional enrichment and network analyses highlighted transcriptional suppression in cancer-related pathways, suggesting potentially novel mechanisms linking OSA with neoplastic signatures.

  7. Inflammation and the Intestinal Barrier: Leukocyte-Epithelial Cell Interactions, Cell Junction Remodeling, and Mucosal Repair.

    PubMed

    Luissint, Anny-Claude; Parkos, Charles A; Nusrat, Asma

    2016-10-01

    The intestinal tract is lined by a single layer of columnar epithelial cells that forms a dynamic, permeable barrier allowing for selective absorption of nutrients, while restricting access to pathogens and food-borne antigens. Precise regulation of epithelial barrier function is therefore required for maintaining mucosal homeostasis and depends, in part, on barrier-forming elements within the epithelium and a balance between pro- and anti-inflammatory factors in the mucosa. Pathologic states, such as inflammatory bowel disease, are associated with a leaky epithelial barrier, resulting in excessive exposure to microbial antigens, recruitment of leukocytes, release of soluble mediators, and ultimately mucosal damage. An inflammatory microenvironment affects epithelial barrier properties and mucosal homeostasis by altering the structure and function of epithelial intercellular junctions through direct and indirect mechanisms. We review our current understanding of complex interactions between the intestinal epithelium and immune cells, with a focus on pathologic mucosal inflammation and mechanisms of epithelial repair. We discuss leukocyte-epithelial interactions, as well as inflammatory mediators that affect the epithelial barrier and mucosal repair. Increased knowledge of communication networks between the epithelium and immune system will lead to tissue-specific strategies for treating pathologic intestinal inflammation. Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.

  8. Differential Gene Expression after Zinc Supplementation and Deprivation in Human Leukocyte Subsets

    PubMed Central

    Haase, Hajo; Mazzatti, Dawn J; White, Andrew; Ibs, Klaus H; Engelhardt, Gabriela; Hebel, Silke; Powell, Jonathan R; Rink, Lothar

    2007-01-01

    An individual’s zinc status has a significant impact on the immune system, and zinc deficiency, as well as supplementation, modulates immune function. To investigate the effects of zinc on different leukocyte subsets, we used microarray technology to analyze and compare the changes in mRNA expression in cell culture models of monocytes (THP-1), T cells (Jurkat), and B cells (Raji), in response to supplementation for 40 h with 50 μM zinc or 2.5 μM of the membrane-permeant zinc chelator TPEN [N,N,N′,N′-tetrakis-(2-pyridyl-methyl)ethylenediamine], respectively. In each cell type, several hundred genes were identified to be zinc sensitive, but only a total of seven genes were commonly regulated in all three cell lines. The majority of those genes were involved in zinc homeostasis, and none in immune function. Nevertheless, further analysis revealed that zinc affects entire functional networks of genes that are related to proinflammatory cytokines and cellular survival. Although the zinc-regulated activities are similar throughout the gene networks, the specific genes that are affected vary significantly between different cell types, a situation that helps to elucidate the disparity of the effects that zinc has on different leukocyte populations. PMID:17622302

  9. Differential roles of ICAM-1 and VCAM-1 in leukocyte-endothelial cell interactions in skin and brain of MRL/faslpr mice.

    PubMed

    Norman, M Ursula; James, Will G; Hickey, Michael J

    2008-07-01

    MRL/fas(lpr) mice, which undergo a systemic autoimmune disease with similarities to systemic lupus erythematosus (SLE), display reduced pathology and prolonged survival if rendered deficient in ICAM-1. However, it remains unclear whether this is a result of the ability of ICAM-1 to promote the immune response or mediate leukocyte recruitment. Therefore, the aim of these studies was to compare the role of ICAM-1 in the elevated leukocyte-endothelial interactions, which affect MRL/fas(lpr) mice. Intravital microscopy was used to compare leukocyte rolling and adhesion in postcapillary venules in the dermal and cerebral (pial) microcirculations of wild-type (ICAM+/+) and ICAM-1-deficient (ICAM-1-/-) MRL/fas(lpr) mice. In the dermal microcirculation of 16-week MRL/fas(lpr) mice, leukocyte adhesion was increased relative to nondiseased MRL+/+ mice. However, this increase was abolished in ICAM-1-/- MRL/fas(lpr) mice. ICAM-1 deficiency was also associated with reduced dermal pathology. In contrast, in the pial microcirculation, the elevation in leukocyte adhesion observed in ICAM+/+ MRL/fas(lpr) mice also occurred in ICAM-1-/- MRL/fas(lpr) mice. VCAM-1 expression was detectable in both vascular beds, but higher levels were detected in the pial vasculature. Furthermore, VCAM-1 blockade significantly reduced leukocyte adhesion and rolling in the cerebral microcirculation of ICAM-1-/- MRL/fas(lpr) mice. Therefore, ICAM-1 was critical for leukocyte adhesion in the skin but not the brain, where VCAM-1 assumed the major function. Given the ongoing development of anti-adhesion molecule therapies and their potential in inflammatory diseases such as SLE, these data indicate that implementation of these therapies in SLE should take into account the potential for tissue-specific functions of adhesion molecules.

  10. Differential roles of ICAM-1 and VCAM-1 in leukocyte-endothelial cell interactions in skin and brain of MRL/faslpr mice

    PubMed Central

    Norman, M. Ursula; James, Will G.; Hickey, Michael J.

    2008-01-01

    MRL/faslpr mice, which undergo a systemic autoimmune disease with similarities to systemic lupus erythematosus (SLE), display reduced pathology and prolonged survival if rendered deficient in ICAM-1. However, it remains unclear whether this is a result of the ability of ICAM-1 to promote the immune response or mediate leukocyte recruitment. Therefore, the aim of these studies was to compare the role of ICAM-1 in the elevated leukocyte-endothelial interactions, which affect MRL/faslpr mice. Intravital microscopy was used to compare leukocyte rolling and adhesion in postcapillary venules in the dermal and cerebral (pial) microcirculations of wild-type (ICAM+/+) and ICAM-1-deficient (ICAM-1−/−) MRL/faslpr mice. In the dermal microcirculation of 16-week MRL/faslpr mice, leukocyte adhesion was increased relative to nondiseased MRL+/+ mice. However, this increase was abolished in ICAM-1−/− MRL/faslpr mice. ICAM-1 deficiency was also associated with reduced dermal pathology. In contrast, in the pial microcirculation, the elevation in leukocyte adhesion observed in ICAM+/+ MRL/faslpr mice also occurred in ICAM-1−/− MRL/faslpr mice. VCAM-1 expression was detectable in both vascular beds, but higher levels were detected in the pial vasculature. Furthermore, VCAM-1 blockade significantly reduced leukocyte adhesion and rolling in the cerebral microcirculation of ICAM-1−/− MRL/faslpr mice. Therefore, ICAM-1 was critical for leukocyte adhesion in the skin but not the brain, where VCAM-1 assumed the major function. Given the ongoing development of anti-adhesion molecule therapies and their potential in inflammatory diseases such as SLE, these data indicate that implementation of these therapies in SLE should take into account the potential for tissue-specific functions of adhesion molecules. PMID:18426970

  11. Depletion of polymorphonuclear leukocytes has no effect on preterm delivery in a mouse model of Escherichia coli-induced labor.

    PubMed

    Filipovich, Yana; Agrawal, Varkha; Crawford, Susan E; Fitchev, Philip; Qu, Xiaowu; Klein, Jeremy; Hirsch, Emmet

    2015-11-01

    The objective of the study was to investigate the role of polymorphonuclear leukocytes (PMNs) in a mouse model of Escherichia coli-induced labor. Intraperitoneal injection of rabbit antimouse PMN antiserum or control was performed in CD-1 mice 29 hours and 5 hours prior to laparotomy and intrauterine injection of either killed E coli or phosphate-buffered saline on day 14.5 of pregnancy. Preterm delivery was defined as delivery of at least 1 pup within 48 hours. Circulating leukocyte counts were determined manually or by flow cytometry at the time of surgery and 8, 24, and 48 hours afterward. Maternal and fetal tissues were analyzed in a separate group of animals 8 hours after surgery. Pretreatment with anti-PMN antiserum significantly decreased the numbers of circulating leukocytes and the proportion of neutrophils among all leukocytes by 70-80% at surgery and at least 8 hours thereafter. Neutrophil depletion significantly reduced 2 markers of neutrophil activation in the uterus and placenta (neutrophil elastase and myeloperoxidase activity) and neutrophil infiltration into gestational tissues in bacterially treated animals to baseline (control) levels but did not affect preterm birth rates. The large E coli-induced increases in uterine inflammatory markers (interleukin-1β, tumor necrosis factor, chemokine ligand-5, cyclooxygenase-2) were not affected or were only minimally affected by neutrophil depletion. Although PMN antiserum reduces both neutrophil number and activity, it does not diminish sensitivity to bacterially induced delivery or meaningfully alter the expression of inflammatory markers in the mouse model. Preterm birth and inflammation in this model are not likely to depend on neutrophil function. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Human leukocyte interferon: structural and biological relatedness to adrenocorticotropic hormone and endorphins.

    PubMed Central

    Blalock, J E; Smith, E M

    1980-01-01

    Anti-alpha-corticotropin [anti-ACTH alpha (1-13)](also alpha-melanotropin) and anti-gamma-endorphin antisera neutralized human leukocyte interferon activity but not fibroblast interferon activity. Human leukocyte interferon was not neutralized by anti-human lutenizing hormone (lutropin) or follicle-stimulating hormone (follitropin) antisra. Conversely, antisera to human leukocyte interferon neutralized ACTH activity. The neturalization of human leukocyte interferon by anti-human leukocyte interferon serum was partially blocked by ACTH. These studies show strong antigenic relatedness among human leukocyte interferon, ACTH, and endorphins, implying that there are underlying structural similarities. Structural relatedness is shown by pepsin cleavage of ACTH activity from human leukocyte interferon. The implication for the natural functions of human leukocyte interferon are discussed. PMID:6160589

  13. 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.

  14. Diagnosis of X-linked adrenoleukodystrophy in blood leukocytes.

    PubMed

    Unterberger, Ursula; Regelsberger, Günther; Sundt, Regina; Bernheimer, Hanno; Voigtländer, Till

    2007-09-01

    Our aim was to replace cultured skin fibroblasts in the diagnosis of X-linked adrenoleukodystrophy (X-ALD) by peripheral blood cells. Very long chain fatty acids (VLCFAs) were analyzed in leukocytes from X-ALD patients, heterozygotes, and controls using gas chromatography-mass spectrometry (GC-MS). Immunofluorescence for adrenoleukodystrophy protein (ALDP) was performed in mononuclear blood cell preparations of X-ALD patients known to be ALDP negative in fibroblasts, heterozygote relatives of these patients, and controls. All X-ALD patients were distinguishable from controls by VLCFA analysis in leukocytes. 91.7% of heterozygotes were identified by combined VLCFA analysis in leukocytes and plasma. All patients investigated lacked ALDP immunoreactivity in mononuclear cells, while heterozygotes showed mosaic patterns of positive and negative cells. Determination of VLCFAs by GC-MS in combination with ALDP immunofluorescence in peripheral blood cells provides a fast and minimally invasive diagnostic method for X-ALD, which, in contrast to plasma analysis, is independent of alimentary influences. Notably, joint evaluation of leukocytes and plasma considerably improves the identification of heterozygotes.

  15. 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 864.7675 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...

  16. Endothelial signaling in paracellular and transcellular leukocyte transmigration

    PubMed Central

    Wittchen, Erika S.

    2008-01-01

    As the primary physical barrier between blood and tissue compartments within the body, blood vessel endothelial cells and integrity of the cell junctions connecting them must be carefully regulated to support leukocyte transendothelial migration only when necessary. Leukocytes utilize two independent routes across the endothelium: the paracellular route involves migration in-between adjacent endothelial cells and requires the transient disassembly of endothelial cell junctions, while the transcellular route occurs directly through an individual endothelial cell, likely requiring the formation of a channel or pore. In this review, I will first summarize the signaling events that are transduced by leukocyte engagement of endothelial cell-surface receptors like ICAM-1 and VCAM-1. Some of these signals include activation of GTPases, production of reactive oxygen species, and phosphorylation of target proteins. These signaling pathways converge to cause junctional disruption, cytoskeletal remodeling, and/or the membrane fusion events that are associated with leukocyte transendothelial migration. The review will conclude with a detailed discussion of the newly characterized transmigratory cup structure, and the recent advances made towards understanding the mechanisms of transcellular transendothelial migration. PMID:19273217

  17. Endothelial signaling in paracellular and transcellular leukocyte transmigration.

    PubMed

    Wittchen, Erika S

    2009-01-01

    As the primary physical barrier between blood and tissue compartments within the body, blood vessel endothelial cells and integrity of the cell junctions connecting them must be carefully regulated to support leukocyte transendothelial migration only when necessary. Leukocytes utilize two independent routes across the endothelium: the paracellular route involves migration in-between adjacent endothelial cells and requires the transient disassembly of endothelial cell junctions, while the transcellular route occurs directly through an individual endothelial cell, likely requiring the formation of a channel or pore. In this review, I will first summarize the signaling events that are transduced by leukocyte engagement of endothelial cell-surface receptors like ICAM-1 and VCAM-1. Some of these signals include activation of GTPases, production of reactive oxygen species, and phosphorylation of target proteins. These signaling pathways converge to cause junctional disruption, cytoskeletal remodeling, and/or the membrane fusion events that are associated with leukocyte transendothelial migration. The review will conclude with a detailed discussion of the newly characterized transmigratory cup structure, and the recent advances made towards understanding the mechanisms of transcellular transendothelial migration.

  18. 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.

  19. 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.

  20. 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

  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. 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

  3. ADRENERGIC NERVES GOVERN CIRCADIAN LEUKOCYTE RECRUITMENT TO TISSUES

    PubMed Central

    Scheiermann, Christoph; Kunisaki, Yuya; Lucas, Daniel; Chow, Andrew; Jang, Jung-Eun; Zhang, Dachuan; Hashimoto, Daigo; Merad, Miriam; Frenette, Paul S.

    2012-01-01

    SUMMARY The multistep sequence leading to leukocyte migration is thought to be locally regulated at the inflammatory site. Here, we show that broad systemic programs involving long-range signals from the sympathetic nervous system (SNS) delivered by adrenergic nerves, regulate rhythmic recruitment of leukocytes in tissues. Constitutive leukocyte adhesion and migration in murine bone marrow (BM) and skeletal muscle microvasculature fluctuated with circadian peak values at night. Migratory oscillations, altered by experimental jetlag, were implemented by perivascular SNS fibers, acting on β-adrenoreceptors expressed on non-hematopoietic cells, and leading to tissue-specific, differential circadian oscillations in the expression of endothelial cell adhesion molecules and chemokines. We showed that these rhythms have physiological consequences by alteration of hematopoietic cell recruitment and overall survival in models of septic shock, sickle cell vaso-occlusion and BM transplantation. These data provide unique insight in the leukocyte adhesion cascade and the potential for time-based therapeutics for transplantation and inflammatory diseases. PMID:22863835

  4. 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.

  5. Biocompatibility of cellulosic and synthetic membranes assessed by leukocyte activation.

    PubMed

    Hernández, Maria Rosa; Galán, Ana Maria; Cases, Aleix; Lopez-Pedret, Jose; Pereira, Arturo; Tonda, Raul; Bozzo, Jordi; Escolar, Gines; Ordinas, Antonio

    2004-01-01

    The contact of blood with artificial surfaces may activate blood leukocytes and platelets and initiate the leukocyte inflammatory response. We have investigated the effect of a hemodialysis (HD) with a cellulosic- and a synthetic-based membrane on circulating leukocyte activation. Samples were obtained from patients with ESRD at baseline, and at 15 and 120 min of a hemodialysis session from both the arterial and venous lines. Leukocyte respiratory burst was analyzed by luminol chemiluminescence. Actin polymerization, expression of CD11b, and heterotypic aggregation were studied by flow cytometry, leukocyte labeling with NBD phallacidin and monoclonal antibodies, respectively. HD with a cellulosic membrane induced a transient fall in neutrophil (1.2 +/- 0.5 x 10(9) vs. 3.6 +/- 0.6 x 10(9) cells/l; p < 0.05) and monocyte counts (0.2 +/- 0.1 x 10(9) vs. 0.7 +/- 0.1 x 10(9) cells/l; p < 0.05). There was also an increase in respiratory burst in the venous line during a HD with a cellulosic membrane, at 15 and 120 min (100 +/- 41 and 143.2 +/- 45.3 vs. 23.8 +/- 15.7; p < 0.05). Polymerized actin, expressed as fluorescence arbitrary units, was increased in baseline samples from uremic patients versus control subjects (327.8 +/- 60.8 for a cellulosic membrane, p < 0.005, and 205 +/- 26.5 for a synthetic one, p < 0.05 vs. 97.8 +/- 27.6 in controls). The percentage of CD11b+ cells increased in samples during a HD with a cellulosic membrane at the venous line at 15 and 120 min (9.6 +/- 4.5 and 18.4 +/- 7.1% vs. 3.3 +/- 1.9%; p < 0.05%). Changes in heterotypic aggregation during HD did not reach statistical significance, but levels were higher in patients treated with a cellulosic membrane at all points than in patients dialyzed with a synthetic one. There is evidence of a priming state of leukocytes from uremic patients, which is more evident in patients dialyzed with a cellulosic membrane. Cellulosic membranes also induce greater leukocyte activation than synthetic membranes

  6. 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.

  7. SMN transcript levels in leukocytes of SMA patients determined by absolute real-time PCR

    PubMed Central

    Tiziano, Francesco Danilo; Pinto, Anna Maria; Fiori, Stefania; Lomastro, Rosa; Messina, Sonia; Bruno, Claudio; Pini, Antonella; Pane, Marika; D'Amico, Adele; Ghezzo, Alessandro; Bertini, Enrico; Mercuri, Eugenio; Neri, Giovanni; Brahe, Christina

    2010-01-01

    Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by homozygous mutations of the SMN1 gene. Three forms of SMA are recognized (type I–III) on the basis of clinical severity. All patients have at least one or more (usually 2–4) copies of a highly homologous gene (SMN2), which produces insufficient levels of functional SMN protein, because of alternative splicing of exon 7. Recently, evidence has been provided that SMN2 expression can be enhanced by pharmacological treatment. However, no reliable biomarkers are available to test the molecular efficacy of the treatments. At present, the only potential biomarker is the dosage of SMN products in peripheral blood. However, the demonstration that SMN full-length (SMN-fl) transcript levels are reduced in leukocytes of patients compared with controls remains elusive (except for type I). We have developed a novel assay based on absolute real-time PCR, which allows the quantification of SMN1-fl/SMN2-fl transcripts. For the first time, we have shown that SMN-fl levels are reduced in leukocytes of type II–III patients compared with controls. We also found that transcript levels are related to clinical severity as in type III patients SMN2-fl levels are significantly higher compared with type II and directly correlated with functional ability in type II patients and with age of onset in type III patients. Moreover, in haploidentical siblings with discordant phenotype, the less severely affected individuals showed significantly higher transcript levels. Our study shows that SMN2-fl dosage in leukocytes can be considered a reliable biomarker and can provide the rationale for SMN dosage in clinical trials. PMID:19603064

  8. Alterations in leukocyte transcriptional control pathway activity associated with major depressive disorder and antidepressant treatment

    PubMed Central

    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

    2016-01-01

    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 dysregulation in subjects with MDD. Using genome-wide transcriptional profiling and promoter-based bioinformatic strategies, we assessed leukocyte transcription factor (TF) activity in leukocytes from 20 unmedicated MDD subjects versus 20 age-, sex- and ethnicity-matched healthy controls, before initiation of antidepressant therapy, and in 17 of the MDD subjects after 8 weeks of sertraline treatment. In leukocytes from unmedicated MDD subjects, bioinformatic analysis of transcription control pathway activity indicated an increased transcriptional activity of cAMP response element-binding/activating TF (CREB/ATF) and increased activity of TFs associated with cellular responses to oxidative stress (nuclear factor erythroid-derived 2-like 2, NFE2l2 or NRF2). Eight weeks of antidepressant therapy was associated with significant reductions in Hamilton Depression Rating Scale scores and reduced activity of NRF2, but not in CREB/ATF activity. Several other transcriptional regulation pathways, including the glucocorticoid receptor (GR), nuclear factor kappa-B cells (NF-κB), early growth response proteins 1–4 (EGR1–4) and interferon-responsive TFs, showed either no significant differences as a function of disease or treatment, or activities that were opposite to those previously hypothesized to be involved in the etiology of MDD or effective treatment. Our results suggest that CREB/ATF and NRF2 signaling may contribute to MDD by activating immune cell transcriptome dynamics that ultimately influence central nervous system (CNS) motivational and affective processes via circulating mediators. PMID:27187237

  9. 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 dysregulation in subjects with MDD. Using genome-wide transcriptional profiling and promoter-based bioinformatic strategies, we assessed leukocyte transcription factor (TF) activity in leukocytes from 20 unmedicated MDD subjects versus 20 age-, sex- and ethnicity-matched healthy controls, before initiation of antidepressant therapy, and in 17 of the MDD subjects after 8 weeks of sertraline treatment. In leukocytes from unmedicated MDD subjects, bioinformatic analysis of transcription control pathway activity indicated an increased transcriptional activity of cAMP response element-binding/activating TF (CREB/ATF) and increased activity of TFs associated with cellular responses to oxidative stress (nuclear factor erythroid-derived 2-like 2, NFE2l2 or NRF2). Eight weeks of antidepressant therapy was associated with significant reductions in Hamilton Depression Rating Scale scores and reduced activity of NRF2, but not in CREB/ATF activity. Several other transcriptional regulation pathways, including the glucocorticoid receptor (GR), nuclear factor kappa-B cells (NF-κB), early growth response proteins 1-4 (EGR1-4) and interferon-responsive TFs, showed either no significant differences as a function of disease or treatment, or activities that were opposite to those previously hypothesized to be involved in the etiology of MDD or effective treatment. Our results suggest that CREB/ATF and NRF2 signaling may contribute to MDD by activating immune cell transcriptome dynamics that ultimately influence central nervous system (CNS) motivational and affective processes via circulating mediators.

  10. Influence of hypothermia and subsequent rewarming upon leukocyte-endothelial interactions and expression of Junctional-Adhesion-Molecules A and B

    PubMed Central

    Bogert, Nicolai V.; Werner, Isabella; Kornberger, Angela; Meybohm, Patrick; Moritz, Anton; Keller, Till; Stock, Ulrich A.; Beiras-Fernandez, Andres

    2016-01-01

    Patients with risks of ischemic injury, e.g. during circulatory arrest in cardiac surgery, or after resuscitation are subjected to therapeutic hypothermia. For aortic surgery, the body is traditionally cooled down to 18 °C and then rewarmed to body temperature. The role of hypothermia and the subsequent rewarming process on leukocyte-endothelial interactions and expression of junctional-adhesion-molecules is not clarified yet. Thus, we investigated in an in-vitro model the influence of temperature modulation during activation and transendothelial migration of leukocytes through human endothelial cells. Additionally, we investigated the expression of JAMs in the rewarming phase. Exposure to low temperatures alone during transmigration scarcely affects leukocyte extravasation, whereas hypothermia during treatment and transendothelial migration improves leukocyte-endothelial interactions. Rewarming causes a significant up-regulation of transmigration with falling temperatures. JAM-A is significantly modulated during rewarming. Our data suggest that transendothelial migration of leukocytes is not only modulated by cell-activation itself. Activation temperatures and the rewarming process are essential. Continued hypothermia significantly inhibits transendothelial migration, whereas the rewarming process enhances transmigration strongly. The expression of JAMs, especially JAM-A, is strongly modulated during the rewarming process. Endothelial protection prior to warm reperfusion and mild hypothermic conditions reducing the difference between hypothermia and rewarming temperatures should be considered. PMID:26912257

  11. In vivo compartmental analysis of leukocytes in mouse lungs

    PubMed Central

    Patel, Brijesh V.; Tatham, Kate C.; Wilson, Michael R.; O'Dea, Kieran P.

    2015-01-01

    The lung has a unique structure consisting of three functionally different compartments (alveolar, interstitial, and vascular) situated in an extreme proximity. Current methods to localize lung leukocytes using bronchoalveolar lavage and/or lung perfusion have significant limitations for determination of location and phenotype of leukocytes. Here we present a novel method using in vivo antibody labeling to enable accurate compartmental localization/quantification and phenotyping of mouse lung leukocytes. Anesthetized C57BL/6 mice received combined in vivo intravenous and intratracheal labeling with fluorophore-conjugated anti-CD45 antibodies, and lung single-cell suspensions were analyzed by flow cytometry. The combined in vivo intravenous and intratracheal CD45 labeling enabled robust separation of the alveolar, interstitial, and vascular compartments of the lung. In naive mice, the alveolar compartment consisted predominantly of resident alveolar macrophages. The interstitial compartment, gated by events negative for both intratracheal and intravenous CD45 staining, showed two conventional dendritic cell populations, as well as a Ly6Clo monocyte population. Expression levels of MHCII on these interstitial monocytes were much higher than on the vascular Ly6Clo monocyte populations. In mice exposed to acid aspiration-induced lung injury, this protocol also clearly distinguished the three lung compartments showing the dynamic trafficking of neutrophils and exudative monocytes across the lung compartments during inflammation and resolution. This simple in vivo dual-labeling technique substantially increases the accuracy and depth of lung flow cytometric analysis, facilitates a more comprehensive examination of lung leukocyte pools, and enables the investigation of previously poorly defined “interstitial” leukocyte populations during models of inflammatory lung diseases. PMID:26254421

  12. Accelerated wound healing in leukocyte-specific, protein 1-deficient mouse is associated with increased infiltration of leukocytes and fibrocytes.

    PubMed

    Wang, JianFei; Jiao, Haiyan; Stewart, Tara L; Lyons, Megan V H; Shankowsky, Heather A; Scott, Paul G; Tredget, Edward E

    2007-12-01

    Wound healing is a complex process involving the integrated actions of numerous cell types, soluble mediators, and ECM. Recently, a newly identified cell type, the fibrocyte, has been reported to contribute to wound healing and fibrotic conditions such as hypertrophic scarring. We previously established leukocyte-specific protein 1 (LSP1) as a marker for fibrocytes. LSP1 is an F-actin binding protein and substrate of p38 mitogen-activated protein kinase and protein kinase C, and has been reported to be important in leukocyte chemotaxis. We examine the biological roles of LSP1 in skin wound healing using Lsp1(-/-) null mice. These animals showed accelerated healing of full-thickness skin wounds, with increased re-epithelialization rates, collagen synthesis, and angiogenesis. Healing wounds in Lsp1(-/-) mice had higher densities of neutrophiles, macrophages, and fibrocytes. Along with increased leukocyte infiltration, levels of macrophage-derived chemokine expression, TGF-beta1, and VEGF were all up-regulated. These results demonstrate that the absence of LSP1 promotes healing of skin wounds. The primary mechanism seems to be an increase in leukocyte infiltration, leading to locally elevated synthesis and release of chemokines and growth factors. Further analysis of Lsp1(-/-) mice may suggest ways to improve wound healing and/or treat fibrotic conditions of skin and other tissue.

  13. Leukocyte profile, gene expression, acute phase response, and metabolite status of cows with sole hemorrhages.

    PubMed

    O'Driscoll, Keelin; McCabe, Matthew; Earley, Bernadette

    2017-08-23

    Sole hemorrhages result from disruption to normal claw horn formation and are caused by a variety of internal and external factors. Evidence suggests that they are painful, although they do not usually cause clinical lameness and are difficult to detect by observing cow gait. Little is known about how or whether sole hemorrhages affect the cow systemically. This study compared hematology profile, leukocyte gene expression, and physiological responses of cows with no/mild hemorrhages (category 1; n = 17), moderate hemorrhages (category 2; n = 18), and severe hemorrhages (category 3; n = 12). At approximately 100 d in milk, all cows in the study herd (n = 374) were locomotion scored before hoof examination. The cows included in the study were not clinically lame and had no other hoof disorder. Blood samples were taken from all cows within 24 h of selection. Leukocyte counts were obtained using an automated cell counter, cortisol and dehydroepiandrosterone (DHEA) concentration by ELISA, and plasma haptoglobin, urea, total protein, creatine kinase and glucose were analyzed on a clinical chemistry analyzer. Expression of 16 genes associated with lameness or stress were estimated using real-time quantitative PCR. Data from cows within each category were compared using the Mixed procedure in SAS (version 9.3; SAS Institute Inc., Cary, NC). Fixed effects included hemorrhage severity category and lactation number, with days in milk and body condition score included as covariates. Locomotion score worsened as sole hemorrhage category worsened. Locomotion score of category 1 cows tended to be lower than that of category 2 cows and was lower than that of category 3 cows. The locomotion score of category 3 cows was also greater than that of categories 1 and 2 combined. Category had no effect on leukocyte number, on any of the individual leukocyte cell numbers or percentages, cortisol or DHEA concentration, cortisol:DHEA ratio, or relative expression of any of the genes

  14. Cardiolipin fingerprinting of leukocytes by MALDI-TOF/MS as a screening tool for Barth syndrome.

    PubMed

    Angelini, Roberto; Lobasso, Simona; Gorgoglione, Ruggiero; Bowron, Ann; Steward, Colin G; Corcelli, Angela

    2015-09-01

    Barth syndrome (BTHS), an X-linked disease associated with cardioskeletal myopathy, neutropenia, and organic aciduria, is characterized by abnormalities of card-iolipin (CL) species in mitochondria. Diagnosis of the disease is often compromised by lack of rapid and widely available diagnostic laboratory tests. The present study describes a new method for BTHS screening based on MALDI-TOF/MS analysis of leukocyte lipids. This generates a "CL fingerprint" and allows quick and simple assay of the relative levels of CL and monolysocardiolipin species in leukocyte total lipid profiles. To validate the method, we used vector algebra to analyze the difference in lipid composition between controls (24 healthy donors) and patients (8 boys affected by BTHS) in the high-mass phospholipid range. The method of lipid analysis described represents an important additional tool for the diagnosis of BTHS and potentially enables therapeutic monitoring of drug targets, which have been shown to ameliorate abnormal CL profiles in cells. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

  15. Effects of radiolabelled monoclonal antibody infusion on blood leukocytes in cancer patients

    SciTech Connect

    Gridley, D.S.; Slater, J.M.; Stickney, D.R. )

    1990-01-01

    This study was undertaken to investigate the effects of a single infusion of radiolabelled murine monoclonal antibody (MAb) on peripheral blood leukocytes in cancer patients. Eleven patients with disseminated colon cancer, malignant melanoma, or lung adenocarcinoma were infused with 111In-labelled anti-ZCE 025, anti-p97 type 96.5c, or LA 20207 MAb, respectively. Blood samples were obtained before infusion, immediately after infusion (1 hr), and at 4 and 7 days postinfusion. Flow cytometry analysis of CD3+, CD4+, CD8+, CD16+, and CD19+ lymphocytes showed increasing CD4:CD8 ratios in seven patients after infusion. This phenomenon was not restricted to antibody subclass or to type of cancer. Two of the remaining patients exhibited a marked post-infusion increase in CD8+ cells. In all three patients with malignant melanoma, decreasing levels of CD16+ lymphocytes were noted after infusion and natural killer cell cytotoxicity showed fluctuations which paralleled the changes in the CD16+ subpopulation. Oxygen radical production by phagocytic cells was markedly affected in three subjects. These results suggest that a single infusion of radiolabelled murine MAb may alter the balance of critical lymphocyte subpopulations and modulate other leukocyte responses in cancer patients.

  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. Leukocyte counts and lymphocyte subsets in relation to pregnancy and HIV infection in Malawian women.

    PubMed

    Mandala, Wilson L; Gondwe, Esther N; Molyneux, Malcolm E; MacLennan, Jenny M; MacLennan, Calman A

    2017-09-01

    We investigated leukocyte and lymphocyte subsets in HIV-infected or HIV-uninfected, pregnant or non-pregnant Malawian women to explore whether HIV infection and pregnancy may act synergistically to impair cellular immunity. We recruited 54 pregnant and 48 non-pregnant HIV-uninfected women and 24 pregnant and 20 non-pregnant HIV-infected Malawian women. We compared peripheral blood leukocyte and lymphocyte subsets between women in the four groups. Parturient HIV-infected and HIV-uninfected women had more neutrophils (each P<.0001), but fewer lymphocytes (P<.0001; P=.0014) than non-pregnant women. Both groups had fewer total T cells (P<.0001; P=.002) and CD8(+) T cells (P<.0001; P=.014) than non-pregnant women. HIV-uninfected parturient women had fewer CD4(+) and γδ T cells, B and NK cells (each P<.0001) than non-pregnant women. Lymphocyte subset percentages were not affected by pregnancy. Malawian women at parturition have an increased total white cell count due to neutrophilia and an HIV-unrelated pan-lymphopenia. © 2017 The Author. American Journal of Reproductive Immunology Published by John Wiley & Sons Ltd.

  18. Flow cytometric leukocyte population patterns in brown bullhead from three Ohio rivers

    SciTech Connect

    Torsella, T.A.; Neiheisel, T.; Cormier, S.M.; Bercz, P.

    1994-12-31

    Brown bullhead (A. nebulosus) were collected from three Ohio rivers: Old Woman Creek (OWC), classified as clean, Cuyahoga (CR) and Black Rivers (BR), documented as polluted. Each river was sampled in April and September 1993. Patterns of leukocyte distribution (prepared via density gradient) by GSH content and oxidative burst capacity, using fluorescent probes were determined by flow cytometry. Sample processing was in blind coded batches. Three principal classes of leukocytes were identified. Type A; large, very granular with high GSH reserves and marked capacity for oxidative burst. Type B were smaller, less granular, contained low GSH and negligible oxidative burst capacity. Type C were small, less granular, possessing intermediate GSH and peroxidative activity. ANOVA of the subset distributions and mean fluorescent intensities by sex, site and season, disclosed: Type C were significantly (p < 0.001) elevated in the spring OWC males, whereas type A dominated in the spring CR and spring BR males. No differences in Type A/Type C patterns were seen in the spring females. In the fall sampling, significant dominance (p < 0.001) of Type A was seen in both sexes of the OWC fish, the CR and BR fish showed a predominance of Type C. These may be explained by chemical stressors, affecting immune competence. Sex differences in the spring were attributed to hormonal (spawning) influences.

  19. Direct visualization of leukocyte/endothelial cell interaction during extracorporeal circulation (ECC) in a new animal model.

    PubMed

    Kamler, M; Jakob, H; Lehr, H A; Gebhard, M M; Hagl, S

    1997-05-01

    The clinical complications of extracorporeal circulation (ECC) have been linked to a systemic activation of cellular and humoral components and to a dysregulation of the microcirculatory compartment. Since to date only in vitro methods exist for evaluation, we developed an animal model to study the effects of ECC on the microcirculation. To establish the model, we assessed whether these effects are dependent on the duration of ECC. Intravital fluorescence microscopy was used on the dorsal skinfold chamber preparation in chronically instrumented, awake Syrian golden hamsters. ECC was realized using a micro-rollerpump and a silicon tube shunting blood between the carotid artery and the jugular vein. ECC was performed in three groups for various times (2, 10 and 20 min) after application of heparin at 300 IU/kg body wt. In hamsters, the application of high-dose heparin releases endothelial bound superoxide dismutase (SOD), a natural scavenger of oxygen-derived free radicals. Protocol II assigned two groups receiving heparin at different doses of 50 and 2000 IU/kg body wt. ECC for 2 min served as control to exclude effects from hemodilution and resulted in a minimal induction of leukocyte/endothelial cell interaction. Isovolemic ECC for 20 min resulted in an increase in rolling (from 11 +/- 3 to 38 +/- 20%, mean +/- S.D., P < 0.05) and adherent leukocytes (from 19 +/- 16 to 215 +/- 145 cells/mm2, mean +/- S.D., P < 0.05) in postcapillary venules. Microhemodynamic parameters and functional capillary density were not significantly affected. Arterial blood pressure and heart rate were stable. Heparin at 2000 IU/kg inhibited post-ECC leukocyte adhesion following ECC, whereas 50 IU/kg showed no protective effects. Leukocyte/endothelial cell interaction, induced by blood contact with synthetic surfaces, was directly visualized in vivo. The number of adherent leukocytes was dependent on the duration of ECC. The application of high-dose heparin followed by release of SOD

  20. The effect of hypothermia on influx of leukocytes in the digital lamellae of horses with oligofructose-induced laminitis.

    PubMed

    Godman, Jennifer D; Burns, Teresa A; Kelly, Carlin S; Watts, Mauria R; Leise, Britta S; Schroeder, Eric L; van Eps, Andrew W; Belknap, James K

    2016-10-01

    Sepsis-related laminitis (SRL) is a common complication in the septic/endotoxemic critically-ill equine patient, in which lamellar injury and failure commonly lead to crippling distal displacement of the distal phalanx. Similar to organ injury in human sepsis, lamellar injury in SRL has been associated with inflammatory events, including the influx of leukocytes into the lamellar tissue and markedly increased expression of a wide array of inflammatory mediators at the onset of Obel grade 1 (OG1) laminitis. The only treatment reported both clinically and experimentally to protect the lamellae in SRL, local hypothermia ("cryotherapy"), has been demonstrated to effectively inhibit lamellar expression of multiple inflammatory mediators when initiated at the time of administration of a carbohydrate overload in experimental models of SRL. However, the effect of hypothermia on leukocyte influx into affected tissue has not been assessed. We hypothesized that cryotherapy inhibits leukocyte emigration into the digital lamellae in SRL. Immunohistochemical staining using leukocyte markers MAC387 (marker of neutrophils, activated monocytes) and CD163 (monocyte/macrophage-specific marker) was performed on archived lamellar tissue samples from an experimental model of SRL in which one forelimb was maintained at ambient temperature (AMB) and one forelimb was immersed in ice water (ICE) immediately following enteral oligofructose administration (10g/kg, n=14 horses). Lamellae were harvested at 24h post-oligofructose administration (DEV, n=7) or at the onset of OG1 laminitis (OG1, n=7). Both MAC387-positive and CD163-positive cells were counted by a single blinded investigator on images [n=10 (40× fields/digit for MAC387 and 20x fields/digit for CD163)] obtained using Aperio microscopy imaging analysis software. Data were assessed for normality and analyzed with a paired t-test and one-way ANOVA with significance set at p<0.05. MAC387-positive cells were present in low numbers in

  1. 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.

  2. 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.

  3. 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.

  4. Big insights from small volumes: deciphering complex leukocyte behaviors using microfluidics

    PubMed Central

    Irimia, Daniel; Ellett, Felix

    2016-01-01

    Inflammation is an indispensable component of the immune response, and leukocytes provide the first line of defense against infection. Although the major stereotypic leukocyte behaviors in response to infection are well known, the complexities and idiosyncrasies of these phenotypes in conditions of disease are still emerging. Novel tools are indispensable for gaining insights into leukocyte behavior, and in the past decade, microfluidic technologies have emerged as an exciting development in the field. Microfluidic devices are readily customizable, provide tight control of experimental conditions, enable high precision of ex vivo measurements of individual as well as integrated leukocyte functions, and have facilitated the discovery of novel leukocyte phenotypes. Here, we review some of the most interesting insights resulting from the application of microfluidic approaches to the study of the inflammatory response. The aim is to encourage leukocyte biologists to integrate these new tools into increasingly more sophisticated experimental designs for probing complex leukocyte functions. PMID:27194799

  5. 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.

  6. 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.

  7. Feature selection and classification of leukocytes using random forest.

    PubMed

    Saraswat, Mukesh; Arya, K V

    2014-12-01

    In automatic segmentation of leukocytes from the complex morphological background of tissue section images, a vast number of artifacts/noise are also extracted causing large amount of multivariate data generation. This multivariate data degrades the performance of a classifier to discriminate between leukocytes and artifacts/noise. However, the selection of prominent features plays an important role in reducing the computational complexity and increasing the performance of the classifier as compared to a high-dimensional features space. Therefore, this paper introduces a novel Gini importance-based binary random forest feature selection method. Moreover, the random forest classifier is used to classify the extracted objects into artifacts, mononuclear cells, and polymorphonuclear cells. The experimental results establish that the proposed method effectively eliminates the irrelevant features, maintaining the high classification accuracy as compared to other feature reduction methods.

  8. Genotoxic effect of ozone in human peripheral blood leukocytes.

    PubMed

    Díaz-Llera, Silvia; González-Hernández, Yanela; Prieto-González, E A; Azoy, Angel

    2002-05-27

    The genotoxic effect of ozone was studied in human leukocytes in vitro, using the single cell gel electrophoresis (SCGE) assay. Cell treatment for 1 h at 37 degrees C with 0.9-5.3 mM O(3) resulted in a dose-dependent increase of DNA damage, comparable to that induced by 4-40 mM of H(2)O(2), used as a positive control. This effect of ozone was reversed by post-treatment incubation of the cells for 45-90 min at 37 degrees C, and prevented by pre-incubation of the cells with catalase (20 microg/ml). These results demonstrate that O(3) induces DNA-damage in primary human leukocytes. The damage is rapidly repaired, and probably mediated by the formation of H(2)O(2).

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. Maternal circulating leukocytes display early chemotactic responsiveness during late gestation

    PubMed Central

    2013-01-01

    Background Parturition has been widely described as an immunological response; however, it is unknown how this is triggered. We hypothesized that an early event in parturition is an increased responsiveness of peripheral leukocytes to chemotactic stimuli expressed by reproductive tissues, and this precedes expression of tissue chemotactic activity, uterine activation and the systemic progesterone/estradiol shift. Methods Tissues and blood were collected from pregnant Long-Evans rats on gestational days (GD) 17, 20 and 22 (term gestation). We employed a validated Boyden chamber assay, flow cytometry, quantitative real time-polymerase chain reaction, and enzyme-linked immunosorbent assays. Results We found that GD20 maternal peripheral leukocytes migrated more than those from GD17 when these were tested with GD22 uterus and cervix extracts. Leukocytes on GD20 also displayed a significant increase in chemokine (C-C motif) ligand 2 (Ccl2) gene expression and this correlated with an increase in peripheral granulocyte proportions and a decrease in B cell and monocyte proportions. Tissue chemotactic activity and specific chemokines (CCL2, chemokine (C-X-C motif) ligand 1/CXCL1, and CXCL10) were mostly unchanged from GD17 to GD20 and increased only on GD22. CXCL10 peaked on GD20 in cervical tissues. As expected, prostaglandin F2α receptor and oxytocin receptor gene expression increased dramatically between GD20 and 22. Progesterone concentrations fell and estradiol-17β concentrations increased in peripheral serum, cervical and uterine tissue extracts between GD20 and 22. Conclusion Maternal circulating leukocytes display early chemotactic responsiveness, which leads to their infiltration into the uterus where they may participate in the process of parturition. PMID:23445935

  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. In-111-leukocyte scanning in inflammatory renal disease

    SciTech Connect

    Fawcett, H.D.; Goodwin, D.A.; Lantieri, R.L.

    1981-06-01

    In-111-leukocyte scanning has recently been introduced as a clinically effective method for detecting inflammatory disease and abscesses. The authors present six cases that demonstrate the usefulness of this new technique in suspected inflammatory renal disease. Two patients had renal abscesses, two had acute pyelonephritis, one had acute focal bacterial nephritis (acute lobar nephronia), and one had a transitional cell carcinoma with associated acute and chronic inflammation.

  19. 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

  20. 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.

  1. 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.

  2. Nonvisualization of sterile surgical incisions with indium-111 labeled leukocytes

    SciTech Connect

    Abdel-Nabi, H.; Hinkle, G.H.; Olsen, J.O.

    1985-09-01

    The localization of In-111 labeled leukocytes (WBCs) in recent surgical incisions was studied in 18 patients. In-111 WBC images correlated well with culture results and clinical findings. No accumulation of In-111 WBCs was detected at the site of noninfected incisions in nine patients. In-111 WBCs did accumulate at incision sites in nine patients with infected surgical incisions. These results indicate that In-111 WBC study can accurately distinguish between normal healing and infection of recent surgical incisions.

  3. 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.

  4. Tetracyclines and host defense mechanisms: interference with leukocyte chemotaxis.

    PubMed

    Belsheim, J; Gnarpe, H; Persson, S

    1979-01-01

    The influence of 2 etracycline preparations, lymecycline and doxycycline, on leukocyte chemotaxis, was investigated in vitro and in vivo. Doxycycline in high concentrations caused a severe depression of both spontaneous and induced migration, an effect which was more pronounced than that caused by similar concentration of lymecycline. Corresponding results were obtained both in experiments on healthy volunteers given recommended dosages of the antibiotics, and in patients treated with doxycycline for postoperative infections.

  5. 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

  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. Phenotypic Characterization of Leukocytes in Prenatal Human Dermis

    PubMed Central

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

    2012-01-01

    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. PMID:22718119

  8. Carbohydrate ligands for endothelial - Leukocyte adhesion molecule 1

    SciTech Connect

    Tiemeyer, M.; Swiedler, S.J.; Ishihara, Masayuki; Moreland, M.; Schweingruber, H.; Hirtzer, P.; Brandley, B.K. )

    1991-02-15

    The acute inflammatory response requires that circulating leukocytes bind to and penetrate the vascular wall to access the site of injury. Several receptors have been implicated in this interaction, including a family of putative carbohydrate-binding proteins. The authors report here the identification of an endogenous carbohydrate ligand for one of these receptors, endothelial-leukocyte adhesion molecule 1 (ELAM-1). Radiolabeled COS cells transfected with a plasmid containing the cDNA for ELAM-1 were used as probes to screen glycolipids extracted from human leukocytes. COS cells transfected with this plasmid adhered to a subset of sialylated glycolipids resolved on TLC plates or adsorbed on polyvinyl chloride microtiter wells. Adhesion to these glycolipids required calcium but was not inhibited by heparin, chondroitin sulfate, keratan sulfate, or yeast phosphomannan. Monosaccharide composition, linkage analysis, and fast atom bombardment mass spectrometry of the glycolipids indicate that the ligands for ELAM-1 are terminally sialylated lactosylceramides with a variable number of N-acetyllactosamine repeats and at least one fucosylated N-acetylglucosamine residue.

  9. Report: Nuclei segmentation of leukocytes in blood smear digital images.

    PubMed

    Abbas, Naveed; Mohamad, Dzulkifli; Abdullah, Abdul Hanan; Saba, Tanzila; Al-Rodhaan, Mznah; Al-Dhelaan, Abdullah

    2015-09-01

    The Leukocytes are differentiated from each other on the basis of their nuclei, demanded in many Medical studies, especially in all types of Leukemia by the Hematologists to note the disorder caused by specific type of Leukocyte. Leukemia is a life threatening disease. The work for diagnosing is manually carried out by the Hematologists involving much labor, time and human errors. The problems mentioned are easily addressed through computer vision techniques, but still accuracy and efficiency are demanded in terms of the basic and challenging step segmentation of Leukocyte's nuclei. The underlying study proposed better method in terms of accuracy and efficiency by designing a dynamic convolution filter for boosting low intensity values in the separated green channel of an RGB image and suppressing the high values in the same channel. The high values in the green channel become 255 (background) while the nuclei always have low values in the green channel and thus clearly appear as foreground. The proposed technique is tested on 365 images achieving an overall accuracy of 95.89%, while improving the efficiency by 10%. The proposed technique achieved its targets in a realistic way by improving the accuracy as well as the efficiency and both are highly required in the area.

  10. Generation of reactive oxygen species by leukocytes of Prochilodus lineatus.

    PubMed

    de Faria, Marcos Tucunduva; Cury-Boaventura, Maria Fernanda; Lopes, Lucia Rossetti; da Silva, José Roberto Machado Cunha

    2014-04-01

    Prochilodus lineatus (curimbatá), from the Procholodontidae family, is a Brazilian freshwater fish, which is important commercially, nutritionally and ecologically. It is encountered in the Rio da Prata Bay in Southern South America. Studies on the immune system of this fish are scarce, but the physiological mechanisms of the species are analogous to those of other vertebrates. Thus, this work discusses the present study, which correlates P. lineatus leukocytes and the generation of reactive oxygen species after modulatory stimuli. Leukocytes were characterized by light and electron transmission microscopy and investigated by the generation of H2O2 and O2 (-), using phenol red, flow-cytometry and electron transmission histochemistry. The study determined that monocytes and neutrophils are the main cells responsible for generating O2 after stimulation with phorbol myristate acetate. Superoxide dismutase successfully inhibited the generation of reactive oxygen species in neutrophils and monocytes, but stimulated generation when in association with phorbol myristate acetate. Fish leukocyte samples from P. lineatus showed cross-reactivity with antibodies directed against human NADPH-oxidase antibody subunits (p47(phox) and p67(phox)). Thus, catalase enhanced the presence of p47(phox). Neutrophil mitochondria were shown to be generators of H2O2 (charged by cerium precipitate), being enlarged and changing their format. The present study contributes to a better understanding of the respiratory burst pathways in this species and suggests mitochondria as the organelle responsible for generation of reactive oxygen species.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. Does human leukocyte elastase degrade intact skin elastin?

    PubMed

    Schmelzer, Christian E H; Jung, Michael C; Wohlrab, Johannes; Neubert, Reinhard H H; Heinz, Andrea

    2012-11-01

    This study aimed to investigate the susceptibility of intact fibrillar human elastin to human leukocyte elastase and cathepsin G. Elastin is a vital protein of the extracellular matrix of vertebrates, and provides exceptional properties including elasticity and tensile strength to many tissues and organs, including the aorta, lung, cartilage, elastic ligaments and skin, and is thus critical for their long-term function. Mature elastin is an insoluble and extremely durable protein that undergoes very little turnover, but sustained exposure to proteases may lead to irreversible and severe damage, and thus to functional loss of the elastic fiber network. Hence, it is a key issue to understand which enzymes actually initiate elastolysis under certain pathological conditions or during intrinsic aging. In this paper, we provide a complete workflow for isolation of pure and intact elastin from very small tissue samples to test enzymes for their elastolytic potential. This workflow was applied to skin samples from variously aged individuals, and it was found that strong differences exist in the degradability of the elastins investigated. In summary, human leukocyte elastase was unable to degrade intact elastin fibers but hydrolyzed elastin derived from the skin of old people. However, cathepsin G cleaved all elastin samples, even those derived from younger individuals. These results indicate that human leukocyte elastase is not a driving force for elastolysis, but may nevertheless promote further breakdown of elastic fibers after the action of other enzymes such as cathepsin G. © 2012 The Authors Journal compilation © 2012 FEBS.

  19. Effects of prepartum stocking density on innate and adaptive leukocyte responses and serum and hair cortisol concentrations.

    PubMed

    Silva, P R B; Lobeck-Luchterhand, K M; Cerri, R L A; Haines, D M; Ballou, M A; Endres, M I; Chebel, R C

    2016-01-01

    Objectives were to evaluate the effects of prepartum stocking density on innate and adaptive leukocyte responses, serum cortisol and haptoglobin concentrations and hair cortisol concentration of Jersey cows. The cows (254 ± 3d of gestation) were balanced for parity (nulliparous vs. parous) and previous lactation projected 305-d mature equivalent milk yield and assigned to one of two treatments: 80SD=80% stocking density (38 animals/48 headlocks) and 100SD=100% stocking density (48 animals/48 headlocks). Pens (n=4) were identical in size and design and each pen received each treatment a total of 2 times (4 replicates; 80SD: n=338; 100SD: n=418). A sub-group of cows (n=48/treatment per parity) was randomly selected on week 1 of each replicate from which blood was sampled weekly from d -14 to 14 (d 0=calving) to determine polymorphonuclear leukocyte (PMNL) phagocytosis, oxidative burst, and expression of CD18 and L-selectin, and hemogram. The same sub-group of cows was treated with chicken egg ovalbumin on d -21, -7, and 7 and had blood sampled weekly from d -21 to 21 for determination of serum IgG anti-ovalbumin concentration. Blood was sampled weekly from d -21 to 21 to determine glucose, cortisol, and haptoglobin concentrations in serum. Hair samples collected at enrollment and within 24h of calving were analyzed for cortisol concentration. The percentage of leukocytes classified as granulocyte and the granulocyte to the lymphocyte ratio were not affected by treatment. Treatment did not affect the percentage of PMNL positive for phagocytosis and oxidative burst or the intensity of phagocytosis and oxidative burst. Similarly, treatment did not affect the percentage of PMNL expressing CD18 and L-selectin or the intensity of expression of CD18 and L-selectin. Concentration of IgG anti-ovalbumin was not affected by treatment. Serum concentrations of haptoglobin and cortisol were not affected by treatment. Similarly, hair cortisol concentration at calving was not

  20. A comparison of leukocyte aggregation, leukocyte migration and skin reactivity to recall antigens in patients with AIDS.

    PubMed Central

    Rouveix, B; Groult, F; Pocidalo, J J

    1986-01-01

    A study was made to evaluate the leukocyte aggregation test (LAT) in patients at risk for acquired immunodeficiency syndrome (AIDS) and with confirmed AIDS using tuberculin, streptococcus and candida as recall antigens. These patients were selected on the basis of their well known absence of delayed hypersensitivity, a fact which should avoid false positive tests. Antigen-induced human peripheral blood leukocyte aggregation was measured quantitatively. The results obtained have been compared with the leukocyte migration inhibition test (LMIT) in patient and control groups. Among the 46 patients, less than 7% were positive for the LAT, whereas more than 50% were LMIT positive (P less than 0.001). These results suggest that there are fewer false positive reactions with LAT which can therefore be considered as a reliable method for assessing CMI in human. Furthermore, a negative LAT correlated well with negative delayed skin tests in more than 90% of the patients for the three antigens. These patients had either an AIDS or an advanced AIDS-related complex (ARC). In only eight of the patients was there a discrepancy in the results of the two tests. A positive LAT and a negative skin test were seen in three cases lacking opportunistic infections (OI), whereas a negative LAT and a positive skin test were found in patients with or without OI. In the latter, a negative LAT could indicate a more advanced stage of disease and hence a poor prognosis. PMID:3568448

  1. Transit time of leukocytes rolling through venules controls cytokine-induced inflammatory cell recruitment in vivo.

    PubMed Central

    Jung, U; Norman, K E; Scharffetter-Kochanek, K; Beaudet, A L; Ley, K

    1998-01-01

    Leukocyte recruitment requires leukocyte rolling, activation, firm adhesion, and transmigration. Injection of the proinflammatory cytokine TNF-alpha induces expression of E-selectin, interleukin-8, and other adhesion molecules and chemoattractants on the endothelial surface. TNF-alpha- treated CD18 null mouse cremaster muscle venules show increased leukocyte rolling velocity and reduced leukocyte recruitment efficiency. Leukocyte recruitment in CD18 null but not wild-type mice is significantly blocked by an mAb to E-selectin. To understand this overlap between adhesion events previously considered separate, we introduce a quantitative analysis of the efficiency of induction of rolling, conversion of rolling to adhesion, and of adhesion to transmigration. We find that CD18 and E-selectin cooperate to control the time a leukocyte needs to roll through an inflamed area and to convert rolling to firm adhesion. Leukocyte rolling time, defined as the time it takes for a rolling leukocyte to pass through a defined length of a vessel segment, emerges as a unifying parameter determining the efficiency of inducing firm adhesion, which is a rate-limiting step controlling leukocyte recruitment in inflammation. We conclude that leukocytes integrate chemoattractant signals while rolling along the endothelial surface until they reach a critical level of activation and become firmly adherent. PMID:9788965

  2. Peripheral blood leukocyte count as an index of defense status in the leukopenic host

    SciTech Connect

    Cawley, S.; Findon, G.; Miller, T.E.

    1988-07-01

    These experimental studies have investigated the reliability of the peripheral blood leukocyte count to predict whether the leukopenic host can contain or eliminate infection. Additionally, we have investigated the possibility that determination of leukocyte recruitment, supplementary to peripheral blood leukocyte counts, might allow individuals with neutropenia at risk from serious infection to be distinguished with greater certainty. Varying doses of radiation, cyclophosphamide, and methylprednisolone were used to induce distinct levels of leukopenia in rats. Leukocyte recruitment was measured by quantifying the response of neutropenic animals to evocative, subcutaneous stimuli, and the results of this assay were then compared with circulating leukocyte counts in the same individuals. Six models of experimentally induced infection were used to compare circulating and recruitable leukocytes as indicators of the susceptibility of the leukopenic host to infection. Response curves relating leukocyte numbers to host resistance were similar when circulating or recruitable leukocytes were used as an index of defense capability. These findings support the use of peripheral blood leukocyte numbers as an index of resistance to infection in individuals with leukopenia and suggest that functional analyses such as leukocyte recruitment are unlikely to provide additional information.

  3. Ischemia-reperfusion injury in the isolated rat lung. Role of flow and endogenous leukocytes.

    PubMed

    Seibert, A F; Haynes, J; Taylor, A

    1993-02-01

    Microvascular lung injury caused by ischemia-reperfusion (IR) may occur via leukocyte-dependent and leukocyte-independent pathways. Leukocyte-endothelial adhesion may be a rate-limiting step in IR lung injury. Leukocyte adhesion to microvascular endothelium occurs when the attractant forces between leukocyte and endothelium are greater than the kinetic energy of the leukocyte and the vascular wall shear rate. We hypothesized (1) that isolated, buffer-perfused rat lungs are not free of endogenous leukocytes, (2) that endogenous leukocytes contribute to IR-induced microvascular injury as measured by the capillary filtration coefficient (Kfc), and (3) that a reduction of perfusate flow rate would potentiate leukocyte-dependent IR injury. Sixty lungs were divided into four groups: (1) low-flow controls, (2) high-flow controls, (3) low-flow IR, and (4) high-flow IR. Microvascular injury was linearly related to baseline perfusate leukocyte concentrations at both low (r = 0.78) and high (r = 0.82) flow rates. Kfc in the high-flow IR group (0.58 +/- 0.03 ml/min/cm H2O/100 g) was less (p < 0.05) than Kfc in the low-flow IR group (0.82 +/- 0.07), and in both groups Kfc values were significantly greater than low-flow (0.34 +/- 0.03) and high-flow (0.31 +/- 0.01) control Kfc values after 75 min. Retention of leukocytes in the lung, evaluated by a tissue myeloperoxidase assay, was greatest in the low-flow IR group. We conclude (1) that isolated, buffer-perfused rat lungs contain significant quantities of leukocytes and that these leukocytes contribute to IR lung injury, and (2) that IR-induced microvascular injury is potentiated by low flow.

  4. Microchannel acoustophoresis does not impact survival or function of microglia, leukocytes or tumor cells.

    PubMed

    Burguillos, Miguel A; Magnusson, Cecilia; Nordin, Maria; Lenshof, Andreas; Augustsson, Per; Hansson, Magnus J; Elmér, Eskil; Lilja, Hans; Brundin, Patrik; Laurell, Thomas; Deierborg, Tomas

    2013-01-01

    The use of acoustic forces to manipulate particles or cells at the microfluidic scale (i.e. acoustophoresis), enables non-contact, label-free separation based on intrinsic cell properties such as size, density and compressibility. Acoustophoresis holds great promise as a cell separation technique in several research and clinical areas. However, it has been suggested that the force acting upon cells undergoing acoustophoresis may impact cell viability, proliferation or cell function via subtle phenotypic changes. If this were the case, it would suggest that the acoustophoresis method would be a less useful tool for many cell analysis applications as well as for cell therapy. We investigate, for the first time, several key aspects of cellular changes following acoustophoretic processing. We used two settings of ultrasonic actuation, one that is used for cell sorting (10 Vpp operating voltage) and one that is close to the maximum of what the system can generate (20 Vpp). We used microglial cells and assessed cell viability and proliferation, as well as the inflammatory response that is indicative of more subtle changes in cellular phenotype. Furthermore, we adapted a similar methodology to monitor the response of human prostate cancer cells to acoustophoretic processing. Lastly, we analyzed the respiratory properties of human leukocytes and thrombocytes to explore if acoustophoretic processing has adverse effects. BV2 microglia were unaltered after acoustophoretic processing as measured by apoptosis and cell turnover assays as well as inflammatory cytokine response up to 48 h following acoustophoresis. Similarly, we found that acoustophoretic processing neither affected the cell viability of prostate cancer cells nor altered their prostate-specific antigen secretion following androgen receptor activation. Finally, human thrombocytes and leukocytes displayed unaltered mitochondrial respiratory function and integrity after acoustophoretic processing. We conclude that

  5. 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

  6. Leukocyte telomere length variation due to DNA extraction method.

    PubMed

    Denham, Joshua; Marques, Francine Z; Charchar, Fadi J

    2014-12-04

    Telomere length is indicative of biological age. Shorter telomeres have been associated with several disease and health states. There are inconsistencies throughout the literature amongst relative telomere length measured by quantitative PCR (qPCR) and different extraction methods or kits used. We quantified whole-blood leukocyte telomere length using the telomere to single copy gene (T/S) ratio by qPCR in 20 young (18-25 yrs) men after extracting DNA using three common extraction methods: Lahiri and Nurnberger (high salt) method, PureLink Genomic DNA Mini kit (Life Technologies) and QiaAmp DNA Mini kit (Qiagen). Telomere length differences of DNA extracted from the three extraction methods was assessed by one-way analysis of variance (ANOVA). DNA purity differed between extraction methods used (P=0.01). Telomere length was impacted by the DNA extraction method used (P=0.01). Telomeres extracted using the Lahiri and Nurnberger method (mean T/S ratio: 2.43, range: 1.57-3.02) and PureLink Genomic DNA Mini Kit (mean T/S ratio: 2.57, range: 2.24-2.80) did not differ (P=0.13). Likewise, QiaAmp and Purelink-extracted telomeres were not statistically different (P=0.14). The Lahiri-extracted telomeres, however, were significantly shorter than those extracted using the QiaAmp DNA Mini Kit (mean T/S ratio: 2.71, range: 2.32-3.02; P=0.003). DNA purity was associated with telomere length. There are discrepancies between the length of leukocyte telomeres extracted from the same individuals according to the DNA extraction method used. DNA purity could be responsible for the discrepancy in telomere length but this will require validation studies. We recommend using the same DNA extraction kit when quantifying leukocyte telomere length by qPCR or when comparing different cohorts to avoid erroneous associations between telomere length and traits of interest.

  7. Leukocyte adhesion molecules as biocompatibility markers for hemodialysis membranes.

    PubMed

    von Appen, K; Goolsby, C; Mehl, P; Goewert, R; Ivanovich, P

    1994-01-01

    Comparative flow cytometric measurement was used to evaluate the significance of leukocyte adhesion molecule (LAM) activity changes during hemodialysis (HD) with different cellulosic and non cellulosic membranes. Six hemodialysis patients (men) who were in a maintenance program for more than 6 months were treated consecutively with five different dialyzers (cuprophan, hemophan, 2 types of cellulose acetate, and polysulfone). During each study HD, blood was sampled from the arterial line at 0, 15, and 60 min and from the venous port at 3 min to harvest leukocytes immediately after the first cell-membrane contact. After whole blood lysis preparation, leukocytes were incubated with fluorescent antibodies to label LAM CD 11A/18 (LFA-1), CD 11B/18 (Mac-1), CD 11C/18 (p150/95), and CD 54 (ICAM-1) (Becton-Dickinson, San Jose, CA). Data were acquired for the granulocyte, monocyte, and lymphocyte population based on forward and 90 degrees scatter light measurements. Accuracy of gating was verified by CD 14/45 staining for all samples. Baseline integrin expression for the selected populations before biomaterial contact was found to be heterogeneous for different patients, but underwent changes for the same patient during HD treatment. The fluorescent intensity corresponding to specific integrins was characterized by different patterns of up/down regulation with maximal deviations occurring at 3 min. Fluorescent intensity of the granulocyte and monocyte populations sampled at 15 min was 40-50% lower as compared with those sampled immediately after the first biomaterial contact. Based on the basal fluorescence levels and values recorded after the first biomaterial contact and those at 15 min, two coefficients were generated to compare membrane properties.

  8. [Chlamydia trachomaatis DNA in leukocytes of peripheral blood from neonates].

    PubMed

    López-Hurtado, Marcela; Cuevas-Recillas, Karla N; Flores-Salazar, Verónica R; Guerra-Infante, Fernando M

    2015-01-01

    Diagnosis of Chlamydia trachomatis infection in newborns is difficult; however, this diagnosis is performed by cell culture or by detection of IgM antibodies against C. trachomatis. Detection of C. trachomatis DNA in peripheral blood leukocytes using polymer chain reaction (PCR) may be a better tool for the diagnosis of infection by this pathogen. A total of 44 premature newborns, all weighing less than 2500g, were included in the study. A blood sample and nasopharyngeal lavages were obtained from each newborn. Leukocyte DNA was obtained by phenol-chloroform extraction technique. Detection of C. trachomatis was performed by amplifying the ompA gene using the PCR endpoint. Cell culture tests and the detection of IgM antibodies against C. trachomatis by microimmunofluorescence assay were also performed. Twenty newborns were PCR-positive (45.5%), with this test being significantly associated with the presence of pneumonia (RR=2.28; 95%CI: 1.01 to 5.17; P=.035). The cell culture of nasopharyngeal lavage was positive in only 7 samples and no significant association was observed with any clinical or laboratory data. The titer of IgM antibodies against C. trachomatis associated with PCR-positive was 1:32 (RR=2.74; 95%CI: 1.21 to 6.23; P=.008), however this titer was not associated with the presence of pneumonia. DNA detection in peripheral blood leukocytes could be useful for diagnosis of C. trachomatis infection. Copyright © 2014 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  9. Leukocyte Telomere Length in the Finnish Diabetes Prevention Study

    PubMed Central

    Hovatta, Iiris; de Mello, Vanessa D. F.; Kananen, Laura; Lindström, Jaana; Eriksson, Johan G.; Ilanne-Parikka, Pirjo; Keinänen-Kiukaanniemi, Sirkka; Peltonen, Markku; Tuomilehto, Jaakko; Uusitupa, Matti

    2012-01-01

    Leukocyte telomere length (TL) is considered a biomarker for biological aging. Shortened TL has been observed in many complex diseases, including type 2 diabetes (T2DM). Lifestyle intervention studies, e.g. the Diabetes Prevention Study (DPS), have shown a decrease in the incidence of T2DM by promoting healthy lifestyles in individuals with impaired glucose tolerance (IGT). Our aim was to study in the DPS the influence of the lifestyle intervention on TL. TL was measured by quantitative PCR-based method at two time points (N = 334 and 343) on average 4.5 years apart during the active intervention and post-intervention follow-up. TL inversely correlated with age. Our main finding was that TL increased in about two thirds of the individuals both in the intervention and in the control groups during follow-up; TL increased most in individuals with the shortest TL at the first measurement. TL was not associated with development of T2DM, nor did lifestyle intervention have an effect on TL. No association between insulin secretion or insulin resistance indices and TL was observed. We did not detect an association between TL and development of T2DM in the DPS participants. It could be due to all participants being overweight and having IGT at baseline, both of which have been found to be independently associated with shorter leukocyte TL in some earlier studies. TL had no substantial role in worsening of glucose tolerance in people with IGT. Our study confirms that leukocyte TL can increase with time even in obese people with impaired glucose metabolism. PMID:22493726

  10. Leukocyte Populations in Human Preterm and Term Breast Milk Identified by Multicolour Flow Cytometry

    PubMed Central

    Trend, Stephanie; de Jong, Emma; Lloyd, Megan L.; Kok, Chooi Heen; Richmond, Peter; Doherty, Dorota A.; Simmer, Karen; Kakulas, Foteini; Strunk, Tobias; Currie, Andrew

    2015-01-01

    Background Extremely preterm infants are highly susceptible to bacterial infections but breast milk provides some protection. It is unknown if leukocyte numbers and subsets in milk differ between term and preterm breast milk. This study serially characterised leukocyte populations in breast milk of mothers of preterm and term infants using multicolour flow cytometry methods for extended differential leukocyte counts in blood. Methods Sixty mothers of extremely preterm (<28 weeks gestational age), very preterm (28–31 wk), and moderately preterm (32–36 wk), as well as term (37–41 wk) infants were recruited. Colostrum (d2–5), transitional (d8–12) and mature milk (d26–30) samples were collected, cells isolated, and leukocyte subsets analysed using flow cytometry. Results The major CD45+ leukocyte populations circulating in blood were also detectable in breast milk but at different frequencies. Progression of lactation was associated with decreasing CD45+ leukocyte concentration, as well as increases in the relative frequencies of neutrophils and immature granulocytes, and decreases in the relative frequencies of eosinophils, myeloid and B cell precursors, and CD16- monocytes. No differences were observed between preterm and term breast milk in leukocyte concentration, though minor differences between preterm groups in some leukocyte frequencies were observed. Conclusions Flow cytometry is a useful tool to identify and quantify leukocyte subsets in breast milk. The stage of lactation is associated with major changes in milk leukocyte composition in this population. Fresh preterm breast milk is not deficient in leukocytes, but shorter gestation may be associated with minor differences in leukocyte subset frequencies in preterm compared to term breast milk. PMID:26288195

  11. Studying the Human Immunome: The Complexity of Comprehensive Leukocyte Immunophenotyping

    PubMed Central

    Biancotto, Angélique

    2014-01-01

    A comprehensive study of the cellular components of the immune system requires both deep and broad immunophenotyping of numerous cell populations in an efficient and practical manner. In this chapter, we describe the technical aspects of studying the human immunome using high-dimensional (15 color) fluorescence-based immunophenotyping. We focus on the technical aspects of polychromatic flow cytometry and the initial stages in developing a panel for comprehensive leukocyte immunophenotyping (CLIP). We also briefly discuss how this panel is being used and the challenges of encyclopedic analysis of these rich data sets. PMID:23975032

  12. Studying the human immunome: the complexity of comprehensive leukocyte immunophenotyping.

    PubMed

    Biancotto, Angélique; McCoy, J Philip

    2014-01-01

    A comprehensive study of the cellular components of the immune system requires both deep and broad immunophenotyping of numerous cell populations in an efficient and practical manner. In this chapter, we describe the technical aspects of studying the human immunome using high-dimensional (15 color) fluorescence-based immunophenotyping. We focus on the technical aspects of polychromatic flow cytometry and the initial stages in developing a panel for comprehensive leukocyte immunophenotyping (CLIP). We also briefly discuss how this panel is being used and the challenges of encyclopedic analysis of these rich data sets.

  13. 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.

  14. 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

  15. Oropouche virus is detected in peripheral blood leukocytes from patients.

    PubMed

    de Souza Luna, Luciano Kleber; Rodrigues, Alcir Humberto; Santos, Rodrigo Ivo Marques; Sesti-Costa, Renata; Criado, Miriã Ferreira; Martins, Ronaldo B; Silva, Maria Lúcia; Delcaro, Luana Sella; Proença-Modena, Jose Luiz; Figueiredo, Luiz Tadeu Moraes; Acrani, Gustavo Olszanski; Arruda, Eurico

    2017-06-01

    Oropouche virus (OROV) is a frequent cause of arboviral febrile disease in the Amazon. The present report describes studies done in two patients, one of them; the first OROV human case acquired outside of the Amazon, which have revealed for the first time the presence of OROV in peripheral blood leukocytes. This novel finding raises important issues regarding pathogenesis of human infections and may offer a new tool, for the rapid diagnosis of this neglected infection. J. Med. Virol. 89:1108-1111, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  16. Production of antibodies which recognize opiate receptors on murine leukocytes

    SciTech Connect

    Carr, D.J.J.; Bost, K.L.; Blalock, J.E.

    1988-01-01

    An antibody has been developed which recognizes opiate receptors on cells of the immune system. This antibody blocks specific binding of the radiolabeled opiate receptor ligand, /sup 3/H-dihydromorphine, to receptors on murine splenocytes. Additionally, the anti-receptor antibody competes with ..beta..-endorphin, meta-enkephalin, and naloxone for the same binding site on the leukocytes. Moreover, the anti-receptor antibody possesses agonist activity similar to ..beta..-endorphin in suppressing cAMP production by lymphocytes. These results suggest the development of an antibody which recognizes classical opiate receptors on cells of the immune system.

  17. The immunocytochemical distribution of leukocytic subpopulations in human endometrium.

    PubMed

    Kamat, B R; Isaacson, P G

    1987-04-01

    Thirty human endometria were selected from women aged 21-54 years who had undergone routine dilation and curettage procedures for tubal ligation, infertility dating, and irregular menstrual cycling. Histologic sections of the cases chosen were examined to exclude any major pathologic condition (including chronic endometritis). The specimens were stained with monoclonal antibodies to a common leukocytic antigen (H Leu-1 and PD7/26), pan-T-cell antigen (UCHT1), T helper/inducer and T suppressor/cytotoxic antigens (Leu-3a and UCHT4, respectively), pan-B cell antigen (To15 and Leu-12), and macrophage antigens (UCHM1 and Leu-M3). Other antibodies used included TAL-1B5 (anti-HLA-DR), Leu-7 (natural killer cell) and Na 1/34 (anti-T6/Langerhans/interdigitating reticulum cell). The endometria contained significant numbers of common leukocyte antigen-positive cells (occupying approximately 10-15% of the stroma), the numbers of which appeared to increase in the late secretory/pre-menstrual phase (20-25% of the stroma). The major leukocyte populations were T cells and macrophages; the latter, with neutrophils, appeared to account for the premenstrual increase in leukocytes. T cells were distributed both diffusely in the stroma and in periglandular stromal aggregates closely applied to the glands. The T8+ suppressor/cytotoxic population was predominant within the stromal nodules. In addition, scattered intraepithelial T suppressor/cytotoxic cells were present. Macrophages (UCHM1 and HLA-DR+) were also distributed diffusely in the stroma and as part of the periglandular stromal aggregates, in areas sending long cell processes into the epithelium. B cells appeared to be limited to scattered cells in the stroma, only increasing in number within lymphoid follicles. Natural killer cells, as defined by Leu-7+ cells, were also present, scattered singly in the stroma and within lymphoid follicles. The demonstration of large mononuclear dendritic-appearing Na 1/34+ cells within the

  18. 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

  19. [Effects of the glucoprotein component of musk on the functions of rat polymorphonuclear leukocytes activated by fMLP in vitro].

    PubMed

    Wang, W; Bai, J; Cheng, G; Zhu, X

    1997-06-01

    To investigate the effects of musk-1, a glucoprotein component isolated from the water extract of musk, on the release of superoxide anion as well as beta-glucuronidase and lysozyme of rat polymorphonuclear leukocytes activated by fMLP. An in vitro incubation system with rat polymorphonuclear leukocytes was used. Superoxide anion production was determined by cytochrome C reduction. beta-glucuronidase and lysozyme release was quantitated by enzyme reactions in which phenolphthaleinglucuronic acid and micrococcus lysodeikticus were as the substrates, respectively. In comparison with control, musk-1 at final concentrations of 1-100 micrograms/ml can increase superoxide anion production by 23.0%-83.6% and decrease beta-glucuronidase and lysozyme release by 7%-47% and 9%-22%, respectively, in rat polymorphonuclear leukocytes. It is concluded that Musk-1 can significantly affect the functions of rat polymorphonuclear leukocytes. Therefore, inhibition of lysosomal enzyme release might be considered as one of the mechanisms of anti-inflammatory role of musk.

  20. 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

  1. 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.

  2. Efficiency of leukocyte removal by filters made of superfine glass fiber membranes.

    PubMed

    Zou, Y; Sun, Q; Li, A; Yao, F; Hu, Z; Li Z, L; Ma, W

    1999-01-01

    To demonstrate the application of leukocyte removal filters made of a new type of filter material - superfine glass fiber - for depleting leukocytes in SAGM red cell suspensions and preventing nonhemolytic transfusion reactions. The extent of leukocyte depletion and red cell recovery was based on cell counts. Trace leukocytes were counted in a 50-microl Nageotte counting chamber or by using a flow cytometer. The chemical stability of the glass fiber membranes was studied by plasma emission spectrometer and by measuring the ion content and weighing nonvolatile matter in water extract. The structural stability of the glass fiber membranes was studied by a micropore-filter membrane method. Leukocyte removal filters made of superfine glass fiber membranes removed more than 99.0% of leukocytes in SAGM red cell suspensions prepared from 400 ml whole blood. Red cell recovery exceeded 90%, and the total number of residual leukocytes was less than 5x10(6). A water extract of the glass fiber membranes contained only traces of Si4+ and Ca2+ and less than 2 mg/100 ml of nonvolatile matter. No broken or loose fibers were found in the filters. Scanning electron microscopy showed that the web structure of the glass fiber membranes was instrumental in trapping and holding leukocytes. A filter made of glass fiber membranes is effective in leukocyte depletion.

  3. 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.

  4. 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)

  5. Influence of 2(3-methyl-cinnamyl-hydrazono)-propionate on glucose and palmitate oxidation in human mononuclear leukocytes. Hydrazonopropionic acids, a new class of hypoglycaemic substances, VII.

    PubMed

    Haeckel, R; Fink, P C; Oellerich, M

    1987-09-01

    2-(3-Methyl-cinnamyl-hydrozono)-propionate stimulated glucose oxidation in human mononuclear leukocytes and the stimulation was similar to that by concanavalin A. Both substances must affect glucose metabolism at two sites, the first site being before the pyruvate dehydrogenase step because of the increase of lactate plus pyruvate concentration. The second site is related to pyruvate oxidation. The hydrazone inhibited the conversion of plamitate to CO2. This effect could have caused an activation of the pyruvate dehydrogenase complex, resulting from a decrease acetyl-CoA/CoA ratio. Concanavalin A did not influence fatty acid oxidation. Both substances did not affect the CO2 formation from acetate. Mononuclear leukocytes appear to be a suitable model for the investigation of the influence of hypoglycaemic substances on glucose and fatty acid metabolism in living human cells.

  6. Age-dependent expression of osteochondrosis-related genes in equine leukocytes

    PubMed Central

    Mendoza, L.; Piquemal, D.; Lejeune, J. P.; Vander Heyden, L.; Noguier, F.; Bruno, R.; Sandersen, C.; Serteyn, D.

    2015-01-01

    Introduction:  Osteochondrosis (OC) is a developmental disease in horses which has a significant impact on the horse's welfare and performance. The early disturbance in the process of endochondral ossification progresses to inflammatory and repair processes in older horses. Previously, differentially expressed genes in leukocytes of OC-affected horses have been identified. The aim of the present study is to detect age-related changes in these differentially expressed genes. Materials and Methods:  The expression of OC-related genes was analysed by real-time PCR and subsequent statistical analysis (ΔΔCT) in the leukocytes of 135 Belgian Warmblood horses divided into three different age groups: <12 months (n=47), 18–24 months (n=50) >30 months (n=38). Results:  Relative expression of genes of horses less than 12 months of age showed significant induction of the genes MGAT4A, PRKCG, MHCI, ApoB, ApoB3G, B4GALT6 and a significantly lower expression of the genes OAS3. Horses of 18–24 months of age, showed a significantly higher expression of the genes TBC1D9, MGAT4A, IFIH1, MHCIIa and MMP1. Horses of more than 30 months of age showed a significantly higher expression of the genes MGAT4A, HP, SECTM1 compared with their age-matched control groups. Conclusions:  The study demonstrates that OC-related genes are differentially expressed in horses of different ages compared with their age-matched controls. Some of the genes may be implicated in cell signalling and differentiation as well as carbohydrate and lipid metabolism and inflammation. However, the causal relationship between the differentially expressed genes and the development and progression of the OC lesions needs to be determined. PMID:26392886

  7. Mitochondrial DNA 4977-base pair common deletion in blood leukocytes and melanoma risk.

    PubMed

    Shen, Jie; Wan, Jie; Huff, Chad; Fang, Shenying; Lee, Jeffrey E; Zhao, Hua

    2016-05-01

    The 4977-base pair common deletion DmtDNA4977 is the most frequently observed mitochondrial DNA mutation in human tissues. Because mitochondrial DNA mutations are mainly caused by reactive oxygen species (ROS), and given that oxidative stress plays an important role in melanoma carcinogenesis, the investigation of DmtDNA4977 may be particularly relevant to the development of melanoma. In this study, we compared DmtDNA4977 levels in blood leukocytes from 206 melanoma patients and 219 healthy controls. Overall, melanoma cases had significantly higher levels of DmtDNA4977 than healthy controls (median: 0.60 vs 0.20, P = 0.008). The difference was evident among individuals who were older than 47 yrs, women, and had pigmentation risk factors (e.g., blond or red hair, blue eye, fair skin, light, or none tanning ability after prolonged sun exposure, and freckling in the sun as a child). The difference was also evident among those who had at least one lifetime sunburn with blistering and had no reported use of a sunlamp. Interestingly, among controls, DmtDNA4977 levels differed by phenotypic index and reported use of a sunlamp. In the risk assessment, increased levels of DmtDNA4977 were associated with a 1.23-fold increased risk of melanoma (odds ratio (OR): 1.23, 95% confidence interval (90% CI): 1.01, 1.50). A significant dose-response relationship was observed in quartile analysis (P = 0.001). In summary, our study suggests that high levels of DmtDNA4977 in blood leukocytes are associated with increased risk of melanoma and that association is affected by both pigmentation and personal history of sun exposure.

  8. In vitro effects of bovine dialyzable leukocyte extract (bDLE) in cancer cells.

    PubMed

    Franco-Molina, M A; Mendoza-Gamboa, E; Miranda-Hernández, D; Zapata-Benavides, P; Castillo-León, L; Isaza-Brando, C; Tamez-Guerra, R S; Rodríguez-Padilla, C

    2006-01-01

    Bovine dialyzable leukocyte extract (bDLE) is a dialyzate of a heterogeneous mixture of low molecular weight substances released from disintegrated blood leukocytes or lymphoid tissue obtained from homogenized bovine spleen. The purpose of this study was to determine if bDLE had cytotoxic effects and modulated apoptosis gene expression in breast cancer cells. The MCF-7, BT-474, MDA-MB-453, A-427, Calu-1, U937 and L5178Y cancer cell lines and PBMC human cells were treated with bDLE (0-0.66 U/mL) for 72 h. The bDLE effect on cell growth proliferation was evaluated by MTT assay, and the MCF-7 was evaluated by ethidium bromide-acridine orange staining; total DNA was evaluated for DNA fragmentation, and total RNA was isolated for p53, bag-1, c-myc, bim, bax, bcl-2 and bad mRNA expression. The bDLE had dose-dependent cytotoxic effects and demonstrated an IC50 at a dosage of 0.06 U/mL (P<0.05). The bDLE did not affect the viability of normal human PBMC. The bDLE induced DNA fragmentation at doses of 0.06 and 0.13 U/mL in MCF-7 breast cancer cells. The bDLE induced cytotoxic effects and suppressed the p53, bag-1, c-myc, bax, bcl-2, and bad mRNA expression that influences apoptosis in MCF-7 breast cancer cells. Bim mRNA expression was not detected. This may open up interesting prospects for the treatment of human breast cancer.

  9. Transcriptomic analyses and leukocyte telomere length measurement in subjects exposed to severe recent stressful life events.

    PubMed

    Lopizzo, N; Tosato, S; Begni, V; Tomassi, S; Cattane, N; Barcella, M; Turco, G; Ruggeri, M; Riva, M A; Pariante, C M; Cattaneo, A

    2017-02-21

    Stressful life events occurring in adulthood have been found able to affect mood and behavior, thus increasing the vulnerability for several stress-related psychiatric disorders. However, although there is plenty of clinical data supporting an association between stressful life events in adulthood and an enhanced vulnerability for psychopathology, the underlying molecular mechanisms are still poorly investigated. Thus, in this study we performed peripheral/whole-genome transcriptomic analyses in blood samples obtained from 53 adult subjects characterized for recent stressful life events occurred within the previous 6 months. Transcriptomic data were analyzed using Partek Genomics Suite; pathway and network analyses were performed using Ingenuity Pathway Analysis and GeneMANIA Software. We found 207 genes significantly differentially expressed in adult subjects who reported recent stressful life experiences (n=21) compared with those without such experiences (n=32). Moreover, the same subjects exposed to such stressful experiences showed a reduction in leukocyte telomere length. A correlation analyses between telomere length and transcriptomic data indicated an association between the exposures to recent stressful life events and the modulation of several pathways, mainly involved in immune-inflammatory-related processes and oxidative stress, such as natural killer cell signaling, interleukin-1 (IL-1) signaling, MIF regulation of innate immunity and IL-6 signaling. Our data suggest an association between exposures to recent stressful life events in adulthood and alterations in the immune, inflammatory and oxidative stress pathways, which could be also involved in the negative effect of stressful life events on leukocyte telomere length. The modulation of these mechanisms may underlie the clinical association between the exposure to recent Stressful life events in adulthood and an enhanced vulnerability to develop psychiatric diseases in adulthood.

  10. DNA integrity of human leukocytes after magnetic resonance imaging.

    PubMed

    Szerencsi, Ágnes; Kubinyi, Györgyi; Váliczkó, Éva; Juhász, Péter; Rudas, Gábor; Mester, Ádám; Jánossy, Gábor; Bakos, József; Thuróczy, György

    2013-10-01

    This study focuses on the effects of high-field (3T) magnetic resonance imaging (MRI) scans on the DNA integrity of human leukocytes in vitro in order to validate the study where genotoxic effects were obtained and published by Lee et al. The scanning protocol and exposure situation were the same as those used under routine clinical brain MRI scan. Peripheral blood samples from healthy non-smoking male donors were exposed to electromagnetic fields (EMF) produced by 3T magnetic resonance imaging equipment for 0, 22, 45, 67, and 89 min during the scanning procedure. Samples of positive control were exposed to ionizing radiation (4 Gy of (60)Co-γ). Single breaks of DNA in leukocytes were detected by single-cell gel electrophoresis (Comet assay). Chromosome breakage, chromosome loss and micronuclei formations were detected by a micronucleus test (MN). Three independent experiments were performed. The data of comet tail DNA%, olive tail moment and micronucleus frequency showed no DNA damages due to MRI exposure. The results of the Comet assay and the micronucleus test indicate that the applied exposure of MRI does not appear to produce breaks in the DNA and has no significant effect on DNA integrity.

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. Physiological roles of voltage-gated proton channels in leukocytes

    PubMed Central

    Demaurex, Nicolas; El Chemaly, Antoun

    2010-01-01

    Voltage-gated proton channels are designed to extrude large quantities of cytosolic acid in response to depolarising voltages. The discovery of the Hvcn1 gene and the generation of mice lacking the channel molecule have confirmed several postulated functions of proton channels in leukocytes. In neutrophils and macrophages, proton channels are required for high-level production of superoxide anions by the phagocytic NADPH oxidase, a bactericidal enzyme essential for host defence against infections. In B lymphocytes, proton channels are required for low-level production of superoxide that boosts the production of antibodies. Proton channels sustain the activity of immune cells in several ways. By extruding excess cytosolic acid, proton channels prevent deleterious acidification of the cytosol and at the same time deliver protons required for chemical conversion of the superoxide secreted by membrane oxidases. By moving positive charges across membranes, proton channels limit the depolarisation of the plasma membrane, promoting the electrogenic activity of NADPH oxidases and the entry of calcium ions into cells. Acid extrusion by proton channels is not restricted to leukocytes but also mediates the intracellular alkalinisation required for the activation of spermatozoids. Proton channels are therefore multitalented channels that control male fertility as well as our innate and adaptive immunity. PMID:20693294

  18. 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

  19. Swine Leukocyte Antigen (SLA) Class II is a Xenoantigen.

    PubMed

    Ladowski, Joseph M; Reyes, Luz M; Martens, Gregory R; Butler, James R; Wang, Zheng-Yu; Eckhoff, Devin E; Tector, Matt; Tector, A Joseph

    2017-08-24

    Over 130 000 patients in the United States alone need a life-saving organ transplant. Genetically modified porcine organs could resolve the donor organ shortage, but human xenoreactive antibodies destroy pig cells and are the major barrier to clinical application of xenotransplantation. The objective of this study was to determine whether waitlisted patients possess preformed antibodies to swine leukocyte antigen (SLA) class II, homologs of the class II human leukocyte antigens (HLA). Sera from people currently awaiting solid organ transplant were tested for IgG binding to class II SLA proteins when expressed on mammalian cells. Pig fibroblasts were made positive by transfection with the class II transactivator (CIITA). As a second expression system, transgenes encoding the alpha and beta chains of class II SLA were transfected into Human embryonic kidney (HEK293) cells. Human sera containing IgG specific for class II HLA molecules exhibited greater binding to class II SLA positive cells than to SLA negative cells. Sera lacking antibodies against class II HLA showed no change in binding regardless of the presence of class II SLA. These antibodies could recognize either SLA-DR or SLA-DQ complexes. Class II SLA proteins may behave as xenoantigens for people with humoral immunity towards class II HLA molecules.

  20. The history of fever, leukocytic pyrogen and interleukin-1

    PubMed Central

    Dinarello, Charles A

    2015-01-01

    There has been great progress in the 30 y since the reporting in 1984 of the cDNA for interleukin1 (IL1) β in the human and IL1α in the mouse. However, the history of IL1 begins in the early 1940s with investigations into the nature of an endogenous fever-producing protein released rabbit peritoneal neutrophils. Most researchers in immunology today are unaware that the field of cytokines, particularly the field of inflammatory cytokines. Toll-like receptors and innate immunity traces back to studies on fever. Researchers in infectious diseases wanted to know about an endogenous protein that caused fever, independent of infection. The endogenous fever-producing protein was called by various names: granulocyte, endogenous or leukocytic pyrogen. It is a fascinating and sometimes controversial story for biology and medicine and for the treatment of inflammatory diseases. Few imagined that this fever-producing protein would play such a major role in nearly every cell and in most diseases. This paper reviews the true background and milestones of interleukin1 from the purification of leukocytic pyrogen to the first cDNA of IL1β and the validation of cytokine biology from ill-defined factors to its present day importance. PMID:27226996

  1. 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

  2. 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.

  3. Leukocyte telomere length predicts SSRI response in major depressive disorder: A preliminary report.

    PubMed

    Hough, Christina M; Bersani, F Saverio; Mellon, Synthia H; Epel, Elissa S; Reus, Victor I; Lindqvist, Daniel; Lin, Jue; Mahan, Laura; Rosser, Rebecca; Burke, Heather; Coetzee, John; Nelson, J Craig; Blackburn, Elizabeth H; Wolkowitz, Owen M

    2016-07-01

    Short leukocyte telomere length (LTL) may be associated with several psychiatric disorders, including major depressive disorder (MDD). Short LTL has previously been associated with poor response to psychiatric medications in bipolar disorder and schizophrenia, but no studies have prospectively assessed the relationship of LTL to SSRI response in MDD. We assessed pre-treatment LTL, depression severity (using the Hamilton Depression Rating Scale [HDRS]), and self-reported positive and negative affect in 27 healthy, unmedicated adults with MDD. Subjects then underwent open-label treatment with a selective serotonin reuptake inhibitor (SSRI) antidepressant for eight weeks, after which clinical ratings were repeated. Analyses were corrected for age, sex and BMI. "Non-responders" to treatment (HDRS improvement <50%) had significantly shorter pre-treatment LTL, compared to "Responders" (p=0.037). Further, shorter pre-treatment LTL was associated with less improvement in negative affect (p<0.010) but not with changes in positive affect (p=0.356). This preliminary study is the first to assess the relationship between LTL and SSRI response in MDD and among the first to prospectively assess its relationship to treatment outcome in any psychiatric illness. Our data suggest that short LTL may serve as a vulnerability index of poorer response to SSRI treatment, but this needs examination in larger samples.

  4. Leukocytes from diabetic patients kill retinal endothelial cells: Effects of berberine

    PubMed Central

    Tian, Pei; Ge, Hongyan; Liu, Haitao; Kern, Timothy S.; Du, Lingling; Guan, Linan; Su, Sheng

    2013-01-01

    Purpose Accumulating evidence in animals suggests that leukocytes are involved in the pathogenesis of diabetic retinopathy. The present study was designed to investigate whether leukocytes from diabetic patients could kill retinal endothelial cells and whether that cytotoxicity could be inhibited in vivo by administration of berberine. Methods Human retinal endothelial cells (HRECs) were cocultured (24 h) with leukocytes freshly isolated from nondiabetic and diabetic patients, and leukocyte-mediated death of HRECs was analyzed with flow cytometry. HRECs or leukocytes were incubated with antibodies against intercellular adhesion molecule-1(ICAM-1) or integrin beta-2, or with various concentrations of berberine. The protein expression levels of inflammatory factors were investigated using western blots, and activities of antioxidant enzymes and malondialdehyde content were examined as markers of oxidative stress. In addition, leukocytes were isolated from 28 diabetic patients with retinopathy and nondiabetic patients before and after 1 month in vivo therapy with berberine. The effects of the berberine on leukocyte-mediated killing of endothelial cells was again assessed. Results Leukocytes from diabetic patients induced more apoptosis of HRECs in a coculture system than did cells from nondiabetic patients, and this killing occurred primarily via direct cell–cell contact. Berberine inhibited the leukocyte-mediated killing of HRECs in vitro, the decrease in antioxidant enzyme activities, the nuclear translocation of nuclear factor kappa B, and the increase in intercellular adhesion molecule-1 and inducible nitric oxide synthase expression and malondialdehyde content in HRECs cultured in high glucose. Berberine also decreased integrin beta-2 expression of leukocytes in vitro and in vivo. Oral consumption of berberine for 1 month likewise inhibited the diabetes-induced increase in leukocyte-mediated killing of HRECs. Conclusions Our findings suggest that leukocytes

  5. Vitamin C prevents cigarette smoke-induced leukocyte aggregation and adhesion to endothelium in vivo.

    PubMed Central

    Lehr, H A; Frei, B; Arfors, K E

    1994-01-01

    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 mm2, P < 0.01; arterioles, 8.5 +/- 4.2 vs. 54.3 +/- 21.6 leukocytes per mm2, P < 0.01; aortas, 0.8 +/- 0.4 vs. 12.4 +/- 5.6 leukocytes per mm2, 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 microM, 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. Images PMID:7519784

  6. Evaluation of cellular immune response during chronic schistosomiasis in humans by the leukocyte aggregation test and the leukocyte migration inhibition test.

    PubMed Central

    Rouveix, B; Derouin, F; Levacher, M

    1985-01-01

    Cellular immune response was evaluated in 31 patients with chronic Schistosoma haematobium and Schistosoma mansoni infections and in 15 healthy normal persons by using S. mansoni soluble worm and egg antigens. Although the leukocyte migration inhibition test demonstrated false-positive reactions, the specificity of the leukocyte aggregation test was confirmed by the negativity of all of the controls. Among the patients, only 10% were positive for the leukocyte aggregation test. This low cellular reactivity was in contrast to markedly elevated specific humoral response determined by an enzyme-linked immunosorbent assay for immunoglobulin G and paper allergosorbent test for immunoglobulin E with soluble worm antigen. These results confirm that the cellular immune reactivity to schistosome antigen as demonstrated by the leukocyte aggregation test is either minimal or absent in chronically infected patients. PMID:3988906

  7. Expression of four canine leukocyte adhesion factors in fresh and stored whole blood samples evaluated using a no-lyse, no-wash method.

    PubMed

    Holst, B Ström; Hagberg, M; Lilliehöök, I; Johannisson, A

    2011-02-15

    Expression of four leukocyte adhesion factors on canine leukocytes was studied by flow cytometry using a no-lyse, no-wash method. The effect of fixation and storage for up to 14 days in 1% paraformaldehyde on labelled samples and within assay variation was evaluated. Monoclonal antibodies directed to monocyte marker CD14, and to adhesion molecules CD11a, CD18, CD32 and CD49d were used. Cell surface marker, cell population, time, and the interactions between time and cell marker significantly affected expression of cell adhesion factors. For CD18, there was a significant difference in mean fluorescence intensity (MFI) between fresh and stored samples (P<0.001), but no significant difference between stored samples. The MFIs of CD11a and CD49d were not significantly affected by fixation and storage. The CVs differed significantly depending on cell marker (P<0.001) and cell population (P=0.005). Fixation and storage did not significantly affect the CV. In conclusion, a no-lyse, no-wash method can be applied to canine leukocytes. The effect of fixation and storage on the resulting MFI differs between monoclonal antibodies, and should be evaluated for each antibody before use. The coefficient of variation was generally acceptable, and high CVs were related to a low MFIs or low numbers of analysed cells. Copyright © 2010 Elsevier B.V. All rights reserved.

  8. 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.

  9. Elevated leukocyte count is associated with periodontitis in Korean adults: the 2012-2014 KNHANES.

    PubMed

    Kwon, Y-J; Jeon, K-J; Chung, T-H; Lee, Y-J

    2017-03-01

    Both an elevated leukocyte count and periodontitis share well-recognized associations with cardiometabolic diseases. This cross-sectional study aimed to identify whether the leukocyte count is associated with periodontitis in a nationally representative Korean adult population. Data from 9391 participants (3659 males and 5732 females) enrolled in 2012-2014 Korean National Health and Nutrition Examination Survey were analyzed. Leukocyte quartiles were categorized as follows: 3000 ≤ Q1 ≤ 4870, 4880 ≤ Q2 ≤5790, 5800 ≤ Q3 ≤ 6840, and 6850 ≤ Q4 ≤ 10000 cells/μl. Periodontitis was defined as scoring greater than or equal to 'code 3' in at least one site according to the WHO's Community Periodontal Index. The odds ratios (ORs) and 95% confidence intervals (95% CIs) for periodontitis in each leukocyte count quartile were calculated using multiple logistic regression analyses. The prevalence of periodontitis was directly correlated with increasing leukocyte quartiles: 19%, 20.4%, 24.3%, and 30.3%. Compared with the lowest leukocyte quartile group, the OR (95% CI) for periodontitis of the highest leukocyte quartile was 1.558 (1.285-1.891) after controlling for confounding factors. An elevated leukocyte count was positively associated with the presence of periodontitis. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. Nitric oxide synthesis inhibition induces leukocyte adhesion via superoxide and mast cells.

    PubMed

    Kubes, P; Kanwar, S; Niu, X F; Gaboury, J P

    1993-10-01

    Recent work has demonstrated that inhibition of nitric oxide production with various nitric oxide synthesis inhibitors (L-NAME, L-NMMA) initiate leukocyte adhesion to postcapillary venules. The objective of this study was to elucidate the mechanism (or mechanisms) that promote the L-NAME-induced leukocyte response. Intravital microscopy was used to examine 25-40 microns venules in the rat mesentery. Nitric oxide synthesis was inhibited with L-NAME and leukocyte adhesion was observed over the first 60 min. The fourfold increase in leukocyte adhesion was independent of alterations in venular red blood cell velocity. The adhesion was superoxide-mediated inasmuch as superoxide dismutase (SOD) abolished the rise in leukocyte adhesion associated with nitric oxide synthesis inhibition. Ketotifen, a mast cell stabilizer, also abolished the rise in leukocyte adhesion induced by L-NAME. Histology revealed that mast cell degranulation occurred only in animals treated with L-NAME but not in animals pretreated with SOD or ketotifen. This observation suggests that mast cells become activated in the absence of nitric oxide production and superoxide contributes to the mast cell activation. The L-NAME-induced leukocyte adhesion could be reproduced by infusing hypoxanthine/xanthine oxidase (a superoxide generating system) or compound 48/80 (an activator of mast cells) and both responses were attenuated by ketotifen. These data suggest that inhibition of nitric oxide synthesis results in a superoxide and mast cell-dependent leukocyte adhesion.

  11. 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.

  12. Seasonal variation of peripheral blood leukocyte telomere length in Costa Rica: a population based observational study

    PubMed Central

    Rehkopf, David H; Dow, William H; Rosero-Bixby, Luis; Lin, Jue; Epel, Elissa S; Blackburn, Elizabeth H

    2014-01-01

    Objectives Peripheral blood leukocyte telomere length is increasingly being used as a biomarker of aging, but its natural variation in human populations is not well understood. Several other biomarkers show seasonal variation, as do several determinants of leukocyte telomere length. We examined whether there was monthly variation in leukocyte telomere length in Costa Rica, a country with strong seasonal differences in precipitation and infection. Methods We examined a longitudinal population based cohort of 581 Costa Rican adults age 60 and above, from which blood samples were drawn between October 2006 and July 2008. Leukocyte telomere length was assayed from these samples using the quantitative PCR method. Multivariate regression models were used to examine correlations between month of blood draw and leukocyte telomere length. Results Telomere length from peripheral blood leukocytes varied by as much as 200 base pairs depending on month of blood draw, and this difference is not likely to be due to random variation. A moderate proportion of this association is statistically accounted for by month and region specific average rainfall. We found shorter telomere length associated with greater rainfall. Conclusions There are two possible explanations of our findings. First, there could be relatively rapid month-to-month changes in leukocyte telomere length. This conclusion would have implications for understanding the natural population dynamics of telomere length. Second, there could be seasonal differences in constituent cell populations. This conclusion would suggest that future studies of leukocyte telomere length use methods to account for the potential impact of constituent cell type. PMID:24615938

  13. Protein-Bound Uremic Toxins Stimulate Crosstalk between Leukocytes and Vessel Wall

    PubMed Central

    Glorieux, Griet; Schepers, Eva; Cohen, Gerald; Gondouin, Bertrand; Van Landschoot, Maria; Eloot, Sunny; Rops, Angelique; Van de Voorde, Johan; De Vriese, An; van der Vlag, Johan; Brunet, Philippe; Van Biesen, Wim; Vanholder, Raymond

    2013-01-01

    Leukocyte activation and endothelial damage both contribute to cardiovascular disease, a major cause of morbidity and mortality in CKD. Experimental in vitro data link several protein-bound uremic retention solutes to the modulation of inflammatory stimuli, including endothelium and leukocyte responses and cardiovascular damage, corroborating observational in vivo data. However, the impact of these uremic toxins on the crosstalk between endothelium and leukocytes has not been assessed. This study evaluated the effects of acute and continuous exposure to uremic levels of indoxylsulfate (IS), p-cresylsulfate (pCS), and p-cresylglucuronide (pCG) on the recruitment of circulating leukocytes in the rat peritoneal vascular bed using intravital microscopy. Superfusion with IS induced strong leukocyte adhesion, enhanced extravasation, and interrupted blood flow, whereas pCS caused a rapid increase in leukocyte rolling. Superfusion with pCS and pCG combined caused impaired blood flow and vascular leakage but did not further enhance leukocyte rolling over pCS alone. Intravenous infusion with IS confirmed the superfusion results and caused shedding of heparan sulfate, pointing to disruption of the glycocalyx as the mechanism likely mediating IS-induced flow stagnation. These results provide the first clear in vivo evidence that IS, pCS, and pCG exert proinflammatory effects that contribute to vascular damage by stimulating crosstalk between leukocytes and vessels. PMID:24009240

  14. Protein-bound uremic toxins stimulate crosstalk between leukocytes and vessel wall.

    PubMed

    Pletinck, Anneleen; Glorieux, Griet; Schepers, Eva; Cohen, Gerald; Gondouin, Bertrand; Van Landschoot, Maria; Eloot, Sunny; Rops, Angelique; Van de Voorde, Johan; De Vriese, An; van der Vlag, Johan; Brunet, Philippe; Van Biesen, Wim; Vanholder, Raymond

    2013-12-01

    Leukocyte activation and endothelial damage both contribute to cardiovascular disease, a major cause of morbidity and mortality in CKD. Experimental in vitro data link several protein-bound uremic retention solutes to the modulation of inflammatory stimuli, including endothelium and leukocyte responses and cardiovascular damage, corroborating observational in vivo data. However, the impact of these uremic toxins on the crosstalk between endothelium and leukocytes has not been assessed. This study evaluated the effects of acute and continuous exposure to uremic levels of indoxylsulfate (IS), p-cresylsulfate (pCS), and p-cresylglucuronide (pCG) on the recruitment of circulating leukocytes in the rat peritoneal vascular bed using intravital microscopy. Superfusion with IS induced strong leukocyte adhesion, enhanced extravasation, and interrupted blood flow, whereas pCS caused a rapid increase in leukocyte rolling. Superfusion with pCS and pCG combined caused impaired blood flow and vascular leakage but did not further enhance leukocyte rolling over pCS alone. Intravenous infusion with IS confirmed the superfusion results and caused shedding of heparan sulfate, pointing to disruption of the glycocalyx as the mechanism likely mediating IS-induced flow stagnation. These results provide the first clear in vivo evidence that IS, pCS, and pCG exert proinflammatory effects that contribute to vascular damage by stimulating crosstalk between leukocytes and vessels.

  15. Possible pitfalls with clinical imaging of indium-111 leukocytes: concise communication

    SciTech Connect

    Coleman, R.E.; Welch, D.

    1980-02-01

    Indium-111 leukocyte imaging is a reliable procedure for detecting abscesses. Problems such as cell clumping can occur before injection, thus altering the normal distribution. Furthermore, accumulation of the labeled leukocytes in the colon, brain infarcts, and accessory spleens has been observed. Thus, these physiologic and pathologic conditions other than abscesses must be borne in mind to avoid false-positive diagnoses of abscess.

  16. Leukocyte Cell Surface Proteinases: Regulation of Expression, Functions, and Mechanisms of Surface Localization

    PubMed Central

    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 proteinase 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. PMID:18329945

  17. Inhibitory effect of FUT-175 on the production of interleukin 8 and polymorphonuclear leukocyte elastase.

    PubMed

    Kikuchi, M; Endo, S; Inada, K; Yamashita, H; Takakuwa, T; Nakae, H; Kasai, T; Baba, N; Yamada, Y

    1995-03-01

    We investigated the inhibitory effects of a protease inhibitor, FUT-175, on the production of interleukin 8 (IL-8) and polymorphonuclear leukocyte elastase (PMNE) by polymorphonuclear leukocytes (PMN) and vascular endothelial cells. IL-8 production by PMN and vascular endothelial cells stimulated with lipopolysaccharide (LPS) was inhibited by FUT-175. This compound also inhibited PMNE production by PMN following LPS stimulation.

  18. 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

  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. 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.

  1. ICAM-1 enrichment near tri-cellular endothelial junctions is preferentially associated with leukocyte transmigration, and signals for reorganization of these junctions to accommodate leukocyte passage

    PubMed Central

    Sumagin, Ronen; Sarelius, Ingrid H

    2010-01-01

    Leukocyte transmigration occurs at specific locations (portals) on the endothelium, but the nature of these portals is not clear. Using intravital confocal microscopy of anesthetized mouse cremaster muscle in combination with immunofluorescence labeling, we showed that in microvessels transmigration is mainly junctional and preferentially occurs at tri-cellular endothelial junctional regions. Our data suggest that enrichment of ICAM-1 near approximately 43% of these junctions makes these locations preferred for transmigration, by signaling the location of a nearby portal, as well as preparing the EC-junctions to accommodate leukocyte passage. Blockade of the extracellular domain of the ICAM-1 significantly reduced transmigration (by 68.8±4.5%), by reducing the ability of leukocytes to get to these portals. In contrast, blockade of the cytoplasmic tail of ICAM-1 reduced transmigration (by 71.1±7.0%) by disabling VE-Cadherin rearrangement. Importantly, venular convergences are optimally equipped to support leukocyte transmigration. Differences in EC morphology result in a significantly higher number of tri-cellular junctions in convergences compared to straight venular regions (20.7±1.2 vs 12.43±1.1/6000μm2, respectively). Consequently leukocyte adhesion and transmigration are significantly higher in convergences compared to straight regions (1.6- and 2.6-fold, respectively). Together, these data identify an important role for EC morphology and expression patterns of ICAM-1 in leukocyte transmigration. PMID:20363969

  2. 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

  3. 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.

  4. 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.

  5. Rosmarinus officinalis L. essential oil inhibits in vivo and in vitro leukocyte migration.

    PubMed

    Nogueira de Melo, Gessilda Alcantara; Grespan, Renata; Fonseca, Jefferson Pitelli; Farinha, Thiago Oliveira; Silva, Expedito Leite; Romero, Adriano Lopes; Bersani-Amado, Ciomar A; Cuman, Roberto Kenji Nakamura

    2011-09-01

    Rosmarinus officinalis L. (Lamiaceae), popularly known as rosemary, is used for food flavoring and in folk medicine as an antispasmodic, analgesic, antirheumatic, diuretic, and antiepileptic agent. Few studies have shown the anti-inflammatory effects of rosemary essential oil (REO). This study evaluated the effects of REO on leukocyte migration through in vivo leukocyte migration and in vitro chemotaxis assay. REO was analyzed by using gas chromatography-mass spectometry, and the main components identified were camphor (27.59%), 1,8-cineole (15.74%), α-pinene (16.58%), and β-myrcene (10.02%). In rats, administration of REO reduced the number of leukocytes that rolled, adhered, and migrated to the scrotal chamber after carrageenan injection. All doses of REO tested significantly inhibited leukocyte chemotaxis induced by casein. The effects of REO on leukocyte migration highlight an important mechanism of the anti-inflammatory action of rosemary.

  6. 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.

  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 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.

  8. 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.

  9. 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.

  10. 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.

  11. The hidden side of SERPINB1/Leukocyte Elastase Inhibitor.

    PubMed

    Torriglia, Alicia; Martin, Elisabeth; Jaadane, Imene

    2017-02-01

    SERPINB1, also called Leukocyte Elastase Inhibitor (LEI) is a member of the clade B of SERPINS. It is an intracellular protein and acts primarily to protect the cell from proteases released into the cytoplasm during stress. Its role in inflammation is clear due to its involvement in the resolution of chronic inflammatory lung and bowel diseases. LEI/SERPINB1 intrinsically possesses two enzymatic activities: an antiprotease activity dependent on its reactive site loop, which is analogous to the other proteins of the family and an endonuclease activity which is unveiled by the cleavage of the reactive site loop. The conformational change induced by this cleavage also unveils a bipartite nuclear localization signal allowing the protein to translocate to the nucleus. Recent data indicate that it has also a role in cell migration suggesting that it could be involved in diverse processes like wound healing and malignant metastases. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Human leukocyte inhibitory factor (LIF): two distinct molecular species.

    PubMed

    Brown, A P; Rocklin, R E

    1979-03-01

    Human leukocyte inhibitory factor or LIF was generated in vitro by stimulating blood lymphocytes with concanavalin A (Con A). The control and Con A active supernatants were partially purified by gel filtration on Sephadex G-100. The fraction containing LIF (68,000 daltons) activity was then subjected to isoelectric focusing (pH 3 to 10 ampholines) in a sucrose gradient. Two LIF activities were reproducibly recovered by this procedure. One molecular form was found to have an isoelectric point of approximately pH 5.0 and the other approximately pH 8.5. Both molecular species were rechromatographed on Sephadex G-75 and found to have the same apparent m.w. (68 to 75,000). Furthermore, the biologic activity of both factors was destroyed after treatment with diisopropylphosphofluoridate, suggesting that they may be esterases.

  13. A1/Bfl-1 in leukocyte development and cell death

    PubMed Central

    Ottina, Eleonora; Tischner, Denise; Herold, Marco J.; Villunger, Andreas

    2012-01-01

    The function of the anti-apoptotic Bcl-2 family member Bcl2a1/Bfl-1/A1 is poorly understood due to the lack of appropriate loss-of-function mouse models and redundant effects with other Bcl-2 pro-survival proteins upon overexpression. Expression analysis of A1 suggests predominant roles in leukocyte development, their survival upon viral or bacterial infection, as well as during allergic reactions. In addition, A1 has been implicated in autoimmunity and the pathology and therapy resistance of hematological as well as solid tumors that may aberrantly express this protein. In this review, we aim to summarize current knowledge on A1 biology, focusing on its role in the immune system and compare it to that of other pro-survival Bcl-2 proteins. PMID:22342458

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. Leukocyte antimicrobial peptides: multifunctional effector molecules of innate immunity.

    PubMed

    Risso, A

    2000-12-01

    Antimicrobial peptides are effector molecules of innate immunity that provide a first line of defense against pathogens. In mammals, they are stored in granules of leukocytes and are present in those sites that are exposed to microbial invasion, such as mucosal surfaces and skin. In the last decade, biochemical investigations and recombinant DNA technology have allowed the identification and characterization of several antimicrobial peptides from various animal and vegetal species. Most of the mammalian peptides have been grouped in two broad families: defensins and cathelicidin-derived peptides. Functional studies have shown that the toxicity mechanisms for many peptides consist of a rapid permeabilization of the target cell membrane. In addition to their microbicidal activity, some members of both families are multifunctional molecules, playing a modulating role in the inflammation and the antigen-driven immune response.

  19. A continuum model of protrusion of pseudopod in leukocytes.

    PubMed Central

    Zhu, C; Skalak, R

    1988-01-01

    The morphology of human leukocytes, the biochemistry of actin polymerization, and the theory of continuum mechanics are used to model the pseudopod protrusion process of leukocytes. In the proposed model, the pseudopod is considered as a porous solid of F-actin network, the pores of which are full of aqueous solution. G-actin is considered as a "solute" transported by convection and diffusion in the fluid phase. The pseudopod grows as actin filaments elongate at their barbed ends at the tip of the pseudopod. The driving force of extension is hypothesized as being provided by the actin polymerization. It is assumed that elongation of actin filaments, powered by chemical energy liberated from the polymerization reaction, does mechanical work against opposing pressure on the membrane. This also gives rise to a pressure drop in the fluid phase at the tip of the pseudopod, which is formulated by an equation relating the work done by actin polymerization to the local state of pressure. The pressure gradient along the pseudopod drives the fluid filtration through the porous pseudopod according to Darcy's Law, which in turn brings more actin monomers to the growing tip. The main cell body serves as a reservoir of G-actin. A modified first-order equation is used to describe the kinetics of polymerization. The rate of pseudopod growth is modulated by regulatory proteins. A one-dimensional moving boundary problem based on the proposed mechanism has been constructed and approximate solutions have been obtained. Comparison of the solutions with experimental data shows that the model is compatible with available observations. The model is also applicable to growth of other cellular systems such as elongation of acrosomal process in sperm cells. Images FIGURE 1 FIGURE 4 PMID:3233268

  20. Cardiac and vascular imaging with labeled platelets and leukocytes

    SciTech Connect

    Dewanjee, M.K.

    1984-07-01

    The contribution of platelets in atherosclerosis and thrombosis in animal models and in clinical studies has been quantified with 111In-platelet scintigraphy. New in vitro quantitative techniques have been developed using 111In-labeled platelets to determine the number of adherent platelets on deendothelialized surfaces of damaged vessel walls and synthetic vascular grafts. In vivo imaging techniques are semi-quantitative in nature; in these studies 111In radioactivity on thrombotic vessels or graft surfaces of iliac, femoral, or popliteal arteries is compared with contralateral vessels. Background 111In radioactivity in the circulating blood pool of venous and capillary networks and radioactivity in marrow decreases the sensitivity of these techniques. Subtraction of blood pool radioactivity with 99mTc-labeled autologous red cells and calculation of 111In radioactivity associated with platelet thrombus on vessel walls also have been performed for coronary, carotid, and femoral arteries. Although platelet concentrates are used frequently after open heart surgery (one to six per patient), consumption of platelets in the artificial lung or oxygenator, lysis of platelets during pumping, and suction of blood only recently have been quantified with the use of 111In-labeled platelets. These studies also demonstrated far less trauma to platelets with the use of a membrane rather than a bubble oxygenator. Further reduction in platelet consumption and trauma was observed with the use of prostacyclin, a short-acting drug with significant beneficial effect on platelet thrombus reduction and disaggregation of aggregated platelets. The role of polymorphonuclear leukocytes in inflammation, infection and myocardial infarction, and in vivo evaluation with 111In-leukocyte scintigraphy in animals and humans has been described.

  1. The multifaceted role of PIP2 in leukocyte biology.

    PubMed

    Tuosto, Loretta; Capuano, Cristina; Muscolini, Michela; Santoni, Angela; Galandrini, Ricciarda

    2015-12-01

    Phosphatidylinositol 4,5-bisphosphate (PIP2) represents about 1 % of plasma membrane phospholipids and behaves as a pleiotropic regulator of a striking number of fundamental cellular processes. In recent years, an increasing body of literature has highlighted an essential role of PIP2 in multiple aspects of leukocyte biology. In this emerging picture, PIP2 is envisaged as a signalling intermediate itself and as a membrane-bound regulator and a scaffold of proteins with specific PIP2 binding domains. Indeed PIP2 plays a key role in several functions. These include directional migration in neutrophils, integrin-dependent adhesion in T lymphocytes, phagocytosis in macrophages, lysosomes secretion and trafficking at immune synapse in cytolytic effectors and secretory cells, calcium signals and gene transcription in B lymphocytes, natural killer cells and mast cells. The coordination of these different aspects relies on the spatio-temporal organisation of distinct PIP2 pools, generated by the main PIP2 generating enzyme, phosphatidylinositol 4-phosphate 5-kinase (PIP5K). Three different isoforms of PIP5K, named α, β and γ, and different splice variants have been described in leukocyte populations. The isoform-specific coupling of specific isoforms of PIP5K to different families of activating receptors, including integrins, Fc receptors, toll-like receptors and chemokine receptors, is starting to be reported. Furthermore, PIP2 is turned over by multiple metabolising enzymes including phospholipase C (PLC) γ and phosphatidylinositol 3-kinase (PI3K) which, along with Rho family small G proteins, is widely involved in strategic functions within the immune system. The interplay between PIP2, lipid-modifying enzymes and small G protein-regulated signals is also discussed.

  2. Leukocyte telomere length, T cell composition and DNA methylation age.

    PubMed

    Chen, Brian H; Carty, Cara L; Kimura, Masayuki; Kark, Jeremy D; Chen, Wei; Li, Shengxu; Zhang, Tao; Kooperberg, Charles; Levy, Daniel; Assimes, Themistocles; Absher, Devin; Horvath, Steve; Reiner, Alexander P; Aviv, Abraham

    2017-09-20

    Both leukocyte telomere length (LTL) and DNA methylation age are strongly associated with chronological age. One measure of DNA methylation age─ the extrinsic epigenetic age acceleration (EEAA)─ is highly predictive of all-cause mortality. We examined the relation between LTL and EEAA. LTL was measured by Southern blots and leukocyte DNA methylation was determined using Illumina Infinium HumanMethylation450 BeadChip in participants in the Women's Health Initiative (WHI; n=804), the Framingham Heart Study (FHS; n=909) and the Bogalusa Heart study (BHS; n=826). EEAA was computed using 71 DNA methylation sites, further weighted by proportions of naïve CD8(+) T cells, memory CD8(+) T cells, and plasmablasts. Shorter LTL was associated with increased EEAA in participants from the WHI (r=-0.16, p=3.1x10(-6)). This finding was replicated in the FHS (r=-0.09, p=6.5x10(-3)) and the BHS (r=-0.07, p=3.8x 10(-2)). LTL was also inversely related to proportions of memory CD8(+) T cells (p=4.04x10(-16)) and positively related to proportions of naive CD8(+) T cells (p=3.57x10(-14)). These findings suggest that for a given age, an individual whose blood contains comparatively more memory CD8(+) T cells and less naive CD8(+) T cells would display a relatively shorter LTL and an older DNA methylation age, which jointly explain the striking ability of EEAA to predict mortality.

  3. Leukocyte Anti-Trafficking Strategies: Current Status and Future Directions.

    PubMed

    Sands, Bruce E

    2017-01-01

    In inflammatory bowel diseases (IBD), a pivotal step in the initiation and perpetuation of mucosal inflammation entails the recruitment of inflammatory leukocytes to the gut. Understanding the carefully coordinated series of molecular events that culminate in the recruitment of leukocytes to the gut has resulted in novel interventions with new capabilities in treating both Crohn's disease and ulcerative colitis. Key Messages: Natalizumab, an anti-α4 integrin antibody, was the first agent to demonstrate the efficacy of this approach for the induction and maintenance of response and remission in Crohn's disease. Widespread adoption was mitigated by the previously unknown risk of progressive multifocal leukoencephalopathy (PML) with this approach. Current approaches employ a more selective inhibition of adhesion molecules targeting the gut to avoid broad suppression of surveillance for JC virus, the causal pathogen of PML. Subsequently, vedolizumab, a humanized anti-α4β7 integrin antibody, has demonstrated efficacy in patients with IBD and has an excellent safety profile. To date, there have been no cases of PML in patients treated with vedolizumab, suggesting that this more selective agent does not have the same risk for PML as natalizumab. Other agents target β7 integrin (etrolizumab) and mucosal addressin cellular adhesion molecule-1, the endothelial ligand of α4β7 integrin. Efforts to inhibit the chemokine receptor CCR9 using the agent CCX282-B in Crohn's disease were not successful. An orally administered anti-α4 integrin compound showed some promise in a phase 2 trial but raises concern for PML. Finally, the S1P1 receptor agonist ozanimod showed promise in early trials in ulcerative colitis. In summary, anti-trafficking agents have the potential to provide safe and effective therapy for IBD, and are a burgeoning class of novel agents. © 2017 S. Karger AG, Basel.

  4. Global methylation of blood leukocyte DNA and risk of melanoma.

    PubMed

    Shen, Jie; Song, Renduo; Wan, Jie; Huff, Chad; Fang, Shenying; Lee, Jeffrey E; Zhao, Hua

    2017-04-01

    Global DNA methylation, possibly influenced by lifestyle and environmental factors, has been suggested to play an active role in carcinogenesis. However, its role in melanoma has rarely been explored. The aims of this study were to evaluate the relationship between melanoma risk and levels of 5-methylcytosine (5-mC), a marker for global DNA methylation, in blood leukocyte DNA, and to determine whether this 5-mC level is influenced by pigmentation and sun exposure. This case-control study included 540 melanoma cases and 540 healthy controls. Overall, melanoma cases had significantly lower levels of 5-mC% than healthy controls (median: 3.24 vs. 3.91, p < 0.001). The significant difference between two groups did not differ by pigmentation or sun exposure. Among healthy controls, however, those who had fair skin color (p = 0.041) or light or no tanning after prolonged sun exposure (p = 0.031) or used a sunlamp (p = 0.028) had lower levels of 5-mC% than their counterparts. In addition, those with an intermediate or high phenotypic index, an indicator of cutaneous cancer susceptibility, had 2.58-fold greater likelihood of having a low level of 5-mC% [odds ratio (OR): 2.58; 95% confidence interval (CI): 1.72, 3.96] than those with a low phenotypic index. Lower levels of 5-mC% were associated with a 1.25-fold greater risk of melanoma (OR: 1.25; 95% CI: 1.08, 1.37). A significant dose-response relationship was observed in quartile analysis (p = 0.001). Our results suggest that global hypomethylation in blood leukocyte DNA is associated with increased risk of melanoma and that the level of methylation is influenced by pigmentation and sun exposure. © 2016 UICC.

  5. Leukocyte DNA methylation and colorectal cancer among male smokers

    PubMed Central

    Gao, Ying; Killian, Keith; Zhang, Hong; Yu, Kai; Li, Qi-Zhai; Weinstein, Stephanie; Virtamo, Jarmo; Tucker, Margaret; Taylor, Philip; Albanes, Demetrius; Meltzer, Paul; Caporaso, Neil

    2012-01-01

    AIM: To explore the association between methylation in leukocyte DNA and colorectal cancer (CRC) risk in male smokers using the α-tocopherol, β-carotene cancer prevention study. METHODS: About 221 incident CRC cases, and 219 controls, frequency-matched on age and smoking intensity were included. DNA methylation of 1505 CpG sites selected from 807 genes were evaluated using Illumina GoldenGate Methylation Cancer Panel I in pre-diagnostic blood leukocytes of study subjects. Tertiles of methylation level classified according to the distribution in controls for each CpG site were used to analyze the association between methylation level and CRC risk with logistic regression. The time between blood draw to cancer diagnosis (classifying cases according to latency) was incorporated in further analyses using proportional odds regression. RESULTS: We found that methylation changes of 31 CpG sites were associated with CRC risk at P < 0.01 level. Though none of these 31 sites remained statistically significant after Bonferroni correction, the most statistically significant CpG site associated with CRC risk achieved a P value of 1.0 × 10-4. The CpG site is located in DSP gene, and the risk estimate was 1.52 (95% CI: 0.91-2.53) and 2.62 (95% CI: 1.65-4.17) for the second and third tertile comparing with the lowest tertile respectively. Taking the latency information into account strengthened some associations, suggesting that the methylation levels of corresponding sites might change over time with tumor progression. CONCLUSION: The results suggest that the methylation level of some genes were associated with cancer susceptibility and some were related to tumor development over time. Further studies are warranted to confirm and refine our results. PMID:22912915

  6. 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. © 2016 International Society for Advancement of Cytometry.

  7. Using milk leukocyte differentials for diagnosis of subclinical bovine mastitis.

    PubMed

    Gonçalves, Juliano Leonel; Lyman, Roberta L; Hockett, Mitchell; Rodriguez, Rudy; Dos Santos, Marcos Veiga; Anderson, Kevin L

    2017-08-01

    This research study aimed to evaluate the use of the milk leukocyte differential (MLD) to: (a) identify quarter milks that are culture-positive; and (b) characterize the milk leukocyte responses to specific groups of pathogens causing subclinical mastitis. The MLD measures the absolute number and relative percentage of inflammatory cells in milk samples. Using the MLD in two dairy herds (170 and 172 lactating cows, respectively), we studied all lactating cows with a most recent monthly Dairy Herd Improvement Association somatic cell count (SCC) >200 × 103 cells/ml. Quarter milk samples from 78 cows meeting study criteria were analysed by MLD and aseptically collected milk samples were subjected to microbiological culture (MC). Based upon automated instrument evaluation of the number and percentage of inflammatory cells in milk, samples were designated as either MLD-positive or - negative for subclinicial mastitis. Positive MC were obtained from 102/156 (65·4%) of MLD-positive milk samples, and 28/135 (20·7%) of MLD-negative milk samples were MC-positive. When MC was considered the gold standard for mastitis diagnosis, the calculated diagnostic Se of the MLD was 65·4% (IC95% = 57·4 to 72·8%) and the Sp was 79·3% (IC95% = 71·4 to 85·7%). Quarter milks positive on MC had higher absolute numbers of neutrophils, lymphocytes and macrophages, with higher neutrophils% and lymphocytes% but lower macrophages%. The Log10 (N/L) ratios were the most useful ratio to differentiate specific subclinical mastitis quarters from healthy quarters. Use of the MLD on cows with monthly composite SCC > 200 × 103 cells/ml for screening at quarter level identified quarters more likely to be culture-positive. In conclusion, the MLD can provide an analysis of mammary quarter status more detailed than provided by SCC alone; however, the MLD response to subclinical mastitis was not found useful to specifically identify the causative pathogen.

  8. Susceptibility of different leukocyte cell types to Vaccinia virus infection

    PubMed Central

    Sánchez-Puig, Juana M; Sánchez, Laura; Roy, Garbiñe; Blasco, Rafael

    2004-01-01

    Background Vaccinia virus, the prototype member of the family Poxviridae, was used extensively in the past as the Smallpox vaccine, and is currently considered as a candidate vector for new recombinant vaccines. Vaccinia virus has a wide host range, and is known to infect cultures of a variety of cell lines of mammalian origin. However, little is known about the virus tropism in human leukocyte populations. We report here that various cell types within leukocyte populations have widely different susceptibility to infection with vaccinia virus. Results We have investigated the ability of vaccinia virus to infect human PBLs by using virus recombinants expressing green fluorescent protein (GFP), and monoclonal antibodies specific for PBL subpopulations. Flow cytometry allowed the identification of infected cells within the PBL mixture 1–5 hours after infection. Antibody labeling revealed that different cell populations had very different infection rates. Monocytes showed the highest percentage of infected cells, followed by B lymphocytes and NK cells. In contrast to those cell types, the rate of infection of T lymphocytes was low. Comparison of vaccinia virus strains WR and MVA showed that both strains infected efficiently the monocyte population, although producing different expression levels. Our results suggest that MVA was less efficient than WR in infecting NK cells and B lymphocytes. Overall, both WR and MVA consistently showed a strong preference for the infection of non-T cells. Conclusions When infecting fresh human PBL preparations, vaccinia virus showed a strong bias towards the infection of monocytes, followed by B lymphocytes and NK cells. In contrast, very poor infection of T lymphocytes was detected. These finding may have important implications both in our understanding of poxvirus pathogenesis and in the development of improved smallpox vaccines. PMID:15555076

  9. Human leukocyte antigen class I and MICA haplotypes in a multicase family with Cladophialophora carrionii chromoblastomycosis.

    PubMed

    Naranjo, F; Márquez, I; Gendzekhadze, K; Zhang, S; Fernández-Mestre, M; Yegres, F; Richard-Yegres, N; Navas, T; Montagnani, S; Ogando, V; Layrisse, Z

    2006-10-01

    Previous studies carried out in an endemic semiarid region northwest of Venezuela at Falcon State have shown a prevalence of 15.4/1000 of chromoblastomycosis following traumatisms with xenophile vegetation infected with Cladophialophora carrionii. We performed high-resolution DNA typing of human leukocyte antigen (HLA)-A, -B and -C and major histocompatibility complex class I chain related gene A (MICA) alleles and segregation analysis in 49 members of one extended family with 12 affected individuals, who have lived for approximately 70 years in this endemic zone. None of the alleles, haplotypes or genotypes is shared by all the patients. No deviation from the expected HLA haplotype distribution or association of chromoblastomycosis with HLA-A, -B and -C haplotypes was observed. Further, a haplotype-sharing transmission/disequilibria testing of 11 nuclear families did not give enough evidence to claim linkage (P = 0.398), suggesting that genes located in the short arm of chromosome 6 may not be relevant in the immune response toward infection with C. carrionii in this Venezuelan endemic zone. Deleted MICA alleles on HLA-B*4802 haplotypes were present among several members of the extended family, but only two of them were affected.

  10. Leukocyte recruitment in the brain in sepsis: involvement of the annexin 1-FPR2/ALX anti-inflammatory system.

    PubMed

    Gavins, Felicity N E; Hughes, Ellen L; Buss, Nicholas A P S; Holloway, Paul M; Getting, Stephen J; Buckingham, Julia C

    2012-12-01

    Unregulated inflammation underlies many diseases, including sepsis. Much interest lies in targeting anti-inflammatory mechanisms to develop new treatments. One such target is the anti-inflammatory protein annexin A1 (AnxA1) and its receptor, FPR2/ALX. Using intravital videomicroscopy, we investigated the role of AnxA1 and FPR2/ALX in a murine model of endotoxin-induced cerebral inflammation [intraperitoneal injection of lipopolysaccharide (LPS)]. An inflammatory response was confirmed by elevations in proinflammatory serum cytokines, increased cerebrovascular permeability, elevation in brain myeloperoxidase, and increased leukocyte rolling and adhesion in cerebral venules of wild-type (WT) mice, which were further exacerbated in AnxA1-null mice. mRNA expression of TLR2, TLR4, MyD-88, and Ly96 was also assessed. The AnxA1-mimetic peptide, AnxA1(Ac2-26) (100 μg/mouse, ∼33 μmol) mitigated LPS-induced leukocyte adhesion in WT and AnxA1-null animals without affecting leukocyte rolling, in comparison to saline control. AnxA1(Ac2-26) effects were attenuated by Boc2 (pan-FPR antagonist, 10 μg/mouse, ∼12 nmol), and by minocycline (2.25 mg/mouse, ∼6.3 nmol). The nonselective Fpr agonists, fMLP (6 μg/mouse, ∼17 nmol) and AnxA1(Ac2-26), and the Fpr2-selective agonist ATLa (5 μg/mouse, ∼11 nmol) were without effect in Fpr2/3(-/-) mice. In summary, our novel results demonstrate that the AnxA1/FPR2 system has an important role in effecting the resolution of cerebral inflammation in sepsis and may, therefore, provide a novel therapeutic target.

  11. Elevated concentration of secretory leukocyte protease inhibitor in the cervical mucus before delivery.

    PubMed

    Samejima, Taiki; Nagamatsu, Takeshi; Schust, Danny J; Itaoka, Nao; Iriyama, Takayuki; Nakayama, Toshio; Komatsu, Atsushi; Kawana, Kei; Osuga, Yutaka; Fujii, Tomoyuki

    2016-06-01

    Cervical remodeling during parturition progresses under exquisite regulation by immunologic mediators and proteases. Secretory leukocyte protease inhibitor is a secretory protein that can function as an antimicrobial peptide, an antiinflammatory molecule, and a protease inhibitor. The involvement of secretory leukocyte protease inhibitor in cervical remodeling before and during parturition is understood poorly. We aimed to reveal the role of secretory leukocyte protease inhibitor in the cervical remodeling process before normal term delivery and to evaluate its utility as a predictive biomarker for timing of delivery. Cervical mucus samples were collected prospectively at weekly prenatal visits from a cohort of pregnant women at term. The secretory leukocyte protease inhibitor concentrations in 95 mucus samples that were obtained from 49 women with uncomplicated pregnancy who subsequently underwent normal vaginal delivery were assessed. Alterations in secretory leukocyte protease inhibitor concentrations at term and the association of secretory leukocyte protease inhibitor levels with the time to delivery were analyzed. A moderate positive correlation with significance was detected between cervical mucus secretory leukocyte protease inhibitor concentrations and days to delivery (r = 0.38; P = .0001). The secretory leukocyte protease inhibitor concentration was significantly higher in samples that were collected within 7 days of delivery when compared with samples that were collected >7 days before delivery (P = .001). Secretory leukocyte protease inhibitor concentrations were also significantly higher in samples from women with premature rupture of membranes when compared with those without premature rupture of membranes (P = .01), all of whom delivered within 7 days. A logistic regression analysis revealed that the cervical secretory leukocyte protease inhibitor level was a significant parameter for the prediction of the onset of delivery. (P = .017; unit

  12. 77 FR 59000 - Guidance for Industry: Pre-Storage Leukocyte Reduction of Whole Blood and Blood Components...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-09-25

    ... HUMAN SERVICES Food and Drug Administration Guidance for Industry: Pre-Storage Leukocyte Reduction of... availability of a document entitled ``Guidance for Industry: Pre- Storage Leukocyte Reduction of Whole Blood... blood establishments with recommendations for pre-storage leukocyte reduction of Whole Blood and...

  13. 76 FR 5386 - Draft Guidance for Industry: Pre-Storage Leukocyte Reduction of Whole Blood and Blood Components...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-31

    ... HUMAN SERVICES Food and Drug Administration Draft Guidance for Industry: Pre-Storage Leukocyte Reduction... availability of a draft document entitled ``Guidance for Industry: Pre- Storage Leukocyte Reduction of Whole... provides blood establishments with recommendations for pre- storage leukocyte reduction of Whole Blood...

  14. The relationship between glycated hemoglobin and polymorphonuclear leukocyte leukotriene B4 release in people with diabetes mellitus.

    PubMed

    Parlapiano, C; Danese, C; Marangi, M; Campana, E; Pantone, P; Giovanniello, T; Zavattaro, E; Sanguigni, S

    1999-10-01

    In order to evaluate polymorphonuclear leukocyte (PMN) activity in diabetes mellitus, leukotriene B4 (LTB4) levels were measured in sixty patients, 31 affected with Type 1 diabetes mellitus and 29 affected with Type 2 diabetes mellitus. The LTB4 levels (12.1+/-0.2 pg/100 microl) in diabetic patients were higher compared to those of the control group (7.9+/-0.1 pg/100 microl) (P < 0.001), and remained significantly higher (P < 0.001) (12.8+/-0.2 pg/100 microl) than in the control group (11.0+/-0.2 pg/100 microl) after stimulation with calcium ionophore. A significant and positive correlation between glycated hemoglobin and LTB4 was demonstrated (P < 0.001, r = 0.80). This study demonstrates that in diabetic patients there is a PMN activation and that this activation is correlated to glycated hemoglobin level.

  15. Deformability based Cell Sorting using Microfluidic Ratchets Enabling Phenotypic Separation of Leukocytes Directly from Whole Blood.

    PubMed

    Guo, Quan; Duffy, Simon P; Matthews, Kerryn; Islamzada, Emel; Ma, Hongshen

    2017-07-26

    The separation of leukocytes from whole blood is a prerequisite for many biological assays. Traditional methods require significant sample volumes and are often undesirable because they expose leukocytes to harsh physical or chemical treatment. Existing microfluidic approaches can work with smaller volumes, but lack selectivity. In particular, the selectivity of microfluidic systems based on microfiltration is limited by fouling due to clogging. Here, we developed a method to separate leukocytes from whole blood using the microfluidic ratchet mechanism, which filters the blood sample using a matrix of micrometer-scale tapered constrictions. Deforming single cells through such constrictions requires directionally asymmetrical forces, which enables oscillatory flow to create a ratcheting transport that depends on cell size and deformability. Simultaneously, oscillatory flow continuously agitates the cells to limit the contact time with the filter microstructure to prevent adsorption and clogging. We show this device is capable of isolating leukocytes from whole blood with 100% purity (i.e. no contaminant erythrocytes) and <2% leukocytes loss. We further demonstrate the potential to phenotypically sort leukocytes to enrich for granulocytes and lymphocytes subpopulations. Together, this process provides a sensitive method to isolate and sort leukocytes directly from whole blood based on their biophysical properties.

  16. 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

  17. 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.

  18. Detection of murine cytomegalovirus DNA in circulating leukocytes harvested during acute infection of mice

    SciTech Connect

    Bale, J.F. Jr.; O'Neil, M.E. )

    1989-06-01

    The authors used virus assay and in situ hybridization with a cloned fragment of the murine cytomegalovirus (MCMV) genome to study MCMV infection of circulating leukocytes harvested from 3-week-old BALB/c, C57BL/6, and C3H mice infected with MCMV intraperitoneally. Infectious virus or MCMV DNA was detected in leukocytes on days 1 through 21 of infection in BALB/c mice and on days 3 through 7 in C57BL/6 mice. On days 5 and 7, MCMV DNA or infectious virus was detected in the leukocytes of 17 (94%) of 18 BALB/c mice and 10 (59%) of 17 C57BL/6 mice. In both strains infection peaked on days 5 and 7, when as many as 0.01 to 0.1% of the circulating leukocytes contained MCMV DNA. In C3H mice, however, infectious virus was rarely recovered from leukocyte fractions and MCMV DNA was detected in the circulating leukocytes of only one animal. Circulating leukocytes may have an important role in the dissemination of CMV infections in susceptible hosts.

  19. 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.

  20. 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

  1. Blocking cyclophilins in the chronic phase of asthma reduces the persistence of leukocytes and disease reactivation.

    PubMed

    Stemmy, Erik J; Balsley, Molly A; Jurjus, Rosalyn A; Damsker, Jesse M; Bukrinsky, Michael I; Constant, Stephanie L

    2011-11-01

    Allergic asthma is characterized by acute influxes of proinflammatory leukocytes in response to allergen stimulation, followed by quiescent (chronic) periods between allergen challenges, during which sustained, low-level inflammation is evident. These chronic phases of disease are thought to be mediated by populations of leukocytes persisting within airways and tissues. The lack of any in situ proliferation by these cells, along with their limited lifespan, suggests that a continual recruitment of leukocytes from the circulation is needed to maintain disease chronicity. The mechanisms regulating this persistent recruitment of leukocytes are unknown. Although classic leukocyte-attracting chemokines are highly elevated after acute allergen challenge, they return to baseline levels within 24 hours, and remain close to undetectable during the chronic phase. In the present study, we investigated whether an alternative family of chemoattractants, namely, extracellular cyclophilins, might instead play a role in regulating the recruitment and persistence of leukocytes during chronic asthma, because their production is known to be more sustained during inflammatory responses. Using a new murine model of chronic allergic asthma, elevated concentrations of extracellular cyclophilin A, but not classic chemokines, were indeed detected during the chronic phase of asthma. Furthermore, blocking the activity of cyclophilins during this phase reduced the number of persisting leukocytes by up to 80%. This reduction was also associated with a significant inhibition of acute disease reactivation upon subsequent allergen challenge. These findings suggest that blocking the function of cyclophilins during the chronic phase of asthma may provide a novel therapeutic strategy for regulating disease chronicity and severity.

  2. 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.

  3. 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.

  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. Early interleukin 6 production by leukocytes during ischemic acute kidney injury is regulated by TLR4

    PubMed Central

    Chen, Jianlin; Hartono, John R.; John, Reji; Bennett, Michael; Zhou, Xin Jin; Wang, Yanxia; Wu, Qingqing; Winterberg, Pamela D.; Nagami, Glenn T.; Lu, Christopher Y.

    2012-01-01

    Although leukocytes infiltrate the kidney during ischemic acute kidney injury (AKI) and release interleukin 6 (IL6), their mechanism of activation is unknown. Here, we tested whether Toll-like receptor 4 (TLR4) on leukocytes mediated this activation by interacting with high-mobility group protein B1 (HMGB1) released by renal cells as a consequence of ischemic kidney injury. We constructed radiation-induced bone marrow chimeras using C3H/HeJ and C57BL/10ScNJ strains of TLR4 (−/−) mice and their respective TLR4 (+/+) wild-type counterparts and studied them at 4 h after an ischemic insult. Leukocytes adopted from TLR4 (+/+) mice infiltrated the kidneys of TLR4 (−/−) mice, and TLR4 (−/−) leukocytes infiltrated the kidneys of TLR4 (+/+) mice but caused little functional renal impairment in each case. Maximal ischemic AKI required both radiosensitive leukocytes and radioresistant renal parenchymal and endothelial cells from TLR4 (+/+) mice. Only TLR4 (−/−) leukocytes produced IL6 in vivo and in response to HMGB1 in vitro. Thus, following infiltration of the injured kidney, leukocytes produce IL6 when their TLR4 receptors interact with HMGB1 released by injured renal cells. This underscores the importance of TLR4 in the pathogenesis of ischemic AKI. PMID:21633411

  6. Detection of acute synthetic vascular graft infection with IN-111 labeled leukocyte imaging

    SciTech Connect

    Alazraki, N.; Dries, D.; Lawrence, P.; Murphy, K.; Kercher, J.; Datz, F.; Christian, P.; Taylor, A.

    1985-05-01

    Synthetic vascular graft infection is characterized by late diagnosis due to indolent and nonspecific symptoms. Reported data on accuracy of In-111 labeled leukocyte imaging to identify vascular graft infection is sparse and conflicting. The purpose of this animal study was to clarify the accuracy of detection of early graft infection using a mixed population of In-111 labeled leukocytes. Twelve mongrel dogs received dacron aortic interposition grafts. Seven grafts were contaminated at surgery by topical ATCC S. aureus, 10/sup 8/ organisms per ml. Six control animals received no graft contamination Mixed population In-111 homologous leukocyte labeling was performed followed by imaging at 24 and 48 hours following intravenous injection of 250 ..mu..Ci In-111 leukocytes. Scans were done on Day 2 post-surgery. Infected dogs were sacrificed following Indium imaging; control dogs were rescanned at 3 weeks postop and sacrificed thereafter. Autopsy results were correlated with scans, yielding sensitivity 71%, specificity 100%, accuracy 85% for In-111 leukocyte imaging to detect early graft infection. False positive leukocyte imaging in the early postop period was not a problem. At autopsy all 5 dogs with infected grafts and positive scans had gross pus. The 2 dogs with false negative scans showed no gross pus at autopsy; cultures were positive for S. aureus in all 7 dogs. Scans at 2 days and 3 weeks post-surgery were true negatives in all 6 control dogs. These data suggest a high level of clinical reliability of leukocyte imaging for early graft infection detection.

  7. 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.

  8. Is Chronic Asthma Associated with Shorter Leukocyte Telomere Length at Midlife?

    PubMed Central

    Shalev, Idan; Sears, Malcolm R.; Hancox, Robert J.; Lee Harrington, Hona; Houts, Renate; Moffitt, Terrie E.; Sugden, Karen; Williams, Benjamin; Poulton, Richie; Caspi, Avshalom

    2014-01-01

    Rationale: Asthma is prospectively associated with age-related chronic diseases and mortality, suggesting the hypothesis that asthma may relate to a general, multisystem phenotype of accelerated aging. Objectives: To test whether chronic asthma is associated with a proposed biomarker of accelerated aging, leukocyte telomere length. Methods: Asthma was ascertained prospectively in the Dunedin Multidisciplinary Health and Development Study cohort (n = 1,037) at nine in-person assessments spanning ages 9–38 years. Leukocyte telomere length was measured at ages 26 and 38 years. Asthma was classified as life-course-persistent, childhood-onset not meeting criteria for persistence, and adolescent/adult-onset. We tested associations between asthma and leukocyte telomere length using regression models. We tested for confounding of asthma-leukocyte telomere length associations using covariate adjustment. We tested serum C-reactive protein and white blood cell counts as potential mediators of asthma-leukocyte telomere length associations. Measurements and Main Results: Study members with life-course-persistent asthma had shorter leukocyte telomere length as compared with sex- and age-matched peers with no reported asthma. In contrast, leukocyte telomere length in study members with childhood-onset and adolescent/adult-onset asthma was not different from leukocyte telomere length in peers with no reported asthma. Adjustment for life histories of obesity and smoking did not change results. Study members with life-course-persistent asthma had elevated blood eosinophil counts. Blood eosinophil count mediated 29% of the life-course-persistent asthma-leukocyte telomere length association. Conclusions: Life-course-persistent asthma is related to a proposed biomarker of accelerated aging, possibly via systemic eosinophilic inflammation. Life histories of asthma can inform studies of aging. PMID:24956257

  9. Is chronic asthma associated with shorter leukocyte telomere length at midlife?

    PubMed

    Belsky, Daniel W; Shalev, Idan; Sears, Malcolm R; Hancox, Robert J; Lee Harrington, Hona; Houts, Renate; Moffitt, Terrie E; Sugden, Karen; Williams, Benjamin; Poulton, Richie; Caspi, Avshalom

    2014-08-15

    Asthma is prospectively associated with age-related chronic diseases and mortality, suggesting the hypothesis that asthma may relate to a general, multisystem phenotype of accelerated aging. To test whether chronic asthma is associated with a proposed biomarker of accelerated aging, leukocyte telomere length. Asthma was ascertained prospectively in the Dunedin Multidisciplinary Health and Development Study cohort (n = 1,037) at nine in-person assessments spanning ages 9-38 years. Leukocyte telomere length was measured at ages 26 and 38 years. Asthma was classified as life-course-persistent, childhood-onset not meeting criteria for persistence, and adolescent/adult-onset. We tested associations between asthma and leukocyte telomere length using regression models. We tested for confounding of asthma-leukocyte telomere length associations using covariate adjustment. We tested serum C-reactive protein and white blood cell counts as potential mediators of asthma-leukocyte telomere length associations. Study members with life-course-persistent asthma had shorter leukocyte telomere length as compared with sex- and age-matched peers with no reported asthma. In contrast, leukocyte telomere length in study members with childhood-onset and adolescent/adult-onset asthma was not different from leukocyte telomere length in peers with no reported asthma. Adjustment for life histories of obesity and smoking did not change results. Study members with life-course-persistent asthma had elevated blood eosinophil counts. Blood eosinophil count mediated 29% of the life-course-persistent asthma-leukocyte telomere length association. Life-course-persistent asthma is related to a proposed biomarker of accelerated aging, possibly via systemic eosinophilic inflammation. Life histories of asthma can inform studies of aging.

  10. Leukocidin, Tetraethylammonium Ions, and the Membrane Acyl Phosphatases in Relation to the Leukocyte Potassium Pump

    PubMed Central

    Woodin, A. M.; Wieneke, Antonnette A.

    1970-01-01

    The response of the leukocyte to leukocidin and its relevance to excitable and secreting tissues are described. New data are presented on the leukocyte membrane phosphatases and the action of tetraethylammonium ion (TEA) on the leukocyte. The leukocyte surface membrane lacks a cation-sensitive ATPase but possesses a potassium- and ouabain-sensitive p-nitrophenyl phosphatase. The p-nitrophenyl phosphatase shows peak activity at three pH values and the pH dependence and potassium sensitivity depend on the state of the membranes. In the presence of magnesium, potassium can stimulate over the range pH 6 to 8. The relation of the leukocyte p-nitrophenyl phosphatase to electrolyte control in the leukocyte and to the properties of cation-sensitive phosphatases in other cells suggests that the leukocyte enzyme is a component of an electrogenic potassium pump. Leukocidin stimulates the leukocyte p-nitrophenyl phosphatase under all the conditions studied. The effect is specific and occurs under conditions that induce cytotoxic effects in the cell. It is concluded that the potassium pump is the site of action of leukocidin. TEA prevents the effects of leukocidin by inhibiting the action of leukocidin and not the responses of the cell to injury. TEA does not inhibit the p-nitrophenyl phosphatase nor prevent its stimulation by leukocidin. The enhancement of leukocidin by diisopropylphosphofluoridate (DFP) is briefly described. It is concluded that TEA acts in the opposite way to DFP and blocks the ion pathway activated by leukocidin in the leukocyte potassium pump. PMID:4327879

  11. House Dust Endotoxin and Peripheral Leukocyte Counts: Results from Two Large Epidemiologic Studies.

    PubMed

    Fessler, Michael B; Carnes, Megan U; Salo, Päivi M; Wilkerson, Jesse; Cohn, Richard D; King, Debra; Hoppin, Jane A; Sandler, Dale P; Travlos, Greg; London, Stephanie; Thorne, Peter; Zeldin, Darryl

    2017-05-31

    The peripheral leukocyte count is a biomarker of inflammation and is associated with human all-cause mortality. Although causes of acute leukocytosis are well-described, chronic environmental determinants of leukocyte number are less well understood. We investigated the relationship between house dust endotoxin concentration and peripheral leukocyte counts in human subjects. The endotoxin–leukocyte relationship was evaluated by linear regression in the National Health and Nutrition Examination Survey (NHANES) 2005–2006 (n=6,254) and the Agricultural Lung Health Study (ALHS; n=1,708). In the ALHS, we tested for a gene [Toll-like Receptor 4 (TLR4), encoding the endotoxin receptor]-by-environment interaction in the endotoxin–leukocyte relationship using regression models with an interaction term. There is a statistically significant, positive association between endotoxin concentration and total leukocyte number [estimated change, 0.186×10(3)/μL (95% CI: 0.070, 0.301×10(3)/μL) per 10-fold change in endotoxin; p=0.004) in the NHANES. Similar positive associations were found for monocytes, lymphocytes, and neutrophils. Stratified analyses revealed possible effect modification by asthma and chronic obstructive pulmonary disease. We observed similar associations in the ALHS. For total leukocytes, there was suggestive evidence in the ALHS of a gene-by-environment interaction for minor allele carrier status at the TLR4 haplotype defined by rs4986790 and rs4986791 (interaction p=0.15). This is, to our knowledge, the first report of an association between house dust endotoxin and leukocyte count in a national survey. The finding was replicated in a farming population. Peripheral leukocyte count may be influenced by residential endotoxin exposure in diverse settings. https://doi.org/10.1289/EHP661.

  12. An agent-based model of leukocyte transendothelial migration during atherogenesis

    PubMed Central

    Bhui, Rita; Hayenga, Heather N.

    2017-01-01

    A vast amount of work has been dedicated to the effects of hemodynamics and cytokines on leukocyte adhesion and trans-endothelial migration (TEM) and subsequent accumulation of leukocyte-derived foam cells in the artery wall. However, a comprehensive mechanobiological model to capture these spatiotemporal events and predict the growth and remodeling of an atherosclerotic artery is still lacking. Here, we present a multiscale model of leukocyte TEM and plaque evolution in the left anterior descending (LAD) coronary artery. The approach integrates cellular behaviors via agent-based modeling (ABM) and hemodynamic effects via computational fluid dynamics (CFD). In this computational framework, the ABM implements the diffusion kinetics of key biological proteins, namely Low Density Lipoprotein (LDL), Tissue Necrosis Factor alpha (TNF-α), Interlukin-10 (IL-10) and Interlukin-1 beta (IL-1β), to predict chemotactic driven leukocyte migration into and within the artery wall. The ABM also considers wall shear stress (WSS) dependent leukocyte TEM and compensatory arterial remodeling obeying Glagov’s phenomenon. Interestingly, using fully developed steady blood flow does not result in a representative number of leukocyte TEM as compared to pulsatile flow, whereas passing WSS at peak systole of the pulsatile flow waveform does. Moreover, using the model, we have found leukocyte TEM increases monotonically with decreases in luminal volume. At critical plaque shapes the WSS changes rapidly resulting in sudden increases in leukocyte TEM suggesting lumen volumes that will give rise to rapid plaque growth rates if left untreated. Overall this multi-scale and multi-physics approach appropriately captures and integrates the spatiotemporal events occurring at the cellular level in order to predict leukocyte transmigration and plaque evolution. PMID:28542193

  13. Synergistic effects of high blood cholesterol and hypertension on leukocyte and platelet recruitment in the cerebral microcirculation.

    PubMed

    Rodrigues, Stephen F; Almeida-Paula, Lidiana D; Granger, Daniel N

    2014-04-01

    Hypertension or hypercholesterolemia can induce a proinflammatory and prothrombogenic phenotype in the microcirculation of the brain; however, less is known about how the combination of these risk factors affects the vasculature. We recently reported that a moderate (60%) increase in plasma cholesterol blunts the recruitment of leukocytes and platelets in the cerebral microvessels elicited by hypertension. In this study, we examined whether larger increments in blood cholesterol (4-fold) exerts a similar modulating influence on the vasculature in the presence of hypertension. Apolipoprotein E-knockout mice with deoxycorticosterone acetate salt-induced hypertension were placed on a high-cholesterol diet and exhibited exaggerated leukocyte and platelet adhesion responses in cerebral microvessels. Intermittent feeding (every fourth day) with high-cholesterol diet yielded similar phenotypic changes in the vasculature. Once the mice were placed on high-cholesterol diet, 4 days on normal diet (ND) were needed to revert to a normal vascular phenotype. Angiotensin II type 1 receptors and reactive oxygen species seem to contribute to the vascular responses induced by hypercholesterolemia and hypertension. Our findings indicate that the combination of hypertension and large increases in plasma cholesterol concentration results in a severe, but reversible, inflammatory and thrombogenic phenotype in the cerebral microvasculature.

  14. Cardiolipin fingerprinting of leukocytes by MALDI-TOF/MS as a screening tool for Barth syndrome[S

    PubMed Central

    Angelini, Roberto; Lobasso, Simona; Gorgoglione, Ruggiero; Bowron, Ann; Steward, Colin G.; Corcelli, Angela

    2015-01-01

    Barth syndrome (BTHS), an X-linked disease associated with cardioskeletal myopathy, neutropenia, and organic aciduria, is characterized by abnormalities of card­iolipin (CL) species in mitochondria. Diagnosis of the disease is often compromised by lack of rapid and widely available diagnostic laboratory tests. The present study describes a new method for BTHS screening based on MALDI-TOF/MS analysis of leukocyte lipids. This generates a “CL fingerprint” and allows quick and simple assay of the relative levels of CL and monolysocardiolipin species in leukocyte total lipid profiles. To validate the method, we used vector algebra to analyze the difference in lipid composition between controls (24 healthy donors) and patients (8 boys affected by BTHS) in the high-mass phospholipid range. The method of lipid analysis described represents an important additional tool for the diagnosis of BTHS and potentially enables therapeutic monitoring of drug targets, which have been shown to ameliorate abnormal CL profiles in cells. PMID:26144817

  15. In Vivo Chemoprotective Activity of Bovine Dialyzable Leukocyte Extract in Mouse Bone Marrow Cells against Damage Induced by 5-Fluorouracil

    PubMed Central

    Coronado-Cerda, Erika Evangelina; Franco-Molina, Moisés Armides; Mendoza-Gamboa, Edgar; Prado-García, Heriberto; Rivera-Morales, Lydia Guadalupe; Zapata-Benavides, Pablo; Rodríguez-Salazar, María del Carmen; Caballero-Hernandez, Diana; Tamez-Guerra, Reyes Silvestre; Rodríguez-Padilla, Cristina

    2016-01-01

    Chemotherapy treatments induce a number of side effects, such as leukopenia neutropenia, peripheral erythropenia, and thrombocytopenia, affecting the quality of life for cancer patients. 5-Fluorouracil (5-FU) is wieldy used as myeloablative model in mice. The bovine dialyzable leukocyte extract (bDLE) or IMMUNEPOTENT CRP® (ICRP) is an immunomodulatory compound that has antioxidants and anti-inflammatory effects. In order to investigate the chemoprotection effect of ICRP on bone marrow cells in 5-FU treated mice, total bone marrow (BM) cell count, bone marrow colony forming units-granulocyte/macrophage (CFU-GM), cell cycle, immunophenotypification, ROS/superoxide and Nrf2 by flow cytometry, and histological and hematological analyses were performed. Our results demonstrated that ICRP increased BM cell count and CFU-GM number, arrested BM cells in G0/G1 phase, increased the percentage of leukocyte, granulocytic, and erythroid populations, reduced ROS/superoxide formation and Nrf2 activation, and also improved hematological levels and weight gain in 5-FU treated mice. These results suggest that ICRP has a chemoprotective effect against 5-FU in BM cells that can be used in cancer patients. PMID:27191003

  16. Leukocyte procoagulant activity: enhancement of production in vitro by IgG and antigen-antibody complexes.

    PubMed Central

    Rothberger, H; Zimmerman, T S; Spiegelberg, H L; Vaughan, J H

    1977-01-01

    In a variety of immunologic diseases, fibrin-fibrinogen and immune complexes deposit in areas of tissue damage. However, the mechanisms which initiate fibrin-fibrinogen deposition have not been clarified. We find that the procoagulant activity of human leukocytes is markedly increased after incubation with immunoglobulin and immune complexes. This procoagulant activity is evident after 4-24 h incubation in the presence of as little as 0.1 mg/ml of autologous, isologous, or heterologous IgG. At least three of the four subclasses of IgG myeloma proteins are effective. Experiments with purified rabbit and rat antibodies demonstrate that enhancement of procoagulant activity is significantly greater with soluble antigen-antibody complexes than with immunoglobulin alone. In contrast, insoluble complexes are less affective than immunoglobulin alone. Artifacts due to endotoxin contamination of the IgG preparations were excluded on the basis of the differential sensitivities of immunoglobulin and endotoxin to heat and polymyxin B. Evidence is also presented which shows that enhancement of procoagulant activity involves the production, rather than a simple release, of leukocyte procoagulant activity in vitro. PMID:190271

  17. 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.

  18. Effect of collection, transport, processing and storage of blood specimens on the activity of lysosomal enzymes in plasma and leukocytes.

    PubMed

    Burin, M; Dutra-Filho, C; Brum, J; Mauricio, T; Amorim, M; Giugliani, R

    2000-09-01

    This study was designed to evaluate the effect of different conditions of collection, transport and storage on the quality of blood samples from normal individuals in terms of the activity of the enzymes ss-glucuronidase, total hexosaminidase, hexosaminidase A, arylsulfatase A and ss-galactosidase. The enzyme activities were not affected by the different materials used for collection (plastic syringes or vacuum glass tubes). In the evaluation of different heparin concentrations (10% heparin, 5% heparin, and heparinized syringe) in the syringes, it was observed that higher doses resulted in an increase of at least 1-fold in the activities of ss-galactosidase, total hexosaminidase and hexosaminidase A in leukocytes, and ss-glucuronidase in plasma. When the effects of time and means of transportation were studied, samples that had been kept at room temperature showed higher deterioration with time (72 and 96 h) before processing, and in this case it was impossible to isolate leukocytes from most samples. Comparison of heparin and acid citrate-dextrose (ACD) as anticoagulants revealed that ss-glucuronidase and hexosaminidase activities in plasma reached levels near the lower normal limits when ACD was used. In conclusion, we observed that heparin should be used as the preferable anticoagulant when measuring these lysosomal enzyme activities, and we recommend that, when transport time is more than 24 h, samples should be shipped by air in a styrofoam box containing wet ice.

  19. In Vivo Chemoprotective Activity of Bovine Dialyzable Leukocyte Extract in Mouse Bone Marrow Cells against Damage Induced by 5-Fluorouracil.

    PubMed

    Coronado-Cerda, Erika Evangelina; Franco-Molina, Moisés Armides; Mendoza-Gamboa, Edgar; Prado-García, Heriberto; Rivera-Morales, Lydia Guadalupe; Zapata-Benavides, Pablo; Rodríguez-Salazar, María Del Carmen; Caballero-Hernandez, Diana; Tamez-Guerra, Reyes Silvestre; Rodríguez-Padilla, Cristina

    2016-01-01

    Chemotherapy treatments induce a number of side effects, such as leukopenia neutropenia, peripheral erythropenia, and thrombocytopenia, affecting the quality of life for cancer patients. 5-Fluorouracil (5-FU) is wieldy used as myeloablative model in mice. The bovine dialyzable leukocyte extract (bDLE) or IMMUNEPOTENT CRP® (ICRP) is an immunomodulatory compound that has antioxidants and anti-inflammatory effects. In order to investigate the chemoprotection effect of ICRP on bone marrow cells in 5-FU treated mice, total bone marrow (BM) cell count, bone marrow colony forming units-granulocyte/macrophage (CFU-GM), cell cycle, immunophenotypification, ROS/superoxide and Nrf2 by flow cytometry, and histological and hematological analyses were performed. Our results demonstrated that ICRP increased BM cell count and CFU-GM number, arrested BM cells in G0/G1 phase, increased the percentage of leukocyte, granulocytic, and erythroid populations, reduced ROS/superoxide formation and Nrf2 activation, and also improved hematological levels and weight gain in 5-FU treated mice. These results suggest that ICRP has a chemoprotective effect against 5-FU in BM cells that can be used in cancer patients.

  20. 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)

  1. Donor-leukocyte imaging in granulocytopenic patients with suspected abscesses: concise communication

    SciTech Connect

    Anstall, H.B.; Coleman, R.E.

    1982-04-01

    /sup 111/In-labeled donor leukocytes were used for the detection of foci of suppuration in eight severely leukopenic patients with marrow suppression, either idiopathic or associated with chemotherapeutic regimens for leukemia. In three patients good correlation was found between the results of imaging and clinical signs or subsequent proof of inflammation. In the other five patients, in whom no evidence of localized suppuration occurred, no abnormal accumulations of radioactivity were demonstrable. Labeled donor leukocytes provide a method for locating suppurative foci in severely leukopenic patients in whom autologous leukocyte labeling is impractical.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Oxidative DNA damage of peripheral blood polymorphonuclear leukocytes, selectively induced by chronic arsenic exposure, is associated with extent of arsenic-related skin lesions

    SciTech Connect

    Pei, Qiuling; Ma, Ning; Zhang, Jing; Xu, Wenchao; Li, Yong; Ma, Zhifeng; Li, Yunyun; Tian, Fengjie; Zhang, Wenping; Mu, Jinjun; Li, Yuanfei; Wang, Dongxing; Liu, Haifang; Yang, Mimi; Ma, Caifeng; Yun, Fen

    2013-01-01

    There is increasing evidence that oxidative stress is an important risk factor for arsenic-related diseases. Peripheral blood leukocytes constitute an important defense against microorganisms or pathogens, while the research on the impact of chronic arsenic exposure on peripheral blood leukocytes is much more limited, especially at low level arsenic exposure. The purpose of the present study was to explore whether chronic arsenic exposure affects oxidative stress of peripheral blood leukocytes and possible linkages between oxidative stress and arsenic-induced skin lesions. 75 male inhabitants recruited from an As-endemic region of China were investigated in the present study. The classification of arsenicosis was based on the degree of skin lesions. Arsenic levels were measured in drinking water and urine by Atomic Fluorescence Spectroscopy. Urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) was tested by Enzyme-Linked Immunosorbent Assay. 8-OHdG of peripheral blood leukocytes was evaluated using immunocytochemical staining. 8-OHdG-positive reactions were only present in polymorphonuclear leukocytes (PMNs), but not in monocytes (MNs). The 8-OHdG staining of PMN cytoplasm was observed in all investigated populations, while the 8-OHdG staining of PMN nuclei was frequently found along with the elevated amounts of cell debris in individuals with skin lesion. Urinary arsenic levels were increased in the severe skin lesion group compared with the normal group. No relationship was observed between drinking water arsenic or urine 8-OHdG and the degree of skin lesions. These findings indicated that the target and persistent oxidative stress in peripheral blood PMNs may be employed as a sensitive biomarker directly to assess adverse health effects caused by chronic exposure to lower levels of arsenic. -- Highlights: ► Male inhabitants were investigated from an As-endemic region of China. ► 8-OHdG-positive reactions were only present in polymorphonuclear leukocytes (PMNs).

  7. Cluster Analysis of Tumor Suppressor Genes in Canine Leukocytes Identifies Activation State

    PubMed Central

    Daly, Julie-Anne; Mortlock, Sally-Anne; Taylor, Rosanne M.; Williamson, Peter

    2015-01-01

    Cells of the immune system undergo activation and subsequent proliferation in the normal course of an immune response. Infrequently, the molecular and cellular events that underlie the mechanisms of proliferation are dysregulated and may lead to oncogenesis, leading to tumor formation. The most common forms of immunological cancers are lymphomas, which in dogs account for 8%–20% of all cancers, affecting up to 1.2% of the dog population. Key genes involved in negatively regulating proliferation of lymphocytes include a group classified as tumor suppressor genes (TSGs). These genes are also known to be associated with progression of lymphoma in humans, mice, and dogs and are potential candidates for pathological grading and diagnosis. The aim of the present study was to analyze TSG profiles in stimulated leukocytes from dogs to identify genes that discriminate an activated phenotype. A total of 554 TSGs and three gene set collections were analyzed from microarray data. Cluster analysis of three subsets of genes discriminated between stimulated and unstimulated cells. These included 20 most upregulated and downregulated TSGs, TSG in hallmark gene sets significantly enriched in active cells, and a selection of candidate TSGs, p15 (CDKN2B), p18 (CDKN2C), p19 (CDKN1A), p21 (CDKN2A), p27 (CDKN1B), and p53 (TP53) in the third set. Analysis of two subsets suggested that these genes or a subset of these genes may be used as a specialized PCR set for additional analysis. PMID:27478369

  8. Inhibitory leukocyte immunoglobulin-like receptors: Immune checkpoint proteins and tumor sustaining factors

    PubMed Central

    Kang, Xunlei; Kim, Jaehyup; Deng, Mi; John, Samuel; Chen, Heyu; Wu, Guojin; Phan, Hiep; Zhang, Cheng Cheng

    2016-01-01

    ABSTRACT Inhibitory leukocyte immunoglobulin-like receptors (LILRBs 1-5) transduce signals via intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that recruit protein tyrosine phosphatase non-receptor type 6 (PTPN6 or SHP-1), protein tyrosine phosphatase non-receptor type 11 (PTPN11 or SHP-2), or Src homology 2 domain-containing inositol phosphatase (SHIP), leading to negative regulation of immune cell activation. Certain of these receptors also play regulatory roles in neuronal activity and osteoclast development. The activation of LILRBs on immune cells by their ligands may contribute to immune evasion by tumors. Recent studies found that several members of LILRB family are expressed by tumor cells, notably hematopoietic cancer cells, and may directly regulate cancer development and relapse as well as the activity of cancer stem cells. LILRBs thus have dual concordant roles in tumor biology – as immune checkpoint molecules and as tumor-sustaining factors. Importantly, the study of knockout mice indicated that LILRBs do not affect hematopoiesis and normal development. Therefore LILRBs may represent ideal targets for tumor treatment. This review aims to summarize current knowledge on expression patterns, ligands, signaling, and functions of LILRB family members in the context of cancer development. PMID:26636629

  9. The endothelial glycocalyx as a barrier to leukocyte adhesion and its mediation by extracellular proteases.

    PubMed

    Lipowsky, Herbert H

    2012-04-01

    The endothelial cell (EC) surface is coated with a layer of polysaccharides linked to membrane-bound and trans-membrane proteoglycans that comprise the glycocalyx, which is augmented by adsorbed proteins derived from the blood stream. This surface layer has been shown to affect hemodynamics in small blood vessels of the microcirculation, the resistance to flow, and leukocyte (WBC) to EC adhesion. Parallel studies of WBC-EC adhesion in response to chemoattractants and cytokines, and shedding of constituents of the glycocalyx, have suggested a role for activation of extracellular proteases in mediating the dynamics of WBC adhesion in response to inflammatory and ischemic stimuli. Likely candidates among the many proteases present are the matrix metalloproteases (MMPs). Inhibition of MMP activation with sub-antimicrobial doses of doxycycline, or zinc chelators, has also inhibited WBC adhesion and shedding of glycans from the EC surface in response to the chemoattractant fMLP. Taken together, these studies suggest that shedding of the EC glycocalyx exposes adhesion receptors and thus enhances WBC-EC adhesion. Future therapeutic strategies for treating pathologies such as the low flow state and inflammation may benefit by further exploration of the mechanics of the glycocalyx in light of protease activation and shear-dependent effects.

  10. Leukocyte chemotactic factor 2 amyloidosis (ALECT2) is a common form of renal amyloidosis among Egyptians

    PubMed Central

    Larsen, Christopher P; Ismail, Wesam; Kurtin, Paul J; Vrana, Julie A; Dasari, Surendra; Nasr, Samih H

    2016-01-01

    Large case series of renal amyloidosis subtypes have recently been published in the United States and Europe showing AL amyloidosis to be the predominant subtype in this part of the world. However, the most common subtypes of renal amyloidosis throughout the rest of the world are unknown. We present here the first large case series detailing the subtypes of renal amyloidosis among Egyptians. In this population, AA amyloidosis was the most common type of amyloidosis on renal biopsy at 48%. The newly described leukocyte chemotactic factor 2 amyloidosis (ALECT2) was the second most common type and represented nearly one-third of renal amyloid cases at 31%. AL accounted for only 20% of cases. The pathologic findings in ALECT2 cases were similar to those previously described in other case series. Thus ALECT2, which was initially thought to affect mainly Hispanics in the United States, appears to represent an important and likely underrecognized etiology of chronic kidney disease among Egyptians and probably in other ethnic groups around the world. PMID:26867784

  11. Oxygen-independent killing of Bacteroides fragilis by granule extracts from human polymorphonuclear leukocytes.

    PubMed Central

    Wetherall, B L; Pruul, H; McDonald, P J

    1984-01-01

    Granule proteins from human neutrophils were prepared by extraction with acetate, and their antibacterial activity against Bacteroides fragilis was determined. Activity was highly dependent on pH; greatest killing occurred at the most acid pH tested (pH 5.0). Optimum activity was observed at physiological ionic strength and low bacterial numbers. Killing was inhibited by incubation temperatures of less than 37 degrees C. Eight times more extract was required to kill 50% of stationary-phase bacteria, compared with those growing in logarithmic phase. The antibacterial effect of granule extract was destroyed by boiling, but some activity was retained after heating to 56 degrees C and 80 degrees C. Granule extract activity was tested under conditions in which oxygen-dependent antibacterial systems were inhibited. The rate and extent of killing was not affected by anaerobiosis, sodium azide, or cysteine hydrochloride. These results suggest that the activity of granule extract is independent of oxidative antibacterial systems, and therefore, under conditions that occur in anaerobic infections, potent leukocyte granule-associated mechanisms exist for the destruction of B. fragilis. PMID:6698601

  12. Inhibitory leukocyte immunoglobulin-like receptors: Immune checkpoint proteins and tumor sustaining factors.

    PubMed

    Kang, Xunlei; Kim, Jaehyup; Deng, Mi; John, Samuel; Chen, Heyu; Wu, Guojin; Phan, Hiep; Zhang, Cheng Cheng

    2016-01-01

    Inhibitory leukocyte immunoglobulin-like receptors (LILRBs 1-5) transduce signals via intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that recruit protein tyrosine phosphatase non-receptor type 6 (PTPN6 or SHP-1), protein tyrosine phosphatase non-receptor type 11 (PTPN11 or SHP-2), or Src homology 2 domain-containing inositol phosphatase (SHIP), leading to negative regulation of immune cell activation. Certain of these receptors also play regulatory roles in neuronal activity and osteoclast development. The activation of LILRBs on immune cells by their ligands may contribute to immune evasion by tumors. Recent studies found that several members of LILRB family are expressed by tumor cells, notably hematopoietic cancer cells, and may directly regulate cancer development and relapse as well as the activity of cancer stem cells. LILRBs thus have dual concordant roles in tumor biology - as immune checkpoint molecules and as tumor-sustaining factors. Importantly, the study of knockout mice indicated that LILRBs do not affect hematopoiesis and normal development. Therefore LILRBs may represent ideal targets for tumor treatment. This review aims to summarize current knowledge on expression patterns, ligands, signaling, and functions of LILRB family members in the context of cancer development.

  13. Leukocyte chemotactic factor 2 amyloidosis (ALECT2) is a common form of renal amyloidosis among Egyptians.

    PubMed

    Larsen, Christopher P; Ismail, Wesam; Kurtin, Paul J; Vrana, Julie A; Dasari, Surendra; Nasr, Samih H

    2016-04-01

    Large case series of renal amyloidosis subtypes have recently been published in the United States and Europe showing AL amyloidosis to be the predominant subtype in this part of the world. However, the most common subtypes of renal amyloidosis throughout the rest of the world are unknown. We present here the first large case series detailing the subtypes of renal amyloidosis among Egyptians. In this population, AA amyloidosis was the most common type of amyloidosis on renal biopsy at 48%. The newly described leukocyte chemotactic factor 2 amyloidosis (ALECT2) was the second most common type and represented nearly one-third of renal amyloid cases at 31%. AL accounted for only 20% of cases. The pathologic findings in ALECT2 cases were similar to those previously described in other case series. Thus ALECT2, which was initially thought to affect mainly Hispanics in the United States, appears to represent an important and likely underrecognized etiology of chronic kidney disease among Egyptians and probably in other ethnic groups around the world.

  14. Personality and gene expression: Do individual differences exist in the leukocyte transcriptome?

    PubMed Central

    Vedhara, Kavita; Gill, Sana; Eldesouky, Lameese; Campbell, Bruce K.; Arevalo, Jesusa M. G.; Ma, Jeffrey; Cole, Steven W.

    2014-01-01

    Background The temporal and situational stability of personality has led generations of researchers to hypothesise that personality may have enduring effects on health, but the biological mechanisms of such relationships remain poorly understood. In the present study, we utilized a functional genomics approach to examine the relationship between the 5 major dimensions of personality and patterns of gene expression as predicted by ‘behavioural immune response’ theory. We specifically focussed on two sets of genes previously linked to stress, threat, and adverse socio-environmental conditions: pro-inflammatory genes and genes involved in Type I interferon and antibody responses. Methods An opportunity sample of 121 healthy individuals was recruited (86 females; mean age 24 years). Individuals completed a validated measure of personality; questions relating to current health behaviours; and provided a 5 ml sample of peripheral blood for gene expression analysis. Results Extraversion was associated with increased expression of pro-inflammatory genes and Conscientiousness was associated with reduced expression of pro-inflammatory genes. Both associations were independent of health behaviours, negative affect, and leukocyte subset distributions. Antiviral and antibody-related gene expression was not associated with any personality dimension. Conclusions The present data shed new light on the long-observed epidemiological associations between personality, physical health, and human longevity. Further research is required to elucidate the biological mechanisms underlying these associations. PMID:25459894

  15. Prepartal Energy Intake Alters Blood Polymorphonuclear Leukocyte Transcriptome During the Peripartal Period in Holstein Cows

    PubMed Central

    Agrawal, A; Khan, MJ; Graugnard, DE; Vailati-Riboni, M; Rodriguez-Zas, SL; Osorio, JS; Loor, JJ

    2017-01-01

    In the dairy industry, cow health and farmer profits depend on the balance between diet (ie, nutrient composition, daily intake) and metabolism. This is especially true during the transition period, where dramatic physiological changes foster vulnerability to immunosuppression, negative energy balance, and clinical and subclinical disorders. Using an Agilent microarray platform, this study examined changes in the transcriptome of bovine polymorphonuclear leukocytes (PMNLs) due to prepartal dietary intake. Holstein cows were fed a high-straw, control-energy diet (CON; NEL = 1.34 Mcal/kg) or overfed a moderate-energy diet (OVE; NEL = 1.62 Mcal/kg) during the dry period. Blood for PMNL isolation and metabolite analysis was collected at −14 and +7 days relative to parturition. At an analysis of variance false discovery rate <0.05, energy intake (OVE vs CON) influenced 1806 genes. Dynamic Impact Approach bioinformatics analysis classified treatment effects on Kyoto Encyclopedia of Genes and Genomes pathways, including activated oxidative phosphorylation and biosynthesis of unsaturated fatty acids and inhibited RNA polymerase, proteasome, and toll-like receptor signaling pathway. This analysis indicates that processes critical for energy metabolism and cellular and immune function were affected with mixed results. However, overall interpretation of the transcriptome data agreed in part with literature documenting a potentially detrimental, chronic activation of PMNL in response to overfeeding. The widespread, transcriptome-level changes captured here confirm the importance of dietary energy adjustments around calving on the immune system. PMID:28579762

  16. Natural micropolymorphism in human leukocyte antigens provides a basis for genetic control of antigen recognition

    SciTech Connect

    Archbold, Julia K.; Macdonald, Whitney A.; Gras, Stephanie; Ely, Lauren K.; Miles, John J.; Bell, Melissa J.; Brennan, Rebekah M.; Beddoe, Travis; Wilce, Matthew C.J.; Clements, Craig S.; Purcell, Anthony W.; McCluskey, James; Burrows, Scott R.; Rossjohn, Jamie

    2009-07-10

    Human leukocyte antigen (HLA) gene polymorphism plays a critical role in protective immunity, disease susceptibility, autoimmunity, and drug hypersensitivity, yet the basis of how HLA polymorphism influences T cell receptor (TCR) recognition is unclear. We examined how a natural micropolymorphism in HLA-B44, an important and large HLA allelic family, affected antigen recognition. T cell-mediated immunity to an Epstein-Barr virus determinant (EENLLDFVRF) is enhanced when HLA-B*4405 was the presenting allotype compared with HLA-B*4402 or HLA-B*4403, each of which differ by just one amino acid. The micropolymorphism in these HLA-B44 allotypes altered the mode of binding and dynamics of the bound viral epitope. The structure of the TCR-HLA-B*4405EENLLDFVRF complex revealed that peptide flexibility was a critical parameter in enabling preferential engagement with HLA-B*4405 in comparison to HLA-B*4402/03. Accordingly, major histocompatibility complex (MHC) polymorphism can alter the dynamics of the peptide-MHC landscape, resulting in fine-tuning of T cell responses between closely related allotypes.

  17. Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

    PubMed Central

    Moraga, Ana; de la Parra, Juan; Lizasoain, Ignacio; Moro, María Ángeles

    2014-01-01

    Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method. PMID:25590380

  18. Leukocyte-specific protein 1 regulates T-cell migration in rheumatoid arthritis

    PubMed Central

    Hwang, Seong-Hye; Jung, Seung-Hyun; Lee, Saseong; Choi, Susanna; Yoo, Seung-Ah; Park, Ji-Hwan; Hwang, Daehee; Shim, Seung Cheol; Sabbagh, Laurent; Kim, Ki-Jo; Park, Sung Hwan; Cho, Chul-Soo; Kim, Bong-Sung; Leng, Lin; Montgomery, Ruth R.; Bucala, Richard; Chung, Yeun-Jun; Kim, Wan-Uk

    2015-01-01

    Copy number variations (CNVs) have been implicated in human diseases. However, it remains unclear how they affect immune dysfunction and autoimmune diseases, including rheumatoid arthritis (RA). Here, we identified a novel leukocyte-specific protein 1 (LSP1) deletion variant for RA susceptibility located in 11p15.5. We replicated that the copy number of LSP1 gene is significantly lower in patients with RA, which correlates positively with LSP1 protein expression levels. Differentially expressed genes in Lsp1-deficient primary T cells represent cell motility and immune and cytokine responses. Functional assays demonstrated that LSP1, induced by T-cell receptor activation, negatively regulates T-cell migration by reducing ERK activation in vitro. In mice with T-cell–dependent chronic inflammation, loss of Lsp1 promotes migration of T cells into the target tissues as well as draining lymph nodes, exacerbating disease severity. Moreover, patients with RA show diminished expression of LSP1 in peripheral T cells with increased migratory capacity, suggesting that the defect in LSP1 signaling lowers the threshold for T-cell activation. To our knowledge, our work is the first to demonstrate how CNVs result in immune dysfunction and a disease phenotype. Particularly, our data highlight the importance of LSP1 CNVs and LSP1 insufficiency in the pathogenesis of RA and provide previously unidentified insights into the mechanisms underlying T-cell migration toward the inflamed synovium in RA. PMID:26554018

  19. LFA-1 integrin antibodies inhibit leukocyte α4β1-mediated adhesion by intracellular signaling.

    PubMed

    Grönholm, Mikaela; Jahan, Farhana; Bryushkova, Ekaterina A; Madhavan, Sudarrshan; Aglialoro, Francesca; Soto Hinojosa, Laura; Uotila, Liisa M; Gahmberg, Carl G

    2016-09-01

    Binding of intercellular adhesion molecule-1 to the β2-integrin leukocyte function associated antigen-1 (LFA-1) is known to induce cross-talk to the α4β1 integrin. Using different LFA-1 monoclonal antibodies, we have been able to study the requirement and mechanism of action for the cross-talk in considerable detail. LFA-1-activating antibodies and those inhibitory antibodies that signal to α4β1 induce phosphorylation of Thr-758 on the β2-chain, which is followed by binding of 14-3-3 proteins and signaling through the G protein exchange factor Tiam1. This results in dephosphorylation of Thr-788/789 on the β1-chain of α4β1 and loss of binding to its ligand vascular cell adhesion molecule-1. The results show that with LFA-1 antibodies, we can activate LFA-1 and inhibit α4β1, inhibit both LFA-1 and α4β1, inhibit LFA-1 but not α4β1, or not affect LFA-1 or α4β1 These findings are important for the understanding of integrin regulation and for the interpretation of the effect of integrin antibodies and their use in clinical applications. © 2016 by The American Society of Hematology.

  20. Secretory Leukocyte Protease Inhibitor Binds to Neisseria gonorrhoeae Outer Membrane Opacity Protein and is Bactericidal

    PubMed Central

    Cooper, Morris D.; Roberts, Melissa H.; Barauskas, Ona L.; Jarvis, Gary A.

    2012-01-01

    Problem Secretory leukocyte protease inhibitor (SLPI) is an innate immune peptide present on the genitourinary tract mucosa which has antimicrobial activity. In this study, we investigated the interaction of SLPI with Neisseria gonorrhoeae. Method of study ELISA and far-western blots were used to analyze binding of SLPI to gonococci. The binding site for SLPI was identified by tryptic digests and mass spectrometry. Antimicrobial activity of SLPI for gonococci was determined using bactericidal assays. SLPI protein levels in cell supernatants were measured by ELISA, and SLPI mRNA levels were assessed by quantitative RT-PCR. Results SLPI bound directly to the gonococcal Opa protein and was bactericidal. Epithelial cells from the reproductive tract constitutively expressed SLPI at different levels. Gonococcal infection of cells did not affect SLPI expression. Conclusion We conclude that SLPI is bactericidal for gonococci and is expressed by reproductive tract epithelial cells and thus is likely to play a role in the pathogenesis of gonococcal infection. PMID:22537232

  1. Ca2+ signaling in airway epithelial cells facilitates leukocyte recruitment and transepithelial migration

    PubMed Central

    Chun, Jarin; Prince, Alice

    2009-01-01

    In airway cells, TLR2 stimulation by bacterial products activates Ca2+ fluxes that signal leukocyte recruitment to the lung and facilitates transepithelial migration into the airway lumen. TLR2 is apically displayed on airway cells, where it senses bacterial stimuli. Biochemical and genetic approaches demonstrate that TLR2 ligands stimulate release of Ca2+ from intracellular stores by activating TLR2 phosphorylation by c-Src and recruiting PI3K and PLCγ to affect Ca2+ release through IP3Rs. This Ca2+ release plays a pivotal role in signaling TLR2-dependent NF-κB activation and chemokine expression to recruit PMNs to the lung. In addition, TLR2-initiated Ca2+ release activates Ca2+-dependent proteases, calpains, which cleave the transmembrane proteins occludin and E-cadherin to promote PMN transmigration. This review highlights recent findings that demonstrate a central role for Ca2+ signaling in airway epithelial cells to induce proinflammatory gene transcription and to initiate junctional changes that accommodate transmigration of recruited PMNs. PMID:19605699

  2. The Difficulty of Eliminating Donor Leukocyte Microchimerism in Rat Recipients Bearing Established Organ Allografts

    PubMed Central

    Kiyomoto, Tetsuma; Toyokawa, Hideyoshi; Nakao, Atsunori; Kaizu, Takashi; Demetris, Anthony J.; Starzl, Thomas E.; Murase, Noriko

    2010-01-01

    Background Unequivocal eradication of donor leukocyte microchimerism from recipients of long-surviving organ transplants has never been reported. Here we describe a drastic attempt to accomplish this objective. Methods In control experiments, a rank order of microchimerism and of associated donor specific nonreactivity was produced in Brown-Norway (BN) rats by transplantation of Lewis (LEW) liver, bone marrow cell (BMC) and heart allografts under a brief course of tacrolimus. The degree of microchimerism at 60 and 110 days was estimated with semiquanitative immunocytochemical and PCR techniques. Tolerance at 110 days was assessed in the different control groups by challenge transplantation of naive LEW hearts. In parallel experimental groups, an attempt was made to eliminate microchimerism from the BN recipients. The animals were submitted at 60 days to 9.5-Gy total body irradiation (TBI), reconstituted immediately with naïve BN BMC, and tested for donor specific nonreactivity by LEW heart transplantation at 110 days. Results After the TBI-reconstitution at 60 days, microchimerism was undetectable in BMC recipients at 110 days, significantly reduced in heart recipients, and least affected in liver recipients. Except in liver recipients, abrogation of LEW-specific nonreactivity was demonstrated by rejection of the priming grafts, or by rejection of the challenge heart grafts, and by in vitro immune assay. Conclusions It is difficult to eliminate microchimerism in organ recipients once the donor cells have settled into tissue niches. PMID:16477232

  3. Contribution of phosphoglucosamine mutase to the resistance of Streptococcus gordonii DL1 to polymorphonuclear leukocyte killing.

    PubMed

    Yajima, Ayako; Takahashi, Yukihiro; Shimazu, Kisaki; Urano-Tashiro, Yumiko; Uchikawa, Yoshimori; Karibe, Hiroyuki; Konishi, Kiyoshi

    2009-08-01

    Phosphoglucosamine mutase (GlmM; EC 5.4.2.10) catalyzes the interconversion of glucosamine-6-phosphate to glucosamine-1-phosphate, an essential step in the biosynthetic pathway leading to the formation of the peptidoglycan precursor uridine 5'-diphospho-N-acetylglucosamine. We have recently identified the gene (glmM) encoding the enzyme of Streptococcus gordonii, an early colonizer on the human tooth and an important cause of infective endocarditis, and indicated that the glmM mutation in S. gordonii appears to influence bacterial cell growth, morphology, and sensitivity to penicillins. In the present study, we assessed whether the glmM mutation also affects escape from polymorphonuclear leukocyte (PMN)-dependent killing. Although no differences in attachment to human PMNs were observed between the glmM mutant and the wild-type S. gordonii, the glmM mutation resulted in increased sensitivity to PMN-dependent killing. Compared with the wild type, the glmM mutant induced increased superoxide anion production and lysozyme release by PMNs. Moreover, the glmM mutant is more sensitive to lysozyme, indicating that the GlmM may be required for synthesis of firm peptidoglycans for resistance to bacterial cell lysis. These findings suggest that the GlmM contributes to the resistance of S. gordonii to PMN-dependent killing. Enzymes such as GlmM could be novel drug targets for this organism.

  4. 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.

  5. 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.

  6. 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

  7. Role of bacteria in leukocyte adhesion deficiency-associated periodontitis.

    PubMed

    Hajishengallis, George; Moutsopoulos, Niki M

    2016-05-01

    Leukocyte adhesion deficiency Type I (LAD-I)-associated periodontitis is an aggressive form of inflammatory bone loss that has been historically attributed to lack of neutrophil surveillance of the periodontal infection. However, this form of periodontitis has proven unresponsive to antibiotics and/or mechanical removal of the tooth-associated biofilm. Recent studies in LAD-I patients and relevant animal models have shown that the fundamental cause of LAD-I periodontitis involves dysregulation of a granulopoietic cytokine cascade. This cascade includes interleukin IL-23 (IL-23) and IL-17 that drive inflammatory bone loss in LAD-I patients and animal models and, moreover, foster a nutritionally favorable environment for bacterial growth and development of a compositionally unique microbiome. Although the lack of neutrophil surveillance in the periodontal pockets might be expected to lead to uncontrolled bacterial invasion of the underlying connective tissue, microbiological analyses of gingival biopsies from LAD-I patients did not reveal tissue-invasive infection. However, bacterial lipopolysaccharide was shown to translocate into the lesions of LAD-I periodontitis. It is concluded that the bacteria serve as initial triggers for local immunopathology through translocation of bacterial products into the underlying tissues where they unleash the dysregulated IL-23-IL-17 axis. Subsequently, the IL-23/IL-17 inflammatory response sustains and shapes a unique local microbiome which, in turn, can further exacerbate inflammation and bone loss in the susceptible host. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. 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

  9. 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.

  10. Association of human leukocyte antigen with postherpetic neuralgia in Koreans.

    PubMed

    Chung, Hye Yoon; Song, Eun Young; Yoon, Jung Ah; Suh, Dae Hun; Lee, Sang Chul; Kim, Yong Chul; Park, Myoung Hee

    2016-10-01

    Postherpetic neuralgia (PHN), the most frequent complication of varicella-zoster virus reactivation, is characterized by pain that persists for more than 3 months, often for years after healing of zoster rash. A few studies revealing the association of human leukocyte antigen (HLA) with PHN have been reported, but only in the Japanese. The aim of this study was to investigate the primary HLA locus associated with PHN susceptibility in Koreans. We compared HLA-A, -B, -C, and DRB1 genotypes of 66 PHN patients with those of 54 herpes zoster (HZ) patients without developing PHN and 235 healthy controls. Frequencies of HLA-B*13, B*44, B*15 (B75), DRB1*10:01, and DRB1*12:02 were increased, and those of HLA-C*01, C*12, and DRB1*01:01 were decreased in PHN patients compared to those in controls (each, p < 0.05). Among these alleles, only the frequency of HLA-B*44 was significantly increased in PHN patients compared to that in HZ patients and the change was due to HLA-B*44:03 (PHN vs controls, p = 0.043; PHN vs HZ, p = 0.012). The results suggest that HLA-B*44:03 or other closely linked gene of the major histocompatibility complex is associated with susceptibility to the development of PHN after HZ, but not with the onset of HZ.

  11. Characterization of Leukocyte Formin FMNL1 Expression in Human Tissues

    PubMed Central

    Heuser, Vanina D.; Iljin, Kristiina; Kampf, Caroline; Uhlen, Mathias; Carpén, Olli

    2014-01-01

    Formins are cytoskeleton regulating proteins characterized by a common FH2 structural domain. As key players in the assembly of actin filaments, formins direct dynamic cytoskeletal processes that influence cell shape, movement and adhesion. The large number of formin genes, fifteen in the human, suggests distinct tasks and expression patterns for individual family members, in addition to overlapping functions. Several formins have been associated with invasive cell properties in experimental models, linking them to cancer biology. One example is FMNL1, which is considered to be a leukocyte formin and is known to be overexpressed in lymphomas. Studies on FMNL1 and many other formins have been hampered by a lack of research tools, especially antibodies suitable for staining paraffin-embedded formalin-fixed tissues. Here we characterize, using bioinformatics tools and a validated antibody, the expression pattern of FMNL1 in human tissues and study its subcellular distribution. Our results indicate that FMNL1 expression is not restricted to hematopoietic tissues and that neoexpression of FMNL1 can be seen in epithelial cancer. PMID:24700756

  12. 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.

  13. Alteration of polymorphonuclear leukocyte activity by viable Candida albicans.

    PubMed Central

    Hilger, A E; Danley, D L

    1980-01-01

    The response of human polymorphonuclear leukocytes (PMN) to blastospores and pseudo-hyphae of the opportunistic fungus Candida albicans has been studied in vitro and in vivo. Of the fungicidal mechanisms elucidated thus far, the myeloperoxidase-hydrogen peroxide-halide system appears to be most effective against cells of this fungus. In our studies on the interaction between murine PMN and blastospores, we assayed the release of H2O2 by PMN incubated with viable or killed, unopsonized or opsonized blastospores by using two assay systems, lysis of murine erythrocytes and oxidation of scopoletin. Our results showed that PMN released increasing amounts of H2O2 when incubated with increasing numbers of opsonized or unopsonized killed blastospores, but released decreasing amounts of H2O2 when incubated with increasing numbers of opsonized or unopsonized viable blastospores. The oxidative metabolic burst by PMN in the presence of viable or killed blastospores was also measured by using reduction of nitroblue tetrazolium and chemiluminescence. Viable blastospores stimulated a stronger metabolic burst than killed blastospores, suggesting that PMN respond to live blastospores more vigorously than killed blastospores; however, live blastospores appear to alter or inhibit the release of H2O2 by PMN. PMID:6991429

  14. Relationship between vitamin D status and leukocytes in hospitalised cats.

    PubMed

    Titmarsh, Helen F; Cartwright, Jennifer A; Kilpatrick, Scott; Gaylor, Donna; Milne, Elspeth M; Berry, Jacqueline L; Bommer, Nicholas X; Gunn-Moore, Danièlle; Reed, Nicola; Handel, Ian; Mellanby, Richard J

    2017-04-01

    Objectives Vitamin D deficiency, as assessed by serum 25-hydroxyvitamin D (25[OH]D) concentrations, has been linked to markers of systemic inflammation in human and canine medicine. However, the relationship between vitamin D status and inflammation has not been previously investigated in cats. The aim of this study was to examine the relationship between serum 25(OH)D concentrations and leukocyte counts in hospitalised sick cats. Methods Serum 25(OH)D concentrations and haematology profiles were measured in 170 consecutive hospitalised sick cats. A binary logistical regression model examined the relationship between serum 25(OH)D concentration, age, sex, breed and neutrophil, monocyte, eosinophil and lymphocyte counts. Results Cats with neutrophilia had lower serum 25(OH)D concentrations than cats with neutrophil concentrations below the upper limit of the reference interval (RI). There were no differences in serum 25(OH)D concentrations in cats with monocyte, lymphocyte or eosinophil counts above their respective RI compared with cats with counts below the upper limit of the RI. Conclusions and relevance Hospitalised cats with a neutrophil count above the RI had lower vitamin D status. There is a need to establish whether lower vitamin D status is a cause or consequence of increased neutrophil counts.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. Role of polymorphonuclear leukocytes in silica-induced pulmonary fibrosis

    SciTech Connect

    Adamson, I.Y.; Bowden, D.H.

    1984-10-01

    Silicosis is usually attributed to fibroblast stimulation by secretion of damaged alveolar macrophages (AMs), but the role of polymorphonuclear leukocytes (PMNs) and of continuing cell injury in the pathogenesis has not been fully studied. Mice given intratracheal injections of 2 mg of silica received 3H-thymidine 1 hour before death at intervals to 20 weeks. Cellular populations and lysosomal content of lavage fluids were correlated with morphology, DNA synthesis, and collagen content of the lung. The initial response involved rapid PMN and AM recruitment to the alveoli. Some free particles crossed Type 1 epithelial cells, and silica was found in interstitial macrophages. Focal Type 1 cell damage was rapidly repaired by Type 2 cell proliferation. Although PMN numbers dropped after a few days, they never reached control levels and rose again after 8 weeks; the number of AMs fell to control values from 2 to 8 weeks, then increased again. Glucosaminidase and glucuronidase levels in the lavage fluid were much higher than control levels throughout the study. Increased DNA synthesis by interstitial cells occurred from 2 days to 20 weeks; increased collagen synthesis was found from 4 weeks onward. The continuing inflammatory response of the lung to silica suggests may contribute to fibroblastic stimulation.

  20. Human polymorphonuclear leukocytes respond to waves of chemoattractant, like Dictyostelium.

    PubMed

    Geiger, Jeremy; Wessels, Deborah; Soll, David R

    2003-09-01

    It has been assumed that the natural chemotactic signal that attracts human polymorphonuclear leukocytes (PMNs) over long distances to sites of infection is in the form of a standing spatial gradient of chemoattractant. We have questioned this assumption on the grounds, first, that standing spatial gradients may not be stable over long distances for long periods of time and, second, that in the one animal cell chemotaxis system in which the natural chemotactic signal has been described in space and time, aggregation of Dicytostelium discoideum, the signal is in the form of an outwardly relayed, nondissipating wave of attractant. Here, it is demonstrated that PMNs alter their behavior in each of the four phases of a wave of PMN chemoattractant, fashioned after the Dictyostelium wave, in a manner similar to Dictyostelium. These results demonstrate that PMNs have all of the machinery to respond to a natural wave of attractant, providing support to the hypothesis that the natural signal that attracts PMNs over large distances to sites of infection in the human body may also be in the form of a wave. Copyright 2003 Wiley-Liss, Inc.

  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. Automatic detection and classification of leukocytes using convolutional neural networks.

    PubMed

    Zhao, Jianwei; Zhang, Minshu; Zhou, Zhenghua; Chu, Jianjun; Cao, Feilong

    2017-08-01

    The detection and classification of white blood cells (WBCs, also known as Leukocytes) is a hot issue because of its important applications in disease diagnosis. Nowadays the morphological analysis of blood cells is operated manually by skilled operators, which results in some drawbacks such as slowness of the analysis, a non-standard accuracy, and the dependence on the operator's skills. Although there have been many papers studying the detection of WBCs or classification of WBCs independently, few papers consider them together. This paper proposes an automatic detection and classification system for WBCs from peripheral blood images. It firstly proposes an algorithm to detect WBCs from the microscope images based on the simple relation of colors R, B and morphological operation. Then a granularity feature (pairwise rotation invariant co-occurrence local binary pattern, PRICoLBP feature) and SVM are applied to classify eosinophil and basophil from other WBCs firstly. Lastly, convolution neural networks are used to extract features in high level from WBCs automatically, and a random forest is applied to these features to recognize the other three kinds of WBCs: neutrophil, monocyte and lymphocyte. Some detection experiments on Cellavison database and ALL-IDB database show that our proposed detection method has better effect almost than iterative threshold method with less cost time, and some classification experiments show that our proposed classification method has better accuracy almost than some other methods.

  3. Swine leukocyte antigen (SLA) diversity in Sinclair and Hanford swine.

    PubMed

    Ho, Chak-Sum; Martens, Gregory W; Amoss, Max S; Gomez-Raya, Luis; Beattie, Craig W; Smith, Douglas M

    2010-03-01

    The swine leukocyte antigen (SLA) haplotype B is associated with increased penetrance of the tumor traits in Sinclair swine cutaneous melanoma (SSCM). We established a series of SinclairxHanford swine crosses to facilitate genetic mapping of the tumor-associated loci. In this study, the SLA diversity in the founding animals was characterized for effective selection of maximum tumor penetrance in the pedigrees. Using the sequence-based typing (SBT) method we identified a total of 29 alleles at five polymorphic SLA loci (SLA-1, SLA-3, SLA-2, DRB1 and DQB1) representing six class I and five class II haplotypes. We subsequently developed a rapid PCR-based typing assay using sequence-specific primers (PCR-SSP) to efficiently follow the SLA types of the crossbred progeny. In a total of 469 animals we identified three crossovers within the class I region and three between the class I and class II regions, which corresponded to recombination frequencies of 0.39% and 0.56%, respectively. We also confirmed the presence of two expressed SLA-1 loci in three of the class I haplotypes and were able to determine the relative chromosomal arrangement of the duplicated loci in two haplotypes. This study furthers our understanding of the allelic architecture and polymorphism of the SLA system and will facilitate the mapping of loci associated with the expression of SSCM. Copyright 2009 Elsevier Ltd. All rights reserved.

  4. Implications for Human Leukocyte Antigen Antibodies After Lung Transplantation

    PubMed Central

    Wang, Ziwei; Chen, Dong-Feng; Reinsmoen, Nancy L.; Finlen-Copeland, C. Ashley; Davis, W. Austin; Zaas, David W.; Palmer, Scott M.

    2013-01-01

    Background: Long-term survival after lung transplant is limited by the development of chronic and progressive airflow obstruction, a condition known as bronchiolitis obliterans syndrome (BOS). While prior studies strongly implicate cellular rejection as a strong risk factor for BOS, less is known about the clinical significance of human leukocyte antigen (HLA) antibodies and donor HLA-specific antibodies in long-term outcomes. Methods: A single-center cohort of 441 lung transplant recipients, spanning a 10-year period, was prospectively screened for HLA antibodies after transplant using flow cytometry-based methods. The prevalence of and predictors for HLA antibodies were determined. The impact of HLA antibodies on survival after transplant and the development of BOS were determined using Cox models. Results: Of the 441 recipients, 139 (32%) had detectable antibodies to HLA. Of these 139, 54 (39%) developed antibodies specific to donor HLA. The detection of posttransplant HLA antibodies was associated with BOS (HR, 1.54; P = .04) and death (HR, 1.53; P = .02) in multivariable models. The detection of donor-specific HLA antibodies was associated with death (HR, 2.42; P < .0001). The detection of posttransplant HLA antibodies was associated with pretransplant HLA-antibody detection, platelet transfusions, and the development of BOS and cytomegalovirus pneumonitis. Conclusions: Approximately one-third of lung transplant recipients have detectable HLA antibodies, which are associated with a worse prognosis regarding graft function and patient survival. PMID:23328795

  5. 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

  6. 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.

  7. 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 fir