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Sample records for blood polymorphonuclear leukocytes

  1. Antibiotic-Enhanced Phagocytosis of ’Borrelia recurrentis’ by Blood Polymorphonuclear Leukocytes.

    DTIC Science & Technology

    1979-11-30

    ANTIBIOTIC-ENHANCED P)4ASOCYTOSIS OF ’ BORRELIA RECURRENT!S* BY B-ETC(U) UCNOV 79 T BUTLER N00014-77-C-0050 W4CLASSIFIEO TR-3 N LIM,1 li 13 2 . 1112...enhanced Phagocytosis of Borrelia recurrentis by Blood Polymorphonuclear Leukocytes 0 ( ---- by rm 046omas / t’e Prepared for Publication in W Infection...jo? Butler 2 Abstract. "The removal of Borrelia spirochetes from the blood in relapsing fever was studied by examining patients’ blood phagocytic

  2. Phagocytosis of bovine blood and milk polymorphonuclear leukocytes after ozone gas administration in vitro.

    PubMed

    Ducusin, Rio John T; Nishimura, Masakazu; Sarashina, Takao; Uzuka, Yuji; Tanabe, Shigeyuki; Otani, Masayuki

    2003-04-01

    To determine the effects of ozone on the phagocytosis of bovine polymorphonuclear leukocytes (PMNs), ozone gas was administered in vitro on the blood and milk of healthy lactating cows, cows with acute mastitis, and cows with milk fever. In the blood of healthy dairy cattle, although there was no significant effect of ozone gas on the viability of the leukocytes, phagocytosis of PMNs significantly decreased. In contrast, ozone gas administration in vitro significantly increased phagocytosis of PMNs from the blood of cows with acute mastitis and milk fever, and from mastitic milk. These findings showed that ozone administration in vitro has positive and negative effects on bovine PMN phagocytosis, depending on the health status of the animal.

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

    PubMed

    Ashkenazi, M; Kohl, S

    1990-06-15

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

  4. Blood Level of Polymorphonuclear Neutrophil Leukocytes and Bronchial Hyperreactivity in Chronic Obstructive Pulmonary Disease

    PubMed Central

    Cukic, Vesna

    2015-01-01

    Introduction: Polymorphonuclear neutrophil leukocytes (PMNL) have an important defensive role against various microorganisms and other agents, but by liberating various substances, first of all the superoxide anion (O 2¯), they can damage the bronchial mucosa and influence the development of bronchial inflammation which is the fundamental of bronchial hyperreactivity (BHR). Objective: to show the role of the PMNL for development and level of BHR in patients with chronic obstructive pulmonary disease (COPD). Material and methods: We observed 160 patients with COPD treated in Clinic for Pulmonary Diseases and TB “Podhrastovi” Sarajevo during three years :from 2012 to 2014. They were divided into groups and subgroups according to the first registration of BHR in the course of illness and to the number of exacerbations of the disease in one year. The number of blood PMNL was measured in a stable state of disease at the begging and at the end of investigation. Results: The number of blood PMNL was significantly greater in patients with 3 or more exacerbations per one year (p <0.01). Patients with BHR had significantly greater number blood PMNL than patients without BHR (p< 0.05). Patients with 3 exacerbations per year had a statistically significant increase of number of PMNL between first and last examination (p<0.01). Conclusion: There is statistically significant correlation between the number of blood PMNL and the level of BHR in COPD, but future examination need to be done to determine real role and mode of action of PMNL for these processes. PMID:26543311

  5. Polymorphonuclear leukocyte and monocyte activation induced by plasma from patients with heparin-induced thrombocytopenia in whole blood.

    PubMed

    Khairy, Mahnouch; Lasne, Dominique; Amelot, Aymeric; Crespin, Malvina; Rendu, Francine; Aiach, Martine; Bachelot-Loza, Christilla

    2004-12-01

    Heparin-induced thrombocytopenia (HIT), a severe complication of heparin therapy, results from platelet activation by heparin-dependent antibodies. Previously, we have shown that plasma from patients with HIT (HIT plasma) induces leukocyteplatelet aggregation in blood. In this report, we examined leukocyte activation by HIT plasma and the contribution of heparin and platelets to this activation, in whole blood. Degranulation of leukocytes from HIT patients was evaluated as a leukocyte activation marker. We showed that polymorphonuclear leukocytes (PMN) and monocytes were the leukocyte subpopulations involved in platelet-leukocyte aggregation induced by HIT plasma in healthy donor blood. PMN and monocyte activation, reflected by increased surface expression of the CD11b adhesion molecule, was induced by HIT plasma in a heparin-dependent manner. The CD11b increase induced by HIT plasma was observed on PMN only when they were associated with platelets. Moreover, the increased CD11b expression on monocytes and PMN correlated strongly with the degree of platelet adhesion to these cells. Degranulation of leukocytes from HIT patients and control subjects (non-HIT heparin-treated patients and healthy subjects) was evaluated in vivo by measuring the plasma myeloperoxidase concentration. HIT plasma contained higher myeloperoxidase concentrations than control plasma, suggesting leukocyte degranulation during HIT. In conclusion, this study provides the first evidence that PMN activation is induced by HIT plasma. HIT plasma induced PMN and monocyte activation in a heparin-dependent manner. In whole blood, platelet association with monocytes and PMN, and the activation of these leukocytes by HIT plasma were interrelated. Finally, leukocyte degranulation could be involved in HIT physiopathology.

  6. The cytokineplast: purified, stable, and functional motile machinery from human blood polymorphonuclear leukocytes

    PubMed Central

    Malawista, SE; De Boisfleury Chevance, A

    1982-01-01

    We examined the formation of motile, chemotactically active, anucleate fragments from human blood polymorphonuclear leukocytes (PMN, granulocytes), induced by the brief application of heat. These granule-poor fragments are former protopods (leading fronts, lamellipodia) that become uncoupled from the main body of the cell and leave it, at first with a connecting filament that breaks and seals itself. The usual random orientation of such filaments can be controlled by preorientation of cells in a gradient of the chemotactic peptide, N-formylmethionylleucylphenylalanine (F-Met-Leu-Phe) (2x10(-9) M- 1x10(-8)). Cytochalsin B, 2.5-5 μg/ml, prevents fragment formation; colchicine, 10(-5) M, does not. In scanning electron micrographs, fragments are ruffled and the cell body rounded up and rather smooth. In transmission electron micrographs, fragments contain microfilaments but lack centrioles and microtubules. Like intact cells, both bound and free fragments can respond chemotactically to an erythrocyte destroyed by laser microirradiation (necrotaxis); the free, anucleate fragments may do so repeatedly, even after having been held overnight at ambient temperatures. We propse the name cytokineplast for the result of this self-purification of motile apparatus. The exodus of the motile machinery from the granulocyte requires anchoring of the bulk of the cell to glass and uncoupling, which may involve heat-induced dysfunction of the centrosome. In ultrastructural studies of the centrosomal region after heat, centriolar structure remains intact, but pericentriolar osmiophilic material appears condensed, and microtubules are sparse. These changes are found in all three blood cell types examined: PMN, eosinophil, and monocyte. Of these, the first two make fragments under our conditions; the more sluggish monocyte does not. Uncoupling is further linked to centrosomal dysfunction by the observation that colchicines-treated granulocytes (10(-5)M, to destroy the centrosome

  7. 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).

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. High affinity capture and concentration of quinacrine in polymorphonuclear neutrophils via vacuolar ATPase-mediated ion trapping: Comparison with other peripheral blood leukocytes and implications for the distribution of cationic drugs

    SciTech Connect

    Roy, Caroline; Gagné, Valérie; Fernandes, Maria J.G.; Marceau, François

    2013-07-15

    Many cationic drugs are concentrated in acidic cell compartments due to low retro-diffusion of the protonated molecule (ion trapping), with an ensuing vacuolar and autophagic cytopathology. In solid tissues, there is evidence that phagocytic cells, e.g., histiocytes, preferentially concentrate cationic drugs. We hypothesized that peripheral blood leukocytes could differentially take up a fluorescent model cation, quinacrine, depending on their phagocytic competence. Quinacrine transport parameters were determined in purified or total leukocyte suspensions at 37 °C. Purified polymorphonuclear leukocytes (PMNLs, essentially neutrophils) exhibited a quinacrine uptake velocity inferior to that of lymphocytes, but a consistently higher affinity (apparent K{sub M} 1.1 vs. 6.3 μM, respectively). However, the vacuolar (V)-ATPase inhibitor bafilomycin A1 prevented quinacrine transport or initiated its release in either cell type. PMNLs capture most of the quinacrine added at low concentrations to fresh peripheral blood leukocytes compared with lymphocytes and monocytes (cytofluorometry). Accumulation of the autophagy marker LC3-II occurred rapidly and at low drug concentrations in quinacrine-treated PMNLs (significant at ≥ 2.5 μM, ≥ 2 h). Lymphocytes contained more LAMP1 than PMNLs, suggesting that the mass of lysosomes and late endosomes is a determinant of quinacrine uptake V{sub max}. PMNLs, however, exhibited the highest capacity for pinocytosis (uptake of fluorescent dextran into endosomes). The selectivity of quinacrine distribution in peripheral blood leukocytes may be determined by the collaboration of a non-concentrating plasma membrane transport mechanism, tentatively identified as pinocytosis in PMNLs, with V-ATPase-mediated concentration. Intracellular reservoirs of cationic drugs are a potential source of toxicity (e.g., loss of lysosomal function in phagocytes). - Highlights: • Quinacrine is concentrated in acidic organelles via V-ATPase-mediated ion

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. High affinity capture and concentration of quinacrine in polymorphonuclear neutrophils via vacuolar ATPase-mediated ion trapping: comparison with other peripheral blood leukocytes and implications for the distribution of cationic drugs.

    PubMed

    Roy, Caroline; Gagné, Valérie; Fernandes, Maria J G; Marceau, François

    2013-07-15

    Many cationic drugs are concentrated in acidic cell compartments due to low retro-diffusion of the protonated molecule (ion trapping), with an ensuing vacuolar and autophagic cytopathology. In solid tissues, there is evidence that phagocytic cells, e.g., histiocytes, preferentially concentrate cationic drugs. We hypothesized that peripheral blood leukocytes could differentially take up a fluorescent model cation, quinacrine, depending on their phagocytic competence. Quinacrine transport parameters were determined in purified or total leukocyte suspensions at 37 °C. Purified polymorphonuclear leukocytes (PMNLs, essentially neutrophils) exhibited a quinacrine uptake velocity inferior to that of lymphocytes, but a consistently higher affinity (apparent KM 1.1 vs. 6.3 μM, respectively). However, the vacuolar (V)-ATPase inhibitor bafilomycin A1 prevented quinacrine transport or initiated its release in either cell type. PMNLs capture most of the quinacrine added at low concentrations to fresh peripheral blood leukocytes compared with lymphocytes and monocytes (cytofluorometry). Accumulation of the autophagy marker LC3-II occurred rapidly and at low drug concentrations in quinacrine-treated PMNLs (significant at ≥2.5 μM, ≥2 h). Lymphocytes contained more LAMP1 than PMNLs, suggesting that the mass of lysosomes and late endosomes is a determinant of quinacrine uptake Vmax. PMNLs, however, exhibited the highest capacity for pinocytosis (uptake of fluorescent dextran into endosomes). The selectivity of quinacrine distribution in peripheral blood leukocytes may be determined by the collaboration of a non-concentrating plasma membrane transport mechanism, tentatively identified as pinocytosis in PMNLs, with V-ATPase-mediated concentration. Intracellular reservoirs of cationic drugs are a potential source of toxicity (e.g., loss of lysosomal function in phagocytes).

  18. 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

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

    NASA Astrophysics Data System (ADS)

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

    1995-05-01

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

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

    PubMed Central

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

    1987-01-01

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

  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. 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. 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

  4. 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.

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

    PubMed

    Matthes, T; Gruler, H

    1988-01-01

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

  6. Pathologic interaction between megakaryocytes and polymorphonuclear leukocytes in myelofibrosis.

    PubMed

    Schmitt, A; Jouault, H; Guichard, J; Wendling, F; Drouin, A; Cramer, E M

    2000-08-15

    Idiopathic myelofibrosis (MF) is a myeloproliferative syndrome characterized by an increase in bone marrow collagen. Megakaryocytes (Mks), which store growth factors in their alpha granules, are known to be involved in the pathogenesis of MF. Previously, mice given bone marrow grafts infected with a retrovirus carrying murine thrombopoietin (TPO) complementary DNA developed a disease resembling human idiopathic MF. In this study, we used this murine model (TPO mice) to determine whether release of alpha granules is responsible for fibroblast activation and development of fibrosis. The intracellular trafficking of several alpha-granule proteins (von Willebrand factor, fibrinogen, and transforming growth factor beta (TGF beta), which are stored in the granule matrix; and alpha(IIb)beta(3) integrin and P-selectin (CD62p), which are located in the alpha-granule membrane) was studied with immune electron microscopy in bone marrow Mks from TPO mice. P-selectin immunolabeling increased consistently and was occasionally found lining the demarcation membrane system. Evidence of extensive emperipolesis was also found in TPO mouse Mks, involving almost exclusively neutrophil and eosinophil polymorphonuclear (PMN) cells with altered morphologic features. In parallel, the host Mks had myeloperoxidase-positive granules scattered in their cytoplasm, associated with marked ultrastructural cytoplasmic alterations and ruptured alpha-granule membranes. Similar observations were made in bone marrow biopsy specimens from 12 patients with idiopathic MF; indeed, there was an increased rate of emperipolesis involving mostly PMN cells, abnormal P-selectin expression, and mutual subcellular PMN and Mk alterations. This study indicates that in idiopathic MF, abnormal P-selectin distribution in Mks induces selective sequestration of PMN cells. This results in a release of alpha-granular proteins and growth factors, which in turn induces fibroblast activation and fibrosis deposition. (Blood

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

    SciTech Connect

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

    1984-05-01

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

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

    PubMed Central

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

    1980-01-01

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

  9. Repeatability of flow cytometric and classical measurement of phagocytosis and respiratory burst in bovine polymorphonuclear leukocytes.

    PubMed

    Kampen, Annette H; Tollersrud, Tore; Larsen, Stig; Roth, James A; Frank, Dagmar E; Lund, Arve

    2004-01-01

    Five methods for measurement of phagocytosis and respiratory burst activity of bovine blood polymorphonuclear leukocytes (PMNs) were evaluated. Eight cows were repeatedly sampled over a two week period and parallel samples tested in all five assays to assess the repeatability and stability of the methods. In the flow cytometric phagocytosis assay, ingestion of fluorescein labeled bacteria was measured, and in the flow cytometric assay for respiratory burst, oxidation of a dye by reactive oxygen species was recorded. In the classical assays, bactericidal effect on opsonized, live bacteria was quantified by the conversion of an indicator substance, superoxide anion production was assayed by the reduction of cytochrome c, whereas myeloperoxidase activity was determined with a radioactive iodination assay. The results showed that the Phagotest, Bursttest, cytochrome c and iodination assays gave repeatable results when samples were run in the same setup on the same day. Although day-to-day variability was significant in all assays, the described methods comprise a panel of useful tests for the evaluation of phagocytosis and respiratory burst activity in bovine PMNs. The flow cytometric methods represent a convenient alternative to the classical methods for measurement of phagocytosis and respiratory burst in bovine blood PMNs.

  10. Experimental bacterial pneumonia in rabbits: polymorphonuclear leukocyte margination and sequestration in rabbit lungs and quantitation and kinetics of /sup 51/Cr-labeled polymorphonuclear leukocytes in E. coli-induced lung lesions

    SciTech Connect

    Cybulsky, M.I.; Movat, H.Z.

    1982-12-01

    A relationship between the circulating and marginal polymorphonuclear leukocyte (PMN) pools was documented using /sup 51/Cr-labeled leukocytes as a marker. /sup 51/Cr-leukocytes marginating in the lungs were found to decrease following a first-order exponential decline, while /sup 51/Cr radioactivity accumulated in the liver and the spleen. Intravenously administered endotoxin caused a rapid selective disappearance of PMNs from the circulation. The percentage of infused /sup 51/Cr cells disappearing was equal to the percentage of disappearance of host cells. The PMNs were found to sequester in the lungs, with peak sequestration of labeled cells occurring 5 min after an endotoxin challenge. Over the next 25 min the /sup 51/Cr radioactivity in the lungs declined. Large numbers of PMNs, probably newly derived from the bone marrow, were observed histologically to be sequestered in the lung vasculature 90 min after an endotoxin dose, while the early sequestration of circulating leukocytes could not be assessed histologically. Pulmonary inflammatory lesions were induced selectively with Escherichia coli in the left lower lobes of rabbits, leaving the right lower lobes as intrinsic controls. PMN-accumulation into the lesions was quantitated using /sup 51/Cr-labeled blood leukocytes. With the aid of /sup 125/I-labeled E. coli, a logarithmic dose-response relationship was found between the number of E. coli and of PMNs. Over a 6-hr period circulating PMNs were found to accumulate in a lesion in the left lower lobe, whereas in the control right lower lobe, leukocyte radioactivity declined. These findings were confirmed with the aid of lavages of the right and left lungs. Two peaks of PMN-accumulation were found by studying leukocyte kinetics: a larger peak between 0 and 6 hr and a smaller peak 18-24 hr after instillation of the microorganisms. Histologic studies confirmed the accumulation of leukocytes, and by 3 weeks showed a complete resolution of the lesions.

  11. Effect of cigarette smoke extract on the polymorphonuclear leukocytes chemiluminescence: influence of a filter containing glutathione.

    PubMed

    Zappacosta, B; Persichilli, S; Minucci, A; Fasanella, S; Scribano, D; Giardina, B; De Sole, P

    2005-01-01

    Cigarette smoking is known to be a risk factor for several chronic and neoplastic diseases. Many compounds formed by cigarette burning, ranging from particulate materials to water solutes and gaseous extracts, are considered to be noxious agents, and many biochemical and molecular mechanisms have been proposed for the toxic effects of cigarette smoke. The oral cavity and the upper respiratory tract represent the first contact areas for smoke compounds; even a single cigarette can produce marked effects on some components of the oral cavity, either chemical compounds, such as glutathione and enzymes, or cellular elements, such as polymorphonuclear leukocytes. Several studies suggest a protective role of glutathione against the noxious effects of tobacco smoke; the sulphydril groups of glutathione, in fact, could react with some smoke products, such as unsaturated aldehydes, leading to the formation of harmless intermediate compounds and simultaneously preventing the inactivation of metabolically essential molecules, such as some enzymes. In this paper we analyse the effect of a filter containing glutathione on the respiratory burst of polymorphonuclear leukocytes exposed to aqueous extract of cigarette smoke, measuring their chemiluminescence activity. The results of this paper indicate that the GSH-containing filter has a likely protective effect against the inhibition of cigarette smoke extract on polymorphonuclear leukocyte activity.

  12. Complement (C5)-derived chemotactic activity accounts for accumulation of polymorphonuclear leukocytes in cerebrospinal fluid of rabbits with pneumococcal meningitis.

    PubMed Central

    Ernst, J D; Hartiala, K T; Goldstein, I M; Sande, M A

    1984-01-01

    Experiments were performed to identify the chemoattractant for polymorphonuclear leukocytes that appears in the cerebrospinal fluid of rabbits with experimental pneumococcal meningitis. Meningitis was induced in anesthetized New Zealand white rabbits by injecting 10(4) cells of stationary-phase Streptococcus pneumoniae type III intracisternally. Before bacteria were injected, cerebrospinal fluid contained neither polymorphonuclear leukocytes nor chemotactic activity. Significant chemotactic activity for rabbit polymorphonuclear leukocytes was detected 12 h after inoculation with bacteria and was maximal after 18 to 20 h. Chemotactic activity appeared in cerebrospinal fluid while concentrations of pneumococci and total protein were increasing but before there was any accumulation of polymorphonuclear leukocytes. The chemotactic activity in cerebrospinal fluid was heat stable (56 degrees C for 30 min), eluted from Sephadex G-75 with a profile identical to that of the chemotactic activity in zymosan-activated rabbit serum, and was inhibited by treatment with antibodies to native human C5. In addition, preincubation of polymorphonuclear leukocytes with partially purified rabbit C5a selectively inhibited their subsequent chemotactic responses to cerebrospinal fluid. These data indicate that complement (C5)-derived chemotactic activity appears in cerebrospinal fluid during the course of experimental pneumococcal meningitis in rabbits and suggest that this activity accounts for the accumulation of polymorphonuclear leukocytes observed in this infection. PMID:6480117

  13. Effect of Phenylbutazone on Phagocytosis and Intracellular Killing by Guinea Pig Polymorphonuclear Leukocytes1

    PubMed Central

    Strauss, Robert R.; Paul, Benoy B.; Sbarra, Anthony J.

    1968-01-01

    The anti-inflammatory drug phenylbutazone has been found to inhibit both engulfment and intracellular killing of E. coli by guinea pig peritoneal polymorphonuclear (PMN) leukocytes. The bactericidal activity of leukocytic homogenates was also inhibited by the drug. Addition of the drug at various time intervals to a phagocytic reacting system caused an almost immediate cessation of bactericidal activity. Metabolic studies showed that the drug sharply curtailed glucose-l-14C and 14C-formate oxidation of both resting and phagocytizing PMN leukocytes. These data indicated an effect upon the hexose monophosphate shunt and H2O2 formation. Further investigation showed that the sites of inhibition were on glucose-6-phosphate and 6-phosphogluconate dehydrogenase. These inhibitions resulted in decreased H2O2 production. It is suggested that H2O2 activates lysosomes and subsequently complexes with the lysosomal enzyme, myeloperoxidase. This complex is a potent bactericidal agent in the phagocyte. PMID:4881700

  14. In Vitro Effect of Tobacco Smoke Components on the Functions of Normal Human Polymorphonuclear Leukocytes

    PubMed Central

    Corberand, Joël; Laharrague, Patrick; Nguyen, Françoise; Dutau, Guy; Fontanilles, Anne Marie; Gleizes, Bernard; Gyrard, Elisabeth

    1980-01-01

    The function of polymorphonuclear leukocytes (PMNs) has previously been shown to be impaired in smokers in comparison with healthy nonsmokers. Potent inhibition of PMN chemotaxis has been achieved with whole tobacco smoke, the gas phase of smoke, and a water-soluble extract of whole smoke. In the present work several aspects of PMN function were studied after exposure to water-soluble fraction of the particle phase of tobacco smoke collected on glass fiber filters. These tests included capillary tube random migration, chemotaxis under agarose, phagocytosis of yeasts, Nitro Blue Tetrazolium dye reduction, and whole-blood bactericidal activity. The water extract of the particle fraction of smoke had a high content of nicotine when compared with the levels achieved in plasma of smokers and a much lower concentration of aldehydes when compared with the gas phase of smoke. It had no cytotoxic effect and did not affect phagocytosis, oxygen consumption, or bactericidal activity. Nitro Blue Tetrazolium reduction of both resting and stimulated PMNs was significantly decreased only with the most concentrated solution. The tested solutions exerted a dose-related depressive effect on capillary tube random migration, whereas the random migration measured in the agarose chemotaxis test was normal. Nevertheless, the chemotactic response to a caseine solution was significantly decreased. The same tests were performed in the presence of several concentrations of a nicotine solution and the only test to be affected was the capillary tube random migration, and, that only at a very high concentration. The results of this study contribute to the more precise delineation of the extent of the dysfunction of PMNs exposed to tobacco smoke components and indicate that deleterious products are released from the particle phase of the smoke, which deposits all along the respiratory tree. PMID:7228386

  15. Effect of thrombopoietin and granulocyte colony-stimulating factor on platelets and polymorphonuclear leukocytes.

    PubMed

    Schattner, M; Pozner, R G; Gorostizaga, A B; Lazzari, M A

    2000-07-15

    Thrombopoietin (TPO) and granulocyte colony-stimulating factor (G-CSF) may be administered together in aplastic patients. We evaluated the effect of both cytokines alone or combined on platelets and polymorphonuclear leukocytes (PMN) functional responses. TPO, G-CSF, or the combination of both cytokines, induced neither platelet nor PMN activation. TPO but not G-CSF synergized with threshold ADP concentrations to induce maximal aggregation and ATP release. The synergistic effect of TPO with ADP was not modified by the presence of G-CSF. Flow cytometry studies have shown that thrombin-induced loss of GPIb from platelet surface was significantly increased by pretreatment of platelets with TPO, G-CSF, or both cytokines. P-selectin expression induced by thrombin was augmented by TPO, but not by G-CSF. Coincubation of the cells with TPO and G-CSF did not modify the values obtained with TPO alone. Expression of CD11b on PMN surface was augmented by G-CSF or fMLP. G-CSF-treated PMN increased the effect of fMLP on CD11b expression. TPO did not modify either basal levels of CD11b or the increased expression induced by G-CSF or fMLP. Incubation of PMN with both cytokines showed no differences compared to G-CSF alone. Platelet-PMN aggregates induced by thrombin in whole blood were augmented by TPO. G-CSF alone neither synergized with thrombin nor changed the results observed with TPO. These data show that in vitro functional responses of platelets, or PMN induced by TPO or G-CSF alone, were neither further increased nor inhibited by treatment of the cells with both cytokines.

  16. Phagocytosis of virulent Porphyromonas gingivalis by human polymorphonuclear leukocytes requires specific immunoglobulin G.

    PubMed Central

    Cutler, C W; Kalmar, J R; Arnold, R R

    1991-01-01

    No studies to date clearly define the interactions between Porphyromonas gingivalis and human peripheral blood polymorphonuclear leukocytes (PMN), nor has a protective role for antibody to P. gingivalis been defined. Using a fluorochrome phagocytosis microassay, we investigated PMN phagocytosis and killing of P. gingivalis as a function of P. gingivalis-specific antibody. Sera from a nonimmune rabbit and a healthy human subject were not opsonic for virulent P. gingivalis A7436, W83, and HG405; phagocytosis of these strains (but not 33277) required opsonization with hyperimmune antiserum (RaPg). Diluting RaPg with a constant complement source decreased proportionally the number of P. gingivalis A7436 cells phagocytosed per phagocytic PMN. Enriching for the immunoglobulin G fraction of RAPg A7436 enriched for opsonic activity toward A7436. An opsonic evaluation of 18 serum samples from adult periodontitis patients revealed that only 3 adult periodontitis sera of 17 with elevated immunoglobulin G to P. gingivalis A7436 were opsonic for A7436 and, moreover, that the serum sample with the highest enzyme-linked immunosorbent assay titer was most opsonic (patient 1). However, the opsonic activity of serum from patient 1 was qualitatively and not just quantitatively different from that of the nonopsonic human sera (but was less effective opsonin than RaPg). Strain variability was observed in resistance of P. gingivalis to phagocytosis, and opsonization was strain specific for some, but not all, strains tested. An evaluation of killing of A7436 revealed that serum killing and extracellular killing of P. gingivalis were less effective alone when compared with intracellular PMN killing alone. PMID:2037370

  17. 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.

  18. Activation of Polymorphonuclear Leukocytes by Candidate Biomaterials for an Implantable Glucose Sensor

    PubMed Central

    Sokolov, Andrey; Hellerud, Bernt Christian; Lambris, John D; Johannessen, Erik A; Mollnes, Tom Eirik

    2011-01-01

    Background Continuous monitoring of glucose by implantable microfabricated devices offers key advantages over current transcutaneous glucose sensors that limit usability due to their obtrusive nature and risk of infection. A successful sensory implant should be biocompatible and retain long-lasting function. Polymorphonuclear leukocytes (PMN) play a key role in the inflammatory system by releasing enzymes, cytokines, and reactive oxygen species, typically as a response to complement activation. The aim of this study was to perform an in vitro analysis of PMN activation as a marker for biocompatibility of materials and to evaluate the role of complement in the activation of PMN. Methods Fifteen candidate materials of an implantable glucose sensor were incubated in lepirudin-anticoagulated whole blood. The cluster of differentiation molecule 11b (CD11b) expression on PMN was analyzed with flow cytometry and the myeloperoxidase (MPO) concentration in plasma was analyzed with enzyme-linked immunosorbent assay. Complement activation was prevented by the C3 inhibitor compstatin or the C5 inhibitor eculizumab. Results Three of the biomaterials (cellulose ester, polyamide reverse osmosis membrane, and polyamide thin film membrane), all belonging to the membrane group, induced a substantial and significant increase in CD11b expression and MPO release. The changes were virtually identical for these two markers. Inhibition of complement with compstatin or eculizumab reduced the CD11b expression and MPO release dose dependently and in most cases back to baseline. The other 12 materials did not induce significant PMN activation. Conclusion Three of the 15 candidate materials triggered PMN activation in a complement-dependent manner and should therefore be avoided for implementation in implantable microsensors. PMID:22226271

  19. Impaired metabolic function of polymorphonuclear leukocytes in glycogen storage disease Ib.

    PubMed

    Gahr, M; Heyne, K

    1983-09-01

    To elucidate the basis for the recurrent infections in patients with glycogen storage disease (GSD) Ib we tested polymorphonuclear leukocyte (PMN) function in one patient. Bactericidal capacity and phagocytosis-induced O2 consumption were reduced. Also, phorbol myristate acetate-stimulated superoxide production and glucose oxidation through the hexose monophosphate shunt were diminished compared to control subjects. Therefore it could be speculated that in PMN of patients with GSD Ib, glucose-6-phosphate has no access to the enzymes of the hexose monophosphate shunt due to a transport-related defect as shown for glucogenesis in hepatocytes.

  20. 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

  1. [Chemiluminescence in a stimulated polymorphonuclear leukocytes--luminol system: suppression by thiols].

    PubMed

    Murina, M A; Roshchupkin, D I; Belakina, N S; Filippov, S V

    2005-01-01

    The effect of some scavengers of thiol nature, which eliminate all reactive oxygen species and oxidants with reactive chlorine, on the luminol-enhanced chemiluminescence of polymorphonuclear leukocytes was studied. The use of two scavengers of this type (penetrating and not penetrating into the cell) made it possible to separate the luminescence of cell structures from the luminescence generated by oxidants in the surrounding medium. It was found that about a half of luminol luminescence is due to its oxidation in the medium surrounding the cell, and it is completely inhibited by the nonpenetrating reduced glutathione. The cell itself is a source of a considerable portion of luminescence, and this luminescence is quenched by penetrating sulfhydryl compounds such as dithiothreitol and N-acethyl cysteine. Reduced glutathione, which penetrates into cells and whose action is due only to the sulfhydryl group, is recommended as a candidate for the selective neutralization of extracellular oxidants.

  2. Shigella flexneri is trapped in polymorphonuclear leukocyte vacuoles and efficiently killed.

    PubMed Central

    Mandic-Mulec, I; Weiss, J; Zychlinsky, A

    1997-01-01

    We examined the bactericidal activity of polymorphonuclear leukocytes (PMN) against an invasive wild-type strain of Shigella flexneri (M90T) and a plasmid-cured noninvasive derivative (BS176). Both Shigella strains, as well as a rough strain of Escherichia coli, were killed with similar efficiencies by intact inflammatory PMN in room air and under N2 (i.e., killing was O2 independent). Bacterial killing by PMN extracts was substantially inhibited by antibodies to the bactericidal/permeability-increasing protein (BPI). Whereas wild-type Shigella escapes from the phagosome to the cytoplasm in epithelial cells and macrophages, wild-type Shigella was trapped in the phagolysosome of PMN as visualized by electron microscopy. The efficient killing of Shigella by PMN suggests that these inflammatory cells may not only contribute initially to the severe tissue damage characteristic of shigellosis but also ultimately participate in clearance and resolution of infection. PMID:8975899

  3. [Chemiluminescence of the polymorphonuclear leukocytes-luminol system in the presence of biogenic chloramines].

    PubMed

    Murina, M A; Belakina, N S; Roshchupkin, D I

    2004-01-01

    It was demonstrated that N-chlorphenylalanine and other chloramines strengthen sharply chemiluminescence in the polymorphonuclear leukocytes (PML)-luminol system without special activation of cells. The intensity of chemiluminescence is higher than the intensity of luminol solution emission induced by N-chlorphenylalanine. But it was nearly equal to chemiluminescence intensity of a mixture of luminol, N-chlorphenylalanine and 20-30 nM H2O2. The increase in chemiluminescence in the PML-luminol system in the presence of N-chlorphenylalanine is not related to PML activation but is the result of direct oxidation of luminol by N-chlorphenylalanine. Chloramine derivatives of amino acids and taurine at final concentrations of 0.01-0.1 mM do not suppress luminol chemiluminescence in suspension of PML stimulated by phorbol-12-myristate-13-acetate. At the same time, hypochlorite inhibits sharply luminol emission induced by stimulated cells.

  4. Determination of phagocytosis of /sup 32/P-labeled Staphylococcus aureus by bovine polymorphonuclear leukocytes

    SciTech Connect

    Dulin, A.M.; Paape, M.J.; Weinland, B.T.

    1984-04-01

    A procedure for the measurement of phagocytosis by bovine polymorphonuclear leukocytes (PMN) of /sup 32/P-labeled Staphylococcus aureus was modified so that a larger number of samples could be compared in a single run, and smaller volumes of sample, PMN, and /sup 32/P-labeled S aureus could be used. Results were highly reproducible, with a coefficient of variation between duplicate determinations of less than or equal to 2%. Lysostaphin was prepared from the supernatant of S staphylolyticus and was compared with a commercially available preparation. Effects of lysostaphin on PMN and influence of incubation media on release of /sup 32/P from /sup 32/P-labeled S aureus by lysostaphin were examined.

  5. Membrane cholesterol modulates the fluid shear stress response of polymorphonuclear leukocytes via its effects on membrane fluidity

    PubMed Central

    Zhang, Xiaoyan; Hurng, Jonathan; Rateri, Debra L.; Daugherty, Alan; Schmid-Schönbein, Geert W.

    2011-01-01

    Continuous exposure of polymorphonuclear leukocytes (PMNLs) to circulatory hemodynamics points to fluid flow as a biophysical regulator of their activity. Specifically, fluid flow-derived shear stresses deactivate leukocytes via actions on the conformational activities of proteins on the cell surface. Because membrane properties affect activities of membrane-bound proteins, we hypothesized that changes in the physical properties of cell membranes influence PMNL sensitivity to fluid shear stress. For this purpose, we modified PMNL membranes and showed that the cellular mechanosensitivity to shear was impaired whether we increased, reduced, or disrupted the organization of cholesterol within the lipid bilayer. Notably, PMNLs with enriched membrane cholesterol exhibited attenuated pseudopod retraction responses to shear that were recovered by select concentrations of benzyl alcohol (a membrane fluidizer). In fact, PMNL responses to shear positively correlated (R2 = 0.96; P < 0.0001) with cholesterol-related membrane fluidity. Moreover, in low-density lipoprotein receptor-deficient (LDLr−/−) mice fed a high-fat diet (a hypercholesterolemia model), PMNL shear-responses correlated (R2 = 0.5; P < 0.01) with blood concentrations of unesterified (i.e., free) cholesterol. In this regard, the shear-responses of PMNLs gradually diminished and eventually reversed as free cholesterol levels in blood increased during 8 wk of the high-fat diet. Collectively, our results provided evidence that cholesterol is an important component of the PMNL mechanotransducing capacity and elevated membrane cholesterol impairs PMNL shear-responses at least partially through its impact on membrane fluidity. This cholesterol-linked perturbation may contribute to dysregulated PMNL activity (e.g., chronic inflammation) related to hypercholesterolemia and causal for cardiovascular pathologies (e.g., atherosclerosis). PMID:21525434

  6. Inhibition of superoxide anion production in guinea pig polymorphonuclear leukocytes by a seleno-organic compound, ebselen.

    PubMed

    Ichikawa, S; Omura, K; Katayama, T; Okamura, N; Ohtsuka, T; Ishibashi, S; Masayasu, H

    1987-10-01

    Production of superoxide anion (O2-) induced by tetradecanoyl phorbol acetate (TPA) in intact guinea pig polymorphonuclear leukocytes (PMNL) was markedly inhibited by a seleno-organic compound, 2-phenyl-1,2-benzisoselenazol-3(2H)-one (Ebselen), with glutathione peroxidase-like activity. The compound almost completely inhibited O2- production by a particulate fraction prepared from TPA-treated PMNL at a concentration as low as 250 nM.

  7. The essential oil of bergamot stimulates reactive oxygen species production in human polymorphonuclear leukocytes.

    PubMed

    Cosentino, Marco; Luini, Alessandra; Bombelli, Raffaella; Corasaniti, Maria T; Bagetta, Giacinto; Marino, Franca

    2014-08-01

    Bergamot (Citrus aurantium L. subsp. bergamia) essential oil (BEO) is used in folk medicine as an antiseptic and anthelminthic and to facilitate wound healing. Evidence indicates that BEO has substantial antimicrobial activity; however its effects on immunity have never been examined. We studied the effects of BEO on reactive oxygen species (ROS) production in human polymorphonuclear leukocytes (PMN) and the role of Ca(2+) in the functional responses evoked by BEO in these cells. Results show that BEO increased intracellular ROS production in human PMN, an effect that required the contribution of extracellular (and, to a lesser extent, of intracellular) Ca(2+) . Bergamot essential oil also significantly increased ROS production induced by the chemotactic peptide N-formyl-Met-Leu-Phe and reduced the response to the protein kinase C activator phorbol myristate acetate. In conclusion, this is the first report showing the ability of BEO to increase ROS production in human PMN. This effect could both contribute to the activity of BEO in infections and in tissue healing as well as underlie an intrinsic proinflammatory potential. The relevance of these findings for the clinical uses of BEO needs careful consideration.

  8. Reduced bioenergetics and toll-like receptor 1 function in human polymorphonuclear leukocytes in aging.

    PubMed

    Qian, Feng; Guo, Xiuyang; Wang, Xiaomei; Yuan, Xiaoling; Chen, Shu; Malawista, Stephen E; Bockenstedt, Linda K; Allore, Heather G; Montgomery, Ruth R

    2014-02-01

    Aging is associated with a progressive decline in immune function (immunosenescence) resulting in an increased susceptibility to viral and bacterial infections. Here we show reduced expression of Toll-like receptor 1 (TLR1) in polymorphonuclear leukocytes (PMN) and an underlying age-dependent deficiency in PMN bioenergetics. In older (>65 years) adults, stimulation through TLR1 led to lower activation of integrins (CD11b and CD18), lower production of the chemokine IL-8, and lower levels of the phosphorylated signaling intermediate p38 MAP kinase than in PMN from younger donors (21-30 years). In addition, loss of CD62L, a marker of PMN activation, was reduced in PMN of older adults stimulated through multiple pathways. Rescue of PMN from apoptosis by stimulation with TLR1 was reduced in PMN from older adults. In seeking an explanation for effects of aging across multiple pathways, we examined PMN energy utilization and found that glucose uptake after stimulation through TLR1 was dramatically lower in PMN of older adults. Our results demonstrate a reduction in TLR1 expression and TLR1-mediated responses in PMN with aging, and reduced efficiency of bioenergetics in PMN. These changes likely contribute to reduced PMN efficiency in aging through multiple aspects of PMN function and suggest potential therapeutic opportunities.

  9. 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.

  10. Killing of gram-negative bacteria by polymorphonuclear leukocytes: role of an O2-independent bactericidal system.

    PubMed

    Weiss, J; Victor, M; Stendhal, O; Elsbach, P

    1982-04-01

    Previous studies have suggested that a cationic bactericidal/permeability-increasing protein (BPI) present in both rabbit and human polymorphonuclear leukocytes is the principal O2-independent bactericidal agent of these cells toward several strains of Escherichia coli and Salmonella typhimurium (1978. J. Biol. Chem. 253: 2664--2672; 1979. J. Biol. Chem. 254: 11000--11009). To further evaluate the possible role of this protein in the killing of gram-negative bacteria by polymorphonuclear leukocytes, we have measured the bactericidal activity of intact rabbit peritoneal exudate leukocytes under aerobic or anaerobic conditions and of intact human leukocytes from a patient with chronic granulomatous disease. Anaerobic conditions were created by flushing the cells under a nitrogen stream. Effective removal of oxygen was demonstrated by the inability of nitrogen-flushed leukocytes to mount a respiratory burst (measured as increased conversion of 1-[14C]glucose leads to 14CO2 or by superoxide production) during bacterial ingestion. At a bacteria/leukocyte ratio of 10:1, killing of gram-positive, BPI-resistant, Staphylococcus epidermidis is markedly impaired in the absence of oxygen (76.4 +/- 3.3% killing in room air, 29.2 +/- 8.2% killing in nitrogen). Essentially all increased bacterial survival is intracellular. In contrast, both a nonopsonized rough strain (MR-10) and an opsonized smooth strain (MS) of S. typhimurium 395 are killed equally well in room air and nitrogen. A maximum of 70--80 MR-10 and 30--40 MS are killed per leukocyte either in the presence or absence of oxygen. There is no intracellular bacterial survival in either condition indicating that intracellular O2-independent bactericidal system(s) of rabbit polymorphonuclear leukocytes can at least match the leukocyte's ingestive capacity. Whole homogenates and crude acid extracts manifest similar bactericidal capacity toward S. typhimurium 395. This activity can be accounted for by the BPI content of these

  11. Killing of gram-negative bacteria by polymorphonuclear leukocytes: role of an O2-independent bactericidal system.

    PubMed Central

    Weiss, J; Victor, M; Stendhal, O; Elsbach, P

    1982-01-01

    Previous studies have suggested that a cationic bactericidal/permeability-increasing protein (BPI) present in both rabbit and human polymorphonuclear leukocytes is the principal O2-independent bactericidal agent of these cells toward several strains of Escherichia coli and Salmonella typhimurium (1978. J. Biol. Chem. 253: 2664--2672; 1979. J. Biol. Chem. 254: 11000--11009). To further evaluate the possible role of this protein in the killing of gram-negative bacteria by polymorphonuclear leukocytes, we have measured the bactericidal activity of intact rabbit peritoneal exudate leukocytes under aerobic or anaerobic conditions and of intact human leukocytes from a patient with chronic granulomatous disease. Anaerobic conditions were created by flushing the cells under a nitrogen stream. Effective removal of oxygen was demonstrated by the inability of nitrogen-flushed leukocytes to mount a respiratory burst (measured as increased conversion of 1-[14C]glucose leads to 14CO2 or by superoxide production) during bacterial ingestion. At a bacteria/leukocyte ratio of 10:1, killing of gram-positive, BPI-resistant, Staphylococcus epidermidis is markedly impaired in the absence of oxygen (76.4 +/- 3.3% killing in room air, 29.2 +/- 8.2% killing in nitrogen). Essentially all increased bacterial survival is intracellular. In contrast, both a nonopsonized rough strain (MR-10) and an opsonized smooth strain (MS) of S. typhimurium 395 are killed equally well in room air and nitrogen. A maximum of 70--80 MR-10 and 30--40 MS are killed per leukocyte either in the presence or absence of oxygen. There is no intracellular bacterial survival in either condition indicating that intracellular O2-independent bactericidal system(s) of rabbit polymorphonuclear leukocytes can at least match the leukocyte's ingestive capacity. Whole homogenates and crude acid extracts manifest similar bactericidal capacity toward S. typhimurium 395. This activity can be accounted for by the BPI content of these

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

    PubMed

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

    2015-04-01

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

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

    PubMed Central

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

    1980-01-01

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

  14. In vivo ultrastructural analysis of the intimate relationship between polymorphonuclear leukocytes and the chlamydial developmental cycle.

    PubMed

    Rank, Roger G; Whittimore, Judy; Bowlin, Anne K; Wyrick, Priscilla B

    2011-08-01

    We utilized a recently developed model of intracervical infection with Chlamydia muridarum in the mouse to elicit a relatively synchronous infection during the initial developmental cycle in order to examine at the ultrastructural level the development of both the chlamydial inclusion and the onset of the inflammatory response. At 18 h after infection, only a few elementary bodies attached to cells were visible, as were an occasional intracellular intermediate body and reticulate body. By 24 h, inclusions had 2 to 5 reticulate bodies and were beginning to fuse. A few polymorphonuclear leukocytes (PMNs) were already present in the epithelium in the vicinity of and directly adjacent to infected cells. By 30 h, the inclusions were larger and consisted solely of reticulate bodies, but by 36 to 42 h, they contained intermediate bodies and elementary bodies as well. Many PMNs were adjacent to or actually inside infected cells. Chlamydiae appeared to exit the cell either (i) through disintegration of the inclusion membrane and rupture of the cell, (ii) by dislodgement of the cell from the epithelium by PMNs, or (iii) by direct invasion of the infected cell by the PMNs. When PMNs were depleted, the number of released elementary bodies was significantly greater as determined both visually and by culture. Interestingly, depletion of PMNs revealed the presence of inclusions containing aberrant reticulate bodies, reminiscent of effects seen in vitro when chlamydiae are incubated with gamma interferon. In vivo evidence for the contact-dependent development hypothesis, a potential mechanism for triggering the conversion of reticulate bodies to elementary bodies, and for translocation of lipid droplets into the inclusion is also presented.

  15. Altered polymorphonuclear leukocyte Fc gamma R expression contributes to decreased candicidal activity during intraabdominal sepsis

    SciTech Connect

    Simms, H.H.; D'Amico, R.; Monfils, P.; Burchard, K.W. )

    1991-03-01

    We investigated the effects of untreated intraabdominal sepsis on polymorphonuclear leukocyte (PMN) candicidal activity. Two groups of swine were studied. Group I (n=6) underwent sham laparotomy, group II (n=7) underwent cecal ligation and incision. Untreated intraabdominal sepsis resulted in a progressive decrease in PMN candicidal activity. Concomitant rosetting and phagocytosis assays demonstrated a decrease in both the attachment and phagocytosis of Candida albicans opsonized with both normal and septic swine serum by PMNs in group II. Iodine 125-labeled swine immunoglobulin G (IgG) and fluorescein isothioalanate (FITC)-labeled swine IgG were used to investigate Fc gamma receptor ligand interactions. Scatchard analyses demonstrated a progressive decline in both the binding affinity constant and number of IgG molecules bound per PMN. Stimulation of the oxidative burst markedly reduced 125I-labeled IgG binding in both group I and group II, with a greater decrement being seen in animals with intraabdominal sepsis. Further, in group II, PMN recycling of the Fc gamma receptor to the cell surface after generation of the oxidative burst was reduced by postoperative day 4. Binding of monoclonal antibodies to Fc gamma receptor II, but not Fc gamma receptor I/III markedly reduced intracellular candicidal activity. Immunofluorescence studies revealed a homogeneous pattern of FITC-IgG uptake by nearly all group I PMNs, whereas by postoperative day 8 a substantial number of PMNs from group II failed to internalize the FITC-IgG. These studies suggest that untreated intraabdominal sepsis reduces PMN candicidal activity and that this is due, in part, to altered PMN Fc gamma receptor ligand interactions.

  16. Effect of piliation on interactions of Haemophilus influenzae type b with human polymorphonuclear leukocytes.

    PubMed Central

    Tosi, M F; Anderson, D C; Barrish, J; Mason, E O; Kaplan, S L

    1985-01-01

    Piliated, adherent (P+) and nonpiliated, nonadherent (P-) strains of Haemophilus influenzae type b (Hib) were compared with respect to their ability to induce polymorphonuclear leukocyte (PMN) chemiluminescence (CL) and superoxide (O2-) generation and their susceptibility to phagocytosis by PMNs. P+ strains opsonized in normal human serum (NHS) induced significantly greater CL than did P- strains (500 X 10(5) +/- 112 X 10(5) versus 242 X 10(5) +/- 65 X 10(5) total counts per 60 min; P less than 0.001) when reacted with normal PMNs. Contributions of immunoglobulin and complement to CL activity in these mixtures were shown by findings of lower overall levels of CL when hypogammaglobulinemic serum or heat-inactivated NHS was used to opsonize either P+ or P- organisms. Results obtained with mixtures of hypogammaglobulinemic plus adsorbed heat-inactivated NHS (with P+ or P- organisms) suggested a role for an antipilus antibody in the enhancement of CL by these strains. NHS-opsonized P+ strains also induced significantly greater (P less than 0.002) O2- generation than did P- strains (2.83 +/- 0.08 versus 1.94 +/- 0.14 nmol of ferricytochrome c reduced per 10 min/10(6) PMN). Comparable ingestion of P+ or P- strains opsonized in NHS by PMNs was demonstrated by a radiolabeled uptake technique and transmission electron microscopy, and primary granule release (beta-glucuronidase) was comparable during ingestion of P+ or P- strains. The basis for the observed enhanced capacity of P+ Hib to stimulate PMN oxidative metabolism as compared with P- organisms is uncertain. Possible clinical implications of these findings deserve further study. Images PMID:2857685

  17. Flow cytometric approach to human polymorphonuclear leukocyte activation induced by gingival crevicular fluid in periodontal disease.

    PubMed

    Biselli, R; Ferlini, C; Di Murro, C; Paolantonio, M; Fattorossi, A

    1995-08-01

    In gingival pockets of patients with periodontal disease, polymorphonuclear leukocytes (PMN) are in contact with a peculiar exudate, the gingival crevicular fluid (GCF). Because of the pivotal role played by PMN in periodontal disease, we evaluated the ability of GCF in modulating normal human PMN. GCF was obtained from two gingival sites with severe periodontitis (SP) and two gingival sites with only mild periodontitis (MP) in 12 patients. Purified PMN were exposed to GCF from SP and MP sites and, as a control, to sterile culture medium. GCF activity was evaluated by monitoring the modulation of membrane molecules relevant to cell function. Compared to control medium, GCF from SP and MP sites was able to induce an activation status in PMN evidenced by an increased CD11b (62 +/- 9% and 28 +/- 7%, respectively) and f-Met-Leu-Phe (56 +/- 5% and 31 +/- 7%, respectively) receptor expression, with a concomitant reduction of CD62L expression (56 +/- 8% and 23 +/- 7%, respectively). Thus, reflecting the clinical status, GCF from SP sites was significantly more efficient in affecting PMN than GCF from MP sites. Cell size modifications, evaluated as an additional indicator of PMN activation, were consistent with membrane molecule modulation. The difference in PMN-activating capacity between SP and MP was abrogated by the successful completion of an appropriate periodontal therapy that dramatically improved clinical status. This is the first direct demonstration that GCF from periodontitis has the capacity to activate normal resting PMN and that this capacity reflects the magnitude of the inflammatory process that takes place in the gingiva.

  18. Putative glycoprotein and glycolipid polymorphonuclear leukocyte receptors for the Actinomyces naeslundii WVU45 fimbrial lectin.

    PubMed Central

    Sandberg, A L; Ruhl, S; Joralmon, R A; Brennan, M J; Sutphin, M J; Cisar, J O

    1995-01-01

    Recognition of receptors on sialidase-treated polymorphonuclear leukocytes (PMNs) by the Gal/GalNAc lectin associated with the type 2 fimbriae of certain strains of actinomyces results in activation of the PMNs, phagocytosis, and destruction of the bacteria. In the present study, plant lectins were utilized as probes to identify putative PMN receptors for the actinomyces lectin. The Gal-reactive lectin from Ricinus communis (RCAI), the Gal/GalNAc-reactive lectins from R. communis (RCAII) and Bauhinia purpurea (BPA), as well as the Gal beta 1-3GalNAc-specific lectins from Arachis hypogaea (PNA) and Agaricus bisporus (ABA) inhibited killing of Actinomyces naeslundii WVU45 by sialidase-treated PMNs. These five lectins detected a 130-kDa surface-labeled glycoprotein on nitrocellulose transfers of PMN extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This glycoprotein was revealed only after treatment of the transfers with sialidase, a condition analogous to the sialidase dependence of the lectin-mediated biological responses of the PMNs to the actinomyces. The mannose-reactive lectin concanavalin A did not inhibit killing of the actinomyces and failed to detect the 130-kDa glycoprotein but did block PMN-dependent killing of Escherichia coli B, a bacterium that possesses mannose-sensitive fimbriae. Therefore, the PMN glycoprotein receptor for A. naeslundii is clearly distinct from those recognized by E. coli. Two major putative glycolipid receptors were also identified by actinomyces and RCAI overlays on sialidase-treated thin-layer chromatograms of PMN gangliosides. Thus, both a 130-kDa glycoprotein and certain gangliosides are implicated in the attachment of the actinomyces to PMNs. PMID:7790078

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

    PubMed Central

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

    1987-01-01

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

  20. Kinetic analysis of microbe opsonification based on stimulated polymorphonuclear leukocyte oxygenation activity.

    PubMed

    Allen, R C; Lieberman, M M

    1984-08-01

    With Pseudomonas aeruginosa as the target microbes and polymorphonuclear leukocytes (PMNL) as effector phagocytes, the microbe-specific, immunoglobulin G (IgG)-dependent opsonic capacities of preimmune and immune sera were measured as the rate of stimulated PMNL dioxygenation of luminol yielding chemiluminescence (CL). When the reactants other than opsonin are present in concentrations that are not rate limiting, the information-effector relationship linking specific opsonin concentration to effector PMNL stimulation is described by the rate equation: L' = k'[IgG]i, where L' is the peak CL velocity (photons per minute), k' is the proportionality constant, [IgG] is the concentration of specific opsonin, and the exponent i is the order of the reaction with respect to opsonin. Since the specific opsonins were polyclonal IgG of unknown absolute serum concentration, the reciprocal rate expression, L' = k'D-i, was employed for data presentation; D is the serum dilution (final volume/initial serum volume), and the sign of i is changed to negative. The relationships of integral, first-derivative, and second-derivative expressions of the CL response to opsonin concentration are illustrated with experimentally obtained data. Based on peak CL velocity or peak CL acceleration measurements taken over different time intervals of testing, the estimated order with respect to opsonin is highest, and probably most accurate, using the shortest test interval allowing reasonably good precision of measurement. As an alternative temporal approach, microbe opsonification kinetics are analyzed based on nodal time (Tn) measurements. The Tn is the time point separating the acceleration and deceleration phases of the PMNL oxygenation response to stimulation and as such satisfies the criterion of a selected condition of PMNL activation.

  1. Polymorphonuclear Leukocytes Restrict Growth of Pseudomonas aeruginosa in the Lungs of Cystic Fibrosis Patients

    PubMed Central

    Kragh, Kasper N.; Alhede, Morten; Jensen, Peter Ø.; Moser, Claus; Scheike, Thomas; Jacobsen, Carsten S.; Seier Poulsen, Steen; Eickhardt-Sørensen, Steffen Robert; Trøstrup, Hannah; Christoffersen, Lars; Hougen, Hans-Petter; Rickelt, Lars F.; Kühl, Michael; Høiby, Niels

    2014-01-01

    Cystic fibrosis (CF) patients have increased susceptibility to chronic lung infections by Pseudomonas aeruginosa, but the ecophysiology within the CF lung during infections is poorly understood. The aim of this study was to elucidate the in vivo growth physiology of P. aeruginosa within lungs of chronically infected CF patients. A novel, quantitative peptide nucleic acid (PNA) fluorescence in situ hybridization (PNA-FISH)-based method was used to estimate the in vivo growth rates of P. aeruginosa directly in lung tissue samples from CF patients and the growth rates of P. aeruginosa in infected lungs in a mouse model. The growth rate of P. aeruginosa within CF lungs did not correlate with the dimensions of bacterial aggregates but showed an inverse correlation to the concentration of polymorphonuclear leukocytes (PMNs) surrounding the bacteria. A growth-limiting effect on P. aeruginosa by PMNs was also observed in vitro, where this limitation was alleviated in the presence of the alternative electron acceptor nitrate. The finding that P. aeruginosa growth patterns correlate with the number of surrounding PMNs points to a bacteriostatic effect by PMNs via their strong O2 consumption, which slows the growth of P. aeruginosa in infected CF lungs. In support of this, the growth of P. aeruginosa was significantly higher in the respiratory airways than in the conducting airways of mice. These results indicate a complex host-pathogen interaction in chronic P. aeruginosa infection of the CF lung whereby PMNs slow the growth of the bacteria and render them less susceptible to antibiotic treatment while enabling them to persist by anaerobic respiration. PMID:25114118

  2. Caffeic Acid Phenethyl Ester and Its Amide Analogue Are Potent Inhibitors of Leukotriene Biosynthesis in Human Polymorphonuclear Leukocytes

    PubMed Central

    Boudreau, Luc H.; Maillet, Jacques; LeBlanc, Luc M.; Jean-François, Jacques; Touaibia, Mohamed; Flamand, Nicolas; Surette, Marc E.

    2012-01-01

    Background 5-lipoxygenase (5-LO) catalyses the transformation of arachidonic acid (AA) into leukotrienes (LTs), which are important lipid mediators of inflammation. LTs have been directly implicated in inflammatory diseases like asthma, atherosclerosis and rheumatoid arthritis; therefore inhibition of LT biosynthesis is a strategy for the treatment of these chronic diseases. Methodology/Principal Findings Analogues of caffeic acid, including the naturally-occurring caffeic acid phenethyl ester (CAPE), were synthesized and evaluated for their capacity to inhibit 5-LO and LTs biosynthesis in human polymorphonuclear leukocytes (PMNL) and whole blood. Anti-free radical and anti-oxidant activities of the compounds were also measured. Caffeic acid did not inhibit 5-LO activity or LT biosynthesis at concentrations up to 10 µM. CAPE inhibited 5-LO activity (IC50 0.13 µM, 95% CI 0.08–0.23 µM) more effectively than the clinically-approved 5-LO inhibitor zileuton (IC50 3.5 µM, 95% CI 2.3–5.4 µM). CAPE was also more effective than zileuton for the inhibition of LT biosynthesis in PMNL but the compounds were equipotent in whole blood. The activity of the amide analogue of CAPE was similar to that of zileuton. Inhibition of LT biosynthesis by CAPE was the result of the inhibition of 5-LO and of AA release. Caffeic acid, CAPE and its amide analog were free radical scavengers and antioxidants with IC50 values in the low µM range; however, the phenethyl moiety of CAPE was required for effective inhibition of 5-LO and LT biosynthesis. Conclusions CAPE is a potent LT biosynthesis inhibitor that blocks 5-LO activity and AA release. The CAPE structure can be used as a framework for the rational design of stable and potent inhibitors of LT biosynthesis. PMID:22347509

  3. Polymorphonuclear leukocyte adhesion triggers the disorganization of endothelial cell-to-cell adherens junctions

    PubMed Central

    1996-01-01

    Polymorphonuclear leukocytes (PMN) infiltration into tissues is frequently accompanied by increase in vascular permeability. This suggests that PMN adhesion and transmigration could trigger modifications in the architecture of endothelial cell-to-cell junctions. In the present paper, using indirect immunofluorescence, we found that PMN adhesion to tumor necrosis factor-activated endothelial cells (EC) induced the disappearance from endothelial cell-to-cell contacts of adherens junction (AJ) components: vascular endothelial (VE)-cadherin, alpha-catenin, beta-catenin, and plakoglobin. Immunoprecipitation and Western blot analysis of the VE- cadherin/catenin complex showed that the amount of beta-catenin and plakoglobin was markedly reduced from the complex and from total cell extracts. In contrast, VE-cadherin and alpha-catenin were only partially affected. Disorganization of endothelial AJ by PMN was not accompanied by EC retraction or injury and was specific for VE- cadherin/catenin complex, since platelet/endothelial cell adhesion molecule 1 (PECAM-1) distribution at cellular contacts was unchanged. PMN adhesion to EC seems to be a prerequisite for VE-cadherin/catenin complex disorganization. This phenomenon could be fully inhibited by blocking PMN adhesion with an anti-integrin beta 2 mAb, while it could be reproduced by any condition that induced increase of PMN adhesion, such as addition of PMA or an anti-beta 2-activating mAb. The effect on endothelial AJ was specific for PMN since adherent activated lymphocytes did not induce similar changes. High concentrations of protease inhibitors and oxygen metabolite scavengers were unable to prevent AJ disorganization mediated by PMN. PMN adhesion to EC was accompanied by increase in EC permeability in vitro. This effect was dependent on PMN adhesion, was not mediated by proteases and oxygen- reactive metabolites, and could be reproduced by EC treatment with EGTA. Finally, immunohistochemical analysis showed that VE

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

    PubMed Central

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

    2011-01-01

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

  5. Nano-layered magnesium fluoride reservoirs on biomaterial surfaces strengthen polymorphonuclear leukocyte resistance to bacterial pathogens.

    PubMed

    Guo, Geyong; Zhou, Huaijuan; Wang, Qiaojie; Wang, Jiaxing; Tan, Jiaqi; Li, Jinhua; Jin, Ping; Shen, Hao

    2017-01-05

    Biomaterial-related bacterial infections cause patient suffering, mortality and extended periods of hospitalization, imposing a substantial burden on medical systems. In this context, understanding of nanomaterials-bacteria-cells interactions is of both fundamental and clinical significance. Herein, nano-MgF2 films were deposited on titanium substrate via magnetron sputtering. Using this platform, the antibacterial behavior and mechanism of the nano-MgF2 films were investigated in vitro and in vivo. It was found that, for S. aureus (CA-MRSA, USA300) and S. epidermidis (RP62A), the nano-MgF2 films possessed excellent anti-biofilm activity, but poor anti-planktonic bacteria activity in vitro. Nevertheless, both the traditional SD rat osteomyelitis model and the novel stably luminescent mouse infection model demonstrated that nano-MgF2 films exerted superior anti-infection effect in vivo, which cannot be completely explained by the antibacterial activity of the nanomaterial itself. Further, using polymorphonuclear leukocytes (PMNs), the critical immune cells of innate immunity, a complementary investigation of MgF2-bacteria-PMNs co-culturing revealed that the nano-MgF2 films improved the antibacterial effect of PMNs through enhancing their phagocytosis and stability. To our knowledge, this is the first time of exploring the antimicrobial mechanism of nano-MgF2 from the perspective of innate immunity both in vitro and in vivo. Based on the research results, a plausible mechanism is put forward for the predominant antibacterial effect of nano-MgF2in vivo, which may originate from the indirect immune enhancement effect of nano-MgF2 films. In summary, this study of surface antibacterial design using MgF2 nanolayer is a meaningful attempt, which can promote the host innate immune response to bacterial pathogens. This may give us a new understanding towards the antibacterial behavior and mechanism of nano-MgF2 films and pave the way towards their clinical applications.

  6. Benidipine, an anti-hypertensive drug, inhibits reactive oxygen species production in polymorphonuclear leukocytes and oxidative stress in salt-loaded stroke-prone spontaneously hypertensive rats.

    PubMed

    Matsubara, Masahiro; Akizuki, Osamu; Ikeda, Jun-ichi; Saeki, Koji; Yao, Kozo; Sasaki, Katsutoshi

    2008-02-02

    Oxidative stress is associated with exacerbation of renal injuries in hypertension. In clinical studies benidipine hydrochloride (benidipine), a dihydropyridine calcium channel blocker with antioxidant activity, reduced oxidative stress. However, the mechanism of suppression of oxidative stress remains to be fully characterized. Reactive oxygen species production by polymorphonuclear leukocyte plays important pathological roles in hypertension. Therefore, we examined the effects of benidipine both on reactive oxygen species production of human polymorphonuclear leukocytes and oxidative stress of an animal model. Human peripheral polymorphonuclear leukocytes or polymorphonuclear leukocyte-like differentiated HL-60 cells were used to examine effects of benidipine (0.1-30 microM) on formyl-Met-Leu-Phe-induced reactive oxygen species production, calcium mobilization, NADPH oxidase activation and phosphorylation of protein kinase C substrates. High-salt (8% NaCl) loaded stroke-prone spontaneously hypertensive rats were treated with or without benidipine (1, 3, 10 mg/kg/day) for 2 weeks, and thiobarbituric acid reactive substances, a plasma oxidative stress marker, and renal expression of oxidative stress-induced genes were measured. Benidipine concentration-dependently suppressed formyl-Met-Leu-Phe-induced reactive oxygen species production in polymorphonuclear leukocytes more potently than other calcium channel blockers such as amlodipine, azelnidipine, nitrendipine and nifedipine. Benidipine partially inhibited all of intracellular Ca(2+) elevation, protein kinase C activation and NADPH oxidase activation. Salt loading in stroke-prone spontaneously hypertensive rats augmented plasma thiobarbituric acid reactive substances levels; renal dysfunction; and renal expression of transforming growth factor-beta, collagen I and collagen III mRNAs; which were attenuated by benidipine treatment. These results indicate that benidipine prevents the polymorphonuclear leukocyte

  7. Chemical, biochemical, pharmacokinetic, and biological properties of L-680,833: a potent, orally active monocyclic beta-lactam inhibitor of human polymorphonuclear leukocyte elastase.

    PubMed Central

    Doherty, J B; Shah, S K; Finke, P E; Dorn, C P; Hagmann, W K; Hale, J J; Kissinger, A L; Thompson, K R; Brause, K; Chandler, G O

    1993-01-01

    A series of potent and highly selective time-dependent monocyclic beta-lactam inhibitors of human polymorphonuclear leukocyte elastase (PMNE, EC 3.4.21.37) is described. The intrinsic potency of these compounds, as exemplified by L-680,833 (k(inactivation)/K(i) of 622,000 M-1.s-1), is reflected at the cellular level where it inhibits generation of the specific N-terminal cleavage product A alpha-(1-21) from the A alpha chain of fibrinogen by enzyme released from isolated polymorphonuclear leukocytes stimulated with fMet-Leu-Phe with an IC50 of 0.06 microM. The inhibitory activity of L-680,833 is also apparent in whole blood stimulated with A23187, where it inhibits formation of A alpha-(1-21) and PMNE-alpha 1-proteinase inhibitor complex formation with IC50 values of 9 microM. Pharmacokinetic studies indicate that after oral dosing L-680,833 is bioavailable in rats and rhesus monkeys. This oral bioavailability is reflected by the inhibition (i) of tissue damage elicited in hamster lungs by intratracheal instillation of human PMNE and (ii) enzyme released from human PMN stimulated after their transfer into the pleural cavity of mice. The properties of L-680,833 allow it to effectively supplement the activity of natural inhibitors of PMNE in vivo, suggesting that this type of low-molecular-weight synthetic inhibitor could have therapeutic value in diseases where PMNE damages tissue. PMID:8378355

  8. Influence of He-Ne laser radiation on biogenic amines content and cytochemical parameters of polymorphonuclear leukocytes in short-term stress

    NASA Astrophysics Data System (ADS)

    Brill, Gregory E.; Dobrovolsky, Gennady A.; Romanova, Tatyana P.; Porozova, Svetlana G.; Brill, Alexander G.

    1997-06-01

    In experiments on white male rats short-term immobilization- sound stress was modelled. Decrease of glycogen content and myeloperoxidase activity, increase of lysosomal cationic proteins level and NBT-test parameters as well as fall of adrenaline, dopamine and 5-hydroxytryptamine amount in polymorphonuclear leukocytes were observed. Preliminary transcutaneous He-Ne laser irradiation modified metabolic reaction of leukocytes to stress and prevented stress- induced decrease of biogenic amines content in cells.

  9. 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

  10. Kinetics of staphylococcal opsonization, attachment, ingestion and killing by human polymorphonuclear leukocytes: a quantitative assay using [3H]thymidine labeled bacteria.

    PubMed

    Verhoef, J; Peterson, P K; Quie, P G

    1977-01-01

    A method has been developed for studying quantitatively the separate processes of bacterial opsonization, phagocytosis, and killing by human polymorphonuclear leukocytes using [3H]thymidine labeled Staphylococcus aureus. Phagocytosis is determined by assaying for leukocytes-associated radioactivity after differential centrifugation and washing the leukocytes. Opsonization is studied by incubating bacteria with an opsonic source for varying durations and then adding leukocytes. By treatment of samples with the muralytic enzyme, lysostaphin, the attachment and ingestion phases of phagocytosis can be separated. Sampling for colony forming units after disruption of the leukocytes permits the measurement of bacterial killing. Using this method, differences in the kinetics of staphylococcal opsonization by normal and C2 deficient sera were defined, opsonic influences on the attachment and ingestion phases of pH agocytosis were delineated, and the influences of different opsonins and leukocyte populations on killing were determined.

  11. Killing of Pseudomonas pseudomallei by polymorphonuclear leukocytes and peritoneal macrophages from chicken, sheep, swine and rabbits.

    PubMed

    Markova, N; Kussovski, V; Radoucheva, T

    1998-07-01

    Differences in the kinetics of Pseudomonas pseudomallei killing by peritoneal macrophages (PM) and polymorphonuclear leucocytes (PMNL) from chickens, sheep, swine and rabbits were found. P. pseudomallei was rapidly killed by porcine PM and PMNL. However the bacterial killing by ovine and lapine PM and PMNL proceeded at a slower rate. In contrast, chicken PM and PMNL ingested and killed the lowest number of P. pseudomallei bacteria. The differences in the bactericidal activity of PM and PMNL from different animal species correlated with the level of their acid phosphatase and glycolytic activity.

  12. Promotion of DNA strand breaks in cocultured mononuclear leukocytes by protein kinase C-dependent prooxidative interactions of benoxaprofen, human polymorphonuclear leukocytes, and ultraviolet radiation

    SciTech Connect

    Schwalb, G.; Beyers, A.D.; Anderson, R.; Nel, A.E.

    1988-06-01

    At concentrations of 5 micrograms/ml and greater the nonsteroidal antiinflammatory drug benoxaprofen caused dose-related activation of lucigenin-enhanced chemiluminescence in human polymorphonuclear leukocytes (PMNL). Benoxaprofen-mediated activation of lucigenin-enhanced chemiluminescence by PMNL was increased by UV radiation and was particularly sensitive to inhibition by the selective protein kinase C inhibitor H-7. To identify the molecular mechanism of the prooxidative activity of benoxaprofen, the effects of the nonsteroidal antiinflammatory drug on the activity of purified protein kinase C in a cell-free system were investigated. Benoxaprofen caused a dose-related activation of protein kinase C by interaction with the binding site for the physiological activator phosphatidylserine, but could not replace diacylglycerol. When autologous mononuclear leukocytes (MNL) were cocultured with PMNL and benoxaprofen in combination, but not individually, the frequency of DNA strand breaks in MNL was markedly increased. UV radiation significantly potentiated damage to DNA mediated by benoxaprofen and PMNL. Inclusion of superoxide dismutase, H-7, and, to a much lesser extent, catalase during exposure of MNL to benoxaprofen-activated PMNL prevented oxidant damage to DNA. These results clearly demonstrate that potentially carcinogenic prooxidative interactions, which are unlikely to be detected by conventional assays of mutagenicity, may occur between phagocytes, UV radiation, and certain pharmacological agents.

  13. Complement C4-derived monocyte-directed chemotaxis-inhibitory factor. A molecular mechanism to cause polymorphonuclear leukocyte-predominant infiltration in rheumatoid arthritis synovial cavities.

    PubMed Central

    Matsubara, S.; Yamamoto, T.; Tsuruta, T.; Takagi, K.; Kambara, T.

    1991-01-01

    To reveal the mechanism of the lesser infiltration of monocytes in synovial cavities with rheumatoid arthritis despite the presence of chronic inflammation, the synovial fluid from 15 rheumatoid arthritis patients was analyzed with respect to leukocyte chemotaxis. The synovial fluid possessed strong chemotactic activity to polymorphonuclear leukocytes but rather suppressed one to monocytes. The synovial fluid contained two different inhibitory activities in monocyte chemotaxis. One, which also suppressed polymorphonuclear leukocyte chemotaxis, was identified as alpha 1 protease inhibitor. The other, with molecular weight of 8 kd, possessed the specificity to monocytes and shared the antigenicity with complement C4 but not with C3 or C5. A similar inhibitor was generated in normal human plasma when the classical pathway of the complement system was initiated with aggregated human IgG, while it was not when alternative pathway was initiated with zymosan. The small size factor in the synovial fluid, apparently derived from C4, seemed to be a cyto-directed factor that might block an early part of signal transduction system of monocytes in the chemotaxis. After removal of the small-size inhibitor, the synovial fluid exhibited chemotactic ability to monocytes. Therefore the apparent C4-derived factor might play a key role in the polymorphonuclear leukocyte-predominant infiltration in the synovial fluid of rheumatoid arthritis. PMID:2024711

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

    SciTech Connect

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

    1990-02-26

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

  15. Harvesting the noncirculating pool of polymorphonuclear leukocytes in rats by hetastarch exchange transfusion (HET): yield and functional assessment

    SciTech Connect

    Williams, J.H. Jr.; Moser, K.M.; Ulich, T.; Cairo, M.S.

    1987-11-01

    Isolation of polymorphonuclear leukocytes (PMN) provides an opportunity to study PMN activity in vitro and to label PMN for study of in vivo kinetics. However, simple phlebotomy (SP) of a small animal frequently yields too few PMN for in vitro handling, while PMN harvested from an induced-peritonitis may not accurately reflect PMN in a less stimulated state. We report a novel method of harvesting PMN from the circulation of rats, using hetastarch exchange transfusion (HET), which is both time and animal sparing. HET harvested 8-fold more PMN than SP. In vitro cell function was examined with assays of adherence, chemotaxis, bacterial killing, and superoxide generation. No significant (p less than 0.05) difference was found between PMN obtained by HET and pooled-PMN obtained by SP. In vivo function was examined following labeling with indium 111-oxine. The kinetics pattern described suggested normal migratory activity when compared to previous reports. The data demonstrate that rats possess a relatively large, noncirculating pool of PMN which is readily accessible by HET.

  16. Reduced intracellular oxidative metabolism promotes firm adhesion of human polymorphonuclear leukocytes to vascular endothelium under flow conditions.

    PubMed

    Montoya, M C; Luscinskas, F W; del Pozo, M A; Aragonés, J; de Landázuri, M O

    1997-08-01

    The interaction of polymorphonuclear leukocytes (PMN) with the vascular endothelium and their subsequent extravasation to the tissues is a key step during different physiological and pathological processes. In certain of these pathologies the oxygen tension becomes very low, leading to reduced cellular oxidative status. To evaluate the effect of lowering the intracellular redox status in the interaction of PMN with the endothelium, exposure to hypoxic conditions as well as treatment with different antioxidant agents was carried out. PMN exposure to hypoxia enhanced beta2 integrin-dependent adhesion to intercellular adhesion molecule-1-coated surfaces, concomitant with a decrease in the intracellular redox status of the cell. As occurs with hypoxia, treatment with antioxidants produced a decrease in the oxidation state of PMN. These agents enhanced adhesion of PMN to human umbilical vein endothelial cells stimulated with tumor necrosis factor-alpha (TNF-alpha), and this effect was also mediated by beta2 integrins LFA-1 and Mac-1. Adhesion studies under defined laminar flow conditions showed that the antioxidant treatment induced an enhanced adhesion mediated by beta2 integrins with a decrease in the fraction of PMN rolling on TNF-alpha-activated endothelial cells. The up-regulated PMN adhesion was correlated to an increase in the expression and activation of integrin Mac-1, without loss of L-selectin surface expression. Altogether, these results demonstrate that a reduction in the intracellular oxidative state produces an enhanced beta2 integrin-dependent adhesion of PMN to stimulated endothelial cells under conditions of flow.

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

    PubMed Central

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

    1991-01-01

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

  18. Purification of the active C5a receptor from human polymorphonuclear leukocytes as a receptor - G sub i complex

    SciTech Connect

    Rollins, T.E.; Siciliano, S.; Kobayashi, S.; Cianciarulo, D.N.; Bonilla-Argudo, V.; Collier, K.; Springer, M.S. )

    1991-02-01

    The authors have isolated, in an active state, the C5a receptor from human polymorphonuclear leukocytes. The purification was achieved in a single step using a C5a affinity column in which the C5a molecule was coupled to the resin through its N terminus. The purified receptor, like the crude solubilized molecule, exhibited a single class of high-affinity binding sites with a K{sub d} of 30 pM. Further, the binding of C5a retained its sensitivity to guanine nucleotides, implying that the purified receptor contained a guanine nucleotide-binding protein (G protein). SDS/PAGE revealed the presence of three polypeptides with molecular masses of 42, 40, and 36 kDa, which were determined to be the C5a-binding subunit and the {alpha} and {beta} subunits of G{sub i}, respectively. The 36- and 40-kDa polypeptides were identified by immunoblotting and by the ability of pertussis toxin to ADP-ribosylate the 40-kDa molecule. These results confirm their earlier hypothesis that the receptor exists as a complex with a G protein in the presence or absence of C5a. The tight coupling between the receptor and G protein should make possible the identification of the G protein(s) involved in the transduction pathways used by C5a to produce its many biological effects.

  19. [Influence of ion pump-inhibiting drugs on the accumulation of ofloxacin and grepafloxacin in human polymorphonuclear leukocytes].

    PubMed

    Orero, A; Cantón, E; Pemán, J; Velert, M M; Bermejo, M V

    2002-12-01

    In this study we tested the influence of three ion pump-inhibiting drugs (digoxin, omeprazole and verapamil) on the accumulation of ofloxacin and grepafloxacin in human polymorphonuclear leukocytes. Two assay conditions were established: cell preincubation with the drug for 30 or 60 minutes before addition of quinolone, or addition of both drugs simultaneously. The maximum I/E for ofloxacin is different depending on the assay conditions: 7.69+/-0.88; 5.64+/-1.91 and 3.56+/-1.04 for the assay without preincubation and with preincubation for 30 or 60 minutes at 37 masculine C, respectively. Similarly, grepafloxacin reached the following maximums: 61.27+/-3.04; 32.18+/-3.25 and 22.52+/-3.86. Digoxin did not significantly modify the accumulation of the quinolones, but it increased the I/E compared with the control. In general, omeprazole reduced the accumulation of both quinolones. When omeprazole and ofloxacin were added together, ofloxacin's I/E was significantly lower; however, for grepafloxacin, 60 minutes of preincubation were necessary. Verapamil induced a significant increase in the I/E for both quinolones when the cells were preincubated at 10 times the plasma concentration.

  20. Effects of Acer okamotoanum sap on the function of polymorphonuclear neutrophilic leukocytes in vitro and in vivo.

    PubMed

    An, Beum-Soo; Kang, Ji-Houn; Yang, Hyun; Yang, Mhan-Pyo; Jeung, Eui-Bae

    2013-02-01

    Sap is a plant fluid that primarily consists of water and small amounts of mineral elements, sugars, hormones and other nutrients. Acer mono (A. mono) is an endemic Korean mono maple which was recently suggested to have health benefits due to its abundant calcium and magnesium ion content. In the present study, we examined the effects of sap from Acer okamotoanum (A. okamotoanum) on the phagocytic response of mouse neutrophils in vivo and rat and canine neutrophils in vitro. We tested the regulation of phagocytic activity, oxidative burst activity (OBA) and the levels of filamentous polymeric actin (F-actin) in the absence and presence of dexamethasone (DEX) in vitro and in vivo. Our results showed that DEX primarily reduced OBA in the mouse neutrophils, and that this was reversed in the presence of the sap. By contrast, the phagocytic activity of the mouse cells was not regulated by either DEX or the sap. Rat and canine polymorphonuclear neutrophilic leukocytes (PMNs) responded in vitro to the sap in a similar manner by increasing OBA. However, regulation of phagocytic activity by the sap was different between the species. In canine PMNs, phagocytic activity was enhanced by the sap at a high dose, while it did not significantly modulate this activity in rat PMNs. These findings suggest that the sap of A. okamotoanum stimulates neutrophil activity in the mouse, rat and canine by increasing OBA in vivo and in vitro, and thus may have a potential antimicrobial effect in the PMNs of patients with infections.

  1. Effects of colchicine, vinblastine and nocodazole on polarity, motility, chemotaxis and cAMP levels of human polymorphonuclear leukocytes.

    PubMed

    Keller, H U; Naef, A; Zimmermann, A

    1984-07-01

    We present evidence for intrinsic polymorphonuclear leukocyte (PMN) polarity manifested in presence of microtubule-disrupting drugs. Polarization in response to colchicine correlated with the known dose-dependent effects of this drug on microtubule disassembly. The response to 10(-5) M colchicine, 10(-5) M vinblastine and 10(-6) M nocodazole was associated with stimulated motility and random locomotion. Responses elicited by microtubule-disrupting drugs differed from f-Met-Leu-Phe (fMLP)-induced polarization by functional and morphological criteria. Polarization, motility and orthokinesis responses were much weaker. Furthermore, ruffling was almost absent in PMNs polarized in response to colchicine, vinblastine or nocodazole. The response was inhibited by cytochalasin B, indicating that it is microfilament-dependent. We suggest that microtubule-disrupting drugs induce motility via structural changes in the cytoskeleton which act as signals for the motor apparatus. The intrinsic polarity manifested in the presence of microtubule-disrupting drugs could be reversed by an extracellular chemotactic gradient. Stimulated locomotion and motility in response to microtubule-disrupting drugs was only observed with initially spherical PMNs but not with initially motile cells. The findings provide an explanation for the numerous conflicting statements on the chemokinetic activities of these drugs. The role of cAMP in stimulated polarization and motility has been studied. Colchicine, vinblastine and nocodazole elicited a transient elevation of cAMP levels within 1 min of stimulation. cAMP elevation and stimulated motility were not quantitatively correlated.

  2. C5a-induced hemodynamic and hematologic changes in the rabbit. Role of cyclooxygenase products and polymorphonuclear leukocytes.

    PubMed Central

    Lundberg, C.; Marceau, F.; Hugli, T. E.

    1987-01-01

    Hemodynamic and hematologic changes occurring after intravascular complement activation have implicated the anaphylatoxins in this response. In this study, the hemodynamic and hematologic effects of purified C5a were investigated in rabbits; and involvement of prostanoids, histamine, and polymorphonuclear leukocytes (PMNs) were examined. The anaphylatoxin C5a induces a reversible systemic arterial hypotension which coincides with an increase in central venous pressure (CVP), decreased cardiac output (CO), increased plasma prostanoid levels, as well as neutropenia. Total peripheral resistance (TPR) remained unchanged. The cyclooxygenase inhibitor indomethacin abolished the C5a-induced hypotension and normalized plasma prostanoid levels without altering the C5a-induced neutropenia. The thromboxane (Tx) A2 synthetase inhibitor dazoxiben reduced TxB2 plasma levels and increased 6-keto-prostaglandin PGF1 alpha and PGE2 levels without altering the hypotensive response. However, with dazoxiben treatment both TPR and CVP decreased. The H2-receptor antagonist cimetidine reduced C5a-induced hypotension and diminished prostanoid release. Both the hypotensive response and elevated prostanoid release were observed after C5a challenge in animals rendered neutropenic prior to challenge. It is concluded that C5a-induced arterial hypotension in the rabbit is a PMN-independent reaction, mediated through cyclooxygenase products and, to some degree, by histamine. The mechanism producing systemic arterial hypotension does not seem to involve peripheral vasodilation but appears to be a secondary effect of pulmonary vasoconstriction, possibly mediated by TxA2. PMID:3115110

  3. The effect of an NADH oxidase inhibitor (hydrocortisone) on polymorphonuclear leukocyte bactericidal activity

    PubMed Central

    Mandell, Gerald L.; Rubin, Walter; Hook, Edward W.

    1970-01-01

    Polymorphonuclear neutrophils (PMN) from patients with chronic granulomatous disease of childhood have impaired bactericidal activity and are deficient in diphosphopyridine nucleotide, reduced form of, (NADH) oxidase. Since hydrocortisone had been shown to inhibit NADH oxidation, experiments were undertaken to determine the effect of hydrocortisone on several parameters of human PMN function. The phagocytic and bactericidal capacity of PMN with or without hydrocortisone (2.1 mM) was determined by quantitation of cell-free, cell-associated, and total bacteria. Phagocytosis of Staphylococcus aureus and several gram-negative rods was unimpaired by the presence of hydrocortisone in the media. In contrast, killing of bacteria was markedly impaired by hydrocortisone. After 30 min of incubation, there were 20-400 times as many bacteria surviving in hydrocortisone-treated PMN as in simultaneously run controls without hydrocortisone. The defect of intracellular killing noted in the presence of hydrocortisone was not related to impaired degranulation. Quantitative kinetic studies of degranulation revealed no difference in the release of granule associated acid phosphatase in hydrocortisone-treated and control PMN after phagocytosis. Electron microscopy of PMN also indicated that the presence of hydrocortisone had no effect on the extent of degranulation after phagocytosis. These observations were confirmed by studies using histochemical techniques to detect lysosomal enzymes. After phagocytosis, hydrocortisone-treated PMN demonstrated less NADH oxidase activity, oxygen consumption, and hydrogen peroxide production than postphagocytic control PMN. In addition, Nitro blue tetrazolium dye reduction was diminished in hydrocortisone-treated PMN. Thus, impairment of NADH oxidase activity in normal human PMN by hydrocortisone results in reduced intracellular killing of bacteria, diminished postphagocytic oxygen consumption, decreased ability to reduce Nitro blue tetrazolium, and

  4. 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.

  5. 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

  6. Effects of niflumic acid on polyphosphoinositide and oxidative metabolism in polymorphonuclear leukocytes from healthy and thermally injured rats.

    PubMed

    Tissot, M; Roch-Arveiller, M; Fontagne, J; Giroud, J P

    1992-12-01

    Thermal injury in rats leads to an impairment of polymorphonuclear leukocyte (PMN) functions, particularly oxidative metabolism and phosphoinositide turnover. As prostaglandin E2, which has immunosuppressive properties, is released in high levels after burn trauma, we investigated the in vitro and in vivo effects of a nonsteroidal antiinflammatory drug, niflumic acid, on oxidative and phosphoinositide metabolism in PMNs from healthy and burned rats. Given the role of fluoride ions on PMN, the influence of niflumic acid was compared with that of sodium fluoride (NaF) at equivalent doses of F-. In vitro, niflumic acid and sodium fluoride had no effect on oxidative metabolism in stimulated by formyl methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan (OZ) or nonstimulated PMNs from healthy and burned rats. Niflumic acid slightly increased the production of inositol phosphate by nonstimulated PMNs from healthy and burned rats. Niflumic acid and NaF partly restored the stimulating effect of FMLP on inositol phosphate production by PMNs from burned rats. In vivo treatment with niflumic acid and NaF increased the oxidative metabolism of PMNs from burned rats but not healthy rats. Niflumic acid, more than NaF, restored the activity of both stimulants on phosphoinositide metabolism in PMNs from burned rats. In conclusion, at non-antiinflammatory doses, while inhibiting cyclooxygenase activity, niflumic acid exerts a complex effect on the burn-induced depression of PMN functions. The fluoride anion induces similar but generally weaker effects and seems to be involved in the restoring effects of niflumic acid on PMN functions in burned rats.

  7. Magnesium-dependent adenosine triphosphatase as a marker enzyme for the plasma membrane of human polymorphonuclear leukocytes.

    PubMed

    Harlan, J; DeChatelet, L R; Iverson, D B; McCall, C E

    1977-02-01

    The adenosine triphosphatase (ATPase) activities of human polymorphonuclear leukocytes (PMNL) were studied with an assay that monitored the release of 32P-labeled inorganic pyrophosphate (32P1) from gamma-[32P]adenosine 5'-triphosphate (ATP). In cell homogenates, (Na+ + K+)-sensitive, ouabain-inhibitable ATPase comprised an insignificant fraction of the total ATPase activity. Additions of p-nitrophenyl phosphate and beta-glycerophosphate (substrates for nonspecific acid and alkaline phosphatases) and of tartrate (inhibitor of acid phosphatase) gave no indication of inhibition. This suggested that the assay was relatively specific for ATP hydrolysis. The activity was found to have a pH optimum of 8.7 and a Km for ATP of 0.6 mM. There was an absolute requirement for Mg2+, with other divalent cations substituting less efficiently. When the Mg2+-dependent ATPase activity of intact cells was compared with that in homogenized cells, no significant difference was observed. The activity in intact cells was linear with respect to incubation time up to at least l0 min. Trypan blue staining and lactate dehydrogenase assays revealed that greater than 92% of the PMNL remained intact and viable during the assay. No soluble ATPase was released from the cells under assay conditions. In following the distribution of gamma[32P]ATP and 32P2 counts became cell associated. Since the experimental evidence supports the observation that PMNL remain intact and viable and that ATP does not penetrate the cell under assay conditions, it is proposed that greater than 90% of the Mg2+-dependent ATPase of the human PMNL is associated with a plasma membrnae enzyme. This would qualify the enzyme for the role of a plasma membrane marker for future fractionation and isolation attempts.

  8. Occurrence of bacteria and polymorphonuclear leukocytes in fetal compartments at parturition; relationships with foal and mare health in the peripartum period.

    PubMed

    Hemberg, E; Einarsson, S; Kútvölgyi, G; Lundeheim, N; Bagge, E; Båverud, V; Jones, B; Morrell, J M

    2015-07-01

    This study investigated the relationship of the health of the newborn foal and (1) number of polymorphonuclear leukocytes (PMNLs) in the amniotic fluid, (2) bacteria present in the amniotic fluid and the venous umbilical blood, and (3) bacteria present in the uterus of the newly foaled mare. A further aim was to investigate relationships between the bacteriologic findings in the amniotic fluid, umbilical blood, and uterus postpartum. Samples were taken from 50 Standardbred trotter foaling mares from a well-managed stud in Sweden. Parturition was spontaneous in all cases. Length of pregnancy, parturition and postpartum complications, health status of the foal, the time between foaling and the expulsion of the placenta, and the number of postfoaling mares becoming pregnant after insemination were recorded. Amniotic fluid was collected when the amniotic vesicle was clearly visible; it was analyzed for bacteriology and occurrence of PMNLs. Umbilical blood was analyzed for the presence of bacteria and the concentration of serum amyloid A. The uterus of the mare was swabbed for bacteriology 6 to 17 hours postpartum. A blood sample was taken from the foal before administering plasma. The foals were divided into two groups: group 1 required up to 2 hours to rise after birth (≤2 hours; 31 foals) and group 2 required more than two hours (>2 hours; 19 foals). The length of gestation varied between 332 and 356 days; there was no significant difference in gestation length between the two foal groups. Partus and postpartum complications occurred in a significantly higher proportion of mares giving birth to group 2 foals than group 1 foals (P = 0.02), although uterine culture postpartum and the subsequent pregnancy rate per season were not different between the groups. Compromised health status was significantly higher among foals belonging to group 2 than group 1 (P = 0.001). Most of the amniotic samples contained 5% or less PMNLs. Only three samples contained more than 30

  9. Production of TNF-alpha by polymorphonuclear leukocytes during mechanical ventilation in the surfactant-depleted rabbit lung.

    PubMed

    Noda, Eri; Hoshina, Hiroaki; Watanabe, Hiroshi; Kawano, Toshio

    2003-12-01

    Previous studies showed that the production of tumor necrosis factor-alpha (TNF-alpha) and the number of recovered cells were much higher in the conventional mechanical ventilation (CMV) group than in the high-frequency oscillation (HFO) group at the end of mechanical ventilation in this model. But the type of cells that generated TNF-alpha in the lungs remained unclear. It was shown that the alveolar macrophage was the source of TNF-alpha in the early stage, but that in the later stage, the cells in the lung lavage fluid contained almost no macrophages. Thus we hypothesized that in the surfactant-depleted lung model, one of the sources of TNF-alpha after 4 hr of CMV is polymorphonuclear leukocyte (PMN), a type of cell which was numerous at that time. We performed the experiment in the same lung lavage model. The results were as follows. All PaO2 values for the HFO group were significantly greater than the corresponding values for the CMV group throughout the experiment (P < 0.05). More than 96% of the recovered cells of the lung lavage fluid at the end of ventilation were PMN. Cell counts after ventilation of HFO and CMV groups were 183.0 +/- 40.8 (mean +/- SD, n = 6)/microl and 1,106.0 +/- 310.0/microl, respectively (P < 0.05). Levels of rabbit TNF-alpha in the lavage fluid before and after 4 hr ventilation were 43.3 +/- 103.7 pg/ml and 2,406.0 +/- 1,525.1 pg/ml, respectively, in the CMV group. In the HFO group, these levels were 26.6 +/- 52.0 pg/ml and 613.3 +/- 362.2 pg/ml, respectively. The level of TNF-alpha was significantly greater in the CMV group after ventilation (P < 0.05). We performed RT-PCR analysis, in which we showed the presence of TNF-alpha mRNA in the intraalveolar cells (PMN) after 4 hr of CMV, and then demonstrated a positive immunofluorescence reaction to anti-TNF-alpha antibody in PMN separated from the lavage fluid. Our conclusion is that in this surfactant-depleted lung model, PMN is one of the sources of TNF-alpha in the lavage fluid

  10. Specificity of immunoglobulin M antibodies in normal human serum that participate in opsonophagocytosis and intracellular killing of Bacteroides fragilis and Bacteroides thetaiotaomicron by by human polymorphonuclear leukocytes.

    PubMed Central

    Bjornson, A B; Bjornson, H S; Kitko, B P

    1980-01-01

    Studies were performed to determine the specificity of immunoglobulin M (IgM) antibodies in normal human serum that participate in opsonophagocytosis and intracellular killing of Bacteroides fragilis 1365 and Bacteroides thetaiotaomicron 1343 by human polymorphonuclear leukocytes. Purified normal human IgM was adsorbed with washed heat-killed cells of the homologous strains and heterologous strains of B. fragilis, B. thetaiotaomicron, Bacteroides vulgatus, Bacteroides distasonis, and Bacteroides asaccharolyticus and with erythrocytes coated with outer membrane complex prepared from the homologous strains. Hypogammaglobulinemic serum was supplemented with the adsorbed IgM preparations, and the ability of the supplemented sera to support opsonophagocytosis and killing of B. fragilis 1365 and B. thetaiotaomicron 1343 by human polymorphonuclear leukocytes was measured in vitro under anaerobic conditions. Normal IgM adsorbed with heat-killed cells of B. fragilis 1365 and B. thetaiotaomicron 1343 or with erythrocytes coated with outer membrane complex prepared from these strains failed to restore the ability of hypogammaglobulinemic serum to support opsonophagocytosis and intracellular killing of the homologous strain. In contrast, adsorption of normal IgM with heat-killed cells of the heterologous strains did not alter its opsonophagocytosis-promoting activity for either test strain. These results indicated that the IgM antibodies in normal human serum that participate in opsonophagocytosis and intracellular killing of B. fragilis 1365 and B. thetaiotaomicron 1343 are directed against strain-specific antigenic determinants contained in the outer membrane complex. Images Fig. 5 Fig. 6 PMID:6160104

  11. 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.

  12. Abnormal mobility of neonatal polymorphonuclear leukocytes. Relationship to impaired redistribution of surface adhesion sites by chemotactic factor or colchicine.

    PubMed Central

    Anderson, D C; Hughes, B J; Smith, C W

    1981-01-01

    To determine the mechanism(s) of diminished, stimulated, and directed migration of neonatal (N) polymorphonuclear leukocytes (PMN), chemotactic factor (CF) sensory and PMN effector functions were studied in healthy N and adult or maternal controls (C). N PMN demonstrated high affinity binding for N-formyl-methionyl-leucyl-[3H]phenylalanine (fMLP), which was saturable between 40 and 100 nM as observed with C PMN. The kinetics of binding and the characteristics of dissociation of binding by N PMN were equivalent to control PMN. Both "threshold" and "peak" concentrations (1 and 10 nM, respectively) of fMLP effected comparable PMN chemiluminescence among neonates and controls. An equivalent threshold concentration (0.05 nM) of fMLP effected N and C PMN shape change in suspension, and a maximally effective concentration (5 nM) induced comparable bipolar configuration, although uropod formation was only 38 +/- 8% of N PMN, compared with 73 +/- 11% of C PMN (P less than 0.01). Striking abnormalities of N PMN adherence were identified: mean +/- SD base-line (unstimulated) N adherence values (39 +/- 8%) were equal to C (38 +/- 9%), but diminished increments in response to single CF stimuli were noted among N (fMLP: 42 +/- 7% (N), 70 +/- 11% (C); C5a: 41 +/- 6% (N), 68 +/- 6% (C); BCF: 41 +/- 6% (N), 63 +/- 9% (C), P less than 0.01 for each CF). On sequential exposure to increasing concentrations of CF N PMN failed to demonstrate expected decreased adherence values; sequential stimuli with fMLP (0.1 nM, 10 nM) or C5a (8 microgram protein/ml, 32 microgram protein/ml) effected mean +/- 1 SD values of 51 +/- 9% (N), 30 +/- 9% (C), and 34 +/- 10 (N), 48 +/- 14% (C), respectively. As demonstrated with a latex bead-binding technique, N PMN failed to redistribute adhesion sites to the cell's tail under the same experimental conditions; in 21 N samples studied, restricted unipolar binding occurred in 33 +/- 8% (fMLP) or 37 +/- 7% (C5a) of PMN in contrast to C values of 70% (f

  13. Formyl peptide-induced chemotaxis of human polymorphonuclear leukocytes does not require either marked changes in cytosolic calcium or specific granule discharge. Role of formyl peptide receptor reexpression (or recycling).

    PubMed Central

    Perez, H D; Elfman, F; Marder, S; Lobo, E; Ives, H E

    1989-01-01

    We examined the role of intracellular and extracellular calcium on the ability of human polymorphonuclear leukocytes to migrate chemotactically and reexpress (or recycle) formyl peptide receptors when challenged with the synthetic chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP). Extracellular calcium was not required for either optimal chemotactic responses or receptor reexpression. Depletion and chelation of intracellular calcium resulted in significant diminution in the ability of polymorphonuclear leukocytes to release the specific granule constituents lactoferrin and vitamin B12-binding protein during the process of chemotaxis, but had no effect on the capability of these cells to respond chemotactically. Similarly, chelation of intracellular calcium did not affect the ability of these cells to reexpress a population of formyl peptide receptors. Inhibition of receptor reexpression, by a nonagglutinating derivative of wheat-germ agglutinin, was associated with inhibition of chemotactic responses to FMLP. Thus, it appears that large changes in cytosolic free calcium are not necessary for formyl peptide-induced polymorphonuclear leukocyte chemotaxis. In contrast, continuous reexpression (or recycling) of formyl peptide receptors is required for polymorphonuclear leukocyte chemotactic responses to FMLP, a process that appears to be independent from specific granule fusion with plasma membrane. PMID:2723068

  14. ALTERATIONS IN BLOOD LEUKOCYTES OF G551D-BEARING CYSTIC FIBROSIS PATIENTS UNDERGOING TREATMENT WITH IVACAFTOR

    PubMed Central

    Bratcher, Preston E.; Rowe, Steven M.; Reeves, Ginger; Roberts, Tambra; Szul, Tomasz; Harris, William T.; Tirouvanziam, Rabindra; Gaggar, Amit

    2015-01-01

    Background Ivacaftor improves clinical outcome by potentiation of mutant G551D CFTR. Due to the presence of CFTR in monocytes and polymorphonuclear neutrophils (PMNs), we hypothesized that ivacaftor may impact leukocyte activation. Methods We examined blood leukocytes from G551D CF subjects prior to and at one and six months after receiving ivacaftor. Blood leukocytes from ivacaftor-naïve G551D, F508del, and healthy controls were also treated with ivacaftor ex vivo to assess mutation-specific effects. Results Compared to healthy controls, G551D CF subjects had significantly higher expression of active CD11b on PMNs and of CD63 on monocytes, which were normalized by in vivo ivacaftor treatment. Ex vivo exposure to ivacaftor of blood cells from G551D, but not F508del and healthy subjects, resulted in changes in CXCR2 and CD16 expression on PMNs. Conclusions In vivo and ex vivo exposure of G551D CF leukocytes to ivacaftor resulted in an altered activation profile, suggesting mutation-specific leukocyte modulation. PMID:25769931

  15. 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

  16. [Luminol-enhanced chemiluminescence of rabbit polymorphonuclear leukocytes: the nature of oxidants that directly induce luminol oxidation].

    PubMed

    Roshchupkin, D I; Belakina, N S; Murina, M A

    2006-01-01

    The present work deals with the reaction pathways, including the formation of hydroxyl radicals and chloroamines, which lead to luminol chemiluminescence caused by hypochlorite generation in a suspension of stimulated rabbit polymorphnonuclear leukocyte. Luminol-enhanced (0.02 mM) chemiluminescence of leukocytes stimulated by phorbol 12-myristate 13-acetate does not change in the presence of dimethyl sulfoxide at moderate concentrations (0.02-2.6 mM) at which it must show the specific ability to scavenge hydroxyl radicals. It suggests that no generation of hydroxyl radical with the participation of hypochlorite and superoxide anion takes place after the stimulation of polymorphnonuclear leukocytes. A high dimethyl sulfoxide concentrations (260 mM) a significant fall in chemiluminescence intensity, due to direct interaction of the scavenger with hypochlorite, is observed. Chemiluminescence intensity rose if luminol was added to a leukocyte suspension preliminary stimulated for 10 min. The effect results from the accumulation of hydrogen peroxide but not chloroamines. Exogenic amino acids and taurin at high concentrations (3-15 mM) weaken the chemiluminescence. The data obtained suggest that chemiluminescence in the system studied results predominantly from the direct initial reaction of hypochlorite with luminol. The chemiluminescence intensity is enhanced by hydrogen peroxide via the oxidation of luminol oxidation products.

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

    PubMed Central

    1983-01-01

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

  18. In vitro effects of aqueous seeds extract of Acacia cyanophylla on the opsonized zymosan-induced superoxide anions production by rat polymorphonuclear leukocytes.

    PubMed

    El Abbouyi, Ahmed; Toumi, Mina; El Hachimi, Youssef; Jossang, Akino

    2004-03-01

    In vitro studies were carried out in rat pleural polymorphonuclear leukocytes (PMNs) activated by opsonized zymosan (OZ) to investigate the effects of aqueous extract from Acacia cyanophylla seeds on superoxide anions generation. PMNs were collected, after induction of an acute inflammatory reaction, by injection in the rat pleural cavity, of a suspension of calcium pyrophosphate (CaPP) crystals (pleurisy with CaPP) or serum (pleurisy with serum). The results obtained indicate that Acacia cyanophylla aqueous seeds extract had, in vitro, a significant stimulatory effect, in a dose dependent manner, on the PMN superoxide anions generation. It also corrected the diminution of superoxide anions production induced by diclofenac pre-treated PMNs. It could be concluded from the results of this study that the stimulatory properties of Acacia cyanophylla seeds aqueous extract may at least be due to the presence of polyphenols such tannins and/or lignins. Further investigations are needed to determine clearly the mechanisms mediating the generation of superoxide radicals in this phenomenon.

  19. Altered postreceptor signal transduction of formyl-Met-Leu-Phe receptors in polymorphonuclear leukocytes of patients with non-insulin-dependent diabetes mellitus.

    PubMed

    Fóris, G; Paragh, G; Dezsõ, B; Keresztes, T; Balogh, Z; Szabó, J

    1998-01-01

    The signal transduction of the formyl-Met-Leu-Phe (FMLP) receptor in polymorphonuclear leukocytes (PMNLs) from patients with non-insulin-dependent diabetes mellitus (NIDDM) was compared to that of PMNLs obtained from healthy volunteers. According to our previous studies in this group of patients neither the decrease in insulin binding capacity nor the enhanced insulin-degrading enzyme activity was involved. In control PMNLs, 10 nM FMLP induced a pertussis toxin-sensitive increase in phosphatidyl inositol (PI) cleavage and a subsequent Ca2+ signaling from the intracellular pools. On the other hand, the FMLP-induced protein kinase C (PKC) activation and translocation into the membrane could not be detected in these cells via the measurement of 32P incorporation into histone. In contrast, in PMNLs of this special group of patients suffering from NIDDM the FMLP stimulus produced a significantly low increase in PI cleavage and Ca2+ signaling from the intracellular pools. Moreover, in resting PMNLs of these patients with NIDDM, not only the [Ca2+]i but also the membrane-bound PKC activity was found to be significantly increased. In addition, PKC translocation into the cell membrane of diabetic PMNLs could be further triggered with FMLP as judged by the measurement of 32P incorporation into histone. Based on these results, it appears that the signaling of FMLP receptors in PMNLs of some NIDDM patients may have an alternative pathway through Ca2+ influx from extracellular medium, arachidonic acid cascade, and PKC activation.

  20. Intravital Förster resonance energy transfer imaging reveals osteopontin-mediated polymorphonuclear leukocyte activation by tumor cell emboli.

    PubMed

    Kamioka, Yuji; Takakura, Kanako; Sumiyama, Kenta; Matsuda, Michiyuki

    2017-02-01

    Myeloid-derived suppressor cells (MDSCs) cause paraneoplastic leukemoid reactions and facilitate tumor cell metastasis. However, the interaction of MDSCs with tumor cells in live tissue has not been adequately visualized. To accomplish this task, we developed an intravital imaging protocol to observe metastasized tumor cells in mouse lungs. For visualization of the activation of MDSCs, bone marrow cells derived from transgenic mice expressing a Förster resonance energy transfer biosensor for ERK were implanted into host mice. Under a two-photon excitation microscope, numerous polymorphonuclear cells (PMNs) were found to infiltrate the lungs of tumor-bearing mice in which 4T1 mammary tumor cells were implanted into the footpads. By Förster resonance energy transfer imaging, we found ERK activation in PMNs around the 4T1 tumor emboli in the lungs. Because antibody array analysis implied the involvement of osteopontin (OPN) in the metastasis of 4T1 cells, we further analyzed the effect of OPN knockdown. The OPN knockdown in 4T1 cells did not affect the cell growth, but markedly suppressed lung metastasis of 4T1 cells and ERK activation in PMNs in the lung. Intravenous injection of recombinant OPN restored the lung metastasis of OPN-deficient 4T1 cells, suggesting that OPN functioned in a paracrine manner. It has been reported that ERK activation of neutrophils causes NETosis and that PMNs promote metastasis of tumor cells by NETosis. In agreement with previous reports, the NETosis inhibitor DNase I inhibited lung metastasis of 4T1 cells. These observations suggest that OPN promotes metastasis of 4T1 cells by activating PMNs and inducing NETosis.

  1. Effect of single oral dose of azithromycin, clarithromycin, and roxithromycin on polymorphonuclear leukocyte function assessed ex vivo by flow cytometry.

    PubMed Central

    Wenisch, C; Parschalk, B; Zedtwitz-Liebenstein, K; Weihs, A; el Menyawi, I; Graninger, W

    1996-01-01

    Azithromycin was given as a single oral dose (20 mg/kg of body weight) to 12 volunteers in a crossover study with roxithromycin (8 to 12 mg/kg) and clarithromycin (8 to 12 mg/kg). Flow cytometry was used to study the phagocytic functions and the release of reactive oxygen products following phagocytosis by neutrophil granulocytes prior to administration of the three drugs, 16 h after azithromycin administration, and 3 h after clarithromycin and roxithromycin administration. Phagocytic capacity was assessed by measuring the uptake of fluorescein isothiocyanate-labeled bacteria. Reactive oxygen generation after phagocytosis of unlabeled bacteria was estimated by the amount of dihydrorhodamine 123 converted to rhodamine 123 intracellularly. Azithromycin resulted in decreased capacities of the cells to phagocytize Escherichia coli (median [range], 62% [27 to 91%] of the control values; P < 0.01) and generate reactive oxygen products (75% [34 to 26%] of the control values; P < 0.01). Clarithromycin resulted in reduced phagocytosis (82% [75 to 98%] of control values; P < 0.01) but did not alter reactive oxygen production (84% [63 to 113%] of the control values; P > 0.05). Roxithromycin treatment did not affect granulocyte phagocytosis (92% [62 to 118%] of the control values; P > 0.05) or reactive oxygen production (94% [66 to 128%] of the control value; P > 0.05). No relation between intra- and/or extracellular concentrations of azithromycin and/or roxithromycin and the polymorphonuclear phagocyte function and/or reactive oxygen production existed (P > 0.05 for all comparisons). These results demonstrate that the accumulation of macrolides in neutrophils can suppress the response of phagocytic cells to bacterial pathogens after a therapeutic dose. PMID:8878577

  2. 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.

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

    PubMed Central

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

    2016-01-01

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

  4. 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.

  5. Differential expression of interleukin-8 by polymorphonuclear leukocytes of two closely related species, Ovis canadensis and Ovis aries, in response to Mannheimia haemolytica infection.

    PubMed

    Herndon, Caroline N; Foreyt, William J; Srikumaran, Subramaniam

    2010-08-01

    The pneumonic lesions and mortality caused by Mannheimia haemolytica in bighorn sheep (BHS; Ovis canadensis) are more severe than those in the related species, domestic sheep (DS; Ovis aries), under both natural and experimental conditions. Leukotoxin (Lkt) and lipopolysaccharide (LPS) are the most important virulence factors of this organism. One hallmark of pathogenesis of pneumonia is the influx of polymorphonuclear leukocytes (PMNs) into the lungs. Lkt-induced cytolysis of PMNs results in the release of cytotoxic compounds capable of damaging lung tissue. Interleukin-8 (IL-8) is a potent PMN chemoattractant. The objective of the present study was to determine if there is differential expression of IL-8 by the macrophages and PMNs of BHS and DS in response to M. haemolytica. Macrophages and PMNs of BHS and DS were stimulated with heat-killed M. haemolytica or LPS. IL-8 expression by the cells was measured by enzyme-linked immunosorbent assays and real-time reverse transcription-PCR (RT-PCR). The PMNs of BHS expressed severalfold higher levels of IL-8 than those of DS upon stimulation. Lesional lung tissue of M. haemolytica-infected BHS contained significantly higher levels of IL-8 than nonlesional tissue. The bronchoalveolar lavage (BAL) fluid of infected BHS also contained higher levels of IL-8 than that of infected DS. Depletion of IL-8 reduced migration of PMNs toward BAL fluid by approximately 50%, indicating that IL-8 is integral to PMN recruitment to the lung during M. haemolytica infection. Excessive production of IL-8, enhanced recruitment of PMNs, and PMN lysis by Lkt are likely responsible for the severity of the lung lesions in M. haemolytica-infected BHS.

  6. Derivative of wheat germ agglutinin specifically inhibits formyl-peptide-induced polymorphonuclear leukocyte chemotaxis by blocking re-expression (or recycling) of receptors

    SciTech Connect

    Perez, H.D.; Elfman, F.; Lobo, E.; Sklar, L.; Chenoweth, D.; Hooper, C.

    1986-03-01

    The mechanism of action of a derivative of wheat germ agglutinin (WGA-D) which specifically and irreversibly inhibits N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced polymorphonuclear leukocyte (PMN) chemotaxis was examined. At a concentration that completely inhibited PMN chemotaxis, WGA-D had no effect on either the uptake or release of (/sup 3/H)-FMLP by PMN. Similarly, WGA-D did not affect either the short-term binding to, or internalization by, PMN of a fluoresceinated FMLP analog. WGA-D did interfere, however, with the re-expression (or recycling) of FMLP receptors by PMN that had been preincubated with 1 ..mu..M FMLP for 10 min at 4/sup 0/C. This effect was specific for WGA-D, because it was not observed when concanavalin A was used. Scatchard plot analysis of FMLP binding to PMN after receptor re-expression demonstrated that WGA-D-treated PMN had a significant diminution in the number of high affinity receptors. WGA-D-mediated inhibition of FMLP receptor re-expression was associated with inhibition of FMLP-induced PMN chemotaxis, but had no effect on either FMLP-induced PMN superoxide anion generation or degranulation. Studies using (/sup 12/%I)-WGA-D demonstrated that PMN did not internalize WGA-D spontaneously. The data indicate that WGA-D perhaps by binding to the FMLP receptor, inhibits FMLP-induced PMN chemotaxis by blocking the re-expression (or recycling) of a population of receptors required for continuous migration.

  7. Mannose-inhibitable adhesins and T3-T7 receptors of Klebsiella pneumoniae inhibit phagocytosis and intracellular killing by human polymorphonuclear leukocytes.

    PubMed Central

    Pruzzo, C; Debbia, E; Satta, G

    1982-01-01

    It has recently been shown that Klebsiella pneumoniae strains adhere to human epithelial cells and that adherence is mediated by mannose-inhibitable adhesins which are also receptors for coliphages T3 and T7. We have now found that Klebsiella strain K59, which adheres to human epithelial cells and carries the receptors for coliphages T3 and T7, adheres to human polymorphonuclear leukocytes (PMN) at 4 degrees C. Strains KRTT1 and KRTT2, which are spontaneous mutants unable to adsorb coliphages T3 and T7 and adhere to human epithelial cells, at this temperature did not adhere to PMN. Adherence of K59 cells to PMN at 4 degrees C was inhibited by D-mannose, by UV-inactivated T7 phages, and by pepsin-digested anti-K59 antibodies absorbed with KRTT1 cells. At 37 degrees C the number of PMN with KRTT bacteria associated was fourfold higher than at 4 degrees C. On the contrary, the number of PMN with K59 bacteria associated at this temperature was fourfold lower than at 4 degrees C. Phagocytosis and intracellular killing experiments performed at 37 degrees C showed that KRTT1 and KRTT2 were phagocytized and killed at a higher rate than K59. After blocking of the mannose-inhibitable adhesins and T3-T7 receptors (MIAT) by D-mannose, UV-inactivated bacteriophage T7, or specific antibodies, K59 cells became more sensitive to phagocytosis and intracellular killing at 37 degrees C. K59 cells lysogenic for prophage AP3 were approximately as sensitive to phagocytosis and intracellular killing by human PMN as strains KRTT1 and KRTT2. Unencapsulated Klebsiella strains isolated from clinical specimens were found to carry MIAT most often. Four such strains were found much more resistant to phagocytosis and intracellular killing than their spontaneous mutants resistant to bacteriophages T3 and T7. PMID:7047402

  8. Maintenance of α1-antitrypsin activity by means of co-application of hypochlorous acid-scavengers in vitro and in the supernatant of polymorphonuclear leukocytes

    PubMed Central

    Schönberg, Maria; Reibetanz, Uta; Rathmann, Sophie; Leßig, Jacqueline

    2012-01-01

    Tissue destruction, pain and loss of function in chronically inflamed tissues can result from noxious agents released from myeloperoxidase (MPO) and its highly reactive product hypochlorous acid (HOCl) or proteases such as neutrophil elastase (NE). Currently there exists a high demand for medications that provide gentle treatments, free from side effects inherent in those prescribed today. One method to circumvent side effects is through the use of locally applied drug delivery. In contrast to systemic therapy, the main advantages of transport systems are the low dosages of drug with a time-controlled delivery. The aim of this study was to ascertain interactions of NE and its inhibitor α1-antitrypsin (AT), the influence of hypochlorous acid (HOCl), as well as its scavengers, in order to define an effective mixture of drugs acting in a synergistic way which can be applied by means of drug delivery systems. These investigations determine the effective amounts of AT/HOCl-scavengers that drug mixtures need for delivery under inflammatory conditions in order to prevent tissue damage. AT was shown to inhibit NE in a dose-dependent manner, whereas a physiological concentration of 1.14 µM AT caused a significant NE inhibition (78%, pH 7.5). The concomitant existence of MPO/HOCl inactivated AT in a dose-dependent manner as well. To regain AT efficacy, HOCl-scavengers, such as l-methionine, α-aminosalicylic acid and cefoperazone were additionally applied. Finally, AT was assembled as surface layer onto layer-by-layer biopolymer-coated microcarriers and carrier phagocytosis by polymorphonuclear leukocytes could be shown. PMID:23507783

  9. IFN-γ regulates xanthine oxidase-mediated iNOS-independent oxidative stress in maneb- and paraquat-treated rat polymorphonuclear leukocytes.

    PubMed

    Singh, Deepali; Kumar, Vinod; Singh, Chetna

    2017-03-01

    Maneb (MB) and paraquat (PQ) provoke oxidative stress-mediated cell damage. Role of xanthine oxidase (XO) in oxidative stress and its association with nitric oxide (NO)/NO synthase (NOS) have been widely reported. While inducible NOS (iNOS) is implicated in MB+PQ-induced toxicity in rat polymorphonuclear leukocytes (PMNs), role of XO and its alliance with iNOS have not yet been established. The study investigated the role of XO in MB+PQ-induced oxidative stress in rat PMNs and its regulation by iNOS and inflammatory cytokines. MB+PQ-augmented reactive oxygen species (ROS), superoxide, nitro-tyrosine, lipid peroxidation (LPO), and nitrite levels along with the catalytic activity of iNOS, superoxide dismutase (SOD), and XO. XO inhibitor, allopurinol (AP), alleviated MB+PQ-induced changes except nitrite content and iNOS activity. Conversely, an iNOS inhibitor, aminoguanidine, mitigated MB+PQ-induced LPO, nitrite, iNOS, and nitro-tyrosine levels; however, no change was observed in ROS, SOD, and XO. Nuclear factor-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), tumor necrosis factor-alpha (TNF-α) inhibitor, pentoxyfylline, and an anti-inflammatory agent, dexamethasone, attenuated MB+PQ-induced increase in XO, superoxide, and ROS with parallel reduction in the expression of interferon-gamma (IFN-γ), TNF-α, and interleukin-1β (IL-1β) in rat PMNs. Exogenous IFN-γ, TNF-α, and IL-1β enhanced superoxide, ROS, and XO in the PMNs of control and MB+PQ-treated rats; however, IFN- γ was found to be the most potent inducer. Moreover, AP ameliorated cytokine-induced free radical generation and restored XO activity towards normalcy. The results thus demonstrate that XO mediates oxidative stress in MB+PQ-treated rat PMNs via iNOS-independent but cytokine (predominantly IFN-γ)-dependent mechanism.

  10. Circulating promyelocytes and low levels of CD16 expression on polymorphonuclear leukocytes accompany early-onset periodontitis.

    PubMed Central

    Nemoto, E; Nakamura, M; Shoji, S; Horiuchi, H

    1997-01-01

    Early-onset periodontitis (EOP) is characterized by rapidly progressive alveolar bone loss, chemotactic defects of neutrophils, and significant familial aggregation. We found immature myeloid lineage cells, defined as promyelocytes, in the peripheral blood in patients with EOP. A hematological examination of peripheral blood cells showed normal reference values regarding cell proportions. Flow cytometry revealed significantly lower expression of CD16, a glycosylphosphatidylinositol (GPI)-anchored protein, on peripheral neutrophils in patients compared with those in age- and sex-matched healthy controls, whereas the levels of CD11a and CD11b expression were similar. The chemotactic response of neutrophils was lower toward not only formyl-methionyl-leucyl-phenylalanine but also complement fragment C5a than that of healthy controls. The expression of another GPI-anchored protein, CD14, was equally expressed by controls and patients. Therefore, the low level of CD16 expression was not due to the incomplete synthesis of the GPI anchor. GPI anchors of CD16 on neutrophils from controls and patients were both partially resistant to phosphatidylinositol-specific phospholipase C. The presence of promyelocytes in peripheral blood, low expression of CD16, and low chemotactic response of neutrophils suggest that patients with EOP have an abnormal maturation system in myeloid lineage cells in the bone marrow, which may be associated with the onset and course of EOP. PMID:9284170

  11. Relationship between zinc malnutrition and alterations in murine peripheral blood leukocytes

    SciTech Connect

    King, L.E.; Morford, L.A.; Fraker, P.J. )

    1991-03-15

    Studies using a murine model have shown that the immune system responds rapidly and adversely to zinc deficiency. The extent of alteration of peripheral blood leukocytes (PBL) and immunoglobulin levels were investigated in four zinc dietary groups: zinc adequate (ZA); restricted fed zinc adequate (RZA); marginal zinc deficient (MZD, 72-76% of ZA mouse weight); and severely zinc deficient. The peripheral white blood cell count was 3.66 {plus minus} 1.08 {times} 10{sup 6} cells/ml for ZA mice decreasing by 21%, 28% and 54% for RZA, MZD and SZD mice respectively. An equally dramatic change in the flow cytometric light scatter profile was found. ZA mice had 66% lymphocytes and 21% polymorphonuclear granulocytes (PMN) in their peripheral blood while MZD and SZD mice contained 43% and 30% lymphocytes and 40% and 60% PMNs respectively. Analysis of the phenotypic distribution of specific classes of lymphocytes revealed ZA blood contained 25% B-cells and 40% T-cells (CD5{sup +}). B-cells decreased 40-50% for RZA and MZD mice and 60-70% for SZD mice. The decline in CD5{sup +} T-cells was more modest at 30% and 45% for MZD and SZD mice. A nearly 40% decline in both T{sub h} and T{sub c/s} cells was noted for both MZD and SZD mice. Radioimmunoassay of serum for changes in IgM and IgG content revealed no change among dietary groups while serum zinc decreased 10% for RZA mice and 50% for both MZD and SZD mice. The authors conclude that peripheral blood differential counts in concert with total B and T-cell phenotype may serve as indicators of zinc status while serum zinc and Ig will not.

  12. The in vivo effect of leukotriene B4 on polymorphonuclear leukocytes and the microcirculation. Comparison with activated complement (C5a des Arg) and enhancement by prostaglandin E2.

    PubMed Central

    Movat, H. Z.; Rettl, C.; Burrowes, C. E.; Johnston, M. G.

    1984-01-01

    The effect of synthetic leukotriene B4 (LTB4) on chemotaxis in vivo (51Cr-polymorphonuclear leukocyte [PMN] accumulation) was examined and its potency compared with that of C5a des Arg-containing zymosan-activated plasma (ZAP). On a molar basis the amount of C5a des Arg calculated to be in our preparation of ZAP was found to be up to approximately 80 times more potent than LTB4, although in vitro the two chemotaxins have been reported to be about equipotent. ZAP is more representative of what may happen in vivo than its principal constituent C5a des Arg, but for a more precise comparison the purified and isolated peptide will have to be compared with synthetic LTB4. Whereas ZAP induced severe PMN-dependent microvascular injury (increase in vessel permeability [125I-albumin] and hemorrhage [59Fe-erythrocytes]), LTB4 only induced an increase in vascular permeability, and this occurred only in the presence of simultaneously injected prostaglandin E2 (PGE2). PGE2 also enhanced substantially the number of PMNs and the amount of exuded plasma at injection sites of the chemotaxins. However, unlike in two other reports, LTB4 did not cause an immediate transient increase in vessel permeability, nor did it enhance the permeability-increasing effect of bradykinin. Furthermore, unlike PGE2 LTB4 did not induce an increase in blood flow, but a decrease (57Co-microspheres). It is concluded that LTB4 may act as a host-derived chemoattractant in vivo, but, compared with that of ZAP (primarily activated complement), its role in acute inflammation is probably less significant than that of the complement-derived chemotaxin(s). Images Figure 8 Figure 9 Figure 10 Figure 11 PMID:6326579

  13. Transient Receptor Potential Ankyrin 1 Channel Expression on Peripheral Blood Leukocytes from Rheumatoid Arthritic Patients and Correlation with Pain and Disability

    PubMed Central

    Pereira, Ione; Mendes, Saulo J. F.; Pereira, Domingos M. S.; Muniz, Thayanne F.; Colares, Valderlane L. P.; Monteiro, Cinara R. A. V.; Martins, Mahiba M. R. de S.; Grisotto, Marcos A. G.; Monteiro-Neto, Valério; Monteiro, Sílvio G.; Calixto, João B.; Brain, Susan D.; Fernandes, Elizabeth S.

    2017-01-01

    Patients with rheumatoid arthritis (RA) suffer from pain and joint disability. The transient receptor potential ankyrin 1 (TRPA1) channel expressed on sensory neurones and non-neuronal cells mediates pain transduction and inflammation and it has been implicated in RA. However, there is little information on the contribution of TRPA1 for human disease. Here, we investigated the expression of TRPA1 on peripheral blood leukocytes and the circulating levels of its endogenous activators 4-hydroxynonenal (4-HNE) and hydrogen peroxide (H2O2) in RA patients treated or not with the anti-rheumatic leflunomide (LFN) or the anti-TNFα adalimumab (ADA). We also assessed whether TRPA1 expression correlates with joint pain and disability, in addition to the immune changes in RA. TRPA1 expression on peripheral blood leukocytes correlated with pain severity and disability. TRPA1 levels on these cells were associated with the numbers of polymorphonuclear and the activation of CD14+ cells. No correlations were found between the lymphocyte population and TRPA1 expression, pain or disability. Patients recently diagnosed with RA expressed increased levels of TRPA1 on their leukocytes whilst treatment with either LFN or ADA down-regulated this receptor probably by reducing the numbers of polymorphonuclears and the activation of CD14+ cells. We suggest that the activation levels of CD14+ cells, the numbers of PMNs in the peripheral blood and the expression of TRPA1 on peripheral blood leukocytes correlate with RA progression, affecting joint pain sensitivity and loss of function. PMID:28239353

  14. Severe microvascular injury induced by lysosomal releasates of human polymorphonuclear leukocytes. Increase in vasopermeability, hemorrhage, and microthrombosis due to degradation of subendothelial and perivascular matrices.

    PubMed Central

    Movat, H. Z.; Wasi, S.

    1985-01-01

    The purpose of this study was to assess the nature of the lesions in the microcirculation of the dermis of rabbits induced with lysosomal releasates of human polymorphonuclear leukocytes (PMNs). No attempt was made in the studies presented in this publication to deal with the offending agent in the releasate. Four parameters of microvascular injury were quantitated: increase in vascular permeability with 125I-labeled serum albumin, hemorrhage with 59Fe-labeled erythrocytes, accumulation (aggregation) of platelets with 111In-labeled platelets. In one experiment accumulation of 51Cr-PMNs was investigated. The lysosomal releasate induced a rapid increase in vasopermeability, but both hemorrhage and exudate formation peaked 1 hour after intradermal injection. Platelet accumulation was also demonstrable in these lesions, and microthrombosis was a very prominent feature. The microvascular injury, including microthrombosis, could be elicited also in animals rendered leukopenic with nitrogen mustard. Simultaneous injection of prostaglandin E2 with the releasate enhanced the microvascular injury. The morphologic changes in the microcirculation of the rabbit's dermis were assessed in lesions 5 minutes to 5 hours old. Several changes were encountered, primarily in the wall of venules and small veins and to a lesser degree in small arteries and capillaries. Ultrastructurally very early lesions (up to 15 minutes) had gaps or spaces in the endothelium, resembling those induced by mediators such as histamine or bradykinin. Older lesions were different, quite characteristic, and represent the hallmark of these lesions. Lysis and disappearance of vascular basement membrane, of perivascular collagen, and of the internal elastic lamina were a frequent finding, best demonstrable when microthrombi did not abut on vessel walls. Cellular components of vessels (endothelium, pericytes, smooth muscle) showed fragmentation, leading to complete disappearance of cellular elements. These

  15. Feed-derived volatile basic nitrogen increases reactive oxygen species production of blood leukocytes in lactating dairy cows.

    PubMed

    Tsunoda, Ei; Gross, Josef J; Kawashima, Chiho; Bruckmaier, Rupert M; Kida, Katsuya; Miyamoto, Akio

    2017-01-01

    The present study investigated over 9 months the changes of fermentative quality of total mixed rations (TMR) containing grass silage (GS) as a major component, associated with changes in the volatile basic nitrogen (VBN) levels in an experimental dairy farm. Effects of VBN levels in TMR on metabolic parameters, reactive oxygen species (ROS) production by blood polymorphonuclear leukocytes (PMNs) and conception rates for dairy cows were analyzed. According to VBN levels in TMR during survey periods, three distinct phases were identified; phase A with low VBN; phase B with high VBN; and phase C with mid-VBN. Metabolic parameters in blood were all within normal range. However, during phases B and C, nitrogen metabolic indices such as blood urea nitrogen and milk urea nitrogen showed higher levels compared to those in phase A, and a simultaneous increase in ROS production by blood PMNs and the load on hepatic function in metabolic parameters was observed in the cows with a lower conception rate. This suggests that feeding TMR with elevated VBN levels due to poor fermented GS results in stimulation of ROS production by PMNs by ammonia, and negatively affects metabolism and reproductive performance in lactating dairy cow.

  16. 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

  17. 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.

  18. 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)

  19. 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.

  20. 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.

  1. Trans-10, cis-12-conjugated linoleic acid increases phagocytosis of porcine peripheral blood polymorphonuclear cells in vitro.

    PubMed

    Kang, Ji-Houn; Lee, Geun-Shik; Jeung, Eui-Bae; Yang, Mhan-Pyo

    2007-01-01

    Trans-10, cis-12-conjugated linoleic acid (t10c12-CLA) has been shown to alter immune function. PPARgamma has been shown to potentially play an important role in regulating inflammatory and immune responses by modulating the activity of monocytes and macrophages. Previous studies have indicated that the phagocytic capacity of porcine peripheral blood polymorphonuclear cells (PMN) was enhanced by the culture supernatant fraction from t10c12-CL-stimulated porcine peripheral blood mononuclear cells (PBMC) but not by t10c12-CLA itself. In the present study, we examined the effects of t10c12-CLA on PPARgamma and TNF-alpha expression of porcine PBMC and the phagocytic capacity of PMN. t10c12-CLA increased TNF-alpha mRNA expression and production by PBMC. The phagocytic capacity of porcine PMN was enhanced by either culture supernatant fraction from PBMC treated with t10c12-CLA or recombinant porcine (rp) TNF-alpha. Anti-rpTNF-alpha polyclonal antibody inhibited the enhancement of PMN phagocytic capacity. t10c12-CLA also up regulated PPARgamma mRNA expression in porcine PBMC. Bisphenol A diglycidyl ether, a PPARgamma antagonist, not only completely negated the t10c12-CLA-stimulating effects on TNF-alpha expression and production by porcine PBMC, but also decreased the enhancement of PMN phagocytic capacity by the t10c12-CLA-stimulated porcine PBMC culture supernatant fraction. These results suggest that t10c12-CLA has an immunostimulating effect on porcine PMN phagocytic capacity, which is mediated by TNF-alpha from PBMC via a PPARgamma-dependent pathway.

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

    PubMed

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

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

  3. 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.

  4. Possible mechanisms for the differential effects of high linoleate safflower oil and high alpha-linolenate perilla oil diets on platelet-activating factor production by rat polymorphonuclear leukocytes.

    PubMed

    Oh-hashi, K; Takahashi, T; Watanabe, S; Kobayashi, T; Okuyama, H

    1997-12-01

    As compared with high dietary linoleate safflower oil, high dietary alpha-linolenate perilla oil decreased platelet-activating factor (PAF) production by nearly half in calcium ionophore (CaI)-stimulated rat polymorphonuclear leukocytes (PMN). In the CaI-stimulated PMN from the perilla oil group, the accumulated amount of arachidonate (AA) plus eicosapentaenoate (EPA) was 30% less and that of lyso-PAF was 50% less, indicating that the decreased availability of lyso-PAF is a factor contributing to the relatively low PAF production. Consistently, eicosatetraynoic acid (ETYA), a dual inhibitor of cyclooxygenase and lipoxygenase, increased free fatty acids (FFA) and decreased PAF production possibly by decreasing the availability of lyso-PAF. Although, leukotrienes (LTs) have been proposed to stimulate PAF production synergistically, a potent LTB4 receptor antagonist, ONO-4057, decreased the formation of free fatty acids and LTB4, but stimulated PAF production somewhat, indicating that LTB4 may not stimulate PAF production in PMN. Lysophospholipid-induced transacylase (CoA-independent transacylase) activity in PMN homogenates was 25-30% lower in the perilla oil group but no significant differences were observed in the lyso-PAF acetyltransferase and PAF acetylhydrolase activities between the two dietary groups. Thus, decreased transacylase activity is another factor associated with the relatively low PAF production in the perilla oil group.

  5. 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 Whole Blood and Blood Components Intended for Transfusion; Availability AGENCY: Food and Drug Administration, HHS. ACTION: Notice. SUMMARY: The Food and Drug Administration (FDA) is announcing...

  6. Flow cytometric detection of bovine viral diarrhea virus in peripheral blood leukocytes of persistently infected cattle.

    PubMed Central

    Qvist, P; Aasted, B; Bloch, B; Meyling, A; Rønsholt, L; Houe, H

    1990-01-01

    Flow cytometry was investigated for detection of bovine viral diarrhea virus (BVDV) in peripheral blood mononuclear leukocytes of persistently infected cattle. The mononuclear leukocytes were purified by sedimentation in a gradient of Ficoll-Paque, fixed, permeabilized, and then labelled by indirect immunofluorescence using biotinylated immunoglobulins from a porcine antiserum to BVDV. Flow cytometric analysis of blood samples obtained from persistently infected cattle revealed virus in 3.0-21.0% (mean +/- SD, 11.2% +/- 6.4%) of the mononuclear leukocytes. Fluorescent cells were not observed in controls. Flow cytometric detection of BVDV in blood cells of persistently infected bovines is a rapid and objective technique which does not require cell culture facilities. PMID:2174298

  7. Interaction of Bovine Peripheral Blood Polymorphonuclear Cells and Leptospira Species; Innate Responses in the Natural Bovine Reservoir Host

    PubMed Central

    Wilson-Welder, Jennifer H.; Frank, Ami T.; Hornsby, Richard L.; Olsen, Steven C.; Alt, David P.

    2016-01-01

    Cattle are the reservoir hosts of Leptospira borgpetersenii serovar Hardjo, and can also be reservoir hosts of other Leptospira species such as L. kirschneri, and Leptospira interrogans. As a reservoir host, cattle shed Leptospira, infecting other animals, including humans. Previous studies with human and murine neutrophils have shown activation of neutrophil extracellular trap or NET formation, and upregulation of inflammatory mediators by neutrophils in the presence of Leptospira. Humans, companion animals and most widely studied models of Leptospirosis are of acute infection, hallmarked by systemic inflammatory response, neutrophilia, and septicemia. In contrast, cattle exhibit chronic infection with few outward clinical signs aside from reproductive failure. Taking into consideration that there is host species variation in innate immunity, especially in pathogen recognition and response, the interaction of bovine peripheral blood polymorphonuclear cells (PMNs) and several Leptospira strains was evaluated. Studies including bovine-adapted strains, human pathogen strains, a saprophyte and inactivated organisms. Incubation of PMNs with Leptospira did induce slight activation of neutrophil NETs, greater than unstimulated cells but less than the quantity from E. coli P4 stimulated PMNs. Very low but significant from non-stimulated, levels of reactive oxygen peroxides were produced in the presence of all Leptospira strains and E. coli P4. Similarly, significant levels of reactive nitrogen intermediaries (NO2) was produced from PMNs when incubated with the Leptospira strains and greater quantities in the presence of E. coli P4. PMNs incubated with Leptospira induced RNA transcripts of IL-1β, MIP-1α, and TNF-α, with greater amounts induced by live organisms when compared to heat-inactivated leptospires. Transcript for inflammatory cytokine IL-8 was also induced, at similar levels regardless of Leptospira strain or viability. However, incubation of Leptospira strains

  8. [The effect of bromantane on the erythro- and leukocytic profile of the peripheral blood in rats].

    PubMed

    Bugaeva, L I; Spasov, A A; Morozov, I S

    1999-01-01

    The specific effect of bromantan on blood tissue is demonstrated in rats. In chronic injection in a dose of 30 mg/kg bromantan raised the level of hemoglobin and leukocytes. In doses of 150 and 600 mg/kg it increased at first (3 months) and then reduced the level of erythrocytes, hemoglobin, and leukocytes. Reversible poikilocytosis, granulocytosis, and agranulocytosis were encountered. Hyperchromatosis and hypertrophy of the hepatocytes were found in the tissues of the liver and hemosiderosis was discovered in the spleen. It is suggested that the blood tissue is a "target" in the toxic effect of bromantan.

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

    PubMed Central

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

    1987-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  11. A New Machine Classification Method Applied to Human Peripheral Blood Leukocytes.

    ERIC Educational Resources Information Center

    Rorvig, Mark E.; And Others

    1993-01-01

    Discusses pattern classification of images by computer and describes the Two Domain Method in which expert knowledge is acquired using multidimensional scaling of judgments of dissimilarities and linear mapping. An application of the Two Domain Method that tested its power to discriminate two patterns of human blood leukocyte distribution is…

  12. α3/4 Fucosyltransferase 3-dependent synthesis of Sialyl Lewis A on CD44 variant containing exon 6 mediates polymorphonuclear leukocyte detachment from intestinal epithelium during transepithelial migration.

    PubMed

    Brazil, Jennifer C; Liu, Renpeng; Sumagin, Ronen; Kolegraff, Keli N; Nusrat, Asma; Cummings, Richard D; Parkos, Charles A; Louis, Nancy A

    2013-11-01

    Polymorphonuclear leukocyte (PMN) migration across the intestinal epithelium closely parallels disease symptoms in patients with inflammatory bowel disease. PMN transepithelial migration (TEM) is a multistep process that terminates with PMN detachment from the apical epithelium into the lumen. Using a unique mAb (GM35), we have previously demonstrated that engagement of the CD44 variant containing exon 6 (CD44v6) blocks both PMN detachment and cleavage of CD44v6. In this article, we report that PMN binding to CD44v6 is mediated by protein-specific O-glycosylation with sialyl Lewis A (sLe(a)). Analyses of glycosyltransferase expression identified fucosyltransferase 3 (Fut3) as the key enzyme driving sLe(a) biosynthesis in human intestinal epithelial cells (IECs). Fut3 transfection of sLe(a)-deficient IECs resulted in robust expression of sLe(a). However, this glycan was not expressed on CD44v6 in these transfected IECs; therefore, engagement of sLe(a) had no effect on PMN TEM across these cells. Analyses of sLe(a) in human colonic mucosa revealed minimal expression in noninflamed areas, with striking upregulation under colitic conditions that correlated with increased expression of CD44v6. Importantly, intraluminal administration of mAb GM35 blocked PMN TEM and attenuated associated increases in intestinal permeability in a murine intestinal model of inflammation. These findings identify a unique role for protein-specific O-glycosylation in regulating PMN-epithelial interactions at the luminal surface of the intestine.

  13. Oxidative DNA Damage to Sperm Cells and Peripheral Blood Leukocytes in Infertile Men

    PubMed Central

    Taken, Kerem; Alp, Hamit Hakan; Eryilmaz, Recep; Donmez, Muhammet Irfan; Demir, Murat; Gunes, Mustafa; Aslan, Rahmi; Sekeroglu, Mehmet Ramazan

    2016-01-01

    Background Oxidative DNA damage is associated with male infertility. The aim of this study was to evaluate the oxidative DNA damage of sperm cells and blood leukocytes and to determine the levels of MDA and NO levels in seminal and blood plasma of idiopathic infertile men. Material/Methods The study enrolled 52 patients, including 30 infertile and 22 fertile men. MDA, NO, and 8-OHdG/106dG were estimated using spectrophotometry and high-pressure liquid chromatography (HPLC)-based methods in seminal and blood plasma. The association with the sperm parameters was assessed, particularly sperm counts and motility. Results The mean sperm concentration and sperm motility of the fertile men were significantly higher than that of the infertile men. The mean MDA and NO concentration in the seminal and blood samples of the infertile men were higher than that of fertile men. Also, the mean numbers of sperm cells and leukocytes 8-OHdG/106dG of the infertile men were significantly higher than that of fertile men (p=0.04 and p<0.001, respectively). Sperm motility and sperm count were negatively correlated with leukocyte and sperm cell 8-OHdG/106dG ratio. However, progressive motility was significantly negatively correlated with sperm cell and leukocyte 8-OHdG/106dG ratio (R=−0.357, p=0.026; R=−0.388, p=0.024, respectively). Conclusions Oxidative stress is an important factor in male infertility. Therefore, biochemical detection of 8-OHdG/106dG in sperm cells and blood leukocytes may be an additional tool in the diagnosis of male infertility. PMID:27837200

  14. Laser-induced priming of human blood leukocytes

    NASA Astrophysics Data System (ADS)

    Chichuk, Tatyana V.; Stranadko, Eugeny P.; Strashkevich, I. A.; Klebanov, Gennady I.

    1999-12-01

    We investigated the influence of He-Ne ((lambda) equals 632.8 nm) laser irradiation (LI) on a functional activity of human blood leucocytes. The method of luminol-dependent chemiluminescence with the zymosan-activated phagocytes was used. The leucocytes were irradiated without and in the presence of autologic human blood plasma, containing of the endogenous (porphyrins) and/or exogenous (phthalocyanine) photosensitizers. The LI initiated a priming of the leucocytes. Priming revealed itself after the activation of the phagocytes by zymosan. The changes of the calcium concentration in leucocytes cytoplasm were studied too. Fluorimetric method with Fura-2AM was used for this. The laser irradiation initiated the changes of the calcium concentration in the leucocytes cytoplasm. All the investigating parameters depended on the irradiation dose and on the concentration of photosensitizers. The results of this work allowed to formulate the main theses of the free radical mechanism of the low intensive laser irradiation action on human blood leucocytes.

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

    PubMed

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

    2013-10-25

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

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

    PubMed

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

    2009-01-01

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

  17. Detection of microparticles of leukocytic origin in the peripheral blood in normal pregnancy and preeclampsia.

    PubMed

    Mikhailova, V A; Ovchinnikova, O M; Zainulina, M S; Sokolov, D I; Sel'kov, S A

    2014-10-01

    Microparticles are microvesicles forming during cell activation and as a result of apoptotic cell death. Normal pregnancy is associated with apoptosis induction in active immune system cells, present in the decidual tissue. Preeclampsia is associated with activation of the peripheral blood leukocytes and more intense apoptosis of the trophoblast cells. As a result, the number of microparticles in the peripheral blood is changing in normal gestation and in preeclampsia. The content of the leukocytic microparticles in the peripheral blood is evaluated in normal pregnancy and preeclampsia. The content of neutrophilic and monocytic microparticles is higher than normally in preeclampsia, this indicating activation of these cells. The number of microparticles formed by NK cells is low in preeclampsia, which can reflect the incompetence of immunological tolerance mechanisms under these conditions.

  18. Influence of hormone supplementation therapy on the incidence of denture stomatitis and on chemiluminescent activity of polymorphonuclear granulocytes in blood of menopausal-aged women

    PubMed Central

    2010-01-01

    Background Menopause is a health and social problem that affects a large number of women. Inadequate quantity of steroid hormones also impacts quality of the mucous membrane of the oral cavity. During menopausal age, many women wear removable prosthetic restorations in order to replace missing teeth. Such restorations may facilitate the development of inflammations in the surface of the oral cavity, referred to as denture stomatitis. Objective The aim of the study was to evaluate the influence of hormone supplementation therapy on the incidence of Candida-associated denture stomatitis and on the metabolic activity of polymorphonuclear granulocytes in peripheral blood of female patients. Materials and methods The study was conducted on a group of women in menopausal age, users of hormone replacement therapy and of removable prosthetic restorations. Female patients were subjected to a clinical study that included interviews and physical examinations. Laboratory microbiological examinations were completed on the basis of direct swabs collected from the mucous membrane of the oral cavity and from the surface of dentures. Metabolic activity of polymorphonuclear granulocytes in peripheral blood of female patients was evaluated by means of a chemiluminescence test. Results Candida-associated denture stomatitis observed was characterized by a strong growth of fungi and a lower chemiluminescent activity of neutrophils in blood of female patients undergoing hormone supplementation therapy. Conclusions Patients using hormone supplementation therapy and removable prosthetic restorations constitute a high-risk group for Candida infections and inflammations of the mucous membrane of the oral cavity; thus they should remain under constant dental control. PMID:21147619

  19. Red blood cell and leukocyte alloimmunization in patients awaiting kidney transplantation

    PubMed Central

    da Silva, Silvia Fernandes Ribeiro; Ferreira, Gláucia Maria; da Silva, Sonia Leite; Alves, Tânia Maria de Oliveira; Ribeiro, Ilana Farias; Ribeiro, Thyciana Rodrigues; Cavalcante, Maria do Carmo Serpa

    2013-01-01

    Objective To determine the rates of red blood cell and leukocyte alloimmunization in patients with chronic kidney disease awaiting kidney transplantation. Methods In this cross-sectional and prospective study, the serum of 393 chronic kidney disease patients on a transplant waiting list in Ceará, Northeastern Brazil were tested for red cell and leukocyte antibodies. In addition, demographic, clinical and laboratory data were collected. Results The average age in the sample of 393 patients was 34.1 ± 14 years. Slightly more than half (208; 52.9%) were male. The average numbers of transfusions and gestations were 3.1 ± 3.3 and 1.6 ± 6, respectively. One third (33.6%) were alloimmunized: 78% with leukocyte antibodies, 9.1% with red cell antibodies and 12.9% with both. Red cell antibodies were detected in 29 cases (7.4%), 17 of whom were women, who had received more transfusions than the males (p-value < 0.0001). The most frequently detected red cell antibodies belonged to the Rh (24.1%) and Kell (13.8%) blood group systems. Leukocyte antibodies were detected in 30.5% of cases, 83 of whom were women, who had received more transfusions than the males (p-value < 0.0001) and were more reactive to panel reactive antibodies (p-value < 0.0001). The mean alloreactivity to panel reactive antibodies was 47.7 ± 31.2%. Conclusion Chronic kidney disease patients on the transplant waiting list in Ceará, Brazil, display high rates of red cell (7.4%) and leukocyte (30.5%) alloimmunization. In this sample, alloimmunization was significantly associated with the number of transfusions and gender. PMID:23904808

  20. Inhalation of ultrafine particles alters blood leukocyte expression of adhesion molecules in humans.

    PubMed

    Frampton, Mark W; Stewart, Judith C; Oberdörster, Günter; Morrow, Paul E; Chalupa, David; Pietropaoli, Anthony P; Frasier, Lauren M; Speers, Donna M; Cox, Christopher; Huang, Li-Shan; Utell, Mark J

    2006-01-01

    Ultrafine particles (UFPs; aerodynamic diameter < 100 nm) may contribute to the respiratory and cardiovascular morbidity and mortality associated with particulate air pollution. We tested the hypothesis that inhalation of carbon UFPs has vascular effects in healthy and asthmatic subjects, detectable as alterations in blood leukocyte expression of adhesion molecules. Healthy subjects inhaled filtered air and freshly generated elemental carbon particles (count median diameter approximately 25nm, geometric standard deviation approximately 1.6), for 2 hr, in three separate protocols: 10 microg/m3 at rest, 10 and 25 microg/m3 with exercise, and 50 microg/m3 with exercise. In a fourth protocol, subjects with asthma inhaled air and 10 microg/m3 UFPs with exercise. Peripheral venous blood was obtained before and at intervals after exposure, and leukocyte expression of surface markers was quantitated using multiparameter flow cytometry. In healthy subjects, particle exposure with exercise reduced expression of adhesion molecules CD54 and CD18 on monocytes and CD18 and CD49d on granulocytes. There were also concentration-related reductions in blood monocytes, basophils, and eosinophils and increased lymphocyte expression of the activation marker CD25. In subjects with asthma, exposure with exercise to 10 microg/m3 UFPs reduced expression of CD11b on monocytes and eosinophils and CD54 on granulocytes. Particle exposure also reduced the percentage of CD4+ T cells, basophils, and eosinophils. Inhalation of elemental carbon UFPs alters peripheral blood leukocyte distribution and expression of adhesion molecules, in a pattern consistent with increased retention of leukocytes in the pulmonary vascular bed.

  1. Reduced Responsiveness of Blood Leukocytes to Lipopolysaccharide Does not Predict Nosocomial Infections in Critically Ill Patients.

    PubMed

    van Vught, Lonneke A; Wiewel, Maryse A; Hoogendijk, Arie J; Scicluna, Brendon P; Belkasim-Bohoudi, Hakima; Horn, Janneke; Schultz, Marcus J; van der Poll, Tom

    2015-08-01

    Critically ill patients show signs of immune suppression, which is considered to increase vulnerability to nosocomial infections. Whole-blood stimulation is frequently used to test the function of the innate immune system. We here assessed the association between whole-blood leukocyte responsiveness to lipopolysaccharide (LPS) and subsequent occurrence of nosocomial infections in critically ill patients admitted to the intensive care unit (ICU). All consecutive critically ill patients admitted to the ICU between April 2012 and June 2013 with two or more systemic inflammatory response syndrome criteria and an expected length of ICU stay of more than 24 h were enrolled. Age- and sex-matched healthy individuals were included as controls. Blood was drawn the first morning after ICU admission and stimulated ex vivo with 100 ng/mL ultrapure LPS for 3 h. Tumor necrosis factor-α, interleukin-1β (IL-1β), and IL-6 were measured in supernatants. Seventy-three critically ill patients were included, of whom 10 developed an ICU-acquired infection. Compared with healthy subjects, whole-blood leukocytes of patients were less responsive to ex vivo stimulation with LPS, as reflected by strongly reduced tumor necrosis factor-α, IL-1β, and IL-6 levels in culture supernatants. Results were not different between patients who did and those who did not develop an ICU-acquired infection. The extent of reduced LPS responsiveness of blood leukocytes in critically ill patients on the first day after ICU admission does not relate to the subsequent development of ICU-acquired infections. These results argue against the use of whole-blood stimulation as a functional test applied early after ICU admission to predict nosocomial infection.

  2. [The influence of geomagnetic field on blood leukocyte radiosensitivity and gender determination in humans].

    PubMed

    Ivanov, S D; Iamshanov, V A; Koshelevskiĭ, V K; Ivanova, A S; Ivanova, T M; Glushkov, R K; Semenov, E V; Petrov, A N

    2003-01-01

    The gender and age features of the geomagnetic field (GMF) influence (K-indexes) on the leukocyte radiosensitivity (S) in human blood in case of normal and enhanced levels of metal-ecotoxicants (Pb, Cd, Hg, Mn, Zn, Cu) in blood was studied, as well as the impact of the GMF intensity alterations on the child gender determination. In the whole studied population (n = 244) it was observed a negative relationship between S- and K-indexes. It was shown that the most changes of blood leukocyte radiosensitivity depended on the GMF oscillations in men older than 45 years. In case of metal-ecotoxicant concentrations in blood near normal level there were observed significant relationships between S- and K-indexes, which was infringed by more than 2-fold excess of these toxicant concentrations in blood. Retrospective analysis showed that in case of the GMF intensity enhancement in the conception moment the girls were born mostly, and in case of the lowering--the boys were.

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

    PubMed

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

    2014-04-01

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

  4. Effect of prenatal arsenic exposure on DNA methylation and leukocyte subpopulations in cord blood.

    PubMed

    Kile, Molly L; Houseman, E Andres; Baccarelli, Andrea A; Quamruzzaman, Quazi; Rahman, Mahmuder; Mostofa, Golam; Cardenas, Andres; Wright, Robert O; Christiani, David C

    2014-05-01

    Prenatal arsenic exposure is associated with increased risk of disease in adulthood. This has led to considerable interest in arsenic's ability to disrupt fetal programming. Many studies report that arsenic exposure alters DNA methylation in whole blood but these studies did not adjust for cell mixture. In this study, we examined the relationship between arsenic in maternal drinking water collected ≤ 16 weeks gestational age and DNA methylation in cord blood (n = 44) adjusting for leukocyte-tagged differentially methylated regions. DNA methylation was quantified using the Infinium HumanMethylation 450 BeadChip array. Recursively partitioned mixture modeling examined the relationship between arsenic and methylation at 473,844 CpG sites. Median arsenic concentration in water was 12 µg/L (range<1- 510 µg/L). Log 10 arsenic was associated with altered DNA methylation across the epigenome (P = 0.002); however, adjusting for leukocyte distributions attenuated this association (P = 0.013). We also observed that arsenic had a strong effect on the distribution of leukocytes in cord blood. In adjusted models, every log 10 increase in maternal drinking water arsenic exposure was estimated to increase CD8+ T cells by 7.4% (P = 0.0004) and decrease in CD4+ T cells by 9.2% (P = 0.0002). These results show that prenatal exposure to arsenic had an exposure-dependent effect on specific T cell subpopulations in cord blood and altered DNA methylation in cord blood. Future research is needed to determine if these small changes in DNA methylation alter gene expression or are associated with adverse health effects.

  5. Effect of prenatal arsenic exposure on DNA methylation and leukocyte subpopulations in cord blood

    PubMed Central

    Kile, Molly L; Houseman, E Andres; Baccarelli, Andrea A; Quamruzzaman, Quazi; Rahman, Mahmuder; Mostofa, Golam; Cardenas, Andres; Wright, Robert O; Christiani, David C

    2014-01-01

    Prenatal arsenic exposure is associated with increased risk of disease in adulthood. This has led to considerable interest in arsenic’s ability to disrupt fetal programming. Many studies report that arsenic exposure alters DNA methylation in whole blood but these studies did not adjust for cell mixture. In this study, we examined the relationship between arsenic in maternal drinking water collected ≤ 16 weeks gestational age and DNA methylation in cord blood (n = 44) adjusting for leukocyte-tagged differentially methylated regions. DNA methylation was quantified using the Infinium HumanMethylation 450 BeadChip array. Recursively partitioned mixture modeling examined the relationship between arsenic and methylation at 473,844 CpG sites. Median arsenic concentration in water was 12 µg/L (range < 1- 510 µg/L). Log10 arsenic was associated with altered DNA methylation across the epigenome (P = 0.002); however, adjusting for leukocyte distributions attenuated this association (P = 0.013). We also observed that arsenic had a strong effect on the distribution of leukocytes in cord blood. In adjusted models, every log10 increase in maternal drinking water arsenic exposure was estimated to increase CD8+ T cells by 7.4% (P = 0.0004) and decrease in CD4+ T cells by 9.2% (P = 0.0002). These results show that prenatal exposure to arsenic had an exposure-dependent effect on specific T cell subpopulations in cord blood and altered DNA methylation in cord blood. Future research is needed to determine if these small changes in DNA methylation alter gene expression or are associated with adverse health effects. PMID:24525453

  6. 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.

  7. Lipids and leukocytes in newborn umbilical vein blood, birth weight and maternal body mass index.

    PubMed

    Brittos, T; de Souza, W B; Anschau, F; Pellanda, L

    2016-12-01

    Maternal obesity during pregnancy may influence fetal development and possibly predispose offspring to cardiovascular disease. The aim of the present study was to evaluate the relationship between maternal pre-pregnancy body mass index (BMI) and weight gain during pregnancy, and newborn birth weight, with lipid profile, high-sensitivity C-reactive protein (hs-CRP) and leukocyte in newborns. We performed a cross-sectional study of 245 mothers and their children. Blood was collected from the umbilical vein and assayed for lipid profile, hs-CRP and leukocyte count. Newborns average weight was 3241 g, total cholesterol 53.9 mg/dl, high-density lipoprotein cholesterol (HDL-c) 21.9 mg/dl, low-density lipoprotein cholesterol (LDL-c) 26.2 mg/dl, triglyceride 29.5 mg/dl and leukocytes 13,777/mm3. There was a direct correlation of pre-pregnancy BMI of overweight mothers with total cholesterol (r=0.220, P=0.037) and LDL-c (r=0.268, P=0.011) of newborns. Total cholesterol, LDL-c and HDL-c were higher in pre-term newborns (66.3±19.7, 35.9±14.6 and 25.2±7.7 mg/dl, respectively) that in full-term (52.4±13.1, 25.0±8.7 and 21.5±6.0 mg/dl), with P=0.001, 0.001 and 0.003, respectively. Leukocyte counts were higher in full-term newborns (14,268±3982/mm3) compared with pre-term (9792±2836/mm3, P<0.0001). There was a direct correlation between birth weight and leukocyte counts of newborns (r=0.282, P<0.0001). These results suggest the possible interaction of maternal weight and fetal growth with lipid metabolism and leukocyte count in the newborn, which may be linked to programming of the immune system.

  8. TNF{alpha} release from peripheral blood leukocytes depends on a CRM1-mediated nuclear export

    SciTech Connect

    Miskolci, Veronika; Ghosh, Chandra C.; Rollins, Janet; Romero, Carlos; Vu, Hai-Yen; Robinson, Staci; Davidson, Dennis; Vancurova, Ivana . E-mail: vancuroi@stjohns.edu

    2006-12-15

    Tumor necrosis factor-{alpha} (TNF{alpha}) is a potent pro-inflammatory cytokine that plays a major role in the pathogenesis of acute and chronic inflammatory disorders such as septic shock and arthritis, respectively. Leukocytes stimulated with inflammatory signals such as lipopolysaccharide (LPS) are the predominant producers of TNF{alpha}, and thus control of TNF{alpha} release from stimulated leukocytes represents a potential therapeutic target. Here, we report that leptomycin B (LMB), a specific inhibitor of CRM1-dependent nuclear protein export, inhibits TNF{alpha} release from LPS-stimulated human peripheral blood neutrophils and mononuclear cells. In addition, immunofluorescence confocal microscopy and immunoblotting analysis indicate that TNF{alpha} is localized in the nucleus of human neutrophils and mononuclear cells. This study demonstrates that the cellular release of TNF{alpha} from stimulated leukocytes is mediated by the CRM1-dependent nuclear export mechanism. Inhibition of CRM1-dependent cellular release of TNF{alpha} could thus provide a novel therapeutic approach for disorders involving excessive TNF{alpha} release.

  9. Toxicogenomic Effects in Rat Blood Leukocytes and Chemoprophylaxis of Radiation-Induced Carcinogenesis.

    PubMed

    Ivanov, S D; Bespalov, V G; Semenov, A L; Kovan'ko, E G; Aleksandrov, V A

    2016-03-01

    Toxicogenomic parameters were studied in the blood of female rats after exposure to ionizing γ-radiation in a dose of 4 Gy and chemoprophylaxis with α-difluoromethylornithine, eleutherococcus or leuzea extracts, which were used in animals with morphological manifestations of tumor growth under conditions of radiation-induced carcinogenesis. Life-time evaluation of toxicogenomic effects was carried out by express method for measurements of blood nucleotid DNA - fluorescent indication. The level of hyperaneu/polyploidy increased in the blood leukocytes of control rats 30 days after radiation exposure. A significant decrease of genotoxicity as a result of drug treatment in comparison with the number and multiplicity of tumors in irradiated animals was found only in the endocrine and reproductive organs of rats treated by eleutherococcus extract.

  10. Prenatal Arsenic Exposure and DNA Methylation in Maternal and Umbilical Cord Blood Leukocytes

    PubMed Central

    Baccarelli, Andrea; Hoffman, Elaine; Tarantini, Letizia; Quamruzzaman, Quazi; Rahman, Mahmuder; Mahiuddin, Golam; Mostofa, Golam; Hsueh, Yu-Mei; Wright, Robert O.; Christiani, David C.

    2012-01-01

    Background: Arsenic is an epigenetic toxicant and could influence fetal developmental programming. Objectives: We evaluated the association between arsenic exposure and DNA methylation in maternal and umbilical cord leukocytes. Methods: Drinking-water and urine samples were collected when women were at ≤ 28 weeks gestation; the samples were analyzed for arsenic using inductively coupled plasma mass spectrometry. DNA methylation at CpG sites in p16 (n = 7) and p53 (n = 4), and in LINE-1 and Alu repetitive elements (3 CpG sites in each), was quantified using pyrosequencing in 113 pairs of maternal and umbilical blood samples. We used general linear models to evaluate the relationship between DNA methylation and tertiles of arsenic exposure. Results: Mean (± SD) drinking-water arsenic concentration was 14.8 ± 36.2 μg/L (range: < 1–230 μg/L). Methylation in LINE-1 increased by 1.36% [95% confidence interval (CI): 0.52, 2.21%] and 1.08% (95% CI: 0.07, 2.10%) in umbilical cord and maternal leukocytes, respectively, in association with the highest versus lowest tertile of total urinary arsenic per gram creatinine. Arsenic exposure was also associated with higher methylation of some of the tested CpG sites in the promoter region of p16 in umbilical cord and maternal leukocytes. No associations were observed for Alu or p53 methylation. Conclusions: Exposure to higher levels of arsenic was positively associated with DNA methylation in LINE-1 repeated elements, and to a lesser degree at CpG sites within the promoter region of the tumor suppressor gene p16. Associations were observed in both maternal and fetal leukocytes. Future research is needed to confirm these results and determine if these small increases in methylation are associated with any health effects. PMID:22466225

  11. 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.

  12. Biochemical and Cellular Changes in Leukocyte-Depleted Red Blood Cells Stored for Transfusion

    PubMed Central

    Nogueira, Diana; Rocha, Susana; Abreu, Estela; Costa, Elísio; Santos-Silva, Alice

    2015-01-01

    Summary Background To evaluate biochemical and cellular changes associated with the storage of leukocyte-depleted red blood cells (RBCs). Methods We investigated 10 leukocyte-depleted RBC units, randomly chosen from volunteer donors. Every week an aliquot was collected for laboratorial evaluation, which included complete cell blood count, glucose-6-phosphate dehydrogenase (G6PD) activity, extracellular sodium, potassium and pH, membrane-bound hemoglobin (MBH), band 3 profile, and quantification of RBC membrane proteins composition. Results We observed an increase in mean cell volume (from 91.86 ± 4.65 fl to 98.10 ± 5.80 fl, day 0 vs. day 21; p < 0.05), red cell distribution width, percentage of macrocytic RBCs, reticulocyte hemoglobin content and a decreased percentage of microcytic RBCs, mean cell volume concentration and G6PD activity. The extracellular concentration of sodium decreased, and that of potassium increased significantly over time. RBC membrane composition revealed an increase in spectrin/ankyrin ratio after 21 days (from 4.84 ± 0.99 to 5.27 ± 0.94, day 0 vs. day 21; p < 0.05). At day 35, a decrease in ankyrin (from 6.44 ± 1.70% to 5.49 ± 1.96%, day 0 vs. day 35; p < 0.05), in protein 4.1/band 3, protein 4.2/band 3, and ankyrin/band 3 ratios and in band 5 was observed. Conclusions Our data show that leukocyte-depleted RBCs present changes in the RBC morphology, membrane protein composition, enzymatic activity, and extracellular electrolyte concentration and pH. PMID:25960715

  13. 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.

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

    PubMed

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

    2002-06-01

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

  15. Blood Leukocyte Count on Admission Predicts Cardiovascular Events in Patients with Acute Non-ST Elevation Myocardial Infarction.

    PubMed

    Dharma, Surya; Hapsari, Rosmarini; Siswanto, Bambang B; van der Laarse, Arnoud; Jukema, J Wouter

    2015-06-01

    We aim to test the hypothesis that blood leukocyte count adds prognostic information in patients with acute non-ST-elevation myocardial infarction (non-STEMI). A total of 585 patients with acute non-STEMI (thrombolysis in myocardial infarction risk score ≥ 3) were enrolled in this cohort retrospective study. Blood leukocyte count was measured immediately after admission in the emergency department. The composite of death, reinfarction, urgent revascularization, and stroke during hospitalization were defined as the primary end point of the study. The mean age of the patients was 61 ± 9.6 years and most of them were male (79%). Using multivariate Cox regression analysis involving seven variables (history of smoking, hypertension, heart rate > 100 beats/minute, serum creatinine level > 1.5 mg/dL, blood leukocyte count > 11,000/µL, use of β-blocker, and use of angiotensin-converting enzyme inhibitor), leukocyte count > 11,000/µL demonstrated to be a strong predictor of the primary end point (hazard ratio = 3.028; 95% confidence interval = 1.69-5.40, p < 0.001). The high blood leukocyte count on admission is an independent predictor of cardiovascular events in patients with acute non-STEMI.

  16. Direct observation of liposome uptake by leukocytes in vivo in skin blood vessels using intravital fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Devoisselle, Jean-Marie; Mordon, Serge R.; Begu, Sylvie; Desmettre, Thomas

    2000-04-01

    This study aimed to observe liposome uptake by leukocytes in vivo. The study was performed on skin by using a dorsal skin-fold chamber implanted in golden hamsters using intravital microscopy. 5,6-CF-encapsulated PEGylated liposomes were injected intravenously. The skin microcirculation was observed with an intravital Eclipse E800 Nikon microscope fitted with a Xenon light source and an epi-fluorescence assembly. An ultra-high sensitivity video-camera mounted on the microscope projected the image onto a monitor, and the images were recorded for playback analysis with a digital video cassette recorder. An acute inflammatory response was obtained by removing one complete layer of skin and the underlying fascia and avascular tissue on the opposing side of the flap corresponding to an area equivalent to the window aperture. Using these model and set-up, leukocyte rolling and adhesion were easily observed and the entry of PEGylated liposomes into hamster blood leukocytes was studied for a period of 6 hours. PEGylated liposomes were clearly identified alone inside the blood flow and inside the leukocytes as soon as the inflammatory reaction appeared. This study shows for the first time that blood leukocytes in their natural milieu of whole blood are capable of interacting with, and taking up liposomes. This observation is in accordance with previous in vitro studies.

  17. Distinct Epigenetic Effects of Tobacco Smoking in Whole Blood and among Leukocyte Subtypes

    PubMed Central

    Porter, Devin K.; Pittman, Gary S.; Bennett, Brian D.; Wan, Ma; Crowl, Christopher L.; Adamski, Kelly N.; Huang, Zhiqing; Murphy, Susan K.; Bell, Douglas A.

    2016-01-01

    Tobacco smoke exposure dramatically alters DNA methylation in blood cells and may mediate smoking-associated complex diseases through effects on immune cell function. However, knowledge of smoking effects in specific leukocyte subtypes is limited. To better characterize smoking–associated methylation changes in whole blood and leukocyte subtypes, we used Illumina 450K arrays and Reduced Representation Bisulfite Sequencing (RRBS) to assess genome-wide DNA methylation. Differential methylation analysis in whole blood DNA from 172 smokers and 81 nonsmokers revealed 738 CpGs, including 616 previously unreported CpGs, genome-wide significantly associated with current smoking (p <1.2x10-7, Bonferroni correction). Several CpGs (MTSS1, NKX6-2, BTG2) were associated with smoking duration among heavy smokers (>22 cigarettes/day, n = 86) which might relate to long-term heavy-smoking pathology. In purified leukocyte subtypes from an independent group of 20 smokers and 14 nonsmokers we further examined methylation and gene expression for selected genes among CD14+ monocytes, CD15+ granulocytes, CD19+ B cells, and CD2+ T cells. In 10 smokers and 10 nonsmokers we used RRBS to fine map differential methylation in CD4+ T cells, CD8+ T cells, CD14+, CD15+, CD19+, and CD56+ natural killer cells. Distinct cell-type differences in smoking-associated methylation and gene expression were identified. AHRR (cg05575921), ALPPL2 (cg21566642), GFI1 (cg09935388), IER3 (cg06126421) and F2RL3 (cg03636183) showed a distinct pattern of significant smoking-associated methylation differences across cell types: granulocytes> monocytes>> B cells. In contrast GPR15 (cg19859270) was highly significant in T and B cells and ITGAL (cg09099830) significant only in T cells. Numerous other CpGs displayed distinctive cell-type responses to tobacco smoke exposure that were not apparent in whole blood DNA. Assessing the overlap between these CpG sites and differential methylated regions (DMRs) with RRBS in 6

  18. Intracerebroventricular infusions of TNF-alpha preferentially recruit blood lymphocytes and induce a perivascular leukocyte infiltrate.

    PubMed

    Seabrook, T J; Hay, J B

    2001-02-01

    Tumour necrosis factor (TNF)-alpha is important in several central nervous system (CNS) inflammatory diseases, however, its role in the recruitment of leukocytes into the cerebral spinal fluid (CSF) and CNS is incompletely understood. Therefore, we examined the effect of intracerebroventricular (icv) and parenchymal infusions of TNF-alpha on the type of leukocyte, the pool and subset of lymphocytes recruited into CSF and brain parenchyma. Parenchymal injections of 500 ng of recombinant human TNF-alpha did not induce inflammation, whereas an icv infusion of TNF-alpha caused CSF leuckocytosis and a perivascular infiltrate. Twenty-four hours after the icv infusion neutrophils predominated, with CD4+ T cells being the major lymphocyte subset in CSF. By 48 h lymphocytes were the dominant cell type with CD8+ cells surpassing CD4+ cells in both the CSF and the perivascular infiltrate. The labeled recirculating lymphocyte pool prevailed in normal CSF, but after the infusion of TNF-alpha, the blood pool of lymphocytes was preferentially recruited. These results have implications for the immune surveillance of the CNS.

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

    PubMed Central

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

    2016-01-01

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

  20. Transfusion-related acute lung injury (TRALI) in graft by blood donor antibodies against host leukocytes.

    PubMed

    Goodwin, Jodi; Tinckam, Kathryn; denHollander, Neal; Haroon, Ayesha; Keshavjee, Shaf; Cserti-Gazdewich, Christine M

    2010-09-01

    It is unknown the extent to which transfusion-related acute lung injury (TRALI) contributes to primary graft dysfunction (PGD), the leading cause of death after lung transplantation. In this case of suspected transfusion-associated acute bilateral graft injury in a 61-year-old idiopathic pulmonary fibrosis patient, recipient sera from before and after transplantation/transfusion, as well as the sera of 22 of the 24 implicated blood donors, were individually screened by Luminex bead assay for the presence of human leukocyte antigen (HLA) antibodies, with recipient and lung donor HLA typing to explore for cognate relationships. A red-cell-unit donor-source anti-Cw6 antibody, cognate with the HLA type of the recipient, was identified. This is the second reported case of TRALI in the setting of lung transplantation, and the first to show an associated interaction between donor antibodies (in a low-plasma volume product) with recipient leukocytes (rather than graft antigens); therefore, it should be considered in the differential diagnosis of PGD.

  1. Gene Expression Patterns in Peripheral Blood Leukocytes in Patients with Recurrent Ciguatera Fish Poisoning: Preliminary Studies.

    PubMed

    Lopez, Maria-Cecilia; Ungaro, Ricardo F; Baker, Henry V; Moldawer, Lyle L; Robertson, Alison; Abbott, Margaret; Roberts, Sparkle M; Grattan, Lynn M; Morris, J Glenn

    2016-07-01

    Ciguatera fish poisoning (ciguatera) is a common clinical syndrome in areas where there is dependence on tropical reef fish for food. A subset of patients develops recurrent and, in some instances, chronic symptoms, which may result in substantial disability. To identify possible biomarkers for recurrent/chronic disease, and to explore correlations with immune gene expression, peripheral blood leukocyte gene expression in 10 ciguatera patients (7 recurrent, 3 acute) from the U.S. Virgin Islands, and 5 unexposed Florida controls were evaluated. Significant differences in gene expression were noted when comparing ciguatera patients and controls; however, it was not possible to differentiate between patients with acute and recurrent disease, possibly due to the small sample sizes involved.

  2. 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.

  3. Methylation of a panel of genes in peripheral blood leukocytes is associated with colorectal cancer

    PubMed Central

    Luo, Xiang; Huang, Rong; Sun, Hongru; Liu, Yupeng; Bi, Haoran; Li, Jing; Yu, Hongyuan; Sun, Jiamei; Lin, Shangqun; Cui, Binbin; Zhao, Yashuang

    2016-01-01

    The relationship between the DNA methylation status of the CpG islands of multiple genes in blood leukocytes in CRC susceptibility and prognosis, as well as possible interactions with dietary factors on CRC risk are unclear. We carried out a case-control study including 421 CRC patients and 506 controls to examine the associations between six genes (AOX-1, RARB2, RERG, ADAMTS9, IRF4, and FOXE-1), multiple CpG site methylation (MCSM) and susceptibility to CRC. High-level MCSM (MCSM-H) was defined as methylation of greater than or equal to 2 of 5 candidate genes (except for RARB2); low-level MCSM (MCSM-L) was when 1 candidate gene was methylated; non-MCSM was when none of the candidate genes were methylated. Blood cell-derived DNA methylation status was detected using methylation-sensitive high-resolution melting analysis. The hypermethylation status of each individual gene was statistically significantly associated with CRC. MCSM status was also associated with CRC (OR = 1.54, 95% CI: 1.15–2.05, P = 0.004). We observed interactions between a high level of dietary intake of cereals, pungent food, and stewed fish with brown sauce, age (older than 60 yrs), smoking and hypermethylation on risk of CRC. MCSM in peripheral blood DNA may be an important biomarker for susceptibility to CRC. PMID:27453436

  4. Effects of spaceflight on rat peripheral blood leukocytes and bone marrow progenitor cells

    NASA Technical Reports Server (NTRS)

    Ichiki, A. T.; Gibson, L. A.; Jago, T. L.; Strickland, K. M.; Johnson, D. L.; Lange, R. D.; Allebban, Z.

    1996-01-01

    The white blood cell (WBC) elements and the bone marrow myeloid progenitor cell populations were analyzed to ascertain adaptation to micro-gravity and subsequent readaptation to 1 G in rats flown on the 14-day Spacelab Life Sciences-2 (SLS-2) mission. Bone marrow cells were harvested from one group of rats killed inflight (FD13) and blood was drawn from three other groups at various times. The WBC level was normal on FD14 with the exception of neutrophilia. On FD13, numbers of colony-forming units-granulocyte (CFU-G), CFU-GM, and CFU-M from flight animals were decreased compared with ground controls when incubated with recombinant rat interleukin-3 (rrIL-3) alone or in combination with recombinant human erythropoietin (rhEpo). On recovery (R + 0), flight rats had decreased numbers of total leukocytes and absolute numbers of lymphocytes and monocytes with elevated neutrophils compared with control rats. They had lower numbers of CD4, CD8, CD2, CD3, and B cells in the peripheral blood but no differences in spleen lymphocytes.

  5. Virus-specific antibodies interfere with avian influenza infection in peripheral blood mononuclear leukocytes from young or aged chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Avian influenza virus (AIV) infection was examined in peripheral blood mononuclear leukocyte cultures (PBMC) that were collected from 1-day-old chicks or from 52-week-old chickens. Virus-specific antibodies were incubated with AIV to model maternal antibody interference in vitro. Interferon-alpha (I...

  6. Inhibited Expression of α4β2 Nicotinic Acetylcholine Receptor in Blood Leukocytes of Chinese Patients with Vascular Dementia and in Blood Leukocytes as Well as the Hippocampus of Brain from Ischemic Rats.

    PubMed

    Xiao, Yan; Zhao, Liang; Kuang, Shi-Xiang; Guan, Zhi-Zhong

    2016-11-01

    Our present aim was to investigate whether changes in the expression of α4β2 nicotinic acetylcholine receptor (nAChR) in patients with vascular dementia (VaD) and ischemic rats are related to cognitive scores. Blood leukocytes for 59 Chinese patients with VaD (diagnosed on the basis of clinical guidelines) and 31 cases as age-matched controls were examined, and the animal model established employing Pulsinelli's four-vessel occlusion. The levels of α4 and β2 subunit mRNA in leukocytes and the hippocampus were analyzed by real-time PCR, and the protein level in the hippocampus by Western blotting. The mini-mental state examination was utilized to characterize the intellectual capacity of the patients with reference to the DSM IV diagnosis and Hachinski Ischemic Scale score, and the Morris Water Maze test to assess the ability of learning and memory of the rats. In patients, the level of α4 mRNA, but not β2, in blood leukocytes was clearly lowered, which was significantly correlated to their clinical cognitive test scores. Smoking exerted no impact on the level of α4 mRNA in the present study. In the blood leukocytes and the hippocampus of the brains of the ischemic rats, the levels of both α4 and β2 mRNA were lowered, and the proteins of these subunits in the hippocampus were decreased. The changes of α4 and β2 mRNA in blood leukocytes, and their protein levels in the hippocampus were significantly correlated with impaired learning and memory. These findings indicate that alterations in expression of the α4β2 subtype of nAChR may be involved in the molecular mechanism(s) underlying the cognitive deficit associated with VaD.

  7. Apoptotic effects of tamoxifen on leukocytes from horse peripheral blood and bronchoalveolar lavage fluid.

    PubMed

    Sarmiento, J; Perez, B; Morales, N; Henriquez, C; Vidal, L; Folch, H; Galecio, J S; Morán, G

    2013-12-01

    A reduction in inflammatory cell apoptosis is an important concept in the maintenance of inflammation and a potential target for the resolution of inflammation in many inflammatory diseases. Dysregulation of apoptosis has been implicated in a range of diseases, including tumors, neurodegenerative disorders and autoimmunity, and may also be implicated in allergic asthma. In horses, recurrent airway obstruction (RAO) is an asthma-like condition that is characterized increased survival neutrophil bronchial. Tamoxifen is a synthetic, non-steroidal, anti-estrogen agent that is widely used for treating all stages of breast cancer and has been approved for the prevention of breast cancer in high-risk women. The observed efficacy of tamoxifen has been attributed to both growth arrest and the induction of apoptosis. Therefore, the aim of our study was to evaluate the ability of tamoxifen to induce apoptosis in vitro in granulocytic cells from peripheral blood and in mononuclear cells from bronchoalveolar lavage fluid (BALF) in horses. Flow cytometry using commercial AnnexinV-FITC and propidium iodide was used to quantify early and late apoptotic leukocytes, respectively. The results showed a significant increase in early apoptosis in peripheral blood and bronchial granulocytic cells treated with tamoxifen. The rate of early apoptosis of mononuclear cells from blood and BALF when incubated with tamoxifen was significantly lower compared with granulocytic cells. We did not observe a direct effect of tamoxifen on late apoptosis in any of the in vitro assays in the cell types used here. These results indicate that the apoptotic mechanisms under these experimental conditions would affect only blood and BALF granulocytic cells, particularly in early apoptosis. Finally, further in vitro and in vivo studies are needed to better understand apoptotic mechanisms because tamoxifen could be used to treat chronic, inflammatory pathologies associated with granulocytes and allergic

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  9. Accurate segmentation of leukocyte in blood cell images using Atanassov's intuitionistic fuzzy and interval Type II fuzzy set theory.

    PubMed

    Chaira, Tamalika

    2014-06-01

    In this paper automatic leukocyte segmentation in pathological blood cell images is proposed using intuitionistic fuzzy and interval Type II fuzzy set theory. This is done to count different types of leukocytes for disease detection. Also, the segmentation should be accurate so that the shape of the leukocytes is preserved. So, intuitionistic fuzzy set and interval Type II fuzzy set that consider either more number of uncertainties or a different type of uncertainty as compared to fuzzy set theory are used in this work. As the images are considered fuzzy due to imprecise gray levels, advanced fuzzy set theories may be expected to give better result. A modified Cauchy distribution is used to find the membership function. In intuitionistic fuzzy method, non-membership values are obtained using Yager's intuitionistic fuzzy generator. Optimal threshold is obtained by minimizing intuitionistic fuzzy divergence. In interval type II fuzzy set, a new membership function is generated that takes into account the two levels in Type II fuzzy set using probabilistic T co norm. Optimal threshold is selected by minimizing a proposed Type II fuzzy divergence. Though fuzzy techniques were applied earlier but these methods failed to threshold multiple leukocytes in images. Experimental results show that both interval Type II fuzzy and intuitionistic fuzzy methods perform better than the existing non-fuzzy/fuzzy methods but interval Type II fuzzy thresholding method performs little bit better than intuitionistic fuzzy method. Segmented leukocytes in the proposed interval Type II fuzzy method are observed to be distinct and clear.

  10. The Activation Pattern of Blood Leukocytes in Head and Neck Squamous Cell Carcinoma Is Correlated to Survival

    PubMed Central

    Millrud, Camilla Rydberg; Månsson Kvarnhammar, Anne; Uddman, Rolf; Björnsson, Sven; Riesbeck, Kristian; Cardell, Lars Olaf

    2012-01-01

    Head and neck squamous cell carcinoma (HNSCC) is known to cause substantial immunosuppression. The present study was designed to characterize blood leukocyte activation in HNSCC and to investigate if the individual activation pattern could be related to tumor progress and survival. The leukocyte activation profile of HNSCC patients and healthy controls was assessed with flow cytometry. HNSCC patients displayed increased numbers of monocytes, neutrophils and total leukocytes as well as an enhanced neutrophil/lymphocyte ratio. In addition, patients had a higher percentage of CD69+, CD71+ and CD98+ T cell subsets and NK cells, and a reduced expression of L-selectin in CD14highCD16+ monocytes and neutrophils, when compared to controls. These changes could be correlated to both tumor burden and spread to lymph nodes. Among the cancer patients an increased neutrophil/lymphocyte ratio, a low neutrophil and CD14high CD16+ monocyte activation state and an elevated CD4/CD8 ratio were related to poor survival. In contrast, a high percentage of CD98+ Th cells appeared to be associated with a better outcome. Taken together, the present data indicate that HNSCC causes activation of blood leukocytes and that the individual activation pattern can be linked to prognosis. PMID:23251433

  11. Systemic administration of a TLR7 ligand leads to transient immune incompetence due to peripheral-blood leukocyte depletion.

    PubMed

    Gunzer, Matthias; Riemann, Helge; Basoglu, Yasmin; Hillmer, Anja; Weishaupt, Carsten; Balkow, Sandra; Benninghoff, Bernd; Ernst, Beat; Steinert, Meike; Scholzen, Thomas; Sunderkötter, Cord; Grabbe, Stephan

    2005-10-01

    Toll-like receptor (TLR) ligands lead to the induction of proinflammatory cytokines and are potent enhancers of specific immune responses. We show here that a single systemic dose of R-848, a ligand for TLR7, potently enhanced hapten sensitization during the induction of contact hypersensitivity (CHS). However, R-848 administration also resulted in a rapid and almost complete depletion of leukocytes from the blood. This effect was transient and was associated with general induction of endothelial adhesiveness. In response to R-848, endothelial cells up-regulated adhesion molecules in vitro and in vivo and leukocytes exhibited increased rolling on endothelia in R-848-treated animals. Adhesion molecule induction appeared to be a direct effect, because endothelial cells expressed TLR7 in vitro and in vivo. After R-848 treatment, the tissue residence time of leukocytes was markedly prolonged in all major peripheral organs. The resulting transiently reduced availability of peripheral-blood leukocytes (PBLs) (TRAP) significantly inhibited otherwise potent CHS responses until the effector cells returned. Thus, although TLR7 ligands are effective adjuvants for the induction of cell-mediated immunity, they can transiently inhibit the elicitation of localized immune responses, possibly due to a systemic endothelial activation throughout the vasculature.

  12. Immunophenotype characteristics of peripheral blood mononuclear leukocytes of chronic idiopathic urticaria patients.

    PubMed

    Garmendia, Jenny V; Zabaleta, Mercedes; Aldrey, Oscar; Rivera, Henry; De Sanctis, Juan B; Bianco, Nicolás E; Blanca, Isaac

    2006-12-01

    The pathogenesis of chronic idiopathic urticaria (CIU) is not completely understood although autoimmunity has been proposed. The aim of the study was to assess the expression of different leukocyte antigens, by flow cytometry, assaying total blood of 29 patients with CIU and of 20 sex and age matched controls. Moreover, we assessed soluble CD154 a marker of immune cell activation, predominantly memory T cells. When patients were divided depending an their response to the autologous serum skin test (ASST), three different groups were encountered: group 1 (n=11): with negative ASST-, group 2 (n=11): positive ASST (ASST+) with normal lymphocyte counts and group 3 (n=7): ASST+ with low lymphocyte counts (< 1500 cells/mm3). A significant increase in CD19+ percentage and not in the absolute count (P < 0.05) was observed in group 1 as compared to controls and to the other groups. In contrast, CD30+, CD45RO+ and CD4+/CD45RO+ percentages and biologically active soluble CD154 levels were significantly higher (P < 0.05) in group 3 as compared to group 1 or to controls. In ASST positive groups, CD45RO+ and CD4+/CD45RO+ positiveness correlates with wheal diameter. In conclusion, memory cells may play a role in these different types of patients and in understanding CIU pathogenesis.

  13. Extracellular Histones Induce Chemokine Production in Whole Blood Ex Vivo and Leukocyte Recruitment In Vivo.

    PubMed

    Westman, Johannes; Papareddy, Praveen; Dahlgren, Madelene W; Chakrakodi, Bhavya; Norrby-Teglund, Anna; Smeds, Emanuel; Linder, Adam; Mörgelin, Matthias; Johansson-Lindbom, Bengt; Egesten, Arne; Herwald, Heiko

    2015-12-01

    The innate immune system relies to a great deal on the interaction of pattern recognition receptors with pathogen- or damage-associated molecular pattern molecules. Extracellular histones belong to the latter group and their release has been described to contribute to the induction of systemic inflammatory reactions. However, little is known about their functions in the early immune response to an invading pathogen. Here we show that extracellular histones specifically target monocytes in human blood and this evokes the mobilization of the chemotactic chemokines CXCL9 and CXCL10 from these cells. The chemokine induction involves the toll-like receptor 4/myeloid differentiation factor 2 complex on monocytes, and is under the control of interferon-γ. Consequently, subcutaneous challenge with extracellular histones results in elevated levels of CXCL10 in a murine air pouch model and an influx of leukocytes to the site of injection in a TLR4 dependent manner. When analyzing tissue biopsies from patients with necrotizing fasciitis caused by Streptococcus pyogenes, extracellular histone H4 and CXCL10 are immunostained in necrotic, but not healthy tissue. Collectively, these results show for the first time that extracellular histones have an important function as chemoattractants as their local release triggers the recruitment of immune cells to the site of infection.

  14. Cell-type specific gene expression profiles of leukocytes in human peripheral blood

    PubMed Central

    Palmer, Chana; Diehn, Maximilian; Alizadeh, Ash A; Brown, Patrick O

    2006-01-01

    Background Blood is a complex tissue comprising numerous cell types with distinct functions and corresponding gene expression profiles. We attempted to define the cell type specific gene expression patterns for the major constituent cells of blood, including B-cells, CD4+ T-cells, CD8+ T-cells, lymphocytes and granulocytes. We did this by comparing the global gene expression profiles of purified B-cells, CD4+ T-cells, CD8+ T-cells, granulocytes, and lymphocytes using cDNA microarrays. Results Unsupervised clustering analysis showed that similar cell populations from different donors share common gene expression profiles. Supervised analyses identified gene expression signatures for B-cells (427 genes), T-cells (222 genes), CD8+ T-cells (23 genes), granulocytes (411 genes), and lymphocytes (67 genes). No statistically significant gene expression signature was identified for CD4+ cells. Genes encoding cell surface proteins were disproportionately represented among the genes that distinguished among the lymphocyte subpopulations. Lymphocytes were distinguishable from granulocytes based on their higher levels of expression of genes encoding ribosomal proteins, while granulocytes exhibited characteristic expression of various cell surface and inflammatory proteins. Conclusion The genes comprising the cell-type specific signatures encompassed many of the genes already known to be involved in cell-type specific processes, and provided clues that may prove useful in discovering the functions of many still unannotated genes. The most prominent feature of the cell type signature genes was the enrichment of genes encoding cell surface proteins, perhaps reflecting the importance of specialized systems for sensing the environment to the physiology of resting leukocytes. PMID:16704732

  15. Perioperative transfusion of leukocyte depleted blood products in gastric cancer patients negatively influences oncologic outcome

    PubMed Central

    Reim, Daniel; Strobl, Andreas N.; Buchner, Christian; Schirren, Rebekka; Mueller, Werner; Luppa, Peter; Ankerst, Donna Pauler; Friess, Helmut; Novotny, Alexander

    2016-01-01

    Abstract The influence of perioperative transfusion (PT) on outcome following surgery for gastric cancer (GC) remains controversial, with randomized trials lacking and observational series confounded by patient risk factors. This analysis determines the association between reception of leukocyte-depleted blood products and post-operative survival for GC. Data from 610 patients who underwent curative surgery for GC in a German tertiary care clinic from 2001 to 2013 were included. Kaplan–Meier survival curves and Cox proportional hazards regression were applied to determine the association of PT and clinical and patient risk factors for overall and relapse-free survival. Propensity score analysis was performed to adjust for observational biases in reception of PT. Higher Union International Contre le Cancer/American Joint Committee on Cancer (UICC/AJCC)-stages (P <0.001), postoperative complications and severity according to the Clavien–Dindo (CD) classification (P <0.001), PT (P = 0.02), higher age (P <0.001), and neoadjuvant chemotherapy (P <0.001) were related to increased mortality rates. Higher UICC-stages (P <0.001), neoadjuvant chemotherapy (P <0.001), and type of surgery (P = 0.02) were independently associated with increased relapse rates. Patients were more likely to receive PT with higher age (P = 0.05), surgical extension to adjacent organs/structures (P = 0.002), tumor location (P = 0.003), and female gender (P = 0.03). In the adjusted propensity score weighted analysis, PT remained associated with an increased risk of death (hazard ratio (HR): 1.31, 95% CI: 1.01–1.69, P = 0.04). Because of the association of PT with negative influence on patient survival following resection for GC, risks from application of blood products should be weighed against the potential benefits. PMID:27442682

  16. [Consequences for labile blood products of leukocyte depletion by whole blood filtration using the Leucoflex LST1 in-line filter. Evaluation of the Leucoflex LST1 filter].

    PubMed

    Janus, G; Samson, T; Faudon, R; Renaudeau, C; Joussemet, M; Fabre, G

    1997-12-01

    The aim of this study is to evaluate the effects of whole blood filtration after a storage time of 20-24 hours at laboratory temperature using the in line filter Leucoflex LST1. The study concerns 49 blood donations in which we studied leukocyte depletion, proteins (IgG, IgA, IgM, haptoglobin, C3, C4), coagulation factors (fibrinogen, factors XII, XI, IX, VIII, V, proteins S and C, plasminogen, tPA, D-Dimers, PDF) at day 1, the parameters of conservation (ATP, 2-3 DPG, extra cellular potassium, haemolysis, pH) of red blood cell concentrates (RCCs) and bacteriological sterility at day 1 and 42. Despite a correct leukocyte depletion (mean depletion of 3.96 log), a 10 fold higher mean level of residual leukocytes/unit than with buffy coat poor RCC filtration (0.514.10(6) vs 0.051.10(6)) is observed. Moreover a lot of concentrates are not in accordance with French regulations (7/42 with more than 1.10(6) leukocytes/unit). The variation of the rates of IgG, IgA, IgM, haptoglobin, C4 and protein C is not significant. For the others there is a slight decrease with a mean level remaining in a physiological range. No sign of activation is noted. The sterility assays remain negative and the RCC conservation is not altered. In conclusion, even if the quality of the leukocyte depletion is not satisfactory in our study and has to be stated more precisely by multicenter studies, the whole blood filtration does not alter the quality of the derived components and allows us obtain RCC in a bigger volume and containing more haemoglobin than with the classical procedure after removing the buffy-coat [10].

  17. Flow cytometry as a tool in the evaluation of blood leukocyte function in Chelonia mydas (Linnaeus, 1758) (Testudines, Cheloniidae).

    PubMed

    Rossi, S; Sá-Rocha, V M; Kinoshita, D; Genoy-Puerto, A; Zwarg, T; Werneck, M R; Sá-Rocha, L C; Matushima, E R

    2009-08-01

    Chelonia mydas is a sea turtle that feeds and nests on the Brazilian coast and a disease called fibropapillomatosis is a threat to this species. Because of this, it is extremely necessary to determine a methodology that would enable the analysis of blood leukocyte function in these sea turtles. In order to achieve this aim, blood samples were collected from C. mydas with or without fibropapillomas captured on the São Paulo north coast. Blood samples were placed in tubes containing sodium heparin and were transported under refrigeration to the laboratory in sterile RPMI 1640 cell culture medium. Leukocytes were separated by density gradient using Ficoll-PaqueTM Plus, Amershan Biociences. The following stimuli were applied in the assessment of leukocyte function: Phorbol Miristate-Acetate (PMA) for oxidative burst activity evaluation and Zymosan A (Saccharomyces cerevisiae) Bio Particles, Alexa Fluor 594 conjugate for phagocytosis evaluation. Three cell populations were identified: heterophils, monocytes and lymphocytes. Monocytes were the cells responsible for phagocytosis and oxidative burst.

  18. Differential expression of intracellular and extracellular CB(2) cannabinoid receptor protein by human peripheral blood leukocytes.

    PubMed

    Castaneda, Julie T; Harui, Airi; Kiertscher, Sylvia M; Roth, Jeffrey D; Roth, Michael D

    2013-03-01

    mRNA encoding for the CB(2) cannabinoid receptor is expressed by many subsets of human peripheral blood leukocytes (PBL), but little is known about the resulting protein expression and function. Employing clones from the A549 and 293T cell lines that were constructed to express both full-length human CB(2) and GFP, we developed a flow cytometry assay for characterizing CB(2) protein expression. A monoclonal antibody directed against human CB(2) selectively stained the surface of transduced but not parental cell lines. When cells were fixed and permeabilized, imaging flow cytometry identified large stores of intracellular protein. Total cellular staining for CB(2) corresponded closely with the level of GFP expression. When exposed to Δ(9)-tetrahydrocannabinol, CB(2)-expressing cells internalized cell surface CB(2) receptors in a time- and dose-dependent manner. Applying these approaches to human PBL, CB(2) protein was identified on the surface of human B cells but not on T cells or monocytes. In contrast, when PBL were fixed and permeabilized, intracellular CB(2) expression was readily detected in all three subsets by both conventional and imaging flow cytometry. Similar to the protein expression pattern observed in fixed and permeabilized PBL, purified B cells, T cells, and monocytes expressed relatively equal levels of CB(2) mRNA by quantitative real-time RT-PCR. Our findings confirm that human PBL express CB(2) protein but that its distribution is predominantly intracellular with only B cells expressing CB(2) protein at the extracellular membrane. The differential role of intracellular and extracellular CB(2) receptors in mediating ligand signaling and immune function remains to be determined.

  19. Fatal TRALI associated with neutrophil antibodies in a recipient of pre-storage leukocyte-reduced blood components.

    PubMed

    Ishii, Kazuyoshi; Kadota, Eiji; Shimizu, Michiomi; Nomura, Shosaku

    2009-01-01

    A 53-year-old man developed possible transfusion-related acute lung injury (TRALI) after red cell component transfusion. The patient developed autoimmune neutropenia with the expression of neutrophil antibodies. Neutrophil aggregation, endothelial damage, and development of a large thrombus containing platelets were observed post mortem in his pulmonary vessels. The patient also had subacute organizing pneumonia. He received blood components treated with universal pre-storage leuko-reduction. Even though leukocytes in the blood components are reduced to a few million by this process, TRALI can be fatal, as was the case for this recipient, who had subacute organizing pneumonia in conjunction with immune-mediated neutropenia.

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

    PubMed

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

    2006-04-01

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

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

    PubMed

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

    2010-01-01

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

  2. [The importance of studying the acid phosphatase of the blood serum and bone marrow lymphoblasts and polymorphonuclear neutrophils in the prognosis of the course of acute lymphoblastic leukemia].

    PubMed

    Vaiuta, N P; Khaĭfets, L M; Mendeleev, I M

    1988-01-01

    The activity of serum acid phosphatase (AP), bone marrow lymphoblasts and polymorphonuclear neutrophils was studied in 45 ALL patients. Cytochemical coefficients (CCC) and the percentage of positively reacting bone marrow cells were determined. All the patients received programmed polychemotherapy. They were investigated before the start of therapy, during recurrence and at different time of remission (from 1 to 60 mos) during each reinduction cycle. At the climax of ALL the activity of serum AP was increased 2.8-fold, a CCC value for lymphoblastic AP--10-fold, for polymorphonuclear neutrophils--3-fold as compared with normal values. A tendency toward the reduction of indices was noted at different time of remission, the approximation to normal values was noted on the 40th-46th months of remission only. In recurrence development the level of the serum and cellular enzyme as well as the percentage of positively reacting cells significantly exceeded normal values and were close to indices at the climax of disease. The above tendency permitted the use of these tests to evaluate the completeness of remission and to predict recurrences during a follow-up of ALL patients.

  3. Leukocyte, red blood cell and morphological adaptation to moderate physical training in rats undernourished in the neonatal period

    PubMed Central

    Viana, Marcelo Tavares; Perez, Manuella Cavalcanti; Ribas, Valdenilson Ribeiro; Martins, Gilberto de Freire; de Castro, Célia Maria Machado Barbosa

    2012-01-01

    Objective To analyze the impact of moderate physical exercise on the total and differential leukocyte counts and red blood cell count of 36 sixty-day-old adult male Wistar rats subjected to early malnourishment. Methods The rats were divided in nourished (N - casein 17%) and malnourished groups (M - casein 8%) and thesegroups were then subdivided in trained (T) untrained (U) creating four groups NT, NU, MT and MU. The NT and MTgroups were submitted to moderate physical exercise using a treadmill (60 min/day, 5 days/week for 8 weeks). Onthe 1st day, before the training started T0 and 24 hours after the last training day of the week (T1 until T8), a 1 mLaliquot of blood was collected from the animals' tails for analysis. The total leukocyte count was evaluated in a cellcounter with an electronic microscope. The cyanmethemoglobin technique was used to measure the hemoglobin level. The hematocrit values were determined as a percentage using the micro-hematocrit technique with a microcapillaryreader and a cell counter was used to determine the red blood cell count. The t-test was used for statistical analysis and a p-value < 0.05 was considered significant. Data are expressed as means ± standard deviation. Results There was a significant difference in the total leukocyte count between the NT (9.1 ± 0.1) and MT groups (8.0 ± 0.1) from T1 and in neutrophils between the NT (22.1 ± 0.6) and MT groups (24.6 ± 1.8) from T7 (p < 0.05). There was no statistical significance in the hemoglobin, hematocrit and red blood cell count from T1. Conclusions According to the results of this study, moderate physical exercise seems to have induced physiologic adaptation in adult rats from T1. PMID:23049442

  4. The modulating effect of royal jelly consumption against radiation-induced apoptosis in human peripheral blood leukocytes.

    PubMed

    Rafat, Navid; Monfared, Ali Shabestani; Shahidi, Maryam; Pourfallah, Tayyeb Allahverdi

    2016-01-01

    The present work was designed to assess the radioprotective effect of royal jelly (RJ) against radiation-induced apoptosis in human peripheral blood leukocytes. In this study, peripheral blood samples were obtained on days 0, 4, 7, and 14 of the study from six healthy male volunteers taking a 1000 mg RJ capsule orally per day for 14 consecutive days. On each sampling day, all collected whole blood samples were divided into control and irradiated groups which were then exposed to the selected dose of 4 Gy X-ray. Percentage of apoptotic cells (Ap %) was evaluated for all samples immediately after irradiation (Ap0) and also after a 24 h postirradiation incubation at 37°C in 5% CO2 (Ap24) by the use of neutral comet assay. Concerning Ap0, collected data demonstrated that the percentage of apoptotic cells in both control and irradiated groups did not significantly change during the study period. However, with respect to Ap24, the percentage of apoptotic cells in irradiated groups gradually reduced during the experiment, according to which a significant decrease was found after 14 days RJ consumption (P = 0.002). In conclusion, the present study revealed the protective role of 14 days RJ consumption against radiation-induced apoptosis in human peripheral blood leukocytes.

  5. Correlation of MLH1 and MGMT methylation levels between peripheral blood leukocytes and colorectal tissue DNA samples in colorectal cancer patients.

    PubMed

    Li, Xia; Wang, Yibaina; Zhang, Zuoming; Yao, Xiaoping; Ge, Jie; Zhao, Yashuang

    2013-11-01

    CpG island methylation in the promoter regions of the DNA mismatch repair gene mutator L homologue 1 (MLH1) and DNA repair gene O(6)-methylguanine-DNA methyltransferase (MGMT) genes has been shown to occur in the leukocytes of peripheral blood and colorectal tissue. However, it is unclear whether the methylation levels in the blood leukocytes and colorectal tissue are correlated. The present study analyzed and compared the levels of MGMT and MLH1 gene methylation in the leukocytes of peripheral blood and colorectal tissues obtained from patients with colorectal cancer (CRC). The methylation levels of MGMT and MLH1 were examined using methylation-sensitive high-resolution melting (MS-HRM) analysis. A total of 44 patients with CRC were selected based on the MLH1 and MGMT gene methylation levels in the leukocytes of the peripheral blood. Corresponding colorectal tumor and normal tissues were obtained from each patient and the DNA methylation levels were determined. The correlation coefficients were evaluated using Spearman's rank test. Agreement was determined by generalized κ-statistics. Spearman's rank correlation coefficients (r) for the methylation levels of the MGMT and MLH1 genes in the leukocytes of the peripheral blood and normal colorectal tissue were 0.475 and 0.362, respectively (P=0.001 and 0.016, respectively). The agreement of the MGMT and MLH1 gene methylation levels in the leukocytes of the peripheral blood and normal colorectal tissue were graded as fair and poor (κ=0.299 and 0.126, respectively). The methylation levels of MGMT and MLH1 were moderately and weakly correlated between the patient-matched leukocytes and the normal colorectal tissue, respectively. Blood-derived DNA methylation measurements may not always represent the levels of normal colorectal tissue methylation.

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

    PubMed

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

    2003-06-15

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

  7. Inhibition of peripheral blood neutrophil oxidative burst in periodontitis patients with a homeopathic medication Traumeel S

    PubMed Central

    žilinskas, Juozas; žekonis, Jonas; žekonis, Gediminas; Šadzevičienė, Renata; Sapragonienė, Marija; Navickaitė, Justina; Barzdžiukaitė, Ingrida

    2011-01-01

    Summary Background The anti-inflammatory effects of a homeopathic remedy, Traumeel S, have been observed in experimental and clinical studies; however, its antioxidant properties have not been elucidated. The aim of the present study was to evaluate the antioxidant effects of Traumeel S on peripheral blood neutrophils in patients with periodontitis. Material/Methods The study was performed using venous blood of 22 individuals with chronic periodontitis and 21 healthy subjects. The antioxidant effects of Traumeel S on the production of reactive oxygen species by unstimulated and stimulated with unopsonized E. coli neutrophils were investigated using luminol- and lucigenin-dependent chemiluminescence (CL). Results Polymorphonuclear leukocytes of periodontitis patients produced higher levels (p<0.01) of light output of lucigenin-dependent chemiluminescence and significantly reduced (p<0.01) light output of luminol-dependent chemiluminescence than analogous cells of healthy subjects. Highly diluted (10−4 of the stem solution) Traumeel S significantly (by approximately 50%) reduced superoxide-induced oxidation of lucigenin by unstimulated and stimulated with unopsonized E. coli polymorphonuclear leukocytes of periodontitis patients and had a tendency to intensify luminol-dependent chemiluminescence. Preincubation of the unstimulated and stimulated with unopsonized E. coli polymorphonuclear leukocytes of healthy subjects with Traumeel S exerts no inhibitory action on the luminol- and lucigenin-dependent chemiluminescence of the above-mentioned cells. Conclusions This study indicates that Traumeel S may significantly reduce production of superoxide anion by unstimulated and stimulated peripheral blood polymorphonuclear neutrophils of periodontitis patients. PMID:21525811

  8. Living T9 glioma cells expressing membrane macrophage colony-stimulating factor produce immediate tumor destruction by polymorphonuclear leukocytes and macrophages via a "paraptosis"-induced pathway that promotes systemic immunity against intracranial T9 gliomas.

    PubMed

    Chen, Yijun; Douglass, Thomas; Jeffes, Edward W B; Xu, Qingcheng; Williams, Christopher C; Arpajirakul, Neary; Delgado, Christina; Kleinman, Michael; Sanchez, Ramon; Dan, Qinghong; Kim, Ronald C; Wepsic, H Terry; Jadus, Martin R

    2002-08-15

    Cloned T9-C2 glioma cells transfected with membrane macrophage colony-stimulating factor (mM-CSF) never formed subcutaneous tumors when implanted into Fischer rats, whereas control T9 cells did. The T9-C2 cells were completely killed within 1 day through a mechanism that resembled paraptosis. Vacuolization of the T9-C2 cell's mitochondria and endoplasmic reticulum started within 4 hours after implantation. By 24 hours, the dead tumor cells were swollen and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL)-positive. Bcl2-transduced T9-C2 cells failed to form tumors in rats. Both T9 and T9-C2 cells produced cytokine-induced neutrophil chemoattractant that recruited the granulocytes into the tumor injection sites, where they interacted with the tumor cells. Freshly isolated macrophages killed the T9-C2 cells in vitro by a mechanism independent of phagocytosis. Nude athymic rats treated with antiasialo GM1 antibody formed T9-C2 tumors, whereas rats treated with a natural killer cell (NK)-specific antibody failed to form tumors. When treated with antipolymorphonuclear leukocyte (anti-PMN) and antimacrophage antibodies, 80% of nude rats formed tumors, whereas only 40% of the rats developed a tumor when a single antibody was used. This suggests that both PMNs and macrophages are involved in the killing of T9-C2 tumor cells. Immunocompetent rats that rejected the living T9-C2 cells were immune to the intracranial rechallenge with T9 cells. No vaccinating effect occurred if the T9-C2 cells were freeze-thawed, x-irradiated, or treated with mitomycin-C prior to injection. Optimal tumor immunization using mM-CSF-transduced T9 cells requires viable tumor cells. In this study optimal tumor immunization occurred when a strong inflammatory response at the injection of the tumor cells was induced.

  9. Cell type-specific release of matrix-metallo-proteinase-9 by bacterial chemoattractant in human blood phagocytic leukocytes.

    PubMed

    Doerner, Astrid M; Chen, Ling-Yu; Ye, Richard D; Yong, Jiang; Huang, Shuang; Pan, Zhixing K

    2011-01-01

    Stimulation of phagocytic leukocytes with bacterial chemoattractant resulted in the release of matrix metal-loproteinases (MMPs). Little is known about the mechanisms of bacterial chemoattractant regulation of MMP in phagocytic leukocytes. We report here that the mechanisms of the bacterial chemotactic peptidefMLP-induced MMP -9 release in monocytes appeared to be different from fMLP-stimulated MMP-9 release in neutrophils. In freshly prepared peripheral blood monocytes, fMLP induces MMP-9 release, starting at 8 h after stimulation. These functions of fMLP is accompanied by an increase in TNFα expression, and mediated through the phosphorylation of ERK1/2 in monocytes. However, neutrophil preparations that responded to fMLP with MMP-9 release did not require activation of ERK1/2 and TNFα expression. These results suggest a different role of fMLP in MMP-9 expression in neutrophils and monocytes, and the signal molecules involved in mediating this effect in human blood monocytes stimulated by bacterial chemoattractant.

  10. Gene expression profiling in peripheral blood leukocytes as a new approach for assessment of human stress response.

    PubMed

    Rokutan, Kazuhito; Morita, Kyoko; Masuda, Kiyoshi; Tominaga, Kumiko; Shikishima, Michiyo; Teshima-Kondo, Shigetada; Omori, Tetsuro; Sekiyama, Atsuo

    2005-08-01

    Stress is the coordinated physiological processes to maintain a dynamic equilibrium under stressful conditions. The equilibrium is threatened by certain physiological and psychological stressors. Stressors trigger physiological, behavioural, and metabolic responses that are aimed at reinstating homeostasis. The hypothalamus-pituitary-adrenal (HPA) axis and the sympathetic nervous system play an essential role in the stress response. Excessive,prolonged, or inadequate response that is termed as "allostasis" or "allostatic load" leads to pathological outcomes. Dysregulation of the HPA axis activity is involved in the pathogenesis of stress-related disorders including major depression. The complex brain-immune-endocrine network regulates the HPA axis, and hereditary predisposition as well as environmental factors such as traumatic experiences in early life also modifies the capacity of an individual to cope. Therefore, it is difficult to correctly assess the complex stress response. We have developed a microarray carrying 1,467 cDNAs that were selected to specifically measure stress response in peripheral blood leukocytes. Using this tool, we have succeeded to objectively assess individual response to acute psychological stress and to detect unique expression profiles in patients with depression. Gene expression profile in peripheral blood leukocytes may be a potentially useful for the detection of disease-associated, abnormal stress responses.

  11. Longitudinal Frequencies of Blood Leukocyte Subpopulations Differ between NOD and NOR Mice but Do Not Predict Diabetes in NOD Mice

    PubMed Central

    Telieps, Tanja; Köhler, Meike; Treise, Irina; Foertsch, Katharina; Adler, Thure; Busch, Dirk H.; Hrabě de Angelis, Martin; Verschoor, Admar; Adler, Kerstin; Bonifacio, Ezio; Ziegler, Anette-Gabriele

    2016-01-01

    Immune phenotyping provides insight into disease pathogenesis and prognostic markers. Trajectories from age of 4 to 36 weeks were modeled for insulin autoantibodies and for leukocyte subpopulations in peripheral blood from female NOD (n = 58) and NOR (n = 22) mice. NOD mice had higher trajectories of insulin autoantibodies, CD4+ and CD8+ T lymphocytes, B lymphocytes, IgD+IgM− B lymphocytes, and NK cells and lower trajectories of CD4+CD25+ T lymphocytes, IgM+ B lymphocytes, granulocytes, and monocytes than NOR mice (all p < 0.001). Of these, only the increased IAA trajectory was observed in NOD mice that developed diabetes as compared to NOD mice that remained diabetes-free. Therefore, the profound differences in peripheral blood leukocyte proportions observed between the diabetes-prone NOD mice and the diabetes-resistant mice do not explain the variation in diabetes development within NOD mice and do not provide markers for diabetes prediction in this model. PMID:26966692

  12. Development of methods to examine the effects of atmospheric particulate matter (PM) on human peripheral blood leukocytes

    NASA Astrophysics Data System (ADS)

    Zussman, Lisa Ann

    In vitro methods to study the effect of atmospheric particulate matter (PM) on leukocyte function using human peripheral blood were developed. These methods were demonstrated using the blood of 1-5 individuals and National Institute of Standards and Technology (NIST) urban PM #1648, diesel PM #1650, silica PM, and a locally collected PM sample (New Jersey PM10). For the blood samples analyzed in this study NIST urban PM and New Jersey PM10 treatment mediated the release of granule contents from peripheral blood leukocytes and induced structural changes associated with degranulation. Flow cytometry revealed PM-induced changes in phagocytosis and cell structure associated with degranulation. Transmission electron microscopy confirmed NIST urban PM-induced cell structure changes were associated with PM internalization. Colorametric and electrophoretic methods showed no PM-induced release of primary granules and a slight PM-induced release of secondary granules associated with only NIST urban PM. Enzyme Immunosorbent Assays detected increased histamine release from basophils treated with NIST urban PM, a locally collected PM, and the soluble and insoluble components of these particles. NIST urban PM was found to be a potent inducer of histamine release in 4 out of 6 individuals tested. Fractionation studies revealed that soluble (aqueous) and insoluble fractions of NIST urban PM contain histamine-releasing activity. This was also demonstrated for the New Jersey PM10 sample for which the soluble fraction exhibited the most activity. Complementary studies with inhibitors of IgE-mediated histamine release conducted on one test subject suggest that PM-induced histamine release was partially mediated by IgE. A new hypothesis has been formed, suggesting that particle toxicity is related to PM-induced histamine release. Due to the bioactive nature of histamine and its association with many cardiopulmonary responses, the PM- mediated release of histamine should be investigated

  13. Anaerobiosis increases resistance of Neisseria gonorrhoeae to O2-independent antimicrobial proteins from human polymorphonuclear granulocytes.

    PubMed Central

    Casey, S G; Shafer, W M; Spitznagel, J K

    1985-01-01

    We investigated the in vitro resistance of Neisseria gonorrhoeae FA19 to the O2-independent antimicrobial systems of human polymorphonuclear leukocytes. Acid extracts of polymorphonuclear leukocyte granules (crude granule extracts) and a purified granule protein (57 kilodaltons) were, at low concentrations, bactericidal for gonococci under aerobic conditions that permitted growth. However, they were less effective under anaerobic conditions that imposed bacteriostasis. We found that adding sodium nitrite to reduced growth media permitted the growth of strain FA19 in an anaerobic environment. Under these conditions with nitrite, anaerobic cultures of strain FA19 were no more resistant to the crude granule extract and the 57-kilodalton protein than aerobic cultures. In contrast, Salmonella typhimurium SL-1004, a facultative anaerobe, was readily killed by both the crude granule extract and the 57-kilodalton antimicrobial protein regardless of the presence or absence of free molecular oxygen. This is the first demonstration that an isolated antimicrobial protein from polymorphonuclear leukocyte granules is active against bacteria under anaerobic conditions. Our results also indicated that the efficacy of human polymorphonuclear leukocyte O2-independent killing of N. gonorrhoeae may, in part, be inhibited by bacteriostatic conditions imposed by hypoxia. Images PMID:3917976

  14. Effect of Very Low Dose Fast Neutrons on the DNA of Rats' Peripheral Blood Mononuclear Cells and Leukocytes.

    PubMed

    Nafee, Sherif S; Saeed, Abdu; Shaheen, Salem A; El Assouli, Sufian M; El Assouli, M-Zaki; Raouf, Gehan A

    2016-01-01

    The effect of very low dose fast neutrons on the chromatin and DNA of rats' peripheral blood mononuclear cells (PBMC) and leukocytes has been studied in the present work using Fourier transform infrared (FTIR) and single-cell gel electrophoresis (comet assay). Fourteen female Wistar rats were used; seven were irradiated with neutrons of 0.9 cGy (Am-Be, 0.02 cGy h(-1)), and seven others were used as control. Second derivative and curve fitting were used to analyze the FTIR spectra. In addition, hierarchical cluster analysis (HCA) was used to classify the group spectra. Meanwhile, the tail moment and percentage of DNA in the tail were used as indicators to sense the breaking and the level of damage in DNA. The analysis of FTIR spectra of the PBMC of the irradiated group revealed a marked increase in the area of phosphodiesters of nucleic acids and the area ratios of RNA/DNA and phosphodiesters/carbohydrates. A sharp significant increase and decrease in the areas of RNA and DNA ribose were recorded, respectively. In the irradiated group, leukocytes with different tail lengths were observed. The distributions of tail moments and the percentage of DNA in the tail of irradiated groups were heterogeneous. The mean value of the percentages of DNA in the tail at 0.5 h post-irradiation represented low-level damage in the DNA. Therefore, one can conclude that very low dose fast neutrons might cause changes in the DNA of PBMC at the submolecular level. It could cause low-level damage, double-strand break, and chromatin fragmentation of DNA of leukocytes.

  15. Neisseria meningitidis and Escherichia coli are protected from leukocyte phagocytosis by binding to erythrocyte complement receptor 1 in human blood.

    PubMed

    Brekke, Ole-Lars; Hellerud, Bernt Christian; Christiansen, Dorte; Fure, Hilde; Castellheim, Albert; Nielsen, Erik Waage; Pharo, Anne; Lindstad, Julie Katrine; Bergseth, Grethe; Leslie, Graham; Lambris, John D; Brandtzaeg, Petter; Mollnes, Tom Eirik

    2011-09-01

    The initial interaction of Gram-negative bacteria with erythrocytes and its implications on leukocyte phagocytosis and oxidative burst in human whole blood were examined. Alexa-labeled Escherichia coli, wild-type H44/76 N. meningitidis and the H44/76lpxA lipopolysaccharide (LPS)-deficient mutant were incubated with whole blood using lepirudin as anticoagulant which has no adverse effects on complement. Bacteria free in plasma, bound to erythrocytes or phagocytized by granulocytes and monocytes were quantified using flow cytometry. The effects of the C3 inhibitor compstatin, a C5a receptor antagonist (C5aRa) and a complement receptor 1 (CR1)-blocking antibody (3D9) were examined. Most bacteria (80%) immediately bound to erythrocytes. The binding gradually declined over time, with a parallel increase in phagocytosis. Complement inhibition with compstatin reduced erythrocyte binding and bacterial C3 opsonization. In contrast, the C5aRa efficiently reduced phagocytosis, but did not affect the binding of bacteria to erythrocytes. The anti-CR1 blocking mAb dose-dependently reduced bacterial binding to erythrocytes to nil, with subsequent increased phagocytosis and oxidative burst. LPS had no effect on these processes since similar results were obtained using an LPS-deficient N. meningitidis mutant. In vivo experiments in a pig model of sepsis showed limited binding of bacteria to erythrocytes, consistent with the facts that erythrocyte CR1 receptors are absent in non-primates and that the bacteria were mainly found in the lungs. In conclusion, complement-dependent binding of Gram-negative bacteria to erythrocyte CR1 decreases phagocytosis and oxidative burst by leukocytes in human whole blood.

  16. Neisseria meningitidis and Escherichia coli are protected from leukocyte phagocytosis by binding to erythrocyte complement receptor 1 in human blood

    PubMed Central

    Brekke, Ole-Lars; Hellerud, Bernt Christian; Christiansen, Dorte; Fure, Hilde; Castellheim, Albert; Nielsen, Erik Waage; Pharo, Anne; Lindstad, Julie Katrine; Bergseth, Grethe; Leslie, Graham; Lambris, John D.; Brandtzaeg, Petter; Mollnes, Tom Eirik

    2011-01-01

    The initial interaction of Gram-negative bacteria with erythrocytes and its implications on leukocyte phagocytosis and oxidative burst in human whole blood were examined. Alexa-labeled Escherichia coli, wild-type H44/76 Neisseria meningitidis (N. meningitidis) and the H44/76lpxA lipopolysaccharide (LPS)-deficient mutant were incubated with whole blood using lepirudin as anticoagulant which has no adverse effects on complement. Bacteria free in plasma, bound to erythrocytes or phagocytized by granulocytes and monocytes were quantified using flow cytometry. The effects of the C3 inhibitor compstatin, a C5a receptor antagonist (C5aRa) and a complement receptor 1 (CR1)-blocking antibody (3D9) were examined. Most bacteria (80%) immediately bound to erythrocytes. The binding gradually declined over time, with a parallel increase in phagocytosis. Complement inhibition with compstatin reduced erythrocyte binding and bacterial C3 opsonization. In contrast, the C5aRa efficiently reduced phagocytosis, but did not affect the binding of bacteria to erythrocytes. The anti-CR1 blocking mAb dose-dependently reduced bacterial binding to erythrocytes to nil, with subsequent increased phagocytosis and oxidative burst. LPS had no effect on these processes since similar results were obtained using an LPS-deficient N. meningitidis mutant. In vivo experiments in a pig model of sepsis showed limited binding of bacteria to erythrocytes, consistent with the facts that erythrocyte CR1 receptors are absent in non-primates and that the bacteria were mainly found in the lungs. In conclusion, complement-dependent binding of Gram-negative bacteria to erythrocyte CR1 decreases phagocytosis and oxidative burst by leukocytes in human whole blood. PMID:21839519

  17. 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.

  18. Cytogenetic observations in human peripheral blood leukocytes following in vitro exposure to THz radiation: a pilot study.

    PubMed

    Zeni, O; Gallerano, G P; Perrotta, A; Romanò, M; Sannino, A; Sarti, M; D'Arienzo, M; Doria, A; Giovenale, E; Lai, A; Messina, G; Scarfì, M R

    2007-04-01

    Emerging technologies are considering the possible use of Terahertz radiation in different fields ranging from telecommunications to biology and biomedicine. The study of the potential effects of Terahertz radiation on biological systems is therefore an important issue in order to safely develop a variety of applications. This paper describes a pilot study devoted to determine if Terahertz radiation could induce genotoxic effects in human peripheral blood leukocytes. For this purpose, human whole blood samples from healthy donors were exposed for 20 min to Terahertz radiation. Since, to our knowledge, this is the first study devoted to the evaluation of possible genotoxic effects of such radiation, different electromagnetic conditions were considered. In particular, the frequencies of 120 and 130 GHz were chosen: the first one was tested at a specific absorption rate (SAR) of 0.4 mW g-1, while the second one was tested at SAR levels of 0.24, 1.4, and 2 mW g-1. Chromosomal damage was evaluated by means of the cytokinesis block micronucleus technique, which also gives information on cell cycle kinetics. Moreover, human whole blood samples exposed to 130 GHz at SAR levels of 1.4 and 2 mW g-1 were also tested for primary DNA damage by applying the alkaline comet assay immediately after exposure. The results obtained indicate that THz exposure, in the explored electromagnetic conditions, is not able to induce either genotoxicity or alteration of cell cycle kinetics in human blood cells from healthy subjects.

  19. A novel splice variant of human gene NPL, mainly expressed in human liver, kidney and peripheral blood leukocyte.

    PubMed

    Wu, Maoqing; Gu, Shaohua; Xu, Jian; Zou, Xianqiong; Zheng, Huarui; Jin, Zhe; Xie, Yi; Ji, Chaoneng; Mao, Yumin

    2005-04-01

    From the human fetal brain cDNA library constructed by our lab, a novel variant cDNA of a human gene was successfully cloned and identified. Because the gene has been named N-acetylneuraminate pyruvate lyase (NPL), accordingly we term our splice variant NPL_v2. The cDNA of NPL_v2 has a full-length open reading frame (ORF) from the nucleotide position 320 to 1225 that encodes a protein comprising 301 amino acids. SMART analysis showed that our hypothetical protein has one dihydrodipicolinate synthase (DHDPS) domain. Phosphorylation analysis of the deduced protein show that there are five phosphorylation sites including three "serine" and two "threonine" at the region that are not found in other splice variant. RT-PCR experiment revealed that our splice variant of the gene is mainly expressed in human placenta, liver, kidney, pancreas, spleen, thymus, ovary, small intestine and peripheral blood leukocyte.

  20. Quantitative analysis of the suppressors of cytokine signaling 1 and 3 in peripheral blood leukocytes of patients with multiple sclerosis.

    PubMed

    Sedeño-Monge, Virginia; Arcega-Revilla, Raúl; Rojas-Morales, Emmanuel; Santos-López, Gerardo; Perez-García, Juan Carlos; Sosa-Jurado, Francisca; Vallejo-Ruiz, Verónica; Solis-Morales, Casandra Lucrecia; Aguilar-Rosas, Salvador; Reyes-Leyva, Julio

    2014-08-15

    Multiple sclerosis (MS) is an autoimmune disease characterized by a triad of inflammation, demyelination and gliosis. Because the suppressors of cytokine signaling (Socs) regulate the immune response, we quantified SOCS1 and SOCS3 transcription in peripheral blood leukocytes of patients with MS. SOCS1 transcription decreased significantly in MS patients compared with neurologically healthy persons (0.08±0.02 vs. 1.02±0.23; p=0.0001); while SOCS3 transcription increased in MS patients compared with controls (2.76±0.66 vs. 1.03±0.27; p=0.0008). Our results showed an imbalance of SOCS1 and SOCS3 transcription in MS patients, and a moderated negative correlation between them (Spearman's r=-0.57; p=0.0003).

  1. The effects of oil exposure on peripheral blood leukocytes and splenic melano-macrophage centers of Gulf of Mexico fishes.

    PubMed

    Ali, Ahmad Omar; Hohn, Claudia; Allen, Peter J; Ford, Lorelei; Dail, Mary Beth; Pruett, Stephen; Petrie-Hanson, Lora

    2014-02-15

    In August and November 2010 we collected and examined peripheral blood and tissues from three species of Gulf of Mexico fish. Findings were compared to non-exposed control fish. The leukocyte counts of exposed alligator gar were not significantly different from controls, while exposed Gulf killifish and sea trout had significantly decreased lymphocyte counts. Liver ethoxyresorufin-O-deethylase (EROD) values from sea trout were significantly greater than control sea trout EROD values, suggesting poly aromatic hydrocarbon exposure. Splenic melano-macrophage centers (MMCs) from exposed sea trout and Gulf killifish showed a significant increase in number compared to non-exposed fish. Sea trout splenic MMCs were also significantly greater in size. These findings suggest that Gulf fish sampled were exposed to crude oil from the Macondo well and were in a lymphopenic or immuno-compromised state.

  2. [DNA extraction from coagulated human blood for application in genotyping techniques for human leukocyte antigen and immunoglobulin-like receptors].

    PubMed

    Cardozo, Daniela Maira; Guelsin, Gláucia Andréia; Clementino, Samaia Laface; Melo, Fabiano Cavalcante de; Braga, Marco Antônio; Souza, Cleonice de; Moliterno, Ricardo Alberto; Visentainer, Jeane Eliete Laguila

    2009-01-01

    The objective of this study was to standardize a method for extracting high-quality DNA from samples of coagulated blood. Forty-eight samples of human coagulated blood were used for DNA extraction by means of the EZ-DNA commercial kit (Biological Industries, Beit Haemek, Israel), the Neoscience column kit (One Lambda Inc., San Diego, CA, USA) and a modified salting-out method. Only the salting-out method was able to extract high concentrations of DNA (mean, 180 ng/(1/4)microl), which were measured using the Qubit fluorescence detector (Invitrogen, USA). This method enabled amplification of HLA (human leukocyte antigen) genes using the Luminex PCR-SSO (polymerase chain reaction - sequence-specific oligonucleotide) technology, which demands good quality DNA, and amplification of KIR (killer-cell immunoglobulin-like receptor) genes using an in-house PCR-SSP (polymerase chain reaction - sequence-specific primer) technique, which demands a specific concentration of DNA (10 ng/(1/4)microl). We concluded that the modified salting-out technique was very efficient, simple and fast for DNA extraction from human coagulated blood samples, with the aim of genotyping the HLA and KIR genes.

  3. Purification and characterization by fast-atom-bombardment mass spectrometry of the polymorphonuclear-leucocyte-elastase-generated A alpha (1-21) fragment of fibrinogen from human blood after incubation with calcium ionophore A23187.

    PubMed Central

    Dewey, R S; Liesch, J M; Williams, H R; Sugg, E E; Dolan, C A; Davies, P; Mumford, R A; Albers-Schönberg, G

    1992-01-01

    The stimulation of human blood with a Ca2+ ionophore, A23187, leads to activation of polymorphonuclear leucocytes (PMN) with release of small amounts of catalyticaly active elastase, as demonstrated by the formation of a characteristic product, the N-terminal A alpha (1-21) peptide of the Aa subunit of fibrinogen. The identity of the peptide was initially established by radioimmunoassay (r.i.a.) with an antibody raised to A alpha (1-21). We now provide independent confirmation of the formation of A alpha (1-21) by fast-atom-bombardment-m.s. analysis of the fractions separated chromatographically after spiking of plasma samples with peptide labelled with [2H8]Phe at position 8. Identity of the peptides was established on the basis of their chromatographic retention time and by the distinct peaks in the mass spectra of these fractions. The relative intensities of the molecular ions of natural and labelled peptides were measured. On the basis of a comparison of the peaks of similar intensities, the concentration of the natural peptide at the time of spiking was close (79%) to the amount obtained by r.i.a. An additional peptide, des-alanyl-A alpha (2-21), was also seen. The total amount of material measured by r.i.a. could be accounted for by the sum of these two provides. The addition of label and assay by m.s. has provided an independent physical-chemical method for identifying A alpha (1-21) as a characteristic product of PMN elastase release in whole blood, but which is absent in freshly drawn blood. PMID:1736899

  4. Modeling leukocyte trafficking at the human blood-nerve barrier in vitro and in vivo geared towards targeted molecular therapies for peripheral neuroinflammation.

    PubMed

    Greathouse, Kelsey M; Palladino, Steven P; Dong, Chaoling; Helton, Eric S; Ubogu, Eroboghene E

    2016-01-06

    Peripheral neuroinflammation is characterized by hematogenous mononuclear leukocyte infiltration into peripheral nerves. Despite significant clinical knowledge, advancements in molecular biology and progress in developing specific drugs for inflammatory disorders such as rheumatoid arthritis, inflammatory bowel disease, and multiple sclerosis, there are currently no specific therapies that modulate pathogenic peripheral nerve inflammation. Modeling leukocyte trafficking at the blood-nerve barrier using a reliable human in vitro model and potential intravital microscopy techniques in representative animal models guided by human observational data should facilitate the targeted modulation of the complex inflammatory cascade needed to develop safe and efficacious therapeutics for immune-mediated neuropathies and chronic neuropathic pain.

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

    PubMed Central

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

    2016-01-01

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

  6. Alterations of p11 in brain tissue and peripheral blood leukocytes in Parkinson’s disease

    PubMed Central

    Green, Holly; Zhang, Xiaoqun; Tiklova, Katarina; Volakakis, Nikolaos; Brodin, Lovisa; Berg, Louise; Perlmann, Thomas; Svenningsson, Per

    2017-01-01

    Individuals with Parkinson’s disease (PD) often suffer from comorbid depression. P11 (S100A10), a member of the S100 family of proteins, is expressed widely throughout the body and is involved in major depressive disorder and antidepressant response. Central p11 levels are reduced in postmortem tissue from depressed individuals; however, p11 has not yet been investigated in PD patients with depression or those without depression. We investigated p11 levels in postmortem PD brains and assessed whether peripheral p11 levels correlate with disease severity. Substantia nigra, putamen, and cortical p11 protein levels were assessed in postmortem brain samples from PD patients and matched controls. In a different set of postmortem brains, p11 mRNA expression was measured in dopaminergic cells from the substantia nigra. Both p11 protein and mRNA levels were decreased in PD patients. Peripheral p11 protein levels were investigated in distinct leukocyte populations from PD patients with depression and those without depression. Monocyte, natural killer (NK) cell, and cytotoxic T-cell p11 levels were positively associated with the severity of PD, and NK cell p11 levels were positively associated with depression scores. Given that inflammation plays a role in both PD and depression, it is intriguing that peripheral p11 levels are altered in immune cells in both conditions. Our data provide insight into the pathological alterations occurring centrally and peripherally in PD. Moreover, if replicated in other cohorts, p11 could be an easily accessible biomarker for monitoring the severity of PD, especially in the context of comorbid depression. PMID:28137881

  7. Phenotyping of leukocytes and granulocyte and monocyte phagocytic activity in the peripheral blood and uterus of cows with endometritis.

    PubMed

    Brodzki, P; Kostro, K; Brodzki, A; Lisiecka, U; Kurek, L; Marczuk, J

    2014-08-01

    This study was a comparative evaluation of selected immunological parameters in peripheral blood and uterine wash samples from cows with a normal postpartum period compared with cows with endometritis. We aimed to determine the usefulness of these parameters in monitoring the puerperium. In total, 40 cows were included in the study: 20 had endometritis (experimental group), and 20 did not have uterine inflammation (control group). Animals were chosen on the basis of cytological and bacteriological test results. The tests were conducted 5, 22, and 40 days postpartum. In both groups, flow cytometric analysis of the surface molecules CD4, CD8, CD21, CD25, and CD14 in the peripheral blood and uterine washings was performed. Granulocyte and monocyte phagocytic activity was determined using a commercial Phagotest kit that was adapted for flow cytometry. The percentage of phagocytic granulocytes and monocytes in both the peripheral blood and the uterine washings was significantly lower for cows in the experimental group compared with the control group (P < 0.01). A significant decrease (P < 0.01) in the percentage of CD4+, CD25+, CD14+, and CD4 + CD25(high) leukocyte subpopulations was also observed in the peripheral blood of cows with endometritis. A significant decrease (P < 0.01) in CD21+ lymphocytes and an increase in CD8+ lymphocytes was detected in uterine washings. The results of this work indicate that cell immunity dysfunction may be the main factor causing advanced inflammation of the uterus in endometritis. Knowledge of the immunological mechanisms observed in cows with endometritis might aid in choosing the correct immunomodulating agent-based adjuvant therapy.

  8. Degeneration and atrophy of the thymus of lethally irradiated dogs, rescued by transfusion of cryopreserved autologous blood leukocytes

    SciTech Connect

    Calvo, W.; Fliedner, T.M.; Herbst, E.W.; Huegl, E.B.; Boedey, B.

    1987-12-01

    Dogs exposed to a fatal radiation dose of 12 Gy were rescued by transfusion of autologous blood leukocytes. A severe acute and long-lasting damage to the thymus was observed. The acute damage, as observed on the tenth day, consisted of a marked reduction in the number of lymphocytes, degeneration of Hassall's bodies, and hemorrhage. Long-term effects, observed several months after irradiation, were partial to total atrophy of the thymus. Regeneration, when it occurred, was limited to a few small isolated areas in which lymphopoiesis was supported by epithelial reticular cells. In contrast, the lymph nodes of all dogs had abundant cortical lymphopoiesis. The abundant hemopoiesis present in the marrow from the tenth day after irradiation until the end of the observation period should have provided sufficient circulating precursor cells to seed the thymus and regenerate the organ to the same extent as that observed in the other blood-forming organs. The impairment of lymphopoietic regeneration in the thymus seems to be due, therefore, to damage caused by irradiation on the specific stroma of the organ, which is not able to support such activity.

  9. Effect of oxidatively modified and non-modified human serum albumin on luminol-dependent chemiluminescence of human peripheral blood leukocytes stimulated with opsonized zymosan.

    PubMed

    Piryazev, A P; Azizova, A P; Aseichev, A V; Sergienko, V I

    2014-07-01

    We studied the effects of native and oxidized human serum albumin on luminol-dependent chemiluminescence of human peripheral blood leukocytes stimulated with opsonized zymosan. Human serum albumin was added simultaneously with opsonized zymosan at the beginning of the chemiluminescent reaction. Otherwise, leukocytes were incubated with human serum albumin at 37°C for various periods before addition of opsonized zymosan. Oxidized human serum albumin was obtained by the method of metal-catalyzed oxidation. In control to non-modified albumin, oxidized albumin produced an inhibitory effect on luminol-dependent chemiluminescence of leukocytes. These changes were observed in experiments with addition of oxidized albumin at the beginning of a chemiluminescent reaction and after incubation of study agent with cells.

  10. Real Time Immunophenotyping of Leukocyte Subsets Early after Double Cord Blood Transplantation Predicts Graft Function.

    PubMed

    Li, Jianqiang; Nicoud, Ian; Blake, Joseph; Oliver, David; Cox, Emily; Heimfeld, Shelly; Milano, Filippo; Imren, Suzan; Delaney, Colleen

    2017-03-01

    Cord blood transplantation (CBT) recipients are at increased risk for delayed engraftment and primary graft failure, complications that are often indistinguishable early post-transplantation. Current assays fail to accurately identify recipients with slow hematopoietic recovery and distinguish them from those with pending graft failure. To address this, we prospectively examined the kinetics of immune cell subset recovery in the peripheral blood of 39 patients on days +7 and +14 after double-unit CBT (dCBT) by multiparametric flow cytometry analysis, which we term real-time immunophenotyping (RTIP). RTIP analysis at day +14 revealed distinctive patterns of reconstitution and, importantly, identified patients with slow hematopoietic recovery who went on to engraft. Strikingly, higher absolute numbers of circulating monocytes and natural killer cells at day +14 were predictive of engraftment, but only the absolute number of circulating monocytes was significantly correlated with time to engraftment. This is the first evidence that RTIP on patient peripheral blood mononuclear cells early after dCBT is technically feasible and can be used as a "signature" for predicting the kinetics of hematopoietic recovery. Furthermore, RTIP is a time- and cost-efficient methodology that has the potential to become a clinically feasible diagnostic tool to guide therapeutic interventions in high-risk patients; therefore, its utility should be evaluated in a large cohort of patients.

  11. An extended convection diffusion model for red blood cell-enhanced transport of thrombocytes and leukocytes

    NASA Astrophysics Data System (ADS)

    Hund, S. J.; Antaki, J. F.

    2009-10-01

    Transport phenomena of platelets and white blood cells (WBCs) are fundamental to the processes of vascular disease and thrombosis. Unfortunately, the dilute volume occupied by these cells is not amenable to fluid-continuum modeling, and yet the cell count is large enough that modeling each individual cell is impractical for most applications. The most feasible option is to treat them as dilute species governed by convection and diffusion; however, this is further complicated by the role of the red blood cell (RBC) phase on the transport of these cells. We therefore propose an extended convection-diffusion (ECD) model based on the diffusive balance of a fictitious field potential, Ψ, that accounts for the gradients of both the dilute phase and the local hematocrit. The ECD model was applied to the flow of blood in a tube and between parallel plates in which a profile for the RBC concentration field was imposed and the resulting platelet concentration field predicted. Compared to prevailing enhanced-diffusion models that dispersed the platelet concentration field, the ECD model was able to simulate a near-wall platelet excess, as observed experimentally. The extension of the ECD model depends only on the ability to prescribe the hematocrit distribution, and therefore may be applied to a wide variety of geometries to investigate platelet-mediated vascular disease and device-related thrombosis.

  12. 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

    ... blood components intended for transfusion, including recommendations for validation and quality control... for transfusion, including recommendations for validation and quality control monitoring of the... have been approved under OMB control number 0910-0052; the collections of information in 21 CFR...

  13. A novel thyroid stimulating hormone beta-subunit isoform in human pituitary, peripheral blood leukocytes, and thyroid.

    PubMed

    Schaefer, Jeremy S; Klein, John R

    2009-07-01

    Thyroid stimulating hormone (TSH) is produced by the anterior pituitary and is used to regulate thyroid hormone output, which in turn controls metabolic activity. Currently, the pituitary is believed to be the only source of TSH used by the thyroid. Recent studies in mice from our laboratory have identified a TSHbeta isoform that is expressed in the pituitary, in peripheral blood leukocytes (PBL), and in the thyroid. To determine whether a human TSHbeta splice variant exists that is analogous to the mouse TSHbeta splice variant, and whether the pattern of expression of the splice variant is similar to that observed in mice, PCR amplification of RNAs from pituitary, thyroid, PBL, and bone marrow was done by reverse-transcriptase PCR and quantitative realtime PCR. Human pituitary expressed a TSHbeta isoform that is analogous to the mouse TSHbeta splice variant, consisting of a 27 nucleotide portion of intron 2 and all of exon 3, coding for 71.2% of the native human TSHbeta polypeptide. Of particular interest, the TSHbeta splice variant was expressed at significantly higher levels than the native form or TSHbeta in PBL and the thyroid. The TSHalpha gene also was expressed in the pituitary, thyroid, and PBL, but not the BM, suggesting that the TSHbeta polypeptide in the thyroid and PBL may exist as a dimer with TSHalpha. These findings identify an unknown splice variant of human TSHbeta. They also have implications for immune-endocrine interactions in the thyroid and for understanding autoimmune thyroid disease from a new perspective.

  14. DUSP1 promoter methylation in peripheral blood leukocyte is associated with triple-negative breast cancer risk.

    PubMed

    Li, Jing; Chen, Yanbo; Yu, Hongyuan; Tian, Jingshen; Yuan, Fengshun; Fan, Jialong; Liu, Yupeng; Zhu, Lin; Wang, Fan; Zhao, Yashuang; Pang, Da

    2017-02-21

    DNA methylation is one of the most common epigenetic alterations, providing important information regarding cancer risk and prognosis. A case-control study (423 breast cancer cases, 509 controls) and a case-only study (326 cases) were conducted to evaluate the association of DUSP1 promoter methylation with breast cancer risk and clinicopathological characteristics. No significant association between DUSP1 methylation in peripheral blood leukocyte (PBL) DNA and breast cancer risk was observed. DUSP1 methylation was significantly associated with ER/PR-negative status; in particular, triple-negative breast cancer patients showed the highest frequency of DUSP1 methylation in both tumour DNA and PBL DNA. Soybean intake was significantly correlated with methylated DUSP1 only in ER-negative (OR 2.978; 95% CI 1.245-7.124) and PR negative (OR 2.735; 95% CI 1.315-5.692) patients. Irregular menstruation was significantly associated with methylated DUSP1 only in ER-positive (OR 3.564; 95% CI 1.691-7.511) and PR-positive (OR 3.902, 95% CI 1.656-9.194) patients. Thus, DUSP1 methylation is a cancer-associated hypermethylation event that is closely linked with triple-negative status. Further investigations are warranted to confirm the association of environmental factors, including fruit and soybean intake, irregular menstruation, and ER/PR status, with DUSP1 methylation in breast tumour DNA.

  15. In vitro effects of 'designer' amphetamines on human peripheral blood mononuclear leukocytes proliferation and on natural killer cell activity.

    PubMed

    Gagnon, L; Lacroix, F; Chan, J; Buttar, H S

    1992-12-01

    Human peripheral blood mononuclear leukocytes (PBML) proliferation was measured in the presence or absence of amphetamines. Proliferation in response to T-cell mitogen PHA was suppressed from 22 to 34% by d- and dl-amphetamine, respectively, contrarily to 1-form which did not affect proliferation of PHA-stimulated PBML. The 'designer' amphetamines appeared to be more potent inhibitors of PBML proliferation induced by both PHA and PWM stimulation than those of the racemic and isomeric forms of amphetamine. A wide variation was seen in the suppressive actions of the 'designer' amphetamines, and the mean percentages of suppression varied from 12 to 45% compared with the control values. 4-Propoxy-amphetamine (4-PA) was found to be the most active among the 'designer' drugs. In vitro effects of d-, 1- and dl-amphetamine were also studied on natural killer (NK) cell activity. A marked increase in the NK cell activity was observed only in the presence of very low concentrations (10(-12) to 10(-10) M) of dl-amphetamine, however, the activity of the NK cell remained within the control limits in the presence of d- or 1-forms. The findings suggest that the abuse of amphetamines, especially the 'designer' drugs, may adversely affect the activity of immunoregulatory cells and might lead to a compromised immune system in amphetamine abusers.

  16. Dry olive leaf extract counteracts L-thyroxine-induced genotoxicity in human peripheral blood leukocytes in vitro.

    PubMed

    Topalović, Dijana Žukovec; Živković, Lada; Čabarkapa, Andrea; Djelić, Ninoslav; Bajić, Vladan; Dekanski, Dragana; Spremo-Potparević, Biljana

    2015-01-01

    The thyroid hormones change the rate of basal metabolism, modulating the consumption of oxygen and causing production of reactive oxygen species, which leads to the development of oxidative stress and DNA strand breaks. Olive (Olea europaea L.) leaf contains many potentially bioactive compounds, making it one of the most potent natural antioxidants. The objective of this study was to evaluate the genotoxicity of L-thyroxine and to investigate antioxidative and antigenotoxic potential of the standardized oleuropein-rich dry olive leaf extract (DOLE) against hydrogen peroxide and L-thyroxine-induced DNA damage in human peripheral blood leukocytes by using the comet assay. Various concentrations of the extract were tested with both DNA damage inducers, under two different experimental conditions, pretreatment and posttreatment. Results indicate that L-thyroxine exhibited genotoxic effect and that DOLE displayed protective effect against thyroxine-induced genotoxicity. The number of cells with DNA damage, was significantly reduced, in both pretreated and posttreated samples (P < 0.05). Comparing the beneficial effect of all tested concentrations of DOLE, in both experimental protocols, it appears that extract was more effective in reducing DNA damage in the pretreatment, exhibiting protective role against L-thyroxine effect. This feature of DOLE can be explained by its capacity to act as potent free radical scavenger.

  17. Attachment, ingestion and intracellular killing of Helicobacter pylori by human peripheral blood mononuclear leukocytes and mouse peritoneal inflammatory macrophages.

    PubMed

    Chmiela, M; Paziak-Domanska, B; Wadström, T

    1995-02-01

    The different steps of phagocytosis, attachment, ingestion and intracellular killing of cells of Helicobacter pylori strain 17874 (expressing sialic acid-specific haemagglutinin) and cells of H. pylori strain 17875 (expressing non-sialic acid-specific haemagglutinin) have been studied. More cells of sialopositive H. pylori strain 17874 have been found attached to human peripheral blood mononuclear leukocytes (PBM) and mouse peritoneal inflammatory macrophages (PIM) than cells of sialonegative H. pylori strain 17875. Binding of cells of H. pylori strain 17874 has been significantly inhibited by treatment of phagocytes with neuraminidase. Inhibition of adhesion of these bacteria preincubated with foetuin to normal phagocytic cells has also been found. Well adhering cells of H. pylori strain 17874 were more resistant to killing mechanisms of human PBM and mouse PIM than cells of strain 17875. Good, probably sialic acid-specific haemagglutinin dependent, adhesion of H. pylori bacteria to phagocytes can be considered as an important virulence factor which facilitates the pathogen to avoid the defence mechanisms.

  18. Dry Olive Leaf Extract Counteracts L-Thyroxine-Induced Genotoxicity in Human Peripheral Blood Leukocytes In Vitro

    PubMed Central

    Žukovec Topalović, Dijana; Živković, Lada; Čabarkapa, Andrea; Djelić, Ninoslav; Bajić, Vladan; Spremo-Potparević, Biljana

    2015-01-01

    The thyroid hormones change the rate of basal metabolism, modulating the consumption of oxygen and causing production of reactive oxygen species, which leads to the development of oxidative stress and DNA strand breaks. Olive (Olea europaea L.) leaf contains many potentially bioactive compounds, making it one of the most potent natural antioxidants. The objective of this study was to evaluate the genotoxicity of L-thyroxine and to investigate antioxidative and antigenotoxic potential of the standardized oleuropein-rich dry olive leaf extract (DOLE) against hydrogen peroxide and L-thyroxine-induced DNA damage in human peripheral blood leukocytes by using the comet assay. Various concentrations of the extract were tested with both DNA damage inducers, under two different experimental conditions, pretreatment and posttreatment. Results indicate that L-thyroxine exhibited genotoxic effect and that DOLE displayed protective effect against thyroxine-induced genotoxicity. The number of cells with DNA damage, was significantly reduced, in both pretreated and posttreated samples (P < 0.05). Comparing the beneficial effect of all tested concentrations of DOLE, in both experimental protocols, it appears that extract was more effective in reducing DNA damage in the pretreatment, exhibiting protective role against L-thyroxine effect. This feature of DOLE can be explained by its capacity to act as potent free radical scavenger. PMID:25789081

  19. DUSP1 promoter methylation in peripheral blood leukocyte is associated with triple-negative breast cancer risk

    PubMed Central

    Li, Jing; Chen, Yanbo; Yu, Hongyuan; Tian, Jingshen; Yuan, Fengshun; Fan, Jialong; Liu, Yupeng; Zhu, Lin; Wang, Fan; Zhao, Yashuang; Pang, Da

    2017-01-01

    DNA methylation is one of the most common epigenetic alterations, providing important information regarding cancer risk and prognosis. A case-control study (423 breast cancer cases, 509 controls) and a case-only study (326 cases) were conducted to evaluate the association of DUSP1 promoter methylation with breast cancer risk and clinicopathological characteristics. No significant association between DUSP1 methylation in peripheral blood leukocyte (PBL) DNA and breast cancer risk was observed. DUSP1 methylation was significantly associated with ER/PR-negative status; in particular, triple-negative breast cancer patients showed the highest frequency of DUSP1 methylation in both tumour DNA and PBL DNA. Soybean intake was significantly correlated with methylated DUSP1 only in ER-negative (OR 2.978; 95% CI 1.245–7.124) and PR negative (OR 2.735; 95% CI 1.315–5.692) patients. Irregular menstruation was significantly associated with methylated DUSP1 only in ER-positive (OR 3.564; 95% CI 1.691–7.511) and PR-positive (OR 3.902, 95% CI 1.656–9.194) patients. Thus, DUSP1 methylation is a cancer-associated hypermethylation event that is closely linked with triple-negative status. Further investigations are warranted to confirm the association of environmental factors, including fruit and soybean intake, irregular menstruation, and ER/PR status, with DUSP1 methylation in breast tumour DNA. PMID:28220843

  20. Effect of antiarrhythmic drugs on In-111-labeled leukocytes: chemotaxis and adherence to nylon wool

    SciTech Connect

    Thakur, M.L.; Walsh, L.J.; Zaret, B.L.; Gottschalk, A.

    1982-02-01

    The influence of lidocaine (L) and procainamide (P) on the chemotactic ability and adherence to nylon wool of In-111-labeled human polymorphonuclear leukocytes (PMNs) was investigated. At the normal therapeutic levels of L (0.022 mM whole blood) or P (0.03 mM whole blood) no change in PMN function was observed. However, at and above five times the aforementioned blood levels of L, significant reduction in the chemotactic ability of PMNs was noted (p less than 0.005). The adverse effects of In-111 radiation appeared insignificant at all L or P concentrations during the 3-hr observation period. The labeled PMNs were resistant to the toxic effects of a higher concentration of P than that of L, and the reduction in PMN chemotaxis and adherence to nylon wool was not apparent until the P concentration reached 1.5 mM.

  1. Effect of antiarrhythmic drugs on In-111-labeled leukocytes: chemotaxis and adherence to nylon wool

    SciTech Connect

    Thakur, M.L.; Walsh, L.J.; Zaret, B.L.; Gottschalk, A.

    1982-02-01

    The influence of lidocaine (L) and procainamide (P) on the chemotactic ability and adherence to nylon wool of In-111-labeled human polymorphonuclear leukocytes (PMNs) was investigated. At the normal therapeutic levels of L (0.022 mM whole blood) or P (0.03 mM whole blood) no change in PMN function was observed. However, at and above five times the aforementioned blood levels of L, significant reduction in the chemotactic ability of PMNs was noted (P <0.005). The adverse effects of In-111 radiation appeared insignificant at all L or P concentrations during the 3-hr observation period. The labeled PMNs were resistant to the toxic effects of a higher concentration of P than that of L, and the reduction in PMN chemotaxis and adherence to nylon wool was not apparent until the P concentration reached 1.5 mM.

  2. In vitro study of interactions between silicon-containing nanoparticles and human peripheral blood leukocytes.

    PubMed

    Andreeva, E R; Rudimov, E G; Gornostaeva, A N; Beklemyshev, V I; Makhonin, I I; Maugeri, U O G; Buravkova, L B

    2013-07-01

    The effects of silicon dioxide-based nanoparticles on the viability and proliferative activity of human peripheral blood cultured lymphocytes were studied. All nanoparticles in a concentration of 100 μg/ml produced a significant cytotoxic effect, its intensity depending on particles' structure: SiO2 nanoparticles were least toxic, while Ce3(+)-intercaled montmorillonite nanoparticles were most toxic. The cells died mainly by apoptosis and postapoptotic necrosis. Incubation with nanoparticles in a concentration of 100 μg/ml for 72 h caused death of all phytohemagglutinin-activated lymphocytes, while in concentrations of 1 and 10 μg/ml the nanoparticles had no effect of proliferative activity of cells. The results suggest that the effects of nanoparticles on cells are determined by the nanoparticle concentration and size, as well as by their structure.

  3. Analysis of DNA methylation of potential age-related methylation sites in canine peripheral blood leukocytes.

    PubMed

    Ito, Genta; Yoshimura, Kuniko; Momoi, Yasuyuki

    2017-04-08

    Reliable methodology for predicting the age of mature dogs is currently unavailable. In this study, amplicon sequencing of 50 blood samples obtained from diseased dogs was used to measure methylation in seven DNA regions. Significant correlations between methylation level and age were identified in four of the seven regions. These four regions were then tested in samples from 31 healthy toy poodles, and correlations were detected in two regions. The age of another 11 dogs was predicted using data from the diseased dogs and the healthy poodles. The mean difference between the actual and calculated ages was 34.3 and 23.1 months, respectively. Further research is needed to identify additional sites of age-related methylation and allow accurate age prediction in dogs.

  4. Flow cytometric assay for quantifying opsonophagocytosis and killing of Staphylococcus aureus by peripheral blood leukocytes.

    PubMed Central

    Martin, E; Bhakdi, S

    1992-01-01

    We describe a novel flow cytometric method for quantifying opsonophagocytosis and killing of Staphylococcus aureus in cell-rich plasma obtained after dextran sedimentation of erythrocytes. To analyze opsonophagocytosis, phagocytes were labeled with a phycoerythrin-conjugated monoclonal antibody and were incubated with viable staphylococci containing carboxyfluorescein as a vital fluorescent dye. Phagocytosing cells assumed a dual, orange-green fluorescence. The relative numbers of bacteria associating with phagocytes could be determined by quantifying the decrease of free green fluorescent particles. A parallel incubation of fluorescent bacteria with unlabeled cell-rich plasma was performed to assess phagocytic killing. Blood cells were lysed with 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate. This detergent spared viable bacteria, and residual green fluorescent particles were counted. The decrease in the number of these particles relative to the controls yielded the degree of killing. At bacteria-to-phagocyte ratios of 1:1 and 10:1, approximately 36 and 75% of the phagocytes participated in opsonophagocytosis, respectively. Over 90% of the staphylococci were phagocyte associated after 30 to 60 min. Killing rates were on the order of 66% +/- 12% and 80% +/- 7% after 1 and 2 h of incubation, respectively. These numbers, which were confirmed by colony countings, were significantly lower than those reported in the majority of past reports. PMID:1400987

  5. [Dynamics of leukocytes' migration degree in oral cavity and parameters of INFgamma and TNFalpha in blood serum of patients suffering from parodontitis on the background of treatment with Unimag].

    PubMed

    Saralidze, M G; Dzhashi, L M; Tskitishvili, T G; Gogebashvili, N N; Surguladze, B V

    2005-12-01

    In the oral cavity of patients with parodontitis before treatment the quantity of migrated leukocytes (ML) was increased. Among them number of living cells was sharply reduced. In the process of treatment on the background of immune modulation with Unimag, the concentration of migrated leukocytes decreases and number of living leukocytes increases. The above-mentioned quantitative and qualitative parameters of migrated leukocytes were normalized after 1-1.5 months. The analogical dynamics has been detected in patients suffering from parodontitis and treated with traditional scheme. However, despite reduced concentration of migrated leukocytes and increased percentage of living leukocytes' content, normalization of the mentioned parameters has not been detected. In the blood serum of patients suffering from parodontitis increased concentration of INFgamma and TNFalpha have been revealed. In the process of treatment, concentration of the mentioned cytokines was decreased, however, in patients treated with Unimag on the background of immune modulation, decrease in concentration of INFgamma and TNFalpha was much more expressed.

  6. Archetypal and rearranged sequences of human polyomavirus JC transcription control region in peripheral blood leukocytes and in cerebrospinal fluid.

    PubMed

    Ciappi, S; Azzi, A; De Santis, R; Leoncini, F; Sterrantino, G; Mazzotta, F; Mecocci, L

    1999-04-01

    Two forms of human polyomavirus JC (JCV) genome are known based upon the structure of the transcriptional control region (TCR) of the virus: the archetypal form, which is commonly detected in urine, and the rearranged form, which was first detected in brain tissue from progressive multifocal leukoencephalopathy (PML) patients. The latter actually includes a group of TCR variants that, relative to the former, are characterized by various deletions and/or duplications. The aim of this study was to establish whether or not a correlation exists among the TCR type, the spreading of the virus within the host and its ability to cause PML. JCV TCR sequences from peripheral blood leukocytes (PBL) and cerebrospinal fluid (CSF) obtained from various groups of patients were compared. JCV with archetypal TCR was detected in CSF and PBL specimens from patients without neurological disorders or who eventually received a diagnosis of a non-PML neurological disorder. Rearranged TCR sequences were detected in all the CSF and PBL specimens from PML patients. The high similarity observed between the TCR structure detected in PBL and CSF specimens from individual patients could strengthen the hypothesis that PBL has a role in spreading JCV to the brain. Moreover, heterogeneous TCR patterns have been shown in individual PBL specimens from PML patients. This supports the hypothesis that, in PBL, JCV may replicate and undergo rearrangements of the TCR. The detection of JCV DNA by PCR in CSF independently from PML, although rare, could suggest that this assay is not sufficient for a virological diagnosis of PML. Further studies are required to assess the usefulness of quantitative assays or TCR typing in combination with PCR for diagnostic purposes.

  7. Immunomodulatory effects upon in vitro exposure of California sea lion and southern sea otter peripheral blood leukocytes to domoic acid.

    PubMed

    Levin, Milton; Joshi, Dhanashree; Draghi, Andrew; Gulland, Frances M; Jessup, David; De Guise, Sylvain

    2010-04-01

    During red tide bloom events, the marine diatom Pseudo-nitzschia produces the toxin domoic acid (DA), which has been associated with stranding and mortality events involving California sea lions (Zalophus californianus) and southern sea otters (Enhydra lutris). In addition to these well-documented DA-induced neurotoxic events, there is increasing concern that DA may exert chronic effects, such as immunomodulation, which may potentially increase an individual's susceptibility to a number of opportunistic infections following nonlethal exposure. We investigated the effects of DA on innate (phagocytosis and respiratory burst) and adaptive (mitogen-induced lymphocyte proliferation) immune functions with the use of peripheral blood leukocytes collected from healthy California sea lions and southern sea otters upon in vitro exposure to 0 (unexposed control), 0.0001, 0.001, 0.01, 0.1, 1.0, 10, and 100 microM DA. Domoic acid did not significantly modulate phagocytosis or respiratory burst in either species. For California sea lions, DA significantly increased ConA-induced T-lymphocyte proliferation upon exposure to DA concentrations ranging from 0.0001 to 10 microM, resulting in a nonlinear dose-response curve. There was no effect on lymphocyte proliferation at the highest concentration of DA tested. No effects on lymphocyte proliferation were observed in southern sea otters. Importantly, the in vitro DA concentrations affecting T-cell proliferation were within or below the range of DA in serum measured in free-ranging California sea lions following natural exposure, suggesting a risk for immunomodulation in free-ranging animals. Understanding the risk for immunomodulation upon DA exposure will contribute in the health assessment and management of California sea lions and southern sea otters, as well as guide veterinarians and wildlife rehabilitators in caring for and treating afflicted animals.

  8. In vitro assessment of blood compatibility: residual and dynamic markers of cellular activation.

    PubMed

    Johnson, Greg; Curry, Benjamin; Cahalan, Linda; Prater, Roni; Beeler, Michael; Gartner, Mark; Biggerstaff, John; Cahalan, Patrick

    2013-05-01

    The blood compatibility of materials and surfaces used for medical device fabrication is a crucial factor in their function and effectiveness. Expansion of device use into more sensitive and longer term applications warrants increasingly detailed evaluations of blood compatibility that reach beyond the customary measures mandated by regulatory requirements. A panel of tests that assess both deposition on the surface and activation of circulating blood in contact with the surface has been developed. Specifically, the ability of a surface to modulate the biological response of blood is assessed by measuring: (1) dynamic thrombin generation; (2) surface-bound thrombin activity after exposure to blood; (3) activation of monocytes, polymorphonuclear leukocytes, lymphocytes, and platelets; (4) activation of complement; and (5) adherent monocytes, polymorphonuclear leukocytes, lymphocytes, and platelets on blood-contacting surfaces. The tests were used to evaluate surfaces modified with immobilized heparin (Ension's proprietary bioactive surface) and demonstrated that the modified surfaces reduced platelet activation, leukocyte activation, and complement activation in flowing human blood. Perfusion of the surfaces with human platelet-rich plasma showed that the immobilized heparin surfaces also reduce both dynamic thrombin levels in the circulating plasma and residual thrombin generated at the material surface.

  9. Simultaneous flow cytometric evaluation of phagocytosis and oxidative burst in human polymorphonuclear cells.

    PubMed

    Horvathova, M; Wsolova, L; Jahnova, E

    2005-01-01

    Phagocytosis and oxidative burst (OXIBURST) activity of human polymorphonuclear cells (PMNs) has been simultaneously measured directly in whole blood samples. The ingestion of yeast was assessed by the phagocytosis activity (FA) and phagocytosis index (FI), and the respiratory burst of PMNs was determined as dihydroethidine (DHE) oxidation. We received comparable results in the ingestion of yeast cells by PMNs using either light microscopy (77.31+/-7.56) or flow cytometry detection method (78.26+/-5.14). The significant differences (p<0.05) in FI and OXIBURST activity were find in the patients (2.29+/-0.29 and 14.67+/-3.99, respectively) when compared to healthy donors (1.64+/-0.21 and 32.38+/-14.94, respectively). The two-color flow cytometric procedure permits measurement of two different functions of neutrophils in one step. This flow cytometric procedure is simple, rapid and has the potential to be an alternative assay to test leukocyte function. (Fig. 3, Ref: 30.)

  10. DNA extraction for short tandem repeat typing from mixed samples using anti-human leukocyte CD45 and ABO blood group antibodies.

    PubMed

    Yano, Shizue; Honda, Katsuya; Kaminiwa, Junko; Nishi, Takeki; Iwabuchi, Yayoi; Sugano, Yukiko; Kurosu, Akira; Suzuki, Yasuhito

    2014-05-01

    DNA testing from mixed cell samples can be difficult to use successfully in criminal investigations. Here, we present a method for the extraction of DNA from mixed bloodstains involving plural contributors, after antibody-microbead captured cell separation. This method, together with the multiplex short tandem repeat typing presented, has proven highly successful in the recovery of DNA profiles corresponding to the ABO blood type. Methodological steps include magnetic separation using leukocyte specific CD45 antibody-coated microbeads and centrifugal separation of leukocyte agglutination by ABO antibody. The detection results of variable mixed ratio showed that the target DNA was detected accurately as low as 1:512 mixed ratio, regardless of the large amount of the background DNA present. The method presented here is applicable to PCR-based identification for various kinds of mixed samples.

  11. The calorically restricted low-fat nutrient-dense diet in Biosphere 2 significantly lowers blood glucose, total leukocyte count, cholesterol, and blood pressure in humans.

    PubMed Central

    Walford, R L; Harris, S B; Gunion, M W

    1992-01-01

    Biosphere 2 is a 3.15-acre space containing an ecosystem that is energetically open (sunlight, electric power, and heat) but materially closed, with air, water, and organic material being recycled. Since September 1991, eight subjects (four women and four men) have been sealed inside, living on food crops grown within. Their diet, low in calories (average, 1780 kcal/day; 1 kcal = 4.184 kJ), low in fat (10% of calories), and nutrient-dense, conforms to that which in numerous animal experiments has promoted health, retarded aging, and extended maximum life span. We report here medical data on the eight subjects, comparing preclosure data with data through 6 months of closure. Significant changes included: (i) weight, 74 to 62 kg (men) and 61 to 54 kg (women); (ii) mean systolic/diastolic blood pressure (eight subjects), 109/74 to 89/58 mmHg (1 mmHg = 133 Pa); (iii) total serum cholesterol, from 191 +/- 11 to 123 +/- 9 mg/dl (mean +/- SD; 36% mean reduction), and high density lipoprotein, from 62 +/- 8 to 38 +/- 5 (risk ratio unchanged); (iv) triglyceride, 139 to 96 mg/dl (men) and 78 to 114 mg/dl (women); (v) fasting glucose, 92 to 74 mg/dl; (vi) leukocyte count, 6.7 to 4.7 x 10(9) cells per liter. We conclude that drastic reductions in cholesterol and blood pressure may be instituted in normal individuals in Western countries by application of a carefully chosen diet and that a low-calorie nutrient-dense regime shows physiologic features in humans similar to those in other animal species. PMID:1454844

  12. Radiation-induced permeability and leukocyte adhesion in the rat blood-brain barrier: modulation with anti-ICAM-1 antibodies.

    PubMed

    Yuan, Hong; Gaber, M Waleed; McColgan, Tamara; Naimark, Michael D; Kiani, Mohammad F; Merchant, Thomas E

    2003-04-18

    We assessed the acute effects of radiation on the rat blood-brain barrier. A cranial window model and intravital microscopy were used to measure changes in permeability and leukocyte adhesion in pial vessels after a localized, single dose of 20 Gy. Permeability was assessed using five sizes of fluorescein isothiocyanate (FITC)-dextran molecules (4.4-, 10-, 38.2-, 70-, and 150-kDa) with measurements performed before and 2, 24, 48, 72 and 96 h after irradiation for the 4.4 and 38.2-kDa molecules and before and 24 h after irradiation for the other three molecules. To demonstrate the nature of blood-brain barrier permeability, we concurrently studied the permeability of microvessels in the cremaster muscle. In both tissues, permeability to FITC-dextran was significantly greater 24 h after irradiation than before (P<0.05). The exception was that radiation did not affect the permeability of pial vessels to the 150-kDa molecule. The particle-size dependence of the permeability changes in the brain were indicative of altered integrity of endothelial tight junctions and occurred concomitantly with an increase in cell adhesion which was determined by fluorescent labeling of leukocytes with rhodamine 6G. An early inflammatory response to irradiation was apparent in the brain 2 h after irradiation. The numbers of rolling and adherent leukocytes increased significantly and peaked at 24 h. Injection with the anti-ICAM-1 mAb significantly reduced leukocyte adhesion and permeability thereby linking the two processes. These findings provide a target to reduce radiation-related permeability and cell adhesion and potentially the side effects of radiation in the CNS.

  13. Density-Gradient Mediated Band Extraction of Leukocytes from Whole Blood Using Centrifugo-Pneumatic Siphon Valving on Centrifugal Microfluidic Discs.

    PubMed

    Kinahan, David J; Kearney, Sinéad M; Kilcawley, Niamh A; Early, Philip L; Glynn, Macdara T; Ducrée, Jens

    2016-01-01

    Here we present retrieval of Peripheral Blood Mononuclear Cells by density-gradient medium based centrifugation for subsequent analysis of the leukocytes on an integrated microfluidic "Lab-on-a-Disc" cartridge. Isolation of white blood cells constitutes a critical sample preparation step for many bioassays. Centrifugo-pneumatic siphon valves are particularly suited for blood processing as they function without need of surface treatment and are 'low-pass', i.e., holding at high centrifugation speeds and opening upon reduction of the spin rate. Both 'hydrostatically' and 'hydrodynamically' triggered centrifugo-pneumatic siphon valving schemes are presented. Firstly, the geometry of the pneumatic chamber of hydrostatically primed centrifugo-pneumatic siphon valves is optimised to enable smooth and uniform layering of blood on top of the density-gradient medium; this feature proves to be key for efficient Peripheral Blood Mononuclear Cell extraction. A theoretical analysis of hydrostatically primed valves is also presented which determines the optimum priming pressure for the individual valves. Next, 'dual siphon' configurations for both hydrostatically and hydrodynamically primed centrifugo-pneumatic siphon valves are introduced; here plasma and Peripheral Blood Mononuclear Cells are extracted through a distinct siphon valve. This work represents a first step towards enabling on disc multi-parameter analysis. Finally, the efficiency of Peripheral Blood Mononuclear Cells extraction in these structures is characterised using a simplified design. A microfluidic mechanism, which we termed phase switching, is identified which affects the efficiency of Peripheral Blood Mononuclear Cell extraction.

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

    PubMed

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

    2008-01-01

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

  15. [The state of the mitochondrial energy-supplying system of blood leukocytes in the dynamics of Guerin's carcinoma growth under the low-level irradiation conditions].

    PubMed

    Marchenko, M M; Voloshchuk, O N

    2014-01-01

    Mitochondrial NADH-dehydrogenase, succinate dehydrogenase and cytochrome oxidase activities of peripheral blood leukocytes of rats with the grafted Guerin's carcinoma were studied in the dynamics of oncogenesis under the conditions of the preliminary low-level irradiation. Tumor growth was accompanied by a decrease in NADH-dehydrogenase activity, an increase of succinate dehydrogenase activity. Cytochrome oxidase activity of leucocytes remained at the control level up to the terminal stages of tumor growth. Preliminary low-level irradiation of the tumor bearing animals caused a tendency to the decrease of enzymatic activities studied. This tendency was observed from the initial stages of oncogenesis.

  16. Effect of irradiation, cyclophosphamide, and etoposide (VP-16) on number of peripheral blood and peritoneal leukocytes in mice under normal conditions and during acute inflammatory reaction

    SciTech Connect

    van't Wout, J.W.; Linde, I.; Leijh, P.C.; van Furth, R.

    1989-02-01

    In order to develop a suitable model for studying the role of granulocytes and monocytes in resistance against pathogenic microorganisms, we investigated the effect of irradiation and cytostatic treatment (cyclophosphamide and VP-16) on the number of both peripheral blood and peritoneal leukocytes in male Swiss mice. Irradiation and cyclophosphamide treatment severely decreased the number of both granulocytes and monocytes in peripheral blood, whereas VP-16 only lowered the number of blood monocytes to a significant degree and had little effect on the number of blood granulocytes or lymphocytes. When normal mice were injected intraperitoneally with newborn calf serum (NBCS) the number of peritoneal granulocytes rose about 100-fold within 6 h. In irradiated and cyclophosphamide-treated mice, this influx of granulocytes into the peritoneal cavity was virtually eliminated, as was the concomitant increase in the number of blood granulocytes; in VP-16-treated mice, on the other hand, the number of peripheral blood and peritoneal granulocytes increased to the same degree as in normal mice. An increase in the number of peripheral blood monocytes and peritoneal macrophages occurred 24-48 h after injection of NBCS in normal mice. This increase was significantly impaired by irradiation as well as by treatment with cyclophosphamide or VP-16.

  17. Biphasic control of polymorphonuclear cell migration by Kupffer cells. Effect of exposure to metabolic products of ethanol

    SciTech Connect

    Fainsilber, Z.; Feinman, L.; Shaw, S.; Lieber, C.S.

    1988-01-01

    In order to investigate the role of the Kupffer cells in the regulation of the inflammatory reaction seen in alcoholic hepatitis, rat liver Kupffer cells were cultured and exposed to products of ethanol metabolism. The resultant supernatants were tested to study their ability to stimulate or inhibit polymorphonuclear cell chemotaxis. Kupffer cells produced increased chemokinetic activity for human polymorphonuclear leukocytes; when incubated with soluble products of microsomal peroxidation, the Kupffer cells engendered more chemokinetic activity than that produced by untreated Kupffer cells. When Kupffer cells were incubated with acetaldehyde, the chemokinetic activity that appeared in the supernatant did not differ from control. Chemotaxis of polymorphonuclear cells was not observed when the Kupffer cell supernatants were tested by checkerboard analysis.

  18. 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.

  19. Label-free in vivo imaging of human leukocytes using two-photon excited endogenous fluorescence

    NASA Astrophysics Data System (ADS)

    Zeng, Yan; Yan, Bo; Sun, Qiqi; Teh, Seng Khoon; Zhang, Wei; Wen, Zilong; Qu, Jianan Y.

    2013-04-01

    We demonstrate that two-photon excited endogenous fluorescence enables label-free morphological and functional imaging of various human blood cells. Specifically, we achieved distinctive morphological contrast to visualize morphology of important leukocytes, such as polymorphonuclear structure of granulocyte and mononuclear feature of agranulocyte, through the employment of the reduced nicotinamide adenine dinucleotide (NADH) fluorescence signals. In addition, NADH fluorescence images clearly reveal the morphological transformation process of neutrophils during disease-causing bacterial infection. Our findings also show that time-resolved NADH fluorescence can be potentially used for functional imaging of the phagocytosis of pathogens by leukocytes (neutrophils) in vivo. In particular, we found that free-to-bound NADH ratios measured in infected neutrophils increased significantly, which is consistent with a previous study that the energy consumed in the phagocytosis of neutrophils is mainly generated through the glycolysis pathway that leads to the accumulation of free NADH. Future work will focus on further developing and applying label-free imaging technology to investigate leukocyte-related diseases and disorders.

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

    SciTech Connect

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

    1982-12-01

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

  1. Density-Gradient Mediated Band Extraction of Leukocytes from Whole Blood Using Centrifugo-Pneumatic Siphon Valving on Centrifugal Microfluidic Discs

    PubMed Central

    Kearney, Sinéad M.; Kilcawley, Niamh A.; Early, Philip L.; Glynn, Macdara T.; Ducrée, Jens

    2016-01-01

    Here we present retrieval of Peripheral Blood Mononuclear Cells by density-gradient medium based centrifugation for subsequent analysis of the leukocytes on an integrated microfluidic “Lab-on-a-Disc” cartridge. Isolation of white blood cells constitutes a critical sample preparation step for many bioassays. Centrifugo-pneumatic siphon valves are particularly suited for blood processing as they function without need of surface treatment and are ‘low-pass’, i.e., holding at high centrifugation speeds and opening upon reduction of the spin rate. Both ‘hydrostatically’ and ‘hydrodynamically’ triggered centrifugo-pneumatic siphon valving schemes are presented. Firstly, the geometry of the pneumatic chamber of hydrostatically primed centrifugo-pneumatic siphon valves is optimised to enable smooth and uniform layering of blood on top of the density-gradient medium; this feature proves to be key for efficient Peripheral Blood Mononuclear Cell extraction. A theoretical analysis of hydrostatically primed valves is also presented which determines the optimum priming pressure for the individual valves. Next, ‘dual siphon’ configurations for both hydrostatically and hydrodynamically primed centrifugo-pneumatic siphon valves are introduced; here plasma and Peripheral Blood Mononuclear Cells are extracted through a distinct siphon valve. This work represents a first step towards enabling on disc multi-parameter analysis. Finally, the efficiency of Peripheral Blood Mononuclear Cells extraction in these structures is characterised using a simplified design. A microfluidic mechanism, which we termed phase switching, is identified which affects the efficiency of Peripheral Blood Mononuclear Cell extraction. PMID:27167376

  2. Anandamide inhibits Theiler's virus induced VCAM-1 in brain endothelial cells and reduces leukocyte transmigration in a model of blood brain barrier by activation of CB1 receptors

    PubMed Central

    2011-01-01

    Background VCAM-1 represents one of the most important adhesion molecule involved in the transmigration of blood leukocytes across the blood-brain barrier (BBB) that is an essential step in the pathogenesis of MS. Several evidences have suggested the potential therapeutic value of cannabinoids (CBs) in the treatment of MS and their experimental models. However, the effects of endocannabinoids on VCAM-1 regulation are poorly understood. In the present study we investigated the effects of anandamide (AEA) in the regulation of VCAM-1 expression induced by Theiler's virus (TMEV) infection of brain endothelial cells using in vitro and in vivo approaches. Methods i) in vitro: VCAM-1 was measured by ELISA in supernatants of brain endothelial cells infected with TMEV and subjected to AEA and/or cannabinoid receptors antagonist treatment. To evaluate the functional effect of VCAM-1 modulation we developed a blood brain barrier model based on a system of astrocytes and brain endothelial cells co-culture. ii) in vivo: CB1 receptor deficient mice (Cnr1-/-) infected with TMEV were treated with the AEA uptake inhibitor UCM-707 for three days. VCAM-1 expression and microglial reactivity were evaluated by immunohistochemistry. Results Anandamide-induced inhibition of VCAM-1 expression in brain endothelial cell cultures was mediated by activation of CB1 receptors. The study of leukocyte transmigration confirmed the functional relevance of VCAM-1 inhibition by AEA. In vivo approaches also showed that the inhibition of AEA uptake reduced the expression of brain VCAM-1 in response to TMEV infection. Although a decreased expression of VCAM-1 by UCM-707 was observed in both, wild type and CB1 receptor deficient mice (Cnr1-/-), the magnitude of VCAM-1 inhibition was significantly higher in the wild type mice. Interestingly, Cnr1-/- mice showed enhanced microglial reactivity and VCAM-1 expression following TMEV infection, indicating that the lack of CB1 receptor exacerbated

  3. HIV-1 Latency-Reversing Agents Prostratin and Bryostatin-1 Induce Blood-Brain Barrier Disruption/Inflammation and Modulate Leukocyte Adhesion/Transmigration.

    PubMed

    Dental, Clélia; Proust, Alizé; Ouellet, Michel; Barat, Corinne; Tremblay, Michel J

    2017-02-01

    A shock-and-kill approach involving the simultaneous treatment of HIV-1-infected patients with latency-reversing agents (LRAs) and combination antiretroviral therapy was proposed as a means to eradicate viral reservoirs. Currently available LRAs cannot discriminate between HIV-1-infected and uninfected cells. Therefore, the risks and benefits of using broad-spectrum LRAs need to be carefully evaluated, particularly in the CNS, where inflammation and leukocyte transmigration must be tightly regulated. We used a real-time impedance-sensing system to dynamically record the impact of different classes of LRAs on the integrity of tight monolayers of the immortalized human cerebral microvascular endothelial cell line hCMEC/D3. Results show that prostratin and bryostatin-1 can significantly damage the integrity of an endothelial monolayer. Moreover, prostratin and bryostatin-1 induce secretion of some proinflammatory cytokines and an increase of ICAM-1 expression. Additional studies demonstrated that prostratin and bryostatin-1 also affect adhesion and transmigration of CD4(+) and CD8(+) T cells as well as monocytes in an in vitro human blood-brain barrier (BBB) model. Prostratin and bryostatin-1 could thus be considered as potent regulators of BBB permeability and inflammation that influence leukocyte transport across the BBB. Altogether, these findings contribute to a better understanding of the potential risks and benefits of using a shock-and-kill approach with LRAs on the normal physiological functions of the BBB.

  4. Influence of In Utero Maternal and Neonate Factors on Cord Blood Leukocyte Telomere Length: Clues to the Racial Disparity in Prostate Cancer?

    PubMed Central

    Weber, Kari A.; Heaphy, Christopher M.; Rohrmann, Sabine; Gonzalez, Beverly; Bienstock, Jessica L.; Agurs-Collins, Tanya; Platz, Elizabeth A.

    2016-01-01

    Background. Modifiable factors in adulthood that explain the racial disparity in prostate cancer have not been identified. Because racial differences in utero that may account for this disparity are understudied, we investigated the association of maternal and neonate factors with cord blood telomere length, as a cumulative marker of cell proliferation and oxidative damage, by race. Further, we evaluated whether cord blood telomere length differs by race. Methods. We measured venous umbilical cord blood leukocyte relative telomere length by qPCR in 38 black and 38 white full-term male neonates. Using linear regression, we estimated geometric mean relative telomere length and tested for differences by race. Results. Black mothers were younger and had higher parity and black neonates had lower birth and placental weights. These factors were not associated with relative telomere length, even after adjusting for or stratifying by race. Relative telomere length in black (2.72) and white (2.73) neonates did not differ, even after adjusting for maternal or neonate factors (all p > 0.9). Conclusions. Maternal and neonate factors were not associated with cord blood telomere length, and telomere length did not differ by race. These findings suggest that telomere length at birth does not explain the prostate cancer racial disparity. PMID:28070423

  5. Interferon-related and other immune genes are downregulated in peripheral blood leukocytes in the luteal phase of the menstrual cycle.

    PubMed

    Dosiou, Chrysoula; Lathi, Ruth B; Tulac, Suzana; Huang, S-T Joseph; Giudice, Linda C

    2004-05-01

    Interaction between the endocrine and the immune systems has been suggested by observations of sexual dimorphism of the immune response, differential susceptibility to autoimmunity between the sexes, changes in autoimmune disease activity during the menstrual cycle and in pregnancy and in vitro studies of hormonal influence on cytokine production.We hypothesized that if there is hormonal regulation of the immune response, this would be manifest in peripheral blood leukocytes (PBLs) at different phases of the menstrual cycle. In this study, we describe gene profiling of PBLs from the follicular and luteal phases of the menstrual cycle. We observe important differences in immune gene expression, with significant down-regulation of the Th1 immune response in the luteal phase. A significant number of interferon (IFN)-related genes are amongst the downregulated genes. These results support significant hormonal regulation of the immune system and may have therapeutic implications in diseases of autoimmunity in women.

  6. Peripheral Blood Leukocyte Gene Expression Patterns and Metabolic Parameters in Habitually Snoring and Non-Snoring Children with Normal Polysomnographic Findings

    PubMed Central

    Khalyfa, Abdelnaby; Gharib, Sina A.; Kim, Jinkwan; Capdevila, Oscar Sans; Kheirandish-Gozal, Leila; Bhattacharjee, Rakesh; Hegazi, Mohamed; Gozal, David

    2011-01-01

    Background: Children who snore but do not have gas exchange abnormalities or alterations of sleep architecture have primary snoring (PS). Since increasing evidence suggest that PS may be associated with morbidity, we hypothesized that assessing genome-wide gene expression in peripheral blood leukocytes (PBL) will identify a distinct signature in PS children. Methods: Children (aged 4–9 years) with and without habitual snoring and a normal PSG were designated as either PS or controls. Whole genome expression profiles of PBL and metabolic parameters in 30 children with PS and 30 age-, gender-, ethnicity-, and BMI-matched controls were compared. Pathway-focused gene network analysis of the PBL transcriptome was performed. Metabolic parameters were measured in an independent follow-up cohort of 98 children (64 PS and 34 controls) to evaluate the computationally derived findings. Results: PS was not associated with a distinct transcriptional signature in PBL. Exploratory functional network analysis of enriched gene sets identified a number of putative pathways—including those mapping to insulin signaling, adipocyte differentiation, and obesity—with significant alterations in glucose metabolism and insulin sensitivity emerging in the follow-up cohort of children with PS, but no differences in lipid profiles. Conclusions: PS children do not exhibit global perturbations in their PBL transcriptional response, suggesting that current normative PSG criteria are overall valid. However, subtle differences in functionally coherent pathways involved in glycemic homeostasis were detected and confirmed in a larger independent pediatric cohort indicating that PS may carry increased risk for end-organ morbidity in susceptible children. Citation: Khalyfa A; Gharib SA; Kim J; Capdevila OS; Kheirandish-Gozal L; Bhattacharjee R; Hegazi M; Gozal D. Peripheral blood leukocyte gene expression patterns and metabolic parameters in habitually snoring and non-snoring children with normal

  7. Immunological changes in canine peripheral blood leukocytes triggered by immunization with first or second generation vaccines against canine visceral leishmaniasis.

    PubMed

    Araújo, Márcio Sobreira Silva; de Andrade, Renata Aline; Sathler-Avelar, Renato; Magalhães, Camila Paula; Carvalho, Andréa Teixeira; Andrade, Mariléia Chaves; Campolina, Sabrina Sidney; Mello, Maria Norma; Vianna, Leonardo Rocha; Mayrink, Wilson; Reis, Alexandre Barbosa; Malaquias, Luiz Cosme Cotta; Rocha, Luciana Morais; Martins-Filho, Olindo Assis

    2011-05-15

    In this study, we summarized the major phenotypic/functional aspects of circulating leukocytes following canine immunization with Leishvaccine and Leishmune®. Our findings showed that Leishvaccine triggered early changes in the innate immunity (neutrophils and eosinophils) with late alterations on monocytes. Conversely, Leishmune(®) induced early phenotypic changes in both, neutrophils and monocytes. Moreover, Leishvaccine triggered mixed activation-related phenotypic changes on T-cells (CD4+ and CD8+ and B-lymphocytes, whereas Leishmune(®) promoted a selective response, mainly associated with CD8+ T-cell activation. Mixed cytokine profile (IFN-γ/IL-4) was observed in Leishvaccine immunized dogs whereas a selective pro-inflammatory pattern (IFN-γ/NO) was induced by Leishmune® vaccination. The distinct immunological profile triggered by Leishvaccine and Leishmune® may be a direct consequence of the distinct biochemical composition of these immunobiological, i.e. complex versus purified Leishmania antigen along with Bacillus Calmette-Guérin (BCG) versus saponin adjuvant. Both immunobiologicals are able to activate phagocytes and CD8+ T-cells and therefore could be considered as a putative vaccines against canine visceral leishmaniasis (CVL).

  8. Evaluation of propolis, honey, and royal jelly in amelioration of peripheral blood leukocytes and lung inflammation in mouse conalbumin-induced asthma model.

    PubMed

    El-Aidy, Waleed K; Ebeid, Ahmad A; Sallam, Abd El-Raouf M; Muhammad, Ibrahim E; Abbas, Ayman T; Kamal, M A; Sohrab, Sayed Sartaj

    2015-11-01

    Bee products have been used since ancient times to treat many diseases, including respiratory ailments. The present study aimed to examine the modulatory effect of honey, royal jelly, and propolis extract on peripheral blood leukocytes and lung inflammation in a mouse conalbumin-induced asthma model. The mice in group I were not sensitised or treated; they were kept as controls. The mice in group II were sensitised and challenged with conalbumin. Twenty-four hours after the first challenge with antigen, the mice in group III received 0.5 mg/kg of dexamethasone intraperitoneally per day for 18 consecutive days and kept as positive controls. The mice in groups IV, V, and VI received 650, 1000, and 30 mg/kg of honey, royal jelly, and propolis (aqueous and ethanolic extract), respectively, once per day for 18 consecutive days. Blood was collected from all of the mice for white blood cell differentiation, and the lungs were removed for histopathological studies. The groups treated with propolis extract exhibited considerable ameliorative effects against asthma, which might be explained by the flavonoids and phenolics found in propolis, which might have antioxidative effects. Otherwise, the sensitised and honey- or royal jelly-treated groups exhibited an increased incidence of asthma cascade events due to increased inflammatory cells. These results might be due to the immunostimulatory and vasodilatory effects of royal jelly and honey, which are antagonistic to bronchial asthma cases. Histopathological examination revealed that the sensitised treated propolis extract groups had significant decreases in inflammatory scores compared with other treatments and the sensitised untreated group. These results confirmed the previous data of peripheral blood cells.

  9. Benidipine, a long-acting calcium channel blocker, inhibits oxidative stress in polymorphonuclear cells in patients with essential hypertension.

    PubMed

    Yasunari, Kenichi; Maeda, Kensaku; Nakamura, Munehiro; Watanabe, Takanori; Yoshikawa, Junichi

    2005-02-01

    To study the relationship between blood pressure and oxidative stress in leukocytes, the effect of benidipine on these variables was compared with that of a placebo. Hypertensive patients were randomly assigned benidipine 4 mg (n=40) or placebo (n=40), and treated for 6 months. Oxidative stress in polymorphonuclear cells (PMNs) was measured by gated flow cytometry. There was a significant relationship between systolic or diastolic arterial pressure and reactive oxygen species (ROS) formation by PMNs in the benidipine group (r=0.61, p<0.01) and in the placebo group (r=0.58, p<0.01). After administration of 4 mg benidipine, ROS formation by PMNs fell by 32 arbitrary units (n=40, p<0.01). After administration of placebo, ROS formation by PMNs decreased by 0.6 arbitrary units (n=40, p=0.31) (p<0.01 for differing treatment effects). There was a significant relationship between the decrease in systolic arterial pressure and the decrease in ROS formation by PMNs in the benidipine group (r=0.52, p<0.01), but not in the placebo group (r=-0.08, p=0.61). There was also a significant relationship between the decrease in diastolic arterial pressure and decrease in ROS formation by PMNs in the benidipine group (r=0.65, p<0.01) but not in the placebo group (r=-0.09, p=0.59). In hypertensive patients, we observed a significant relationship between systolic or diastolic blood pressure and ROS formation by PMNs, and found that benidipine decreased oxidative stress in PMNs of hypertensive patients, at least in part by decreasing blood pressure.

  10. Comparative study of peripheral blood leukocytes in healthy dogs and in dogs with cancer and inflammatory diseases.

    PubMed

    García-Sancho, Mercedes; Villaescusa, Alejandra; Rodríguez-Franco, Fernando; Sainz, Angel

    2014-03-01

    In the present study, the peripheral blood lymphocyte subset distribution was compared between healthy dogs and dogs with chronic gastrointestinal disease, dental and skin conditions, and cancer. The immunophenotype of the group with chronic gastrointestinal disease and the group with dental and skin conditions showed no statistically significant differences with other groups of healthy or diseased dogs. When compared with healthy dogs, animals with cancer showed significantly lower absolute values of T cytotoxic cells (CD3+, CD8+) and lymphocytes that express major histocompatibility complex (MHC) class II (MHC-II+) in peripheral blood. The results suggest that peripheral blood immunophenotype is mainly altered in dogs with cancer but not in other diseases. Further studies are required to evaluate the clinical relevance of these findings.

  11. 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.

  12. Human T-Lymphotropic Virus Type 1-Induced Overexpression of Activated Leukocyte Cell Adhesion Molecule (ALCAM) Facilitates Trafficking of Infected Lymphocytes through the Blood-Brain Barrier

    PubMed Central

    Curis, Céline; Percher, Florent; Jeannin, Patricia; Montange, Thomas; Chevalier, Sébastien A.; Seilhean, Danielle; Cartier, Luis; Couraud, Pierre-Olivier; Gout, Olivier; Gessain, Antoine; Ceccaldi, Pierre-Emmanuel

    2016-01-01

    ABSTRACT Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of a slowly progressive neurodegenerative disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). This disease develops upon infiltration of HTLV-1-infected lymphocytes into the central nervous system, mostly the thoracic spinal cord. The central nervous system is normally protected by a physiological structure called the blood-brain barrier (BBB), which consists primarily of a continuous endothelium with tight junctions. In this study, we investigated the role of activated leukocyte cell adhesion molecule (ALCAM/CD166), a member of the immunoglobulin superfamily, in the crossing of the BBB by HTLV-1-infected lymphocytes. We demonstrated that ALCAM is overexpressed on the surface of HTLV-1-infected lymphocytes, both in chronically infected cell lines and in primary infected CD4+ T lymphocytes. ALCAM overexpression results from the activation of the canonical NF-κB pathway by the viral transactivator Tax. In contrast, staining of spinal cord sections of HAM/TSP patients showed that ALCAM expression is not altered on the BBB endothelium in the context of HTLV-1 infection. ALCAM blockade or downregulation of ALCAM levels significantly reduced the migration of HTLV-1-infected lymphocytes across a monolayer of human BBB endothelial cells. This study suggests a potential role for ALCAM in HAM/TSP pathogenesis. IMPORTANCE Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of a slowly progressive neurodegenerative disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). This disease is the consequence of the infiltration of HTLV-1-infected lymphocytes into the central nervous system (CNS), mostly the thoracic spinal cord. The CNS is normally protected by a physiological structure called the blood-brain barrier (BBB), which consists primarily of a continuous endothelium with tight junctions. The mechanism of migration of lymphocytes into

  13. Evaluation of the in vivo genotoxic effects of gamma radiation on the peripheral blood leukocytes of head and neck cancer patients undergoing radiotherapy.

    PubMed

    Kadam, Samit B; Shyama, Soorambail K; Almeida, Valentine G

    2013-04-15

    The present study aimed to evaluate the genotoxic effects of ionizing radiation on non-target cells of Head and Neck Squamous Cell Carcinoma (HNSCC) patients exposed to various cumulative doses of gamma rays during radiotherapy. The ten patients (P1-P10) were treated with cobalt 60 gamma radiation (External Beam Radiotherapy) for a period of five to six weeks with a daily fraction of 2Gy for 5 days each week. The genotoxic effects of radiation (single strand breaks - SSBs) in these patients were analyzed using the alkaline single cell gel electrophoresis (SCGE) technique, with the Olive Tail Moment (OTM) as the critical parameter. A sample of each patient's peripheral blood before starting with radiotherapy (pre-therapy) served as the control, and blood collected at weekly time intervals during the course of the radiotherapy served as treated (10, 20, 30, 40, 50 and 60Gy) samples. In vivo radiosensitivity of these patients, as indicated by SSB's after the cumulative radiation doses at the various times, was assessed using Student's t-test. Significant DNA damage relative to the individual patient's pre-therapy baseline data was observed in all patients. Inter-individual variation of the genotoxic effects was analyzed using two-way ANOVA. The correlation between doses for the means of smoker and non-smoker patients was calculated using the Pearson test. The results of this study may indicate the need to reduce the daily radiotherapy dose further to prevent genotoxic effects on non-target cells, thus improving safety. Furthermore, these results may indicate that the estimation of DNA damage following exposure to a gamma radiation, as measured by the comet assay in whole blood leukocytes, can be used to screen human populations for radiation-induced genetic damage at the molecular level.

  14. Effects of recombinant trout leptin in superoxide production and NF-κB/MAPK phosphorylation in blood leukocytes.

    PubMed

    Mariano, Giovanna; Stilo, Romania; Terrazzano, Giuseppe; Coccia, Elena; Vito, Pasquale; Varricchio, Ettore; Paolucci, Marina

    2013-10-01

    Studies in mammals indicate that leptin is a multifunctional cytokine involved in regulation of energy metabolism and the modulation of the immune function. However, evidence for an immunomodulatory effect of leptin in fish is still missing. At least in part, this lack of knowledge is due to the absence of materials and models. In this study, we produced trout recombinant leptin (rt-lep) and tested its capacity to trigger cellular pathways, usually active in mammal immune system cells. STAT3, NF-κB, and the three major MAPK cascades (JNK, p38 and ERK), were activated by rt-lep in in vitro incubations with blood leucocytes of the rainbow trout Oncorhynchus mykiss. We also showed that rt-lep causes a decrease in superoxide anion production in trout blood leucocytes. Thus our data indicate that as in mammals also in teleosts leptin plays pleiotropic activities. Importantly, its actions in fishes do not always conform to the picture emerging for mammals.

  15. Release of cyclooxygenase-2 and lipoxin A4 from blood leukocytes in aspirin-exacerbated respiratory disease

    PubMed Central

    Heinemann, Akos; Keck, Tilman

    2016-01-01

    Background: The release of cyclooxygenase-2 (COX-2) and lipoxin A4 (LXA4) from blood mononuclear cells in patients with aspirin-exacerbated respiratory disease (AERD) is only partially understood. Objective: To investigate the presence of COX-2 and LXA4 in peripheral blood mononuclear cells (PBMC) derived from patients with AERD and with nasal polyps (NP) (designated as the AERD-NP group), patients with NP without AERD (the NP group), and healthy controls without sinus disease (the control group). Methods: Blood was taken from 14 patients in the AERD-NP group, 6 patients in the NP group, and 8 healthy subjects in the control group. After culturing of human PBMC, the presence of COX-2 protein and LXA4 (ELISA) was detected in the supernatant, and the results were compared among the groups. Results: COX-2 and LXA4 were detectable after culturing of PBMC in all patients in the AERD-NP and NP groups and in the control subjects. COX-2 was highest in the patients in the AERD-NP group, but the difference was not significant compared with patients with non-AERD polyp and with the control subjects. LXA4 was also highest in the AERD-NP group, but the difference was also not significant compared with the patients who were non-AERD polyp and the control subjects. Conclusion: Neither the release of COX-2 or LXA4 was different between the patients with AERD and with NPs, the patients without AERD and with NPs, and the healthy control group. The release of these proteins in AERD needs further investigation. PMID:28107149

  16. Increased expression of TLR-2, COX-2, and SOD-2 genes in the peripheral blood leukocytes of opisthorchiasis patients induced by Opisthorchis viverrini antigen.

    PubMed

    Yongvanit, Puangrat; Thanan, Raynoo; Pinlaor, Somchai; Sithithaworn, Paiboon; Loilome, Watcharin; Namwat, Nisana; Techasen, Anchalee; Dechakhamphu, Somkid

    2012-05-01

    Re-infection with liver fluke, Opisthorchis viverrini, increases proinflammatory molecules involved in inflammation-mediated disease and carcinogenesis in an animal model. To clarify whether these genes respond to parasite antigen in peripheral blood leukocytes (PBL) of opisthorchiasis patients, we examined the transcriptional level of oxidant-generating (toll-like receptor 2 (TLR-2), nuclear factor-kappa B (NF-KB), and cyclooxygenase 2 (COX-2)), anti-oxidant-generating (manganese superoxide dismutase 2 (SOD-2) and catalase (CAT)), proinflammatory cytokine (interleukin (IL)-1β), and anti-inflammatory cytokine (IL-10), in PBL exposed to parasite antigen in O. viverrini-infected patients compared with healthy individuals in an in vitro experiment. After O. viverrini antigen-treated PBL, quantitative RT-PCR analysis revealed that increased expression of cytokines and oxidant-generating genes in PBL was similar between O. viverrini-infected and healthy groups. Interestingly, compared with healthy subjects, increase of TLR-2, COX-2, and SOD-2 and decreased CAT mRNA expression levels were observed in O. viverrini-infected group. The results indicate that O. viverrini antigen induces upregulation of TLR-2, COX-2, and SOD-2 and downregulation of CAT genes in opisthorchiasis patients, suggesting that imbalance of oxidant/anti-oxidant transcripts during re-infection may be involved in the inflammatory-driven carcinogenesis. These molecules may be used as the chemopreventive target for intervention of opisthorchiasis patients in an endemic area.

  17. The effect of core and lanthanide ion dopants in sodium fluoride-based nanocrystals on phagocytic activity of human blood leukocytes

    NASA Astrophysics Data System (ADS)

    Sojka, Bartlomiej; Liskova, Aurelia; Kuricova, Miroslava; Banski, Mateusz; Misiewicz, Jan; Dusinska, Maria; Horvathova, Mira; Ilavska, Silvia; Szabova, Michaela; Rollerova, Eva; Podhorodecki, Artur; Tulinska, Jana

    2017-02-01

    Sodium fluoride-based β-NaLnF4 nanoparticles (NPs) doped with lanthanide ions are promising materials for application as luminescent markers in bio-imaging. In this work, the effect of NPs doped with yttrium (Y), gadolinium (Gd), europium (Eu), thulium (Tm), ytterbium (Yb) and terbium (Tb) ions on phagocytic activity of monocytes and granulocytes and the respiratory burst was examined. The surface functionalization of <10-nm NPs was performed according to our variation of patent pending ligand exchange method that resulted in meso-2,3-dimercaptosuccinic acid (DMSA) molecules on their surface. Y-core-based NCs were doped with Eu ions, which enabled them to be excited with UV light wavelengths. Cultures of human peripheral blood ( n = 8) were in vitro treated with five different concentrations of eight NPs for 24 h. In summary, neither type of nanoparticles is found toxic with respect to conducted test; however, some cause toxic effects (they have statistically significant deviations compared to reference) in some selected doses tested. Both core types of NPs (Y-core and Gd-core) impaired the phagocytic activity of monocytes the strongest, having minimal or none whatsoever influence on granulocytes and respiratory burst of phagocytic cells. The lowest toxicity was observed in Gd-core, Yb, Tm dopants and near-infrared nanoparticles. Clear dose-dependent effect of NPs on phagocytic activity of leukocytes and respiratory burst of cells was observed for limited number of samples.

  18. Impact on outcomes of human leukocyte antigen matching by allele-level typing in adults with acute myeloid leukemia undergoing umbilical cord blood transplantation.

    PubMed

    Sanz, Jaime; Jaramillo, Francisco J; Planelles, Dolores; Montesinos, Pau; Lorenzo, Ignacio; Moscardó, Federico; Martin, Guillermo; López, Francisca; Martínez, Jesús; Jarque, Isidro; de la Rubia, Javier; Larrea, Luis; Sanz, Miguel A; Sanz, Guillermo F

    2014-01-01

    This retrospective study analyzed the impact of directional donor-recipient human leukocyte antigen (HLA) disparity using allele-level typing at HLA-A, -B, -C, and -DRB1 in 79 adults with acute myeloid leukemia (AML) who received single-unit umbilical cord blood (UCB) transplant at a single institution. With extended high-resolution HLA typing, the donor-recipient compatibility ranged from 2/8 to 8/8. HLA disparity showed no negative impact on nonrelapse mortality (NRM), graft-versus-host (GVH) disease or engraftment. Considering disparities in the GVH direction, the 5-year cumulative incidence of relapse was 44% and 22% for patients receiving an UCB unit matched ≥ 6/8 and < 6/8, respectively (P = .04). In multivariable analysis, a higher HLA disparity in the GVH direction using extended high-resolution typing (Risk ratio [RR] 2.8; 95% confidence interval [CI], 1.5 to 5.1; P = .0009) and first complete remission at time of transplantation (RR 2.1; 95% CI, 1.2 to 3.8; P = .01) were the only variables significantly associated with an improved disease-free survival. In conclusion, we found that in adults with AML undergoing single-unit UCBT, an increased number of HLA disparities at allele-level typing improved disease-free survival by decreasing the relapse rate without a negative effect on NRM.

  19. In vitro assessment of the effects of temperature on phagocytosis, reactive oxygen species production and apoptosis in bovine polymorphonuclear cells.

    PubMed

    Lecchi, Cristina; Rota, Nicola; Vitali, Andrea; Ceciliani, Fabrizio; Lacetera, Nicola

    2016-12-01

    Heat stress exerts a direct negative effect on farm animal health, triggering physiological responses. Environmental high temperature induces immunosuppression in dairy cows, increasing the risk of mastitis and milk somatic cell counts. The influence of heat stress on leukocytes activities has not been fully elucidated. The present in vitro study was aimed at assessing whether the exposure to temperature simulating conditions of severe whole body hyperthermia affects defensive functions of bovine blood polymorphonuclear cells. Blood was collected from seven clinically healthy, multiparous, late lactating Holstein cows. After isolation, PMN were incubated at either 39 or 41°C. Phagocytosis, respiratory burst and apoptosis were then investigated. The selected temperatures of 39°C or 41°C mimicked conditions of normothermia or severe heat stress, respectively. Phagocytosis assay was carried out by measuring the fluorescence of phagocyted fluorescein-labelled E. coli bioparticles. The modulation of oxidative burst activity was studied by the cytochrome C reduction method. Apoptosis was determined by measuring the activities of two enzymes that play an effector role in the process, namely Caspase-3 and Caspase-7. Statistical analyses were performed using SPSS 22.0. A Student t-test for paired samples and a Generalised Estimating Equation were used based on data distribution. The phagocytosis rate was reduced (-37%, P<0.01) when PMN were incubated for 2h at 41°C, when compared to phagocytosis rate measured at 39°C. The oxidative burst, as determined by extracellular production of reactive oxygen species (ROS), was also reduced by the exposure of cells to 41°C compared to 39°C. Such reduction ranged between -2 and -21% (P<0.05). Apoptosis rate was not affected by different temperatures. The results reported in this study suggest that phagocytosis and ROS production in PMN exposed to severe high temperature are impaired, partially explaining the higher occurrence of

  20. West Nile virus-induced cell adhesion molecules on human brain microvascular endothelial cells regulate leukocyte adhesion and modulate permeability of the in vitro blood-brain barrier model.

    PubMed

    Roe, Kelsey; Orillo, Beverly; Verma, Saguna

    2014-01-01

    Characterizing the mechanisms by which West Nile virus (WNV) causes blood-brain barrier (BBB) disruption, leukocyte infiltration into the brain and neuroinflammation is important to understand the pathogenesis of WNV encephalitis. Here, we examined the role of endothelial cell adhesion molecules (CAMs) in mediating the adhesion and transendothelial migration of leukocytes across human brain microvascular endothelial cells (HBMVE). Infection with WNV (NY99 strain) significantly induced ICAM-1, VCAM-1, and E-selectin in human endothelial cells and infected mice brain, although the levels of their ligands on leukocytes (VLA-4, LFA-1and MAC-1) did not alter. The permeability of the in vitro BBB model increased dramatically following the transmigration of monocytes and lymphocytes across the models infected with WNV, which was reversed in the presence of a cocktail of blocking antibodies against ICAM-1, VCAM-1, and E-selectin. Further, WNV infection of HBMVE significantly increased leukocyte adhesion to the HBMVE monolayer and transmigration across the infected BBB model. The blockade of these CAMs reduced the adhesion and transmigration of leukocytes across the infected BBB model. Further, comparison of infection with highly neuroinvasive NY99 and non-lethal (Eg101) strain of WNV demonstrated similar level of virus replication and fold-increase of CAMs in HBMVE cells suggesting that the non-neuropathogenic response of Eg101 is not because of its inability to infect HBMVE cells. Collectively, these results suggest that increased expression of specific CAMs is a pathological event associated with WNV infection and may contribute to leukocyte infiltration and BBB disruption in vivo. Our data further implicate that strategies to block CAMs to reduce BBB disruption may limit neuroinflammation and virus-CNS entry via 'Trojan horse' route, and improve WNV disease outcome.

  1. Assessing Immune Function by Profiling Cytokine Release from Stimulated Blood Leukocytes and the Risk of Infection in Rheumatoid Arthritis

    PubMed Central

    Krause, Megan L.; Davis, John M.; Knutson, Keith L.; Strausbach, Michael A.; Crowson, Cynthia S.; Therneau, Terry M.; Wettstein, Peter J.; Matteson, Eric L.; Gabriel, Sherine E.

    2011-01-01

    Persons with rheumatoid arthritis (RA) suffer a high burden of infections, but currently no biomarkers are available to identify individuals at greatest risk. A prospective longitudinal study was therefore conducted to determine the association between the responsiveness of ex vivo cytokine production and 6-month risk of infections. Infections were identified by billing codes and validated by medical record review. At baseline, the release of 17 cytokines by peripheral blood mononuclear cells in response to stimulation, or media alone, was measured using multiplexed cytokine analysis. Production of IL-2, IL-8, IL-10, IL-17, TNF-α, IFN-γ, and GM-CSF, induced by various conditions, was significantly associated with the occurrence of infections. A multivariable prediction model based on these data provided new information on the risk of infection beyond standard assessments of disease activity, severity, and treatment. Future studies could utilize this information to devise new biomarkers for the prediction of infection in patients with RA. PMID:21703930

  2. The expression of GPR109A, NF-kB and IL-1β in peripheral blood leukocytes from patients with type 2 diabetes.

    PubMed

    Liu, Fengxiu; Fu, Yucai; Wei, Chiju; Chen, Yongru; Ma, Shuhua; Xu, Wencan

    2014-01-01

    This study was designed to explore the association between the G protein-coupled receptor 109A (GPR109A) expression in peripheral blood leukocytes (PBLs) and type 2 diabetes (T2DM) and to discuss the regulation of inflammatory factors by GPR109A signaling. GPR109A signaling has been confirmed to be associated with homeostasis of glucose/lipid metabolism, but the role of signaling in T2DM is still poorly understood. Peripheral blood samples and biochemical data were collected from healthy individuals (normal controls) and T2DM patients. Immunocytochemical staining was used to detect the expression of GPR109A in PBLs. Reverse transcription polymerase chain reaction (RT-PCR) was used to measure mRNA levels of GPR109A, NF-κB, and IL-1β in PBLs. Immunocytochemical staining showed that the GPR109A protein is localized in the nucleus and cytoplasm of granulocytes, monocytes, and lymphocytes. RT-PCR showed that mRNA levels of GPR109A, NF-κB, and IL-1β were higher in the T2DM group than in the control group (P<0.05). Correlation analysis showed a positive correlation both between GPR109A/NF-κB (r=0.376, P<0.05), and GPR109A/IL-1β (r=501, P<0.05) and between GPR109A and fasting plasma glucose (FPG) (r=0.179, P<0.05) and NF-κB /FPG (r=0.358, p<0.05). Our results suggest that GPR109A signaling is associated with T2DM, playing a role in regulation of the inflammatory cytokines.

  3. [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.

  4. Simple and Reliable Method to Quantify the Hepatitis B Viral Load and Replicative Capacity in Liver Tissue and Blood Leukocytes

    PubMed Central

    Minosse, Claudia; Coen, Sabrina; Visco Comandini, Ubaldo; Lionetti, Raffaella; Montalbano, Marzia; Cerilli, Stefano; Vincenti, Donatella; Baiocchini, Andrea; Capobianchi, Maria R.; Menzo, Stefano

    2016-01-01

    Background A functional cure of chronic hepatitis B (CHB) is feasible, but a clear view of the intrahepatic viral dynamics in each patient is needed. Intrahepatic covalently closed circular DNA (cccDNA) is the stable form of the viral genome in infected cells, and represents the ideal marker of parenchymal colonization. Its relationships with easily accessible peripheral parameters need to be elucidated in order to avoid invasive procedures in patients. Objectives The goal of this study was to design, set up, and validate a reliable and straightforward method for the quantification of the cccDNA and total DNA of the hepatitis B virus (HBV) in a variety of clinical samples. Patients and Methods Clinical samples from a cohort of CHB patients, including liver biopsies in some, were collected for the analysis of intracellular HBV molecular markers using novel molecular assays. Results A plasmid construct, including sequences from the HBV genome and from the human gene hTERT, was generated as an isomolar multi-standard for HBV quantitation and normalization to the cellular contents. The specificity of the real-time assay for the cccDNA was assessed using Dane particles isolated on a density gradient. A comparison of liver tissue from 6 untreated and 6 treated patients showed that the treatment deeply reduced the replicative capacity (total DNA/cccDNA), but had limited impact on the parenchymal colonization. The peripheral blood mononuclear cells (PBMCs) and granulocytes from the treated and untreated patients were also analyzed. Conclusions A straightforward method for the quantification of intracellular HBV molecular parameters in clinical samples was developed and validated. The widespread use of such versatile assays could better define the prognosis of CHB, and allow a more rational approach to time-limited tailored treatment strategies. PMID:27882060

  5. Disorders of sex development expose transcriptional autonomy of genetic sex and androgen-programmed hormonal sex in human blood leukocytes

    PubMed Central

    Holterhus, Paul-Martin; Bebermeier, Jan-Hendrik; Werner, Ralf; Demeter, Janos; Richter-Unruh, Annette; Cario, Gunnar; Appari, Mahesh; Siebert, Reiner; Riepe, Felix; Brooks, James D; Hiort, Olaf

    2009-01-01

    Background Gender appears to be determined by independent programs controlled by the sex-chromosomes and by androgen-dependent programming during embryonic development. To enable experimental dissection of these components in the human, we performed genome-wide profiling of the transcriptomes of peripheral blood mononuclear cells (PBMC) in patients with rare defined "disorders of sex development" (DSD, e.g., 46, XY-females due to defective androgen biosynthesis) compared to normal 46, XY-males and 46, XX-females. Results A discrete set of transcripts was directly correlated with XY or XX genotypes in all individuals independent of male or female phenotype of the external genitalia. However, a significantly larger gene set in the PBMC only reflected the degree of external genital masculinization independent of the sex chromosomes and independent of concurrent post-natal sex steroid hormone levels. Consequently, the architecture of the transcriptional PBMC-"sexes" was either male, female or even "intersex" with a discordant alignment of the DSD individuals' genetic and hormonal sex signatures. Conclusion A significant fraction of gene expression differences between males and females in the human appears to have its roots in early embryogenesis and is not only caused by sex chromosomes but also by long-term sex-specific hormonal programming due to presence or absence of androgen during the time of external genital masculinization. Genetic sex and the androgen milieu during embryonic development might therefore independently modulate functional traits, phenotype and diseases associated with male or female gender as well as with DSD conditions. PMID:19570224

  6. Secretion of 10-kDa and 12-kDa thioredoxin species from blood monocytes and transformed leukocytes.

    PubMed

    Sahaf, B; Rosén, A

    2000-01-01

    Thioredoxins (TRX) are ubiquitous, small redox-active proteins with multiple functions, including antioxidant, cytoprotective, and chemoattractant activities. In addition to a 12-kDa intracellular form, extracellular 10-kDa and 12-kDa TRX have been defined. The biological activities of the 10-kDa TRX were previously measured as eosinophil cytotoxicity enhancing activity or B-cell stimulatory activity. Cytotrophoblastic cell lines also release a 10-kDa TRX form. To study the biological role of 10-kDa TRX, we established two highly sensitive enzyme-linked immuno-spot assays (ELISPOT), which detect secreted truncated 10-kDa and full-length 12-kDa TRX at the single cell level. TRX secretion was investigated in several cell lines including the T-helper cell hybridoma MP6, the Jurkat T-cell leukemia, the U-937 myelomonocytic leukemia, and the 3B6, EBV-transformed, lymphoblastoid B-cell line. The highest number of secreting cells was found in 3B6 cultures, median = 34 (quartiles, 27-39) per well (10(5) cells). Peripheral blood monocytes isolated from healthy donors secreted significantly more TRX after stimulation with ionomycin, phorbol myristate acetate (PMA), fMLP, and lipopolysaccharide (LPS), compared to unstimulated cells. Oxidative stress induced by thioloxidant diamide also induced the secretion of both truncated and full-length TRX measured in ELISPOT (p = 0.047 and p = 0.031, respectively). The biological activity of the truncated and full-length forms was tested in a cell migration assay. Truncated TRX was devoid of protein disulfide reductase activity, but retained strong chemoattractant activity for human monocytes, in the same range as full-length TRX, as previously reported (Bertini et al., 1999).

  7. Leukocytes, cytokines, growth factors and hormones in human skeletal muscle and blood after uphill or downhill running

    PubMed Central

    Malm, Christer; Sjödin, Bertil; Sjöberg, Berit; Lenkei, Rodica; Renström, Per; Lundberg, Ingrid E; Ekblom, Björn

    2004-01-01

    Muscular adaptation to physical exercise has previously been described as a repair process following tissue damage. Recently, evidence has been published to question this hypothesis. The purpose of this study was to investigate inflammatory processes in human skeletal muscle and epimysium after acute physical exercise with large eccentric components. Three groups of subjects (n= 19) performed 45 min treadmill running at either 4 deg (n= 5) or 8 deg (n= 9) downhill or 4 deg uphill (n= 5) and one group served as control (n= 9). One biopsy was taken from each subject 48 h post exercise. Blood samples were taken up to 7 days post exercise. Compared to the control group, none of the markers of inflammation in muscle and epimysium samples was different in any exercised group. Only subjects in the Downhill groups experienced delayed onset of muscle soreness (DOMS) and increased serum creatine kinase activity (CK). The detected levels of immunohistochemical markers for T cells (CD3), granulocytes (CD11b), leukaemia inhibitory factor (LIF) and hypoxia-inducible factor 1β (HIF-1β) were greater in epimysium from exercised subjects with DOMS ratings >3 (0–10 scale) compared to exercised subjects without DOMS but not higher than controls. Eccentric physical exercise (downhill running) did not result in skeletal muscle inflammation 48 h post exercise, despite DOMS and increased CK. It is suggested that exercise can induce DOMS by activating inflammatory factors present in the epimysium before exercise. Repeated physical training may alter the content of inflammatory factors in the epimysium and thus reduce DOMS. PMID:14766942

  8. Hyperreactivity of Blood Leukocytes in Patients with NAFLD to Ex Vivo Lipopolysaccharide Treatment Is Modulated by Metformin and Phosphatidylcholine but Not by Alpha Ketoglutarate

    PubMed Central

    Daniluk, Jadwiga; Słabczyńska, Olga; Kandefer-Szerszeń, Martyna

    2015-01-01

    Introduction and Aims Toll-like receptor 4 and proinflammatory cytokines play a central role in the progression of nonalcoholic fatty liver disease. We investigated IL-1, IL-6 and TNFα production and toll-like receptor 4 in both—obese and lean patients with non-alcoholic fatty liver disease who met different sets of metabolic syndrome criteria and linked the results with the disease burden. Materials and Methods 95 subjects were divided into four groups depending on the following criteria: presence or absence of metabolic syndrome and/or non-alcoholic fatty liver disease, glucose tolerance (prediabetes or normoglycemia) and BMI value (obese or lean). We determined the levels of IL-1β, IL-6, TNFα, and monocyte toll-like receptor 4 expression in fresh blood as well as in blood cultures treated with lipopolysaccharide with or without metformin, alphaketoglutarate or phosphatidylcholine supplementation. Results The blood leukocytes of patients with non-alcoholic fatty liver disease are hypersensitive to lipopolysaccharide treatment and produce elevated levels of pro-inflammatory cytokines in response to ex vivo treatment with lipopolysaccharide. Moreover, they overexpress toll-like receptor-4. Hyperreactivity was typical mainly for obese patients with non-alcoholic fatty liver disease together with metabolic syndrome and decreased with the severity of disease. Metformin was the most effective in attenuation of hyperreactivity in all groups of patients with non-alcoholic fatty liver disease, but in obese patients the effectiveness of metformin was weaker than in lean. The reduction of cytokine level by metformin was accompanied by the decrease in toll-like receptor-4 expression. phosphatidylcholine also attenuated hyperreactivity to lipopolysaccharide but mainly in obese patients. Alpha ketoglutarate did not modulate cytokines’ level and toll-like receptor 4 expression in non-alcoholic fatty liver disease patients. Conclusions Metformin and phosphatidylcholine

  9. Leukocyte abnormalities.

    PubMed

    Gabig, T G

    1980-07-01

    Certain qualitative abnormalities in neutrophils and blood monocytes are associated with frequent, severe, and recurrent bacterial infections leading to fatal sepsis, while other qualitative defects demonstrated in vitro may have few or no clinical sequelae. These qualitative defects are discussed in terms of the specific functions of locomotion, phagocytosis, degranulation, and bacterial killing.

  10. The role of G-CSF and IL-6 in the granulopoiesis-stimulating activity of murine blood serum induced by perorally administered ultrafiltered pig leukocyte extract, IMUNOR.

    PubMed

    Vacek, Antonín; Hofer, Michal; Holá, Jirina; Weiterová, Lenka; Streitová, Denisa; Svoboda, Jaroslav

    2007-05-01

    IMUNOR, a low-molecular weight (< 12 kD) ultrafiltered pig leukocyte extract, has been previously found to have significant stimulatory effects on murine hematopoiesis supressed by ionizing radiation or cytotoxic drugs. This communication shows data on the mechanisms of these effects. Using ELISA assay, significantly increased levels of granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6) were observed. On the contrary, no detectable levels of granulocyte-macrophage colony-stimulating factor (GM-CFC) and interleukin-3 (IL-3) have been found in blood serum of IMUNOR-treated mice. Incubation of the serum from IMUNOR-treated mice with antibodies against G-CSF caused abrogation of the ability of the sera to stimulate in vitro growth of colonies originating from granulocyte-macrophage progenitor cells (GM-CFC). In contrast, incubation of the serum with antibodies against IL-6 did not change its colony-stimulating activity. It may be inferred from these findings that G-CSF is probably the main cytokine responsible for the granulopoiesis-stimulating effects of IMUNOR. When the serum from IMUNOR-treated mice with G-CSF inactivated by anti-G-CSF antibodies (but with elevated IL-6) was added to cultures of bone marrow cells together with a suboptimum concentration of IL-3, a significant increase in the numbers of GM-CFC colonies was found. Moreover, conjoint inactivation of G-CSF and IL-6 significantly decreased the numbers of GM-CFC colonies in comparison with those observed when only G-CSF was inactivated. This observation strongly suggests that though IMUNOR-induced IL-6 is not able to induce the growth of GM-CFC colonies alone, it is able to potentiate the hematopoiesis-stimulating effect of IL-3. These findings represent a new knowledge concerning the hematopoiesis-stimulating action of IMUNOR, a promising immunomodulatory agent.

  11. Separation of uncompromised whole blood mixtures for single source STR profiling using fluorescently-labeled human leukocyte antigen (HLA) probes and fluorescence activated cell sorting (FACS).

    PubMed

    Dean, Lee; Kwon, Ye Jin; Philpott, M Katherine; Stanciu, Cristina E; Seashols-Williams, Sarah J; Dawson Cruz, Tracey; Sturgill, Jamie; Ehrhardt, Christopher J

    2015-07-01

    Analysis of biological mixtures is a significant problem for forensic laboratories, particularly when the mixture contains only one cell type. Contributions from multiple individuals to biologic evidence can complicate DNA profile interpretation and often lead to a reduction in the probative value of DNA evidence or worse, its total loss. To address this, we have utilized an analytical technique that exploits the intrinsic immunological variation among individuals to physically separate cells from different sources in a mixture prior to DNA profiling. Specifically, we applied a fluorescently labeled antibody probe to selectively bind to one contributor in a mixture through allele-specific interactions with human leukocyte antigen (HLA) proteins that are expressed on the surfaces of most nucleated cells. Once the contributor's cells were bound to the probe, they were isolated from the mixture using fluorescence activated cell sorting (FACS)-a high throughput technique for separating cell populations based on their optical properties-and then subjected to STR analysis. We tested this approach on two-person and four-person whole blood mixtures where one contributor possessed an HLA allele (A*02) that was not shared by other contributors to the mixture. Results showed that hybridization of the mixture with a fluorescently-labeled antibody probe complimentary to the A*02 allele's protein product created a cell population with a distinct optical profile that could be easily differentiated from other cells in the mixture. After sorting the cells with FACS, genetic analysis showed that the STR profile of this cell population was consistent with that of the contributor who possessed the A*02 allele. Minor peaks from the A*02 negative contributor(s) were observed but could be easily distinguished from the profile generated from A*02 positive cells. Overall, this indicates that HLA antibody probes coupled to FACS may be an effective approach for generating STR profiles of

  12. Leukocyte Margination in a Model Microvessel

    NASA Astrophysics Data System (ADS)

    Freund, Jonathan

    2006-11-01

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

  13. 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.

  14. The effect of cyanide on the uptake of gold by red blood cells.

    PubMed

    Graham, G G; Haavisto, T M; Jones, H M; Champion, G D

    1984-04-15

    Cyanide markedly increased the rate of uptake of gold by red blood cells when incubated with sodium aurothiomalate, a polymeric gold complex. Thiocyanate had no significant effect on gold uptake. The effect of cyanide was demonstrated to be due to the conversion of aurothiomalate to the complexion, aurocyanide, which is rapidly taken up by red blood cells. At a low ratio (1:20) of cyanide to aurothiomalate, cyanide appeared to act as a shuttle to carry gold into red blood cells. Tobacco smoking is known to increase the concentrations of gold in red blood cells in patients treated with aurothiomalate. The present data indicate that this effect of smoking is most likely due to cyanide inhaled in tobacco smoke and not to thiocyanate, a circulating metabolite of cyanide. An effect of cyanide on the uptake of polymeric gold complexes to target cells such as polymorphonuclear leukocytes and monocytes is suggested.

  15. Expression Profile of Cytokines and Enzymes mRNA in Blood Leukocytes of Dogs with Leptospirosis and Its Associated Pulmonary Hemorrhage Syndrome

    PubMed Central

    Maissen-Villiger, Carla A.; Schweighauser, Ariane; van Dorland, H. Anette; Morel, Claudine; Bruckmaier, Rupert M.; Zurbriggen, Andreas; Francey, Thierry

    2016-01-01

    Background Dogs with leptospirosis show similar organ manifestations and disease course as human patients, including acute kidney injury and pulmonary hemorrhage, making this naturally-occurring infection a good animal model for human leptospirosis. Expression patterns of cytokines and enzymes have been correlated with disease manifestations and clinical outcome in humans and animals. The aim of this study was to describe mRNA expression of pro- and anti-inflammatory mediators in canine leptospirosis and to compare it with other renal diseases to identify patterns characterizing the disease and especially its pulmonary form. Methodology and Principal Findings The mRNA abundance of cytokines (IL-1α, IL-1β, IL-8, IL-10, TNF-α, TGF-β) and enzymes (5-LO, iNOS) was measured prospectively in blood leukocytes from 34 dogs with severe leptospirosis and acute kidney injury, including 22 dogs with leptospirosis-associated pulmonary hemorrhages. Dogs with leptospirosis were compared to 14 dogs with acute kidney injury of other origin than leptospirosis, 8 dogs with chronic kidney disease, and 10 healthy control dogs. Canine leptospirosis was characterized by high 5-LO and low TNF-α expression compared to other causes of acute kidney injury, although the decreased TNF-α expression was also seen in chronic kidney disease. Leptospirosis-associated pulmonary hemorrhage was not characterized by a specific pattern, with only mild changes noted, including increased IL-10 and decreased 5-LO expression on some days in affected dogs. Fatal outcome from pulmonary hemorrhages was associated with low TNF-α, high IL-1β, and high iNOS expression, a pattern possibly expressed also in dogs with other forms of acute kidney injury. Conclusion The patterns of cytokine and enzyme expression observed in the present study indicate a complex pro- and anti-inflammatory response to the infection with leptospires. The recognition of these signatures may be of diagnostic and prognostic relevance

  16. Allogeneic transplantation of CD34(+) selected cells from peripheral blood from human leukocyte antigen-identical siblings: detrimental effect of a high number of donor CD34(+) cells?

    PubMed

    Urbano-Ispizua, A; Carreras, E; Marín, P; Rovira, M; Martínez, C; Fernández-Avilés, F; Xicoy, B; Hernández-Boluda, J C; Montserrat, E

    2001-10-15

    Clinical results after T-cell-depleted allografts might be improved by modifying the graft content of progenitor and accessory cells. Although the association of the number of donor T cells with the clinical outcome has been studied extensively, the optimum number of progenitor cells that should be administered to patients is unknown. The characteristics of 84 consecutive human leukocyte antigen (HLA)-identical sibling transplants of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells depleted of T cells by CD34(+) positive selection (allo-PBT/CD34(+)) were analyzed for their effect on clinical outcome. After a median follow-up of 24 months (range, 1-70 months), 50 patients remain alive (59.5%) and 34 have died (21 [25%] as a result of the transplant and 13 [15.5%] due to disease relapse). The median number of CD34(+) cells administered to the patients was 3.9 x 10(6)/kg (range, 1.2-14.3 x 10(6)/kg). A number of CD34(+) cells in the inoculum of 1 x 10(6)/kg to 3 x 10(6)/kg was associated with increased survival: 21 of 28 (75%) patients are alive, as compared with 29 of 56 (52%) patients receiving more than 3 x 10(6)/kg (actuarial probability 75% vs. 42%, respectively; P =.01). In the multivariate analysis, the independent prognostic variables for survival were CD34(+) cell dose 1 x 10(6)/kg to 3 x 10(6)/kg (RR = 4.8; P =.0008), sex-pairing match (RR = 3.2; P =.002), and early stage of disease (RR = 2.8; P =.007). From these results it appears that, in allo-PBT/CD34(+) from HLA-identical siblings, a number of CD34(+) cells in the inoculum between 1 x 10(6)/kg to 3 x 10(6)/kg is an important factor for better survival, and that higher CD34(+) cell doses might be associated with a poorer outcome.

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

    PubMed Central

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

    2013-01-01

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

  18. Influence of SkQ1 on Expression of Nrf2 Gene, ARE-Controlled Genes of Antioxidant Enzymes and Their Activity in Rat Blood Leukocytes under Oxidative Stress.

    PubMed

    Vnukov, V V; Gutsenko, O I; Milutina, N P; Kornienko, I V; Ananyan, A A; Danilenko, A O; Panina, S B; Plotnikov, A A; Makarenko, M S

    2015-12-01

    The study demonstrated that oxidative stress induced by hyperoxia (0.5 MPa for 90 min) resulted in reduction of mRNA levels of transcription factor Nrf2 and Nrf2-induced genes encoding antioxidant enzymes (SOD1, CAT, GPx4) in peripheral blood leukocytes of rats. The changes in gene expression profiles under hyperoxia were accompanied by disbalance of activity of antioxidant enzymes in the leukocytes, namely activation of superoxide dismutase and inhibition of catalase, glutathione peroxidase, and glutathione-S-transferase. Pretreatment of rats with SkQ1 (50 nmol/kg for five days) significantly increased mRNA levels of transcription factor Nrf2 and Nrf2-induced genes encoding antioxidant enzymes SOD2 and GPx4 and normalized the transcriptional activity of the SOD1 and CAT genes in the leukocytes in hyperoxia-induced oxidative stress. At the same time, the activity of catalase and glutathione peroxidase was increased, and the activity of superoxide dismutase and glutathione-S-transferase returned to the control level. It is hypothesized that protective effect of SkQ1 in hyperoxia-induced oxidative stress can be realized via a direct antioxidant property and the stimulation of the Keap1/Nrf2 redox-sensitive signaling system.

  19. Substitution of Aspartate for glycine 1018 in the Type III procollagen (COL3AI) gene causes type IV Ehlers-Danlos Syndrome: The mutated allele is present in most blood leukocytes of the asymptomatic and mosaic mother

    SciTech Connect

    Kontusaari, S.; Tromp, G.; Kuivaniemi, H.; Prockop, D.J. ); Stolle, C. ); Pope, F.M.

    1992-09-01

    A proband with arterial ruptures and skin changes characteristic of the type IV variant of Ehlers-Danlos syndrome was found to have a single-base mutation in the type III procollagen gene, which converted the codon for glycine at amino position 1018 to a codon for aspartate. (Amino acid positions are numbered by the standard convention in which the first glycine of the triple-helical domain of an [alpha] chain is number 1. The numbers of positions in the [alpha]1(III) chains can be converted to positions in the human pro[alpha](III) chain by adding 167.). Nucleotide sequencing of overlapping PCR products in which the two alleles were distinguished demonstrated that the mutation of glycine 1018 was the only mutation that changed the primary structure of type III procollagen. The glycine substitution markedly decreased the amount of type III procollagen secreted into the medium by cultured skin fibroblasts from the proband. It is surprising that the same mutation was found in about 94% of the peripheral blood leukocytes from the proband's asymptomatic 72-year-old mother. Other tissues from the mother contained the mutated allele; it was present in 0%-100% of different samples of hair cells and in about 40% of cells from the oral epithelium. Therefore, the mother was a mosaic for the mutation. Since the mutated allele was present in cells derived from all three germ layers, the results indicated that the mutation arose by the late blastocyst stage of development. The results also indicate that assays of blood leukocytes do not always reveal mosaicism or predict phenotypic involvement of tissues, such as blood vessels, that are derived from the same embryonic cells as are leukocytes. 66 refs., 6 figs., 1 tab.

  20. Chemotactic activity of Helicobacter pylori sonicate for human polymorphonuclear leucocytes and monocytes.

    PubMed Central

    Nielsen, H; Andersen, L P

    1992-01-01

    The immunopathology of Helicobacter pylori associated active chronic gastritis, which is characterised by predominance of polymorphonuclear leucocyte infiltration, is largely unknown. To evaluate the role of bacterial components as inflammatory mediators ultracentrifuged sonicated preparations were made of clinical isolates of Helicobacter pylori. The crude sonicates were shown to exhibit chemotactic activity for human polymorphonuclear leucocytes and blood monocytes in a concentration dependent fashion. The potency was comparable with previously described bacterial derived cytotaxins. The cytotaxin(s) was non-dialysable and completely destroyed by proteinase. Heat treatment did not decrease the chemotactic activity, but in sonicate subjected to 100 degrees C for 15 minutes all activity disappeared after dialysis suggesting the breakdown of a larger protein to small fragments that are still biological active. By ammonium sulphate precipitation at increasing concentrations the cytotaxin(s) was selectively found in 10% ammonium sulphate saturation, and by further molecular gel separation the chemotactic activity was found in the molecular size range from 25 to 35 kDa. The demonstration of a polymorphonuclear leucocyte and monocyte cytotaxin from Helicobacter pylori sonicate may help in understanding the mucosal immune response in gastric inflammatory diseases. PMID:1624151

  1. Regulation of polymorphonuclear cell activation by thrombopoietin.

    PubMed Central

    Brizzi, M F; Battaglia, E; Rosso, A; Strippoli, P; Montrucchio, G; Camussi, G; Pegoraro, L

    1997-01-01

    Thrombopoietin (TPO) regulates early and late stages of platelet formation as well as platelet activation. TPO exerts its effects by binding to the receptor, encoded by the protooncogene c-mpl, that is expressed in a large number of cells of hematopoietic origin. In this study, we evaluated the expression of c-Mpl and the effects of TPO on human polymorphonuclear cells (PMN). We demonstrate that PMN express the TPO receptor c-Mpl and that TPO induces STAT1 tyrosine phosphorylation and the formation of a serum inducible element complex containing STAT1. The analysis of biological effects of TPO on PMN demonstrated that TPO, at concentrations of 1-10 ng/ml, primes the response of PMN to n-formyl-met-leu-phe (FMLP) by inducing an early oxidative burst. TPO-induced priming on FMLP-stimulated PMN was also detected on the tyrosine phosphorylation of a protein with a molecular mass of approximately 28 kD. Moreover, we demonstrated that TPO by itself was able to stimulate, at doses ranging from 0.05 to 10 ng/ml, early release and delayed synthesis of interleukin 8 (IL-8). Thus, our data indicate that, in addition to sustaining megakaryocytopoiesis, TPO may have an important role in regulating PMN activation. PMID:9120001

  2. 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

  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. Replication and persistence of measles virus in defined subpopulations of human leukocytes.

    PubMed Central

    Joseph, B S; Lampert, P W; Oldstone, M B

    1975-01-01

    Replication of Edmonston strain measles virus was studied in several human lymphoblast lines, as well as in defined subpopulations of circulating human leukocytes. It was found that measles virus can productively infect T cells, B cells, and monocytes from human blood. These conclusions were derived from infectious center studies on segregated cell populations, as well as from ultrastructural analyses on cells labeled with specific markers. In contrast, mature polymorphonuclear cells failed to synthesize measles virus nucleocapsids even after infection at a relatively high multiplicity of infection. Measles virus replicated more efficiently in lymphocytes stimulated with mitogens than in unstimulated cells. However, both phytohemagglutinin and pokeweed mitogen had a negligible stimulatory effect on viral synthesis in purified populations of monocytes. In all instances the efficiency of measles virus replication by monocytes was appreciably less than that of mitogenically stimulated lymphocytes or of continuously culture lymphoblasts. Under standard conditions of infection, all of the surveyed lymphoblast lines produced equivalent amounts of measles virus regardless of the major histocompatibility (HL-A) haplotype. Hence, no evidence was found that the HL-A3,7 haplotype conferred either an advantage or disadvantage with respect to measles virus synthesis in an immunologically neutral environment. A persistent infection with measles virus could be established in both T and B lymphoblasts. The release of infectious virus from such persistently infected cells was stable over a period of several weeks and was approximately 100-fold less than peak viral titers obtained in each respective line after acute infection. Images PMID:1081602

  5. Micron-scale positioning of features influences the rate of polymorphonuclear leukocyte migration.

    PubMed Central

    Tan, J; Shen, H; Saltzman, W M

    2001-01-01

    Microfabrication technology was used to create regular arrays of micron-size holes (2 microm x 2 microm x 210 nm) on fused quartz and photosensitive polyimide surfaces. The patterned surfaces, which possessed a basic structural element of a three-dimensional (3-D) network (i.e., spatially separated mechanical edges), were used as a model system for studying the effect of substrate microgeometry on neutrophil migration. The edge-to-edge spacing between features was systematically varied from 6 microm to 14 microm with an increment of 2 microm. In addition, collagen was used to coat the patterned quartz surfaces in an attempt to change the adhesive properties of the surfaces. A radial flow detachment assay revealed that cell adhesion was the strongest on the quartz surface (approximately 50% cell attached), whereas it was relatively weaker on polyimide and collagen-coated quartz (approximately 25% cell attached). Cell adhesion to each substrate was not affected either by the presence of holes or by the spacing between holes. A direct visualization assay showed that neutrophil migration on each patterned surface could be characterized as a persistent random walk; the dependence of the random motility coefficient (mu) as a function of spacing was biphasic with the optimal spacing at approximately 10 microm on each substrate. The presence of evenly distributed holes at the optimal spacing of 10 microm enhanced mu by a factor of 2 on polyimide, a factor of 2.5 on collagen-coated quartz, and a factor of 10 on uncoated quartz. The biphasic dependence on the mechanical edges of neutrophil migration on 2-D patterned substrate was strikingly similar to that previously observed during neutrophil migration within 3-D networks, suggesting that microfabricated materials provide relevant models of 3-D structures with precisely defined physical characteristics. In addition, our results demonstrate that the microgeometry of a substrate, when considered separately from adhesion, can play a significant role in cell migration. PMID:11606271

  6. Fate of surface proteins of rabbit polymorphonuclear leukocytes during phagocytosis. I. Identification of surface proteins

    PubMed Central

    1979-01-01

    To study the fate of external membrane proteins during phagocytosis, rabbit peritoneal neutrophils were labeled by enzymatic iodination. Iodine was incorporated into at least 13 proteins ranging in size from approximately 250,000 to 18,000 daltons as judged from autoradiography of gels after SDS-polyacrylamide gel electrophoresis of labeled cells. The major contractile proteins of neutrophils, actin and myosin, were not labeled when intact cells were iodinated but were labeled when homogenates of these cells were iodinated. Nine of the iodinated proteins were released by mild protease treatment of intact cells. A plasma membrane-rich fraction was isolated by density centrifugation. This fraction was enriched at least 10-fold for lactoperoxidase-labeled acid-insoluble proteins. It was enriched to the same extent for the presence of iodinated wheat germ agglutinin that had been bound to intact cells at 4 degrees C before homogenization. Analysis of SDS- polyacrylamide gel electrophoresis revealed that the proteins of this fraction were predominantly of high molecular weight. However, only 8 of the 13 proteins iodinated on intact cells were found in this fraction. The remaining five were enriched in a dense fraction containing nuclei, intact cells, and membranous vesicles, and may represent a specialized segment of the neutrophil cell surface. PMID:479301

  7. Phospholipase C Activity in Human Polymorphonuclear Leukocytes: Partial Characterization and Effect of Indomethacin

    DTIC Science & Technology

    1988-12-01

    phospholipase C activity alone, and in the presence of 0.5 mM and I mM indomethacin, is plotted according to Lineweaver and Burke as described previously...The data were plotted according to the method of Lineweaver and Burke (26). The values represent the mean + S.E.M. of values derived from neutrophils of 4 subjects. 18

  8. 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

  9. Interaction of human leukocytes and Entamoeba histolytica. Killing of virulent amebae by the activated macrophage.

    PubMed Central

    Salata, R A; Pearson, R D; Ravdin, J I

    1985-01-01

    Capable effector mechanisms in the human immune response against the cytolytic, protozoan parasite Entamoeba histolytica have not been described. To identify a competent human effector cell, we studied the in vitro interactions of normal human polymorphonuclear neutrophils, peripheral blood mononuclear cells (PBMC), monocytes (MC), and MC-derived macrophages with virulent axenic amebae (strain HMI-IMSS). Amebae killed neutrophils, PBMC, MC, and MC-derived macrophages (P less than 0.001), without loss of parasite viability. The addition of heat-inactivated immune serum did not enable leukocytes to kill amebae, nor did it protect these host cells from amebae. MC-derived macrophages, activated with lymphokine elicited by the mitogens conconavalin A, phytohemagglutinin, or an amebic soluble protein preparation (strain HK9), killed 55% of amebae by 3 h in a trypan blue exclusion assay (P less than 0.001); during this time, 40% of the activated macrophages died. Lysis of amebae was confirmed using 111Indium oxine radiolabeled parasites and was antibody independent. Macrophage death appeared to be due to the deleterious effect of lysed amebae rather than the contact-dependent effector mechanisms of E. histolytica. Adherence between activated macrophages and amebae was greater than that between other leukocytes and amebae (P less than 0.001). Microscopic observations, kinetic analysis of the killing of amebae by activated macrophages, and suspension of amebae with adherent activated macrophages in a 10% dextran solution indicated that contact by activated macrophages was necessary to initiate the killing of amebae. Catalase but not superoxide dismutase inhibited the amebicidal capacity of activated macrophages (P less than 0.001). However, activated macrophages from an individual with chronic granulomatous disease were able to kill amebae, but not as effectively as normal cells (P less than 0.01). In summary, activated MC-derived macrophages killed virulent E. histolytica

  10. Protective effect of quercetin in gentamicin-induced oxidative stress in vitro and in vivo in blood cells. Effect on gentamicin antimicrobial activity.

    PubMed

    Bustos, Pamela Soledad; Deza-Ponzio, Romina; Páez, Paulina Laura; Albesa, Ines; Cabrera, José Luis; Virgolini, Miriam Beatriz; Ortega, María Gabriela

    2016-12-01

    We have evaluated the effect of gentamicin and gentamicin plus quercetin on ROS production, endogenous antioxidant defenses (SOD and CAT) and lipid peroxidation in vitro on human leukocytes and in vivo on whole rat blood. Gentamicin generated ROS production in human leukocytes, produced a dual effect on both enzymes dosage-dependent and generated an increase in lipid peroxidation. Quercetin, in leukocytes stimulated by gentamicin, showed more inhibitory capacity in ROS production than the reference inhibitor (vitaminC) in mononuclear cells and a similar protective behavior at this inhibitor in polymorphonuclear cells. Quercetin, in both cellular systems, tend to level SOD and CAT activities, reaching basal values and could prevent lipidic peroxidation induced by gentamicin. The results in Wistar rats confirmed that therapeutic doses of gentamicin can induce oxidative stress in whole blood and that the gentamicin treatment plus quercetin can suppress ROS generation, collaborate with SOD and CAT and diminish lipid peroxidation. Finally, flavonoid and antibiotic association was evaluated on the antimicrobial activity in S. aureus and E. coli, showing that changes were not generated in the antibacterial activity of gentamicin against E. coli strains, while for strains of S. aureus a beneficial effect observes. Therefore, we have demonstrated that gentamicin could induce oxidative stress in human leukocytes and in whole blood of Wistar rats at therapeutic doses and that quercetin may to produce a protective effect on this oxidative stress generated without substantially modifying the antibacterial activity of gentamicin against E. coli strains, and it contributes to this activity against S. aureus strains.

  11. 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.

  12. Human peripheral blood granulocytes and myeloid leukemic cell lines express both transcripts encoding for stem cell factor.

    PubMed

    Ramenghi, U; Ruggieri, L; Dianzani, I; Rosso, C; Brizzi, M F; Camaschella, C; Pietsch, T; Saglio, G

    1994-09-01

    Stem cell factor (SCF), the ligand for the c-kit proto-oncogene, has been shown to play a critical role in the migration of melanocytes and germ cells during embryogenesis as well as in the proliferative control of the hematopoietic compartment. In this study we investigated the expression of both the soluble and transmembrane SCF forms in purified peripheral blood populations and in several hematopoietic cell lines. Expression of both transcripts, though in different ratios, was identified in whole bone marrow, in bone marrow stromal cells and in human peripheral blood. In peripheral blood, SCF expression could be ascribable to polymorphonuclear leukocytes (PMN), whereas no SCF expression was detected in isolated lymphocytes, monocytes and in some T lymphoid cell lines. Conversely, some hematopoietic myeloid cell lines, such as HL-60, KG1 and K562, express SCF with similar patterns.

  13. Human Leukocytes Kill Brugia malayi Microfilariae Independently of DNA-Based Extracellular Trap Release

    PubMed Central

    McCoy, Ciaran J.; Reaves, Barbara J.; Giguère, Steeve; Coates, Ruby; Rada, Balázs

    2017-01-01

    Background Wuchereria bancrofti, Brugia malayi and Brugia timori infect over 100 million people worldwide and are the causative agents of lymphatic filariasis. Some parasite carriers are amicrofilaremic whilst others facilitate mosquito-based disease transmission through blood-circulating microfilariae (Mf). Recent findings, obtained largely from animal model systems, suggest that polymorphonuclear leukocytes (PMNs) contribute to parasitic nematode-directed type 2 immune responses. When exposed to certain pathogens PMNs release extracellular traps (NETs) in the form of chromatin loaded with various antimicrobial molecules and proteases. Principal findings In vitro, PMNs expel large amounts of NETs that capture but do not kill B. malayi Mf. NET morphology was confirmed by fluorescence imaging of worm-NET aggregates labelled with DAPI and antibodies to human neutrophil elastase, myeloperoxidase and citrullinated histone H4. A fluorescent, extracellular DNA release assay was used to quantify and observe Mf induced NETosis over time. Blinded video analyses of PMN-to-worm attachment and worm survival during Mf-leukocyte co-culture demonstrated that DNase treatment eliminates PMN attachment in the absence of serum, autologous serum bolsters both PMN attachment and PMN plus peripheral blood mononuclear cell (PBMC) mediated Mf killing, and serum heat inactivation inhibits both PMN attachment and Mf killing. Despite the effects of heat inactivation, the complement inhibitor compstatin did not impede Mf killing and had little effect on PMN attachment. Both human PMNs and monocytes, but not lymphocytes, are able to kill B. malayi Mf in vitro and NETosis does not significantly contribute to this killing. Leukocytes derived from presumably parasite-naïve U.S. resident donors vary in their ability to kill Mf in vitro, which may reflect the pathological heterogeneity associated with filarial parasitic infections. Conclusions/Significance Human innate immune cells are able to

  14. White Blood Cell Count

    MedlinePlus

    ... limited. Home Visit Global Sites Search Help? White Blood Cell Count Share this page: Was this page helpful? ... Count; Leukocyte Count; White Count Formal name: White Blood Cell Count Related tests: Complete Blood Count , Blood Smear , ...

  15. 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.

  16. 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.

  17. 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.

  18. [Oxidative stress in blood leukocytes, pro/antioxidant status and fatty acids composition of pancreas lipids at experimental acute pancreatitis in rats].

    PubMed

    Pryvrots'ka, I B; Kuchmerovs'ka, T M

    2013-01-01

    In an experimental model of acute pancreatitis (AP) in rats no alteration in leukocyte's viability was found by flow cytometry as compared to control. After 1 day of AP production of reactive oxygen forms in granulocytes was increased more than 5 times, but after 3 days their level was decreased. Alterations of pro/antioxidant status and specific changes in the fatty acid composition in the pancreas were established. With the development of AP, the processes of lipids peroxidation were intensified while antioxidant system was altered, that was evidenced by inflammation in the pancreas. In these conditions, the increase of phospholipase A2 activity was accompanied by significant changes of fatty acid composition of the total lipids in the pancreas. This increased relative total content of saturated fatty acids, in particular myristic, palmitic and stearic acid increased, while the total content of polyunsaturated essential fatty acids omega-3 (linolenic, eicosapentaenoic, dokozapentayenoic, docosahexaenoic) decreased. The preparation containing omega-3 polyunsaturated fatty acids partially normalized the lipid and fatty acids composition as well as prooxidant-antioxidant system.

  19. Coupled Flow-Structure-Biochemistry Simulations of Dynamic Systems of Blood Cells Using an Adaptive Surface Tracking Method.

    PubMed

    Hoskins, M H; Kunz, R F; Bistline, J E; Dong, C

    2009-07-01

    A method for the computation of low Reynolds number dynamic blood cell systems is presented. The specific system of interest here is interaction between cancer cells and white blood cells in an experimental flow system. Fluid dynamics, structural mechanics, six-degree-of freedom motion control and surface biochemistry analysis components are coupled in the context of adaptive octree-based grid generation. Analytical and numerical verification of the quasi-steady assumption for the fluid mechanics is presented. The capabilities of the technique are demonstrated by presenting several three-dimensional cell system simulations, including the collision/interaction between a cancer cell and an endothelium adherent polymorphonuclear leukocyte (PMN) cell in a shear flow.

  20. Coupled flow-structure-biochemistry simulations of dynamic systems of blood cells using an adaptive surface tracking method

    NASA Astrophysics Data System (ADS)

    Hoskins, M. H.; Kunz, R. F.; Bistline, J. E.; Dong, C.

    2009-07-01

    A method for the computation of low-Reynolds number dynamic blood cell systems is presented. The specific system of interest here is interaction between cancer cells and white blood cells in an experimental flow system. Fluid dynamics, structural mechanics, six-degree-of-freedom motion control, and surface biochemistry analysis components are coupled in the context of adaptive octree-based grid generation. Analytical and numerical verification of the quasi-steady assumption for the fluid mechanics is presented. The capabilities of the technique are demonstrated by presenting several three-dimensional cell system simulations, including the collision/interaction between a cancer cell and an endothelium adherent polymorphonuclear leukocyte (PMN) cell in a shear flow.

  1. The effect of depth of centrifuged synovial fluid on leukocyte esterase test for periprosthetic joint infection.

    PubMed

    Ruangsomboon, Pakpoom; Chinprasertsuk, Sriprapa; Khejonnit, Varanya; Chareancholvanich, Keerati

    2017-03-17

    Centrifugation of aspirated synovial fluid before leukocytes esterase (LE) testing for diagnosing periprosthetic joint infection (PJI) may make blood tinged specimens interpretable. We aimed to establish the proper sampling depth of centrifuged specimens for LE testing as one diagnostic criterion and also AS-D chloroacetate esterase (CAE) staining testing as an adjunctive tool. A definite PJI knee joint group and an aseptic primary total knee arthroplasty control group were studied quasi-experimentally (N = 46). At 2000 g for 15 minutes, 3 ml of synovial fluid was centrifuged. LE strip testing and median synovial WBC count were performed at 2, 4, and 6 mm depths. CAE staining test characterized LE particles. ROC curve, area under the curve, and significant differences were determined. The proper predictive depth to diagnose PJI was sought by forward stepwise logistic regression. All fresh blood-tinged specimens had uncertain interpretations. Centrifugation increased interpretability (55% to 100%). ROC curve and area under the curve at 2, 4, and 6 mm depths were 0.822, 0.804, and 0.786, respectively. The cut point of ++ to diagnose PJI was statistically significant (p < 0.05) at all depths. P-values of forward stepwise logistic regression at 2, 4, and 6 mm were 0.001, 0.752, and 0.756, respectively. CAE staining confirmed extracellular LE release by polymorphonuclear neutrophils (PMN). A specimen at < 2 mm from the surface of centrifuged synovial fluid at a grading of ++ or more for PJI diagnosis is proper for LE testing. CAE staining testing adjunctively characterizes LE particles and cell morphology. This article is protected by copyright. All rights reserved.

  2. 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.

  3. 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

  4. Relationship between somatic cell count, polymorphonuclear leucocyte count and quality parameters in bovine bulk tank milk.

    PubMed

    Wickström, Erik; Persson-Waller, Karin; Lindmark-Månsson, Helena; Ostensson, Karin; Sternesjö, Ase

    2009-05-01

    The somatic cell count (SCC) in bovine bulk tank milk is presently used as an indicator of raw milk quality, reflecting the udder health status of the herd. During mastitis, SCC increases, mostly owing to an influx of polymorphonuclear leucocytes (PMN) from blood into milk, with a concomitant change in milk composition. Bulk tank milk samples were categorized according to their SCC, as well as polymorphonuclear leucocyte count (PMNC), to study relationships between SCC, PMNC and various raw milk quality traits, i.e. contents of total protein, whey protein, casein, fat and lactose, casein number, proteolysis and rheological properties. The proportion of PMN, obtained by direct microscopy, was significantly higher in samples with high SCC compared with low SCC samples. SCC and PMNC were strongly correlated, yielding a correlation coefficient of 0.85. High SCC samples had lower lactose and casein contents, lower casein number and more proteolysis than low SCC samples. Samples with high PMNC had a lower casein number than low PMNC samples. Samples with high and low SCC or PMNC did not differ in respect to rheological properties. Our results do not indicate that PMNC is a better biomarker than SCC for raw bulk tank milk quality, as previously proposed.

  5. 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.

  6. Hypomethylation of the IL17RC Promoter in Peripheral Blood Leukocytes is Not A Hallmark of Age-Related Macular Degeneration

    PubMed Central

    Oliver, Verity F; Franchina, Maria; Jaffe, Andrew E; Branham, Kari E; Othman, Mohammad; Heckenlively, John R; Swaroop, Anand; Campochiaro, Betsy; Vote, Brendan J; Craig, Jamie E; Saffery, Richard; Mackey, David A; Qian, Jiang; Zack, Donald J; Hewitt, Alex W; Merbs, Shannath L

    2014-01-01

    SUMMARY Age-related macular degeneration (AMD) is a leading cause of visual impairment worldwide. Aberrant DNA methylation within the promoter of IL17RC in peripheral blood mononuclear cells has recently been reported in AMD. To validate this association, we examined DNA methylation of the IL17RC promoter in peripheral blood. First, we used Illumina Human Methylation450 Bead Arrays, a widely-accepted platform for measuring global DNA methylation. Second, methylation status at multiple sites within the IL17RC promoter was determined by bisulfite pyrosequencing in two cohorts. Third, a methylation-sensitive QPCR-based assay was performed on a subset of samples. In contrast to previous findings, we did not find evidence of differential methylation between AMD cases and age-matched controls. We conclude that hypomethylation within the IL17RC gene promoter in peripheral blood is not suitable for use as a clinical biomarker of AMD. This study highlights the need for considerable replication of epigenetic association studies prior to clinical application. PMID:24373284

  7. 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.

  8. Expression of IL-1β, IL-2, IL-10, TNF-β and GM-CSF in peripheral blood leukocytes of rabbits experimentally infected with rabbit haemorrhagic disease virus.

    PubMed

    Trzeciak-Ryczek, Alicja; Tokarz-Deptuła, Beata; Deptuła, Wiesław

    2016-04-15

    Rabbit haemorrhagic disease (RHD) is a highly morbid and mortal viral infection of European rabbits. This disease is one of the main causes of death in wild rabbits, and results in large economic losses in farms of rabbits worldwide. Although the first outbreak of this disease was noted in 1984, the pathogenesis of RHD and mechanisms of RHDV (rabbit haemorrhagic disease virus) pathogenecity have still not been fully elucidated. Recent studies indicate a role of the immune response, especially peripheral blood leukocytes (PBL), in the pathogenesis of this disease. Thus, in the present study we investigated the expression of IL-1β, IL-2, IL-10, TNF-β and GM-CSF genes in PBL of RHDV-infected rabbits. We also compared the expression of genes encoding these cytokines in rabbits with different course of RHDV infection (in animals that died 36h postinfection or survived until 60th h after infection). The study revealed that three (IL-10, TNF-β and GM-CSF) out of five investigated genes encoding cytokines showed increased expression in PBL of RHDV-infected rabbits, and the level of expression depended on the course of RHD. The results indicate the potential role of these cytokines in RHDV infection and their influence on the survival time of infected rabbits.

  9. Renal cell carcinoma alters endothelial receptor expression responsible for leukocyte adhesion.

    PubMed

    Juengel, Eva; Krueger, Geraldine; Rutz, Jochen; Nelson, Karen; Werner, Isabella; Relja, Borna; Seliger, Barbara; Fisslthaler, Beate; Fleming, Ingrid; Tsaur, Igor; Haferkamp, Axel; Blaheta, Roman A

    2016-04-12

    Renal cell carcinoma (RCC) escapes immune recognition. To elaborate the escape strategy the influence of RCC cells on endothelial receptor expression and endothelial leukocyte adhesion was evaluated. Human umbilical vein endothelial cells (HUVEC) were co-cultured with the RCC cell line, Caki-1, with and without tumor necrosis factor (TNF)-alpha. Intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), endothelial (E)-selectin, standard and variants (V) of CD44 were then analysed in HUVEC, using flow cytometry and Western blot analysis. To determine which components are responsible for HUVEC-Caki-1 interaction causing receptor alteration, Caki-1 membrane fragments versus cell culture supernatant were applied to HUVECS. Adhesion of peripheral blood lymphocytes (PBL) and polymorphonuclear neutrophils (PMN) to endothelium was evaluated by co-culture adhesion assays. Relevance of endothelial receptor expression for adhesion to endothelium was determined by receptor blockage. Co-culture of RCC and HUVECs resulted in a significant increase in endothelial ICAM-1, VCAM-1, E-selectin, CD44 V3 and V7 expression. Previous stimulation of HUVECs with TNF-alpha and co-cultivation with Caki-1 resulted in further elevation of endothelial CD44 V3 and V7 expression, whereas ICAM-1, VCAM-1 and E-selectin expression were significantly diminished. Since Caki-1 membrane fragments also caused these alterations, but cell culture supernatant did not, cell-cell contact may be responsible for this process. Blocking ICAM-1, VCAM-1, E-selectin or CD44 with respective antibodies led to a significant decrease in PBL and PMN adhesion to endothelium. Thus, exposing HUVEC to Caki-1 results in significant alteration of endothelial receptor expression and subsequent endothelial attachment of PBL and PMN.

  10. 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.

  11. [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.

  12. 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.

  13. Temporarily decreasing progesterone after timed artificial insemination decreased expression of interferon-tau stimulated gene 15 (ISG15) in blood leukocytes, serum pregnancy-specific protein B concentrations, and embryo size in lactating Holstein cows.

    PubMed

    Carvalho, P D; Consentini, C C; Weaver, S R; Barleta, R V; Hernandez, L L; Fricke, P M

    2017-04-01

    Our objective was to evaluate the effects of temporarily decreasing progesterone (P4) after timed artificial insemination (TAI) on embryonic growth in dairy cows. Lactating Holstein cows (n = 80) were submitted to a Double-Ovsynch protocol for first TAI and were assigned randomly to receive 12.5 mg of PGF2α 5 d after the last GnRH treatment (LowP4) or remain untreated (control). Blood samples were collected thrice weekly from 5 to 29 d after TAI for all cows and from 32 to 67 d for pregnant cows, and were analyzed for P4 and pregnancy-specific protein B concentrations. Expression of interferon-tau stimulated gene 15 (ISG15) was assessed in blood leukocyte mRNA 18 and 20 d after TAI. Pregnancy diagnosis was performed weekly using ultrasound from 32 to 67 d after TAI, and embryonic crown-rump length was measured 32, 39, and 46 d after TAI. Data were analyzed by ANOVA and logistic regression using the MIXED and GLIMMIX procedures of SAS. The LowP4 cows had less P4 than control cows from 6 to 11 d after TAI; however, pregnancy outcomes 32 d after TAI and pregnancy loss from 32 to 67 d after TAI did not differ between treatments. Control cows diagnosed pregnant 32 d after TAI had greater expression of ISG15 20 d after TAI than LowP4 cows diagnosed pregnant 32 d after TAI, and pregnant control cows had greater pregnancy-specific protein B concentrations from 25 to 67 d after TAI than pregnant LowP4 cows. Embryo size did not differ between treatments 32 and 39 d after TAI, but control cows had larger embryos 46 d after TAI. In conclusion, temporarily decreasing P4 after TAI decreased embryonic growth during early pregnancy in lactating Holstein cows but did not affect pregnancies per artificial insemination or pregnancy loss.

  14. The Fruiting Bodies, Submerged Culture Biomass, and Acidic Polysaccharide Glucuronoxylomannan of Yellow Brain Mushroom Tremella mesenterica Modulate the Immunity of Peripheral Blood Leukocytes and Splenocytes in Rats with Impaired Glucose Tolerance.

    PubMed

    Hsu, Tai-Hao; Lee, Chien-Hsing; Lin, Fang-Yi; Wasser, Solomon P; Lo, Hui-Chen

    2014-01-01

    The prevalence of diabetes mellitus (DM), a chronic disease with hyperglycemia and impaired immune function, is increasing worldwide. Progression from impaired glucose tolerance (IGT) to type 2 DM has recently become a target for early intervention. The fruiting bodies (FB) and submerged culture mycelium (CM) of Tremella mesenterica, an edible and medicinal mushroom, have been demonstrated to have antihyperglycemic and immunomodulatory activities in type 1 DM rats. Herein, we investigated the effects of acidic polysaccharide glucuronoxylomannan (GX) extracted from CM on the immunocyte responses. Male Wistar rats were injected with streptozotocin (65 mg/kg) plus nicotinamide (200 mg/kg) for the induction of IGT, and gavaged daily with vehicle, FB, CM, or GX (1 g/kg/day). Rats injected with saline and gavaged vehicle were used as controls. Two weeks later, peripheral blood leukocytes (PBLs) and splenocytes were collected. Ingestion of FB, CM, and GX significantly decreased blood glucose levels in the postprandial period and in oral glucose tolerance test, and partially reversed T-splenocytic proliferation in IGT rats. CM significantly decreased T-helper lymphocytes in the PBLs and B-splenocytes. In addition, FB, CM, and GX significantly reversed the IGT-induced decreases in tumor necrosis factor-α production; GX significantly increased interleukin-6 production in T-lymphocytes in the PBLs and splenocytes; and CM and GX significantly reversed IGT-induced decrease in interferon-γ production in T-lymphocytes in the spleen. In conclusion, FB, CM, and acidic polysaccharide GX of T. mesenterica may increase T-cell immunity via the elevation of proinflammatory and T-helper cytokine production in rats with impaired glucose tolerance.

  15. The Fruiting Bodies, Submerged Culture Biomass, and Acidic Polysaccharide Glucuronoxylomannan of Yellow Brain Mushroom Tremella mesenterica Modulate the Immunity of Peripheral Blood Leukocytes and Splenocytes in Rats with Impaired Glucose Tolerance

    PubMed Central

    Hsu, Tai-Hao; Lee, Chien-Hsing; Lin, Fang-Yi; Wasser, Solomon P.; Lo, Hui-Chen

    2014-01-01

    The prevalence of diabetes mellitus (DM), a chronic disease with hyperglycemia and impaired immune function, is increasing worldwide. Progression from impaired glucose tolerance (IGT) to type 2 DM has recently become a target for early intervention. The fruiting bodies (FB) and submerged culture mycelium (CM) of Tremella mesenterica, an edible and medicinal mushroom, have been demonstrated to have antihyperglycemic and immunomodulatory activities in type 1 DM rats. Herein, we investigated the effects of acidic polysaccharide glucuronoxylomannan (GX) extracted from CM on the immunocyte responses. Male Wistar rats were injected with streptozotocin (65 mg/kg) plus nicotinamide (200 mg/kg) for the induction of IGT, and gavaged daily with vehicle, FB, CM, or GX (1 g/kg/day). Rats injected with saline and gavaged vehicle were used as controls. Two weeks later, peripheral blood leukocytes (PBLs) and splenocytes were collected. Ingestion of FB, CM, and GX significantly decreased blood glucose levels in the postprandial period and in oral glucose tolerance test, and partially reversed T-splenocytic proliferation in IGT rats. CM significantly decreased T-helper lymphocytes in the PBLs and B-splenocytes. In addition, FB, CM, and GX significantly reversed the IGT-induced decreases in tumor necrosis factor-α production; GX significantly increased interleukin-6 production in T-lymphocytes in the PBLs and splenocytes; and CM and GX significantly reversed IGT-induced decrease in interferon-γ production in T-lymphocytes in the spleen. In conclusion, FB, CM, and acidic polysaccharide GX of T. mesenterica may increase T-cell immunity via the elevation of proinflammatory and T-helper cytokine production in rats with impaired glucose tolerance. PMID:24872934

  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. 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

  18. Hydrogen peroxide signals E. coli phagocytosis by human polymorphonuclear cells; up-stream and down-stream pathway.

    PubMed

    Petropoulos, Michalis; Karamolegkou, Georgia; Rosmaraki, Eleftheria; Tsakas, Sotiris

    2015-12-01

    Hydrogen peroxide (Η2Ο2) is produced during a variety of cellular procedures. In this paper, the regulatory role of Η2Ο2, in Escherichia coli phagocytosis by the human polymorphonuclears, was investigated. White blood cells were incubated with dihydrorhodamine (DHR) in order to study H2O2 synthesis and E. coli-FITC to study phagocytosis. Flow cytometry revealed increased synthesis of H2O2 in polymorphonuclears which incorporated E. coli-FITC. The blocking of H2O2 synthesis by specific inhibitors, N-ethylmaleimide (ΝΕΜ) for NADPH oxidase and diethyldithiocarbamate (DDC) for superoxide dismutase (SOD), decreased E. coli phagocytosis, as well. Immunoblot analysis of white blood cell protein extracts revealed that the blocking of NADPH oxidase and SOD decreased ERK-1/2 phosphorylation, while it had no effect on JNK and p38. Confocal microscopy showed that phosphorylation of MAPKs and phagocytosis solely occur in the polymorphonuclear and not in mononuclear cells. The use of specific MAPKs inhibitors showed that all of them are necessary for phagocytosis, but only phospho-p38 affects H2O2 synthesis. The blocking of JNK phosphorylation, in the presence of E. coli, evoked a further decrease of cytoplasmic p47 thus increasing its translocation onto the plasma membrane for the assembly of NADPH oxidase. It appears that newly synthesised H2O2 invigorates the phosphorylation and action of ERK-1/2 in E. coli phagocytosis, while phospho-JNK and phospho-p38 appear to regulate H2O2 production.

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

    PubMed Central

    Goswami, Debashree; Vestweber, Dietmar

    2016-01-01

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

  20. Effect of an extract based on the medicinal mushroom Agaricus blazei murill on release of cytokines, chemokines and leukocyte growth factors in human blood ex vivo and in vivo.

    PubMed

    Johnson, E; Førland, D T; Saetre, L; Bernardshaw, S V; Lyberg, T; Hetland, G

    2009-03-01

    An immunostimulatory extract based on the medicinal mushroom Agaricus blazei Murill (AbM) has been shown to stimulate mononuclear phagocytes in vitro to produce pro-inflammatory cytokines, and to protect against lethal peritonitis in mice. The present aim was to study the effect of AbM on release of several cytokines in human whole blood both after stimulation ex vivo and in vivo after oral intake over several days in healthy volunteers. The 17 signal substances examined were; T helper 1 (Th1) cytokines [interleukin (IL)-2, interferon (IFN)-gamma and IL-12], T helper 2 cytokines (IL-4, IL-5 and IL-13), pleiotropic (IL-7, IL-17), pro-inflammatory [IL-1beta, IL-6, tumour necrosis factor (TNF)-alpha (mainly produced by Th1 cells)]--and anti-inflammatory (IL-10) cytokines, chemokines [IL-8, macrophage inhibitory protein (MIP)-1beta and monocyte chemoattractant protein (MCP)-1] and leukocyte growth factors [granulocyte colony-stimulating factor (G-CSF), granulocyte/macrophage colony stimulating factor]. After stimulation of whole blood ex vivo with 0.5-5.0% of a mushroom extract, AndoSan mainly containing AbM, there was a dose-dependent increase in all the cytokines studied, ranging from two to 399-fold (TNF-alpha). However, in vivo in the eight volunteers who completed the daily intake (60 ml) of this AbM extract for 12 days, a significant reduction was observed in levels of IL-1beta (97%), TNF-alpha (84%), IL-17 (50%) and IL-2 (46%). Although not significant, there was a trend towards reduced levels for IL-8, IFN-gamma and G-CSF, whilst those of the remaining nine cytokines tested, were unaltered. The discrepant results on cytokine release ex vivo and in vivo may partly be explained by the antioxidant activity of AbM in vivo and limited absorption of its large, complex and bioactive beta-glucans across the intestinal mucosa to the reticuloendothelial system and blood.

  1. Polymorphonuclear leucocyte motility in men with ankylosing spondylitis.

    PubMed Central

    Pease, C T; Fennell, M; Brewerton, D A

    1989-01-01

    The polymorphonuclear leucocyte (PMN) response to a chemotactic or chemokinetic stimulus is enhanced in men with ankylosing spondylitis (AS). This effect does not parallel the severity of disease activity or the size of the acute phase response, and it is independent of non-steroidal anti-inflammatory drug treatment. Polymorph function is normal in HLA-B27 positive brothers of probands with AS and in other HLA-B27 positive individuals in the absence of disease. Polymorph motility is also normal in patients with psoriasis vulgaris or Crohn's disease, indicating that enhanced PMN motility is not a non-specific consequence of all inflammatory disorders. PMID:2784306

  2. Effects of Himatanthus lancifolius on human leukocyte chemotaxis and their adhesion to integrins.

    PubMed

    Nardin, Jeanine Marie; de Souza, Wesley Maurício; Lopes, Juliano Ferreira; Florão, Angela; de Moraes Santos, Cid Aimbiré; Weffort-Santos, Almeriane Maria

    2008-08-01

    The aim of this work was to investigate the anti-inflammatory activities of the uleine-rich fraction extracted from the barks of Himatanthus lancifolius (Muell. Arg.) Woodson (Apocynaceae). To achieve this, we focused on its in vitro effects on some steps of the inflammatory response using peripheral human leukocytes. The results presented herein show that the uleine-rich fraction significantly inhibits the migration of casein-induced granulocytes and their adhesion to fibronectin and vitronectin, along with mononuclear cells, by down-regulating the expression of alpha 4beta1 and alpha5beta1 integrins. The data suggest that H. LANCIFOLIUS has the potential of interferring with leukocyte trafficking through its uleine-rich fraction, emphasizing its usefulness in inflammatory conditions. DEXA:dexamethasone disodium phosphate FN:fibronectin PMN:polymorphonuclear URF:uleine-rich fraction VN:vitronectin.

  3. Expression of selected proteins of the extrinsic and intrinsic pathways of apoptosis in human leukocytes exposed to N-nitrosodimethylamine.

    PubMed

    Iwaniuk, A; Jabłońska, E; Jabłoński, J; Ratajczak-Wrona, W; Garley, M

    2015-06-01

    N-nitrosodimethylamine (NDMA) is a xenobiotic widespread in human environment capable of regulating the lifespan of immune cells. In this study, we examined the roles of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/death receptor 5 (DR5) complex and the Fas molecule in the induction of the extrinsic apoptosis pathway in human neutrophils (polymorphonuclear neutrophils (PMNs)) and peripheral blood mononuclear cells (PBMCs) exposed to NDMA. Also we assessed these proteins ability to trigger the intrinsic apoptosis pathway in those cells. For this purpose, we examined the expression of Fas-associated protein with death domain, truncated Bid (tBid) proteins, and apoptogenic factors such as apoptosis-inducing factor, Smac/Diablo, Omi/HtrA2, and caspase-3 as an indication of accomplished apoptosis phenomenon. PMNs and PBMCs were isolated from whole blood by density gradient centrifugation using Polymorphrep. Apoptotic cells were assessed with flow cytometry using a ready-made kit. The expression of proapoptotic molecules was investigated by Western blot analysis of PMNs and PBMCs treated with NDMA and/or rhTRAIL. The obtained results confirm the proapoptotic effects of NDMA on the examined human leukocytes and indicate an active participation of the TRAIL/DR5 complex and Fas protein in the process of apoptosis. Moreover, the research revealed distinct mechanisms of intrinsic apoptosis pathway activation between PMNs and PBMCs exposed to NDMA, as confirmed by the different levels of tBid, Smac/Diablo, Omi/HtrA2, and caspase-3 expression in those cells.

  4. 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

  5. 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.

  6. Chorioretinitis with a combined defect in T and B lymphocytes and granulocytes. A new syndrome successfully treated with dialyzable leukocyte extracts (transfer factor).

    PubMed

    Kyong, C U; Wilson, G B; Fudenberg, H H; Goust, J M; Richardson, P; Echerd, J

    1980-06-01

    A patient with immune deficiency, recurrent pyogenic infections and active chorioretinitis is described; in addition to agammaglobulinemia, both quantitative and qualitative T-cell deficiencies were documented. Furthermore, the patient's granulocytes (polymorphonuclear leukocytes), although normal in their bactericidal capacity for Staphylococcus, responded poorly to both leukocyte migration inhibition factor and neutrophil immobilizing factor obtained from normal cells. The immunologic features of this patient appear to comprise a new syndrome. Remarkable diminution of the ocular lesions and increased visual acuity occurred within two months after the initiation of therapy with dialyzable leukocyte extracts (transfer factor). Concurrent testing of the patient's cell-mediated immunity showed increased numbers of circulating T lymphocytes and improved T-cell function following dialyzable leukocyte extract [DLE] therapy. The dramatic clinical results indicate that similar therapy may prove to be beneficial in other patients with chorioretinitis and T-cell deficiency.

  7. The anti-inflammatory pharmacology of Pycnogenol in humans involves COX-2 and 5-LOX mRNA expression in leukocytes.

    PubMed

    Canali, Raffaella; Comitato, Raffaella; Schonlau, Frank; Virgili, Fabio

    2009-09-01

    We investigated the effects of Pycnogenol supplementation on the arachidonic acid pathway in human polymorphonuclear leukocytes (PMNL) in response to an inflammatory stimulus. Pycnogenol is a standardised extract of French maritime pine bark consisting of procyanidins and polyphenolic monomers. Healthy volunteers aged 35 to 50 years were supplemented with 150 mg Pycnogenol a day for five days. Before and after the final day of supplementation, blood was drawn and PMNL were isolated. PMNL were primed with lipopolysaccharide (LPS) and stimulated with the receptor-mediated agonist formyl-methionyl-leucyl-phenylalanine (fMLP) to activate the arachidonic acid pathway and the biosynthesis of leukotrienes, thromboxane and prostaglandins. Pycnogenol supplementation inhibited 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) gene expression and phospholipase A2 (PLA2) activity. This effect was associated with a compensatory up-regulation of COX-1 gene expression. Interestingly, Pycnogenol suspended the interdependency between 5-LOX and 5-lipoxygenase activating protein (FLAP) expression. Pycnogenol supplementation reduced leukotriene production but did not leave prostaglandins unaltered, which we attribute to a decline of COX-2 activity in favour of COX-1. Here we show for the first time that Pycnogenol supplementation simultaneously inhibits COX-2 and 5-LOX gene expression and reduces leukotriene biosynthesis in human PMNL upon pro-inflammatory stimulation ex vivo.

  8. 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.

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

    PubMed

    Kreuger, Johan; Phillipson, Mia

    2016-02-01

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

  10. Blood

    MedlinePlus

    ... The liquid part, called plasma, is made of water, salts, and protein. Over half of your blood is plasma. The solid part of your blood contains red blood cells, white blood cells, and platelets. Red ...

  11. Radiographic evaluation of destructive periodontal disease in blue mink in relation to age and blood morphology

    PubMed Central

    2005-01-01

    Abstract In this study, blood samples and jaws were collected from 2 genotypes of blue mink (n = 289) in order to examine phenotypic expression of specific characteristics of Chediak-Higashi Syndrome (C-HS). Blood samples were subjected to differential counts to assess the proportion of abnormal polymorphonuclear leukocytes characteristic for CH-S (C-HS-leukocytes). Abnormal leukocytes with characteristic signs of C-HS were found in blood smears from all mink included in this study. Four teeth in one half of the mandible (P3, P4, M1, M2) were subjected to quantitative radiographic evaluation of alveolar bone loss and tooth loss. There was a high prevalence of destructive periodontal disease among blue mink included in this study. Mild to moderate periodontal disease (defined by less than 50% alveolar bone loss related to 1 or more teeth) affected 73.7% of young mink (age = 7 mo) and 67.9% of older animals (age ≥ 19 mo). Severe periodontal disease (defined by more than 50% bone loss related to one or more teeth) was not detected in mink aged 7 mo, but affected 15.3% of mink aged 19 mo and 39.6% of mink aged 31 mo. The positive relationship between age and periodontal disease was statistically significant (P < 0.01). The prevalence of tooth loss was found to be high among blue mink aged >19 mo (21.6%) and was also significantly related to age (P < 0.01). A significant positive interaction between alveolar bone loss and tooth loss (P < 0.01), implies that the highly prevalent tooth loss in the mink was related to and possibly caused by destructive periodontal disease. There was no significant difference in the prevalence of periodontal disease between the 2 genotypes and age was found to be the only statistical predictor of poor production results (P < 0.01) in blue mink. PMID:15971677

  12. 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

  13. 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.

  14. Glucose supplementation has minimal effects on blood neutrophil functionand gene expression in vitro

    Technology Transfer Automated Retrieval System (TEKTRAN)

    During early lactation, glucose availability is low and the effect of glucose supply on bovine polymorphonuclear leukocyte (PMNL) function is poorly understood. The objective of this study was to determine the effect of glucose supplementation on the function and transcriptomic inflammatory respons...

  15. The effect of citrus-derived oil on bovine blood neutrophil response in vitro

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Research on the use of natural products to treat or prevent microbial invasion as alternatives to antibiotic use is growing.Polymorphonuclear leukocytes (PMNL) play a vital role with regard to the innate immune response that affects severity and or duration of mastitis. To our knowledge, effect of c...

  16. Polymorphonuclear Neutrophils Are Necessary for the Recruitment of CD8+ T Cells in the Liver in a Pregnant Mouse Model of Chlamydophila abortus (Chlamydia psittaci Serotype 1) Infection

    PubMed Central

    de Oca, Roberto Montes; Buendía, Antonio J.; Del Río, Laura; Sánchez, Joaquín; Salinas, Jesús; Navarro, Jose A.

    2000-01-01

    The role of polymorphonuclear neutrophils (PMNs) in the development of the specific immune response against Chlamydophila abortus (Chlamydia psittaci serotype 1) infection was studied in a pregnant mouse model involving treatment with RB6-8C5 monoclonal antibody. PMN depletion significantly affected the immune response in the liver, in which the T-lymphocyte and F4/80+ cell populations decreased, particularly the CD8+ T-cell population. A Th1-like response, characterized by high levels of gamma interferon without detectable levels of interleukin 4 (IL-4) in serum, was observed in both depleted and nondepleted mice, although an increased production of IL-10 was detected in the depleted group. Our results suggest that PMNs play a very important role in the recruitment of other leukocyte populations to the inflammatory foci but have little influence in the polarization of the immune specific response toward a Th1-like response. PMID:10679002

  17. Leukocytes in chemotactic-fragment-induced lung inflammation. Vascular emigration and alveolar surface migration.

    PubMed Central

    Shaw, J. O.

    1980-01-01

    Lung inflammation was induced in rabbits by intratracheal injections of chemotactic fragments obtained from zymosan-activated serum (CF-ZAS), and the route of vascular emigration and alveolar surface interaction of polymorphonuclear leukocytes (PMNs) and monocytes migrating into the lung was characterized by transmission (TEM) and scanning (SEM) electron-microscopic examination. Leukocytes migrated from capillaries and venules into the alveolar wall interstitium by adherence to the vascular endothelium and migration through the endothelial intracellular junction to attain a position between a reapposed endothelial cell junction and the vascular basement membrane. The cells then migrated into the interstitium through a narrow opening in the basement membrane. Leukocyte entrance into the alveolar space from the interstitium appeared to occur through small openings in the epithelial basement membrane at or near the Type I epithelial intercellular junction. Once in the alveolus, PMNs and macrophages demonstrated surface adherence and spreading along with evidence of migration, pseudopod extension, interalveolar pore transit, and retraction fiber formation. This study indicates the leukocyte influx into the alveolus in acute chemotactic-factor-induced inflammation is via a continuum of migrational activity, beginning at the pulmonary capillary endothelial surface and persisting on the alveolar epithelial surface. Images Figure 10 Figure 11 Figure 12 Figure 1 Figure 2 Figure 3 Figure 13 Figure 14 Figure 4 Figure 5 Figure 6 Figure 15 Figure 7 Figure 8 Figure 16 Figure 9 PMID:7435538

  18. Temporal Expression of Peripheral Blood Leukocyte Biomarkers in a Macaca fascicularis Infection Model of Tuberculosis; Comparison with Human Datasets and Analysis with Parametric/Non-parametric Tools for Improved Diagnostic Biomarker Identification

    PubMed Central

    Wareham, Alice; Lewandowski, Kuiama S.; Williams, Ann; Dennis, Michael J.; Sharpe, Sally; Vipond, Richard; Silman, Nigel; Ball, Graham

    2016-01-01

    A temporal study of gene expression in peripheral blood leukocytes (PBLs) from a Mycobacterium tuberculosis primary, pulmonary challenge model Macaca fascicularis has been conducted. PBL samples were taken prior to challenge and at one, two, four and six weeks post-challenge and labelled, purified RNAs hybridised to Operon Human Genome AROS V4.0 slides. Data analyses revealed a large number of differentially regulated gene entities, which exhibited temporal profiles of expression across the time course study. Further data refinements identified groups of key markers showing group-specific expression patterns, with a substantial reprogramming event evident at the four to six week interval. Selected statistically-significant gene entities from this study and other immune and apoptotic markers were validated using qPCR, which confirmed many of the results obtained using microarray hybridisation. These showed evidence of a step-change in gene expression from an ‘early’ FOS-associated response, to a ‘late’ predominantly type I interferon-driven response, with coincident reduction of expression of other markers. Loss of T-cell-associate marker expression was observed in responsive animals, with concordant elevation of markers which may be associated with a myeloid suppressor cell phenotype e.g. CD163. The animals in the study were of different lineages and these Chinese and Mauritian cynomolgous macaque lines showed clear evidence of differing susceptibilities to Tuberculosis challenge. We determined a number of key differences in response profiles between the groups, particularly in expression of T-cell and apoptotic makers, amongst others. These have provided interesting insights into innate susceptibility related to different host `phenotypes. Using a combination of parametric and non-parametric artificial neural network analyses we have identified key genes and regulatory pathways which may be important in early and adaptive responses to TB. Using comparisons

  19. Human leukocyte antigen supertype matching after myeloablative hematopoietic cell transplantation with 7/8 matched unrelated donor allografts: a report from the Center for International Blood and Marrow Transplant Research

    PubMed Central

    Lazaryan, Aleksandr; Wang, Tao; Spellman, Stephen R.; Wang, Hai-Lin; Pidala, Joseph; Nishihori, Taiga; Askar, Medhat; Olsson, Richard; Oudshoorn, Machteld; Abdel-Azim, Hisham; Yong, Agnes; Gandhi, Manish; Dandoy, Christopher; Savani, Bipin; Hale, Gregory; Page, Kristin; Bitan, Menachem; Reshef, Ran; Drobyski, William; Marsh, Steven GE; Schultz, Kirk; Müller, Carlheinz R.; Fernandez-Viña, Marcelo A.; Verneris, Michael R.; Horowitz, Mary M.; Arora, Mukta; Weisdorf, Daniel J.; Lee, Stephanie J.

    2016-01-01

    The diversity of the human leukocyte antigen (HLA) class I and II alleles can be simplified by consolidating them into fewer supertypes based on functional or predicted structural similarities in epitope-binding grooves of HLA molecules. We studied the impact of matched and mismatched HLA-A (265 versus 429), -B (230 versus 92), -C (365 versus 349), and -DRB1 (153 versus 51) supertypes on clinical outcomes of 1934 patients with acute leukemias or myelodysplasia/myeloproliferative disorders. All patients were reported to the Center for International Blood and Marrow Transplant Research following single-allele mismatched unrelated donor myeloablative conditioning hematopoietic cell transplantation. Single mismatched alleles were categorized into six HLA-A (A01, A01A03, A01A24, A02, A03, A24), six HLA-B (B07, B08, B27, B44, B58, B62), two HLA-C (C1, C2), and five HLA-DRB1 (DR1, DR3, DR4, DR5, DR9) supertypes. Supertype B mismatch was associated with increased risk of grade II–IV acute graft-versus-host disease (hazard ratio =1.78, P=0.0025) compared to supertype B match. Supertype B07-B44 mismatch was associated with a higher incidence of both grade II–IV (hazard ratio=3.11, P=0.002) and III–IV (hazard ratio=3.15, P=0.01) acute graft-versus-host disease. No significant associations were detected between supertype-matched versus -mismatched groups at other HLA loci. These data suggest that avoiding HLA-B supertype mismatches can mitigate the risk of grade II–IV acute graft-versus-host disease in 7/8-mismatched unrelated donor hematopoietic cell transplantation when multiple HLA-B supertype-matched donors are available. Future studies are needed to define the mechanisms by which supertype mismatching affects outcomes after alternative donor hematopoietic cell transplantation. PMID:27247320

  20. 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.

  1. 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.

  2. Blood

    MedlinePlus

    ... that die or are lost from the body. White Blood Cells White blood cells (WBCs, and also ... of severe pain. previous continue Diseases of the White Blood Cells Neutropenia (pronounced: new-truh-PEE-nee- ...

  3. Biomaterials differentially regulate Src kinases and phosphoinositide 3-kinase-γ in polymorphonuclear leukocyte primary and tertiary granule release.

    PubMed

    Cohen, Hannah Caitlin; Frost, Dustin C; Lieberthal, Tyler Jacob; Li, Lingjun; Kao, W John

    2015-05-01

    In the foreign body response, infiltrating PMNs exocytose granule subsets to influence subsequent downstream inflammatory and wound healing events. In previous studies, we found that PMNs cultured on poly(ethylene glycol) (PEG)-containing hydrogels (i.e., PEG and gelatin + PEG hydrogels) had enhanced primary granule release, yet similar tertiary granule release compared with PMNs cultured on polydimethylsiloxane or tissue culture polystyrene. PMN primary granules contain microbicidal proteins and proteases, which can potentially injure bystander cells, degrade the extracellular matrix, and promote inflammation. Here, we sought to understand the mechanism of the enhanced primary granule release from PMNs on PEG hydrogels. We found that primary granule release from PMNs on PEG hydrogels was adhesion mediated and involved Src family kinases and PI3K-γ. The addition of gelatin to PEG hydrogels did not further enhance PMN primary granule release. Using stable-isotope dimethyl labeling-based shotgun proteomics, we identified many serum proteins - including Ig gamma constant chain region proteins and alpha-1-acid glycoprotein 1 - that were absorbed/adsorbed in higher quantities on PEG hydrogels than on TCPS, and may be involved in mediating PMN primary granule release. Ultimately, this mechanistic knowledge can be used to direct inflammation and wound healing following biomaterial implantation to promote a more favorable healing response.

  4. Leukocyte Esterase as a Biomarker in the Diagnosis of Periprosthetic Joint Infection

    PubMed Central

    Wang, Chi; Li, Rui; Wang, Qi; Duan, Jinyan; Wang, Chengbin

    2017-01-01

    Background Total joint arthroplasty (TJA) has been one of the most rewarding interventions for treating patients suffering from joint disorders. However, periprosthetic joint infection (PJI) is a serious complication that frequently accompanies TJA. Our study aimed to investigate the application of the leukocyte esterase (LE) strip in the diagnosis of PJI. Material/Methods From October 2014 to July 2015, 72 patients who had undergone joint puncture after arthroplasty in our hospital were enrolled in this trial. One drop of synovial fluid from each available patient was applied to the LE strip, and the results were observed after 1–3 min. If the color turned to dark purple, we recognized this as a positive result, while other colors were regarded as negative results. Centrifugation was used when the synovial fluid was mixed with blood. The Musculoskeletal Infection Society (MSIS) definition was used as the standard reference to identify whether PJI was found in patients or not. The results of diagnosis and LE strips test were compared, and indicators reflecting diagnostic value were calculated. Correlation of the LE data with erythrocyte sedimentation rate (ESR), elevated C-reactive protein (CRP), synovial white blood cell (WBC) counts, and polymorphonuclear neutrophil (PMN) percentage was calculated. Results By MSIS criteria, 38 patients were diagnosed with PJI and 34 patients were not infected. Two types of LE strip presented the same results with sensitivity of 84.21% (95% confidence interval [CI]: 68.75~93.98%), specificity of 97.06% (95% CI: 84.67~99.93%), positive predictive value (PPV) of 96.97% (95% CI: 84.24~99.92%), and negative predictive value (NPV) of 84.62% (95% CI: 69.47~94.14%). There were one false-positive case and six false-negative cases in this trial. There is a strong correlation between LE strip and synovial fluid PMN percentage. Conclusions The sensitivity and specificity of the LE strip in the diagnosis of PJI are quite high, which means

  5. 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)

  6. 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

  7. 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.

  8. Transmigration of polymorphnuclear neutrophils and monocytes through the human blood-cerebrospinal fluid barrier after bacterial infection in vitro

    PubMed Central

    2013-01-01

    Background Bacterial invasion through the blood-cerebrospinal fluid barrier (BCSFB) during bacterial meningitis causes secretion of proinflammatory cytokines/chemokines followed by the recruitment of leukocytes into the CNS. In this study, we analyzed the cellular and molecular mechanisms of polymorphonuclear neutrophil (PMN) and monocyte transepithelial transmigration (TM) across the BCSFB after bacterial infection. Methods Using an inverted transwell filter system of human choroid plexus papilloma cells (HIBCPP), we studied leukocyte TM rates, the migration route by immunofluorescence, transmission electron microscopy and focused ion beam/scanning electron microscopy, the secretion of cytokines/chemokines by cytokine bead array and posttranslational modification of the signal regulatory protein (SIRP) α via western blot. Results PMNs showed a significantly increased TM across HIBCPP after infection with wild-type Neisseria meningitidis (MC58). In contrast, a significantly decreased monocyte transmigration rate after bacterial infection of HIBCPP could be observed. Interestingly, in co-culture experiments with PMNs and monocytes, TM of monocytes was significantly enhanced. Analysis of paracellular permeability and transepithelial electrical resistance confirmed an intact barrier function during leukocyte TM. With the help of the different imaging techniques we could provide evidence for para- as well as for transcellular migrating leukocytes. Further analysis of secreted cytokines/chemokines showed a distinct pattern after stimulation and transmigration of PMNs and monocytes. Moreover, the transmembrane glycoprotein SIRPα was deglycosylated in monocytes, but not in PMNs, after bacterial infection. Conclusions Our findings demonstrate that PMNs and monoctyes differentially migrate in a human BCSFB model after bacterial infection. Cytokines and chemokines as well as transmembrane proteins such as SIRPα may be involved in this process. PMID:23448224

  9. 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.

  10. 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.

  11. 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.

  12. Myocellular enzyme leakage, polymorphonuclear neutrophil activation and delayed onset muscle soreness induced by isokinetic eccentric exercise.

    PubMed

    Croisier, J L; Camus, G; Deby-Dupont, G; Bertrand, F; Lhermerout, C; Crielaard, J M; Juchmès-Ferir, A; Deby, C; Albert, A; Lamy, M

    1996-01-01

    To address the question of whether delayed onset muscular soreness (DOMS) following intense eccentric muscle contraction could be due to increased production of the arachidonic acid derived product prostaglandin E2 (PGE2). 10 healthy male subjects were submitted to eccentric and concentric isokinetic exercises on a Kin Trex device at 60 degrees/s angular velocity. Exercise consisted of 8 stages of 5 maximal contractions of the knee extensor and flexor muscle groups of both legs separated by 1 min rest phases. There was an interval of at least 30 days between eccentric and concentric testing, and the order of the two exercise sessions was randomly assigned. The subjective presence and intensity of DOMS was evaluated using a visual analogue scale, immediately, following 24 h and 48 h after each test. Five blood samples were drawn from an antecubital vein: at rest before exercise, immediately after, after 30 min recovery, 24 h and 48 h after the tests. The magnitude of the acute inflammatory response to exercise was assessed by measuring plasma levels of polymorphonuclear elastase ([EL]), myeloperoxidase ([MPO]) and PGE2 ([PGE2]). Using two way analysis of variance, it appeared that only eccentric exercise significantly increased [EL] and DOMS, especially of the hamstring muscles. Furthermore, a significant decrease in eccentric peak torque of this muscle group only was observed on day 2 after eccentric work (- 21%; P < 0.002). Serum activity of creatine kinase and serum concentration of myoglobin increased significantly 24 and 48 h after both exercise tests. However, these variables reached significantly higher values following eccentric contractions 48 h after exercise. Mean [PGE2] in the two exercise modes remained unchanged over time and were practically equal at each time point. On the basis of these findings, we conclude that the magnitude of polymorphonuclear (PMN) activation, muscle damage, and DOMS are greater after eccentric than after concentric muscle

  13. Assessment of the functional capacity for intracellular death and phagocytosis of polymorphonuclear cells in healthy neonates.

    PubMed

    Del Rey-Pineda, G; Gómez-González, M V; Solórzano-Santos, F; Arredondo-García, J L

    1997-01-01

    The objective of this study was to assess the functional capacity for intracellular death (ID) and/or phagocytic index (PI) of polymorphonuclear cells of 24-h-old healthy newborns with respect to the PMN cells of adults using the same standard exogenous source of opsonins. The ID and PI techniques were standardized and 2-3 ml of blood were used. No differences were found in the percentages of ID, P, PI among the PMNs of the newborns and those of the adults: 43.95 +/- 15.70 vs. 44.56 +/- 8.43 for ID; 38.96 +/- 14.34 vs. 39.00 +/- 14.54 for P; 1.71 +/- 0.54 vs. 1.73 0.45 for PI, respectively. It was concluded that the PMNs of 24-h newborns have an ID, P, PI functionality comparable to adult PMNs; differences observed in PMN function in newborns may be due to humoral deficiencies (opsonins).

  14. [Role of polymorphonuclear neutrophil in exogenous hydrogen sulfide attenuating endotoxin-induced acute lung injury].

    PubMed

    Huang, Xin-Li; Zhou, Xiao-Hong; Zhou, Jun-Lin; Ding, Chun-Hua; Xian, Xiao-Hui

    2009-08-25

    The animal model of acute lung injury (ALI) caused by intravenous injection of lipopolysaccharides (LPS) and cultured human peripheral blood polymorphonuclear neutrophil (PMN) were used to study the effects of sodium hydrosulfide (NaHS), hydrogen sulfide (H2S) donor, on LPS-induced PMN accumulation, microvascular permeability and PMN apoptosis. Control group, NaHS group, LPS group and LPS + NaHS group were established both in in vivo and in vitro studies. Microvascular permeability, PMN accumulation in lung and apoptosis of PMN were detected. The results showed that: (1) In in vivo study, PMN accumulation in lung, the protein content in bronchoalveolar lavage fluid (BALF) and the Evans blue dye in lung tissue of LPS group were markedly higher than those of both sham operation group and LPS + NaHS group (P<0.05, P<0.01); (2) In in vitro study, the apoptotic rates of PMN in LPS group and NaHS group were significantly higher than that in control group (P<0.01), while compared with LPS group, LPS + NaHS group showed significantly higher apoptotic rate (P<0.01). These results suggest that NaHS attenuates LPS-induced microvascular permeability and alleviates ALI. PMN apoptosis induced by NaHS is possibly one of the potential mechanisms underlying the decrease of PMN accumulation in lung tissue.

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

    SciTech Connect

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

    1985-05-01

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

  16. Quantitative analysis of human herpesvirus-6 and human cytomegalovirus in blood and saliva from patients with acute leukemia.

    PubMed

    Nefzi, Faten; Ben Salem, Nabil Abid; Khelif, Abderrahim; Feki, Salma; Aouni, Mahjoub; Gautheret-Dejean, Agnès

    2015-03-01

    Human herpesvirus-6 (HHV-6) and human cytomegalovirus (HCMV) DNAs were quantified by real-time PCR assays in blood and saliva obtained from 50 patients with acute leukemia at the time of diagnosis (50 of each matrix), aplasia (65 of each matrix), remission (55 of each matrix), and relapse (20 of each matrix) to evaluate which biological matrix was more suitable to identify a viral reactivation, search for a possible link between HHV-6 and HCMV reactivations, and evaluate the relations between viral loads and count of different leukocyte types in blood. The median HHV-6 loads were 136; 219; 226, and 75 copies/million cells in blood at diagnosis, aplasia, remission and relapse, respectively. The HCMV loads were 193 and 317 copies/million cells in blood at diagnosis and remission. In the saliva samples, the HHV-6 loads were 22,165; 15,238; 30,214, and 17,454 copies/million cells at diagnosis, aplasia, remission, and relapse, respectively. The HCMV loads were 8,991; 1,461; 2,980, and 4,283 copies/million cells at diagnosis, aplasia, remission, and relapse, respectively. The HHV-6 load in the blood was correlated to the counts of polymorphonuclear leukocytes (R(2)  = 0.5; P < 0.0001) and lymphocytes (R(2)  = 0.4; P = 0.001) and was not correlated to the monocyte counts (R(2)  = 0.07; P = 0.7). Saliva appears to be a more sensitive biological matrix than whole blood in the detection of HHV-6 or HCMV reactivations. The HHV-6 and HCMV reactivations were linked only in saliva.

  17. MicroRNA-941 Expression in Polymorphonuclear Granulocytes Is Not Related to Granulomatosis with Polyangiitis

    PubMed Central

    Svendsen, Jesper Brink; Baslund, Bo; Cramer, Elisabeth Præstekjær; Rapin, Nicolas; Borregaard, Niels

    2016-01-01

    Jumonji Domain-Containing Protein 3 (JMJD3)/lysine demethylase 6B (KDM6B) is an epigenetic modulator that removes repressive histone marks on genes. Expression of KDM6B mRNA is elevated in leukocytes from patients with ANCA-associated vasculitis (AAV) and has been suggested to be the reason for higher proteinase 3 (PR3) mRNA expression in these cells due to derepression of PRTN3 gene transcription. MicroRNA-941 (miR-941) has been shown to target KDM6B mRNA and inhibit JMJD3 production. We therefore investigated whether polymorphonuclear granulocytes (PMNs) from patients suffering from granulomatosis with polyangiitis (GPA) have lower expression of miR-941 than healthy control donors as a biological cause for higher JMJD3 levels. We found no significant difference in the degree of maturation of PMNs from GPA patients (n = 8) and healthy controls (n = 11) as determined from cell surface expression of the neutrophil maturation marker CD16 and gene expression profile of FCGR3B. The expression of PRTN3 and KDM6B mRNAs and miR-941 was not significantly different in GPA patients and healthy controls. Transfection of pre-miR-941 into the neutrophil promyelocyte cell line PLB-985 cells did not result in reduction of the KDM6B mRNA level as shown previously in a hepatocellular carcinoma cell line. The amount of PR3 in PMNs from GPA patients and healthy controls was comparable. In conclusion, we found that PRTN3 mRNA, KDM6B mRNA, and miR-941 expression levels in PMNs do not differ between GPA patients and healthy controls, and that miR-941 does not uniformly regulate KDM6B mRNA levels by inducing degradation of the transcript. Thus, decreased miR-941 expression in PMNs cannot be part of the pathogenesis of GPA. PMID:27755585

  18. Dietary Fiber Intake is Associated with Increased Colonic Mucosal GPR43+ Polymorphonuclear Infiltration in Active Crohn's Disease.

    PubMed

    Zhao, Mingli; Zhu, Weiming; Gong, Jianfeng; Zuo, Lugen; Zhao, Jie; Sun, Jing; Li, Ning; Li, Jieshou

    2015-07-01

    G protein-coupled receptor 43/free fatty acid receptor 2 (GPR43/FFAR2) is essential for polymorphonuclear (PMN) recruitment. We investigated the expression of GPR43/FFAR2 in the colon from Crohn's disease patients and whether dietary fiber in enteral nutrition increases GPR43+ polymorphonuclear infiltration in mucosa. Segments of ascending colon and white blood cells from peripheral blood were obtained from 46 Crohn's disease patients and 10 colon cancer patients. The Crohn's disease patients were grouped by the activity of disease (active or remission) and enteral nutrition with or without dietary fiber. Histological feature, expression and location of GPR43/FFAR2 and level of tumor necrosis factor-α (TNF-α), interleukine-6 (IL-6) and myeloperoxidase were assessed. The results of hematoxylin-eosin and immunohistochemistry staining revealed that the infiltration of immune cells, including GPR43+ PMN, was more severe in active Crohn's disease patients who consumed normal food or enteral nutrition with dietary fiber than in remission patients and colon cancer patients. This finding was supported by the results of GPR43 and myeloperoxidase expression. Active Crohn's disease (CD) patients who consumed enteral nutrition without dietary fiber exhibited severe immune cell infiltration similar to the other active CD patients, but GPR43+ PMNs were rarely observed. The level of TNF-α mRNA in active Crohn's disease patients was higher than those of the other patients. In conclusion, the use of dietary fiber in enteral nutrition by active Crohn's disease patients might increase GPR43+ PMNs infiltration in colon mucosa. This effect was not observed in Crohn's disease patients in remission.

  19. Dietary Fiber Intake is Associated with Increased Colonic Mucosal GPR43+ Polymorphonuclear Infiltration in Active Crohn’s Disease

    PubMed Central

    Zhao, Mingli; Zhu, Weiming; Gong, Jianfeng; Zuo, Lugen; Zhao, Jie; Sun, Jing; Li, Ning; Li, Jieshou

    2015-01-01

    G protein-coupled receptor 43/free fatty acid receptor 2 (GPR43/FFAR2) is essential for polymorphonuclear (PMN) recruitment. We investigated the expression of GPR43/FFAR2 in the colon from Crohn’s disease patients and whether dietary fiber in enteral nutrition increases GPR43+ polymorphonuclear infiltration in mucosa. Segments of ascending colon and white blood cells from peripheral blood were obtained from 46 Crohn’s disease patients and 10 colon cancer patients. The Crohn’s disease patients were grouped by the activity of disease (active or remission) and enteral nutrition with or without dietary fiber. Histological feature, expression and location of GPR43/FFAR2 and level of tumor necrosis factor-α (TNF-α), interleukine-6 (IL-6) and myeloperoxidase were assessed. The results of hematoxylin-eosin and immunohistochemistry staining revealed that the infiltration of immune cells, including GPR43+ PMN, was more severe in active Crohn’s disease patients who consumed normal food or enteral nutrition with dietary fiber than in remission patients and colon cancer patients. This finding was supported by the results of GPR43 and myeloperoxidase expression. Active Crohn’s disease (CD) patients who consumed enteral nutrition without dietary fiber exhibited severe immune cell infiltration similar to the other active CD patients, but GPR43+ PMNs were rarely observed. The level of TNF-α mRNA in active Crohn’s disease patients was higher than those of the other patients. In conclusion, the use of dietary fiber in enteral nutrition by active Crohn’s disease patients might increase GPR43+ PMNs infiltration in colon mucosa. This effect was not observed in Crohn’s disease patients in remission. PMID:26140540

  20. Extravasation of leukocytes in comparison to tumor cells

    PubMed Central

    Strell, Carina; Entschladen, Frank

    2008-01-01

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

  1. Platelets Guide Leukocytes to Their Sites of Extravasation

    PubMed Central

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

    2016-01-01

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

  2. 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.

  3. 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.

  4. Impact of ambient air pollution on the differential white blood cell count in patients with chronic pulmonary disease.

    PubMed

    Brüske, Irene; Hampel, Regina; Socher, Martin M; Rückerl, Regina; Schneider, Alexandra; Heinrich, Joachim; Oberdörster, Günter; Wichmann, H-Erich; Peters, Annette

    2010-02-01

    Epidemiologic studies report associations between particulate air pollution and increased mortality from pulmonary diseases. This study was performed to examine whether the exposure to ambient gaseous and particulate air pollution leads to an alteration of the differential white blood cell count in patients with chronic pulmonary diseases like chronic bronchitis, chronic obstructive pulmonary disease, and asthma. A prospective panel study was conducted in Erfurt, Eastern Germany, with 12 repeated differential white blood cell counts in 38 males with chronic pulmonary diseases. Hourly particulate and gaseous air pollutants and meteorological data were acquired. Mixed models with a random intercept adjusting for trend, meteorology, weekday, and other risk variables were used. In this explorative analysis, we found an immediate decrease of polymorphonuclear leukocytes in response to an increase of most gaseous and particulate pollutants. Lymphocytes increased within 24 h in association with all gaseous pollutants but showed only minor effects in regard to particulate air pollution. Monocytes showed an increase associated with ultrafine particles, and nitrogen monoxide. The effect had two peaks in time, one 0-23 h before blood withdrawal and a second one with a time lag of 48-71 h. The increase of particulate and gaseous air pollution was associated with multiple changes in the differential white blood cell count in patients with chronic pulmonary diseases.

  5. Scaling deterministic lateral displacement arrays for high throughput and dilution-free enrichment of leukocytes

    NASA Astrophysics Data System (ADS)

    Inglis, David W.; Lord, Megan; Nordon, Robert E.

    2011-05-01

    A disposable device for fractionation of blood into its components that is simple to operate and provides throughput of greater than 1 mL min-1 is highly sought after in medical diagnostics and therapies. This paper describes a device with parallel deterministic lateral displacement devices for enrichment of leukocytes from blood. We show capture of 98% and approximately ten-fold enrichment of leukocytes in whole blood. We demonstrate scaling up through the integration of six parallel devices to achieve a flow rate of 115 µL of undiluted blood per minute per atmosphere of applied pressure.

  6. Short communication: amino acid supplementation and stage of lactation alter apparent utilization of nutrients by blood neutrophils from lactating dairy cows in vitro

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glutamine is the preferred AA used by polymorphonuclear leukocytes (PMN) during the inflammatory response. However, the effect of other AA on bovine PMN response during inflammation and how this is altered by stage of lactation has not been fully elucidated. The objective of this study was to dete...

  7. 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.

  8. In vitro evaluation of the behaviour of human polymorphonuclear neutrophils in direct contact with chitosan-based membranes.

    PubMed

    Santos, T C; Marques, A P; Silva, S S; Oliveira, J M; Mano, J F; Castro, A G; Reis, R L

    2007-10-31

    Several novel biodegradable materials have been proposed for wound healing applications in the past few years. Taking into consideration the biocompatibility of chitosan-based biomaterials, and that they promote adequate cell adhesion, this work aims at investigating the effect of chitosan-based membranes, over the activation of human polymorphonuclear neutrophils (PMNs). The recruitment and activation of polymorphonuclear neutrophils (PMNs) reflects a primary reaction to foreign bodies. Activation of neutrophils results in the production of reactive oxygen species (ROS) such as O(2)(-) and HO(-) and the release of hydrolytic enzymes which are determinant factors in the inflammatory process, playing an essential role in the healing mechanisms. PMNs isolated from human peripheral blood of healthy volunteers were cultured in the presence of chitosan or chitosan/soy newly developed membranes. The effect of the biomaterials on the activation of PMNs was assessed by the quantification of lysozyme and ROS. The results showed that PMNs, in the presence of the chitosan-based membranes secrete similar lysozyme amounts, as compared to controls (PMNs without materials) and also showed that the materials do not stimulate the production of either O(2)(-) or HO(-). Moreover, PMNs incubated with the biomaterials when stimulated with phorbol 12-myristate 13-acetate (PMA) or formyl-methionyl-leucyl-phenylalanine (fMLP) showed a chemiluminescence profile with a slightly lower intensity, to that observed for positive controls (cells without materials and stimulated with PMA), which reflects the maintenance of their stimulation capacity. Our data suggests that the new biomaterials studied herein do not elicit activation of PMNs, as assessed by the low lysozyme activity and by the minor detection of ROS by chemiluminescence. These findings reinforce previous statements supporting the suitability of chitosan-based materials for wound healing applications.

  9. Polymorphonuclear counts in ascitic fluid and microorganisms producing spontaneous bacterial peritonitis: an under-recognized relationship.

    PubMed

    Ariza, Xavier; Lora-Tamayo, Jaime; Castellote, José; Xiol, Xavier; Ariza, Javier

    2013-10-01

    BACKGROUND AND AIMS. In cirrhotic patients with spontaneous bacterial peritonitis (SBP) higher polymorphonuclear (PMN) count in ascitic fluid have been reported in infections caused by Gram-negative bacilli (GNB) as opposed to Gram-positive cocci (GPC). However, the influence of other associated factors on the PMN count, such as the specific microorganism causing the episode of SBP, has not been well established. METHODS. Retrospective observational study of 194 episodes of positive ascitic and/or blood culture SBP in 159 patients with liver cirrhosis (2001-2009). Parameters associated with PMN count in ascitic fluid at diagnosis were evaluated. RESULTS. The multivariate analysis (model 1) showed that a virulent etiology of the infection [coefficient 3.941 (95% confidence interval (95 CI): 0.421-7.461)] and the model for end-stage liver disease (MELD) score [coefficient 0.196 (95 CI: 0.007-0.384)] were positively associated with the PMN count in ascites, while a nosocomial acquisition was inversely associated [coefficient -3.546 (95 CI: -6.855 - -0.238)]. A nonsignificant trend toward higher PMN count was found in GNB versus GPC, but there were differences between groups of microorganisms: pyogenic streptococci [median (p25-p75): 3211 (1615-8004)], Enterobacteriaceae [2958 (917-7690)], Vibrionaceae [9215 (375-17280)], nonfermenting GNB [1384 (565-3865)], viridans group streptococci [1044 (503-2354)] and enterococci [1050 (476-4655)](p = 0.005). No clear cut-offs of ascitic PMN count predicting a particular etiology could be calculated out of these data. CONCLUSIONS. In cirrhotic patients with SBP, the causing microorganism, the place of acquisition of the infection and the host liver condition were the main factors determining PMN count in ascitic fluid. Third-generation cephalosporin resistance was associated with low PMN count probably because this group included bacteria with inherent low virulence.

  10. 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

  11. Spermadhesin PSP-I/PSP-II heterodimer induces migration of polymorphonuclear neutrophils into the uterine cavity of the sow.

    PubMed

    Rodriguez-Martinez, H; Saravia, F; Wallgren, M; Martinez, E A; Sanz, L; Roca, J; Vazquez, J M; Calvete, J J

    2010-01-01

    Seminal plasma (SP) is a complex fluid which exerts biological actions in the female reproductive tract. In pigs, SP elicits endometrial inflammation and consequent immune changes after mating. This study tested whether heparin-binding spermadhesins (HBPs) and the heterodimer of porcine sperm adhesions I and II (PSP-I/PSP-II) in SP recruit different lymphocyte subsets (CD2(+), CD4(+) and CD8(+) T cells) or polymorphonuclear leukocytes (PMNs) to the superficial endometrium or luminal epithelium and lumen, respectively, of oestrous sows. In Experiment 1, endometrial biopsies were taken between 2 and 120 min after infusion of uterine horns with HBPs, PSP-I/PSP-II or saline and evaluated by immunohistochemistry or histology. In Experiment 2, the uterus of oestrous sows was infused with PSP-I/PSP-II or saline to assess PMN numbers in the uterine lumen 3h later. PSP-I/PSP-II elicited CD2+ T cell recruitment from 10 min, and CD8(+) T cells from 60 min after infusion, while HBPs increased CD4(+) T cell recruitment by 120 min. PSP-I/PSP-II but not HBPs induced PMN migration to the surface epithelium by 10 min. PMN numbers were elevated 5-fold by 30 min and 7-fold from 60 min, with PMNs detectable in the lumen from 30 min after infusion. Six-fold more PMNs were collected from the uterine lumen of PSP-I/PSP-II-infused sows compared to controls at 3h after infusion. These data show that PSP-I/PSP-II heterodimer in seminal plasma has a predominant role in triggering the recruitment of uterine PMNs and T cells after mating, initiating a cascade of transient and long-lasting immunological events.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. Blood and inflammatory cells of the lungfish Lepidosiren paradoxa.

    PubMed

    Ribeiro, Maria Lucia da S; DaMatta, Renato A; Diniz, José A P; de Souza, Wanderley; do Nascimento, Jose Luiz M; de Carvalho, Tecia Maria U

    2007-07-01

    A special interest exists concerning lungfish because they may possess characteristics of the common ancestor of land vertebrates. However, little is known about their blood and inflammatory cells; thus the fine structure, cytochemistry and differential cell counts of coelomic exudate and blood leucocytes were studied in Lepidosiren paradoxa. Blood smear analyses revealed erythrocytes, lymphocytes, monocytes, polymorphonuclear agranulocytes, thrombocytes and three different granulocytes. Blood monocytes and lymphocytes had typical vertebrate morphology. Thrombocytes had large vacuoles filled with a myelin rich structure. The polymorphonuclear agranulocyte had a nucleus morphologically similar to the human neutrophil with no apparent granules. Types I and II granulocytes had eosinophilic granules. Type I granulocytes had round or elongated granules heterogeneous in size, while type II had granules with an electron dense core. Type III granulocyte had many basophilic granules. The order of frequency was: type I granulocyte, followed by lymphocyte, type II granulocyte, monocyte, polymorphonuclear agranulocyte and type III granulocyte. Peroxidase localized mainly at the periphery of the granules from type II granulocytes, while no peroxidase expression was detected in type I granulocytes. Alkaline phosphatase was localized in the granules of type II granulocyte and acid phosphatase cytochemistry also labelled a few vacuoles of polymorphonuclear agranulocyte. About 85% of the coelomic inflammatory exudate cell population was type II granulocyte, 10% polymorphonuclear agranulocyte and 5% macrophages as judged by the nucleus and granule morphology. These results indicate that this lungfish utilises type II granulocytes as its main inflammatory granulocytes and that the polymorphonuclear agranulocyte may also be involved in the inflammatory response. The other two granulocytes appear similar to the mammalian eosinophil and basophil. In summary, this lungfish appears to

  17. Changes in blood pressure, heart rate, and blood profile in mares during the last 3 months of gestation and the peripartum period.

    PubMed

    Nagel, Christina; Trenk, Lisa; Aurich, Jörg; Wulf, Manuela; Aurich, Christine

    2016-10-15

    In this study, peripartum changes in complete blood count, plasma electrolyte concentrations, blood pressure, heart rate, and heart rate variability (HRV) were determined in pregnant Warmblood mares (n = 10). Blood was collected from Days 245 to 330 of gestation (phase A), 2Days 3 to 1 before foaling (phase B), repeatedly within 12 hours after foaling (phase C), and on Days 1 to 3 postpartum (phase D). On the same days as blood collection, blood pressure and cardiac beat-to-beat intervals were measured and time domain HRV variables were calculated. Blood pressure decreased during the past 3 months of gestation, reached a nadir at foaling and increased rapidly thereafter (P < 0.001). Heart rate increased in phase A, reached a maximum in phase B, and decreased directly after foaling (P < 0.001) while HRV increased transiently after foaling (P < 0.001). The number of polymorphonuclear granulocytes was higher during phases B, C, and D than that during phase A (P < 0.001) and total leukocyte numbers peaked in phase C (P < 0.001). Erythrocyte counts and hematocrit increased immediately after foaling with a rapid decrease thereafter (P < 0.001), while plasma total protein was highest 1 hour after foaling (P < 0.001). Sodium and chloride concentrations increased from phases A to C and decreased in phase D (P < 0.001). Calcium concentrations were lowest immediately after foaling. In conclusion, changes in cardiovascular function and a transient hemoconcentration occur in peripartum mares. These changes may favor adequate uterine perfusion.

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

    PubMed

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

    2005-08-01

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

  19. Characterization of rag1 mutant zebrafish leukocytes

    PubMed Central

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

    2009-01-01

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

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

    DTIC Science & Technology

    2014-07-01

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

  1. 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

  2. Leukocyte driven-decidual angiogenesis in early pregnancy.

    PubMed

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

    2014-11-01

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

  3. Blood

    MedlinePlus

    ... increased red blood cell destruction can affect teens: G6PD deficiency. G6PD is an enzyme that helps to protect ... can cause red cells to hemolyze, or burst. G6PD deficiency is a common hereditary disease among people of ...

  4. 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

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

    PubMed

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

    2015-01-01

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

  6. Lipoproteins of Treponema denticola: their effect on human polymorphonuclear neutrophils.

    PubMed

    Sela, M N; Bolotin, A; Naor, R; Weinberg, A; Rosen, G

    1997-07-01

    The presence of lipoproteins and lipooligosaccharides in Treponema denticola, an oral spirochaete associated with periodontal diseases, was investigated. T. denticola ATCC 35404 and the clinical isolate GM-1 were metabolically labeled with [3H]-cis-9-octadecenoic acid and extracted with the non-ionic detergent Triton X-114. The extract was phase separated, precipitated with acetone and delipidated to remove non-covalently bound lipid (dLPP). In T. denticola ATCC 35404, sodium dodecyl sulfate polyacrylamide electrophoretic separation followed by autoradiography showed [3H]-cis-9-octadecenoic acid incorporation in bands with apparent molecular masses of 14, 20, 26, 31, 38, 72 and 85 kDa and a broad band running from 113 kDa to the top of the gel. This last band resolved into a 53 kDa [3H]-cis-9-octadecenoic acid band upon heating for 10 min, at 100 degrees C. The structural relationship of the outer sheath major oligomeric polypeptide of strain ATCC 35404 and the 53 kDa protein was demonstrated immunologically. Antibodies against the 113 kDa component of the oligomer cross-reacted with the 53 kDa protein. Proteinase K degraded the [3H]-cis-9-octadecenoic acid bands with the exception of the 14 kDa. The 14 kDa was also the major [3H]-fatty acid labeled compound found in the water phase following phenol-water extraction of whole T. denticola ATCC 35404 cells. This compound was purified from the water phase by gel filtration followed by hydrophobic chromatography. Chemical analysis showed that hexadecanoic acid was the predominant fatty acid bound to T. denticola lipoproteins. In the GM-1 strain [3H]-cis-9-octadecenoic acid incorporation was observed in the 116 kDa and 14 kDa bands. dLPP from strain ATCC 35404 caused an enhanced (0.8-8 micrograms/ml) luminol dependent chemiluminiscence (LDCL) effect in human polymorphonuclear neutrophils (PMN) which could be related to protein concentration. The addition of dLPP to PMN together with FMLP at submaximal concentration (1

  7. 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

  8. Association between Snoring and Leukocyte Telomere Length

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Chang, Xiaojian; Gorbet, Maud

    2013-09-01

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

  10. The expression and localization of RNase and RNase inhibitor in blood cells and vascular endothelial cells in homeostasis of the vascular system

    PubMed Central

    Ohashi, Ayaka; Murata, Aya; Cho, Yuichiro; Ichinose, Shizuko; Sakamaki, Yuriko; Nishio, Miwako; Hoshi, Osamu; Fischer, Silvia; Preissner, Klaus T.; Koyama, Takatoshi

    2017-01-01

    RNA may be released from vascular cells including endothelial cells in the event of injury and in vascular disease. Extracellular RNAs have been recognized as novel procoagulant and permeability-increasing factors. Extracellular RNA may function as inflammatory host alarm signals that serve to amplify the defense mechanism, but it may provide important links to thrombus formation. Extracellular RNA is degraded by RNase. We propose that RNase and its inhibitor RNase inhibitor (RI) act as modulators of homeostasis in the vasculature to control the functions of extracellular RNA. We aimed to investigate the expression and localization of RNase 1 and RI in cells that contact blood, such as platelets, mononuclear cells, polymorphonuclear cells, and red blood cells. RNase 1 and RI expression and localization in blood cells were compared with those in the human umbilical vein endothelial cell line, EAhy926. Additionally, we further investigated the effect of thrombin on the expression of RNase 1 and RI in platelets. We used an RNase activity assay, reverse transcription-polymerase chain reaction, western blot, immunocytochemistry, transmission electron microscopy, and immunoelectron microscopy (pre- and post-embedding methods). RNase activity in the supernatant from EAhy926 cells was 50 times than in blood cells (after 60 min). RNase 1 mRNA and protein expression in EAhy926 cells was highest among the cells examined. However, RI mRNA and protein expression was similar in most cell types examined. Furthermore, we observed that RNase 1 and von Willebrand factor were partially colocalized in EAhy926 cells and platelets. In conclusion, we propose that high RNase activity is ordinarily released from endothelial cells to support anticoagulation in the vasculature. On the other hand, platelets and leukocytes within thrombi at sites of vascular injury show very low RNase activity, which may support hemostatic thrombus formation. However, activated platelets and leukocytes may

  11. Leukocyte chemoattractant receptors in human disease pathogenesis.

    PubMed

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

    2015-01-01

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

  12. Visualization of a prosthetic vascular graft due to platelet contamination during /sup 111/Indium-labeled leukocyte scintigraphy

    SciTech Connect

    Oates, E.; Ramberg, K.

    1988-09-01

    A prosthetic axillo-femoral bypass graft was visualized during /sup 111/In-labeled leukocyte scintigraphy in a patient referred for possible abdominal abscess. The presence of significant cardiac blood-pool activity raised the possibility that this uptake was due to deposition of contaminating labeled platelets rather than labeled leukocytes. An analysis of a small sample of the patient's blood confirmed that the circulating activity was due to labeled platelets. Increased activity along prosthetic vascular grafts in patients undergoing /sup 111/In-labeled leukocyte scintigraphy may be due to adherent platelet, and not indicative of infection.

  13. 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.

  14. A stochastic model for leukocyte random motility and chemotaxis based on receptor binding fluctuations

    PubMed Central

    1988-01-01

    Two central features of polymorphonuclear leukocyte chemosensory movement behavior demand fundamental theoretical understanding. In uniform concentrations of chemoattractant, these cells exhibit a persistent random walk, with a characteristic "persistence time" between significant changes in direction. In chemoattractant concentration gradients, they demonstrate a biased random walk, with an "orientation bias" characterizing the fraction of cells moving up the gradient. A coherent picture of cell movement responses to chemoattractant requires that both the persistence time and the orientation bias be explained within a unifying framework. In this paper, we offer the possibility that "noise" in the cellular signal perception/response mechanism can simultaneously account for these two key phenomena. In particular, we develop a stochastic mathematical model for cell locomotion based on kinetic fluctuations in chemoattractant/receptor binding. This model can simulate cell paths similar to those observed experimentally, under conditions of uniform chemoattractant concentrations as well as chemoattractant concentration gradients. Furthermore, this model can quantitatively predict both cell persistence time and dependence of orientation bias on gradient size. Thus, the concept of signal "noise" can quantitatively unify the major characteristics of leukocyte random motility and chemotaxis. The same level of noise large enough to account for the observed frequency of turning in uniform environments is simultaneously small enough to allow for the observed degree of directional bias in gradients. PMID:3339093

  15. Minimal amounts of kindlin-3 suffice for basal platelet and leukocyte functions in mice.

    PubMed

    Klapproth, Sarah; Moretti, Federico A; Zeiler, Marlis; Ruppert, Raphael; Breithaupt, Ute; Mueller, Susanna; Haas, Rainer; Mann, Matthias; Sperandio, Markus; Fässler, Reinhard; Moser, Markus

    2015-12-10

    Hematopoietic cells depend on integrin-mediated adhesion and signaling, which is induced by kindlin-3 and talin-1. To determine whether platelet and polymorphonuclear neutrophil (PMN) functions require specific thresholds of kindlin-3, we generated mouse strains expressing 50%, 10%, or 5% of normal kindlin-3 levels. We report that in contrast to kindlin-3-null mice, which die perinatally of severe bleeding and leukocyte adhesion deficiency, mice expressing as little as 5% of kindlin-3 were viable and protected from spontaneous bleeding and infections. However, platelet adhesion and aggregation were reduced in vitro and bleeding times extended. Similarly, leukocyte adhesion, extravasation, and bacterial clearance were diminished. Quantification of protein copy numbers revealed stoichiometric quantities of kindlin-3 and talin-1 in platelets and neutrophils, indicating that reduction of kindlin-3 in our mouse strains progressively impairs the cooperation with talin-1. Our findings show that very low levels of kindlin-3 enable basal platelet and neutrophil functions, whereas in stress situations such as injury and infection, platelets and neutrophils require a maximum of functional integrins that is achieved with high and stoichiometric quantities of kindlin-3 and talin-1.

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

    PubMed

    Merchant, Mark; Williams, Stetson; Hardy, Ross

    2009-01-01

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

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

    PubMed Central

    1996-01-01

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

  18. 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.

  19. Vimentin, a Novel NF-κB Regulator, Is Required for Meningitic Escherichia coli K1-Induced Pathogen Invasion and PMN Transmigration across the Blood-Brain Barrier

    PubMed Central

    Zhang, Bao; Liu, Li-Qun; Wu, Xuedong; Mor-Vaknin, Nirit; Markovitz, David M.; Cao, Hong; Zhou, Yan-Hong

    2016-01-01

    Background NF-κB activation, pathogen invasion, polymorphonuclear leukocytes (PMN) transmigration (PMNT) across the blood-brain barrier (BBB) are the pathogenic triad hallmark features of bacterial meningitis, but the mechanisms underlying these events remain largely unknown. Vimentin, which is a novel NF-κB regulator, is the primary receptor for the major Escherichia coli K1 virulence factor IbeA that contributes to the pathogenesis of neonatal bacterial sepsis and meningitis (NSM). We have previously shown that IbeA-induced NF-κB signaling through its primary receptor vimentin as well as its co-receptor PTB-associated splicing factor (PSF) is required for pathogen penetration and leukocyte transmigration across the BBB. This is the first in vivo study to demonstrate how vimentin and related factors contributed to the pathogenic triad of bacterial meningitis. Methodology/Principal Findings The role of vimentin in IbeA+ E. coli K1-induced NF-κB activation, pathogen invasion, leukocyte transmigration across the BBB has now been demonstrated by using vimentin knockout (KO) mice. In the in vivo studies presented here, IbeA-induced NF-κB activation, E. coli K1 invasion and polymorphonuclear neutrophil (PMN) transmigration across the BBB were significantly reduced in Vim-/- mice. Decreased neuronal injury in the hippocampal dentate gyrus was observed in Vim-/- mice with meningitis. The major inflammatory regulator α7 nAChR and several signaling molecules contributing to NF-κB activation (p65 and p-CamKII) were significantly reduced in the brain tissues of the Vim-/- mice with E. coli meningitis. Furthermore, Vim KO resulted in significant reduction in neuronal injury and in α7 nAChR-mediated calcium signaling. Conclusion/Significance Vimentin, a novel NF-κB regulator, plays a detrimental role in the host defense against meningitic infection by modulating the NF-κB signaling pathway to increase pathogen invasion, PMN recruitment, BBB permeability and neuronal

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

    PubMed

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

    2003-03-01

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

  1. Initial blood storage experiment

    NASA Technical Reports Server (NTRS)

    Surgenor, Douglas MACN.

    1988-01-01

    The possibility of conducting experiments with the formed elements of the blood under conditions of microgravity opens up important opportunities to improve the understanding of basic formed element physiology, as well as, contribution to improved preservation of the formed elements for use in transfusion. The physiological, biochemical, and physical changes of the membrane of the erythrocyte, platelet, and leukocyte was studied during storage under two specific conditions: standard blood bank conditions and microgravity, utilizing three FDA approved plastic bags. Storage lesions; red cell storage on Earth; platelet storage on Earth; and leukocyte storage Earth were examined. The interaction of biomaterials and blood cells was studied during storage.

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

    SciTech Connect

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

    1990-05-01

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

  3. TT virus (TTV) loads associated with different peripheral blood cell types and evidence for TTV replication in activated mononuclear cells.

    PubMed

    Maggi, F; Fornai, C; Zaccaro, L; Morrica, A; Vatteroni, M L; Isola, P; Marchi, S; Ricchiuti, A; Pistello, M; Bendinelli, M

    2001-06-01

    TT virus (TTV) loads associated with the peripheral blood cells of seven patients known to carry the virus in plasma were investigated by real-time PCR. Whereas red cells/platelets were uniformly negative, six and four patients yielded positive peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes, respectively, but viral titres were generally low. Fractionation of PBMCs into monocyte- and B, T4, and T8 lymphocyte-enriched subpopulations showed no pattern in the viral loads that might suggest the preferential association of TTV to one or more specific cell types. TTV-negative PBMCs absorbed measurable amounts of virus when incubated with infected plasma at 4 degrees C. Furthermore, cultures of TTV-negative phytohaemagglutinin-stimulated PBMCs exposed in vitro to virus-positive plasma and faecal extracts released considerable levels of infectious TTV into the supernatant fluid and the same was true for TTV-positive stimulated PBMCs. These results indicate that, whereas freshly harvested resting PBMCs seem to produce little, if any TTV, stimulated PBMCs actively replicate the virus.

  4. The effect of spermatozoa and seminal plasma on leukocyte migration into the uterus of gilts.

    PubMed

    Rozeboom, K J; Troedsson, M H; Molitor, T W; Crabo, B G

    1999-08-01

    Yorkshire x Landrace gilts were used to determine the effect of spermatozoa and seminal plasma on postbreeding uterine leukocyte influx. Estrus detection was performed with a boar at 12-h intervals following synchronization with 400 IU eCG and 200 IU of hCG. All gilts were AI once, 24 h after the detection of estrus following random assignment to a 2x2x3 factorial arrangement of treatments (sperm or sperm-free AI doses), AI dose medium (seminal plasma or PBS), and lavage time following AI. Gilts were treated with sperm (5x10(9) spermatozoa; SPZ; n = 30) or sperm-free (SF; n = 30) doses containing either 100 mL of seminal plasma (SP; n = 15/treatment) or PBS (n = 15/treatment). Uterine lavage was performed once on each gilt (n = 20/time) at one of three times after AI (6, 12, or 36 h) to determine the total number of uterine leukocytes. The leukocytes consisted predominately (92 to 99%) of polymorphonuclear neutrophilic granulocytes (PMN). There was an AI x medium interaction on uterine PMN numbers. The number of uterine PMN recovered from gilts inseminated with sperm suspended in PBS was greater than the number of PMN recovered from the uterine lumen of gilts inseminated with sperm in SP, SP alone, or PBS alone (P<.05). Furthermore, SP accelerated the rate of uterine clearance when suspended with sperm cells during the first 36 h following AI (P<.05). These results indicate that seminal plasma suppresses PMN migration into the uterus following breeding and enhances the rate of disappearance of uterine inflammation.

  5. Endogenous thrombospondin-1 regulates leukocyte recruitment and activation and accelerates death from systemic candidiasis.

    PubMed

    Martin-Manso, Gema; Navarathna, Dhammika H M L P; Galli, Susana; Soto-Pantoja, David R; Kuznetsova, Svetlana A; Tsokos, Maria; Roberts, David D

    2012-01-01

    Disseminated Candida albicans infection results in high morbidity and mortality despite treatment with existing antifungal drugs. Recent studies suggest that modulating the host immune response can improve survival, but specific host targets for accomplishing this goal remain to be identified. The extracellular matrix protein thrombospondin-1 is released at sites of tissue injury and modulates several immune functions, but its role in C. albicans pathogenesis has not been investigated. Here, we show that mice lacking thrombospondin-1 have an advantage in surviving disseminated candidiasis and more efficiently clear the initial colonization from kidneys despite exhibiting fewer infiltrating leukocytes. By examining local and systemic cytokine responses to C. albicans and other standard inflammatory stimuli, we identify a crucial function of phagocytes in this enhanced resistance. Subcutaneous air pouch and systemic candidiasis models demonstrated that endogenous thrombospondin-1 enhances the early innate immune response against C. albicans and promotes activation of inflammatory macrophages (inducible nitric oxide synthase⁺, IL-6(high), TNF-α(high), IL-10(low)), release of the chemokines MIP-2, JE, MIP-1α, and RANTES, and CXCR2-driven polymorphonuclear leukocytes recruitment. However, thrombospondin-1 inhibited the phagocytic capacity of inflammatory leukocytes in vivo and in vitro, resulting in increased fungal burden in the kidney and increased mortality in wild type mice. Thus, thrombospondin-1 enhances the pathogenesis of disseminated candidiasis by creating an imbalance in the host immune response that ultimately leads to reduced phagocytic function, impaired fungal clearance, and increased mortality. Conversely, inhibitors of thrombospondin-1 may be useful drugs to improve patient recovery from disseminated candidiasis.

  6. Both IL-1β and TNF-α Regulate NGAL Expression in Polymorphonuclear Granulocytes of Chronic Hemodialysis Patients

    PubMed Central

    Arena, Adriana; Stassi, Giovanna; Iannello, Daniela; Gazzara, Domenica; Calapai, Maria; Bisignano, Carlo; Bolignano, Davide; Lacquaniti, Antonio; Buemi, Michele

    2010-01-01

    Background. NGAL is involved in modulation of the inflammatory response and is found in the sera of uremic patients. We investigated whether hemodiafiltration (HDF) could influence the ability of polymorphonuclear granulocytes (PMGs) to release NGAL. The involvement of interleukin- (IL-)1β and tumor necrosis factor- (TNF-)α on NGAL release was evaluated. Methods. We studied end-stage renal disease (ESRD) patients at the start of dialysis (Pre-HDF) and at the end of treatment (Post-HDF) and 18 healthy subjects (HSs). Peripheral venous blood was taken from HDF patients at the start of dialysis and at the end of treatment. Results. PMGs obtained from ESRD patients were hyporesponsive to LPS treatment, with respect to PMG from HS. IL-1β and TNF-α produced by PMG from post-HDF patients were higher than those obtained by PMG from pre-HDF. Neutralization of IL-1β, but not of TNF-α, determined a clear-cut production of NGAL in PMG from healthy donors. On the contrary, specific induction of NGAL in PMG from uremic patients was dependent on the presence in supernatants of IL-1β and TNF-α. Conclusion. Our data demonstrate that in PMG from healthy subjects, NGAL production was supported solely by IL-1β, whereas in PMG from HDF patients, NGAL production was supported by IL-1β, TNF-α. PMID:21403867

  7. Recruitment of 99m-technetium- or 111-indium-labelled polymorphonuclear leucocytes in experimentally induced pyogranulomas in lambs

    SciTech Connect

    Guilloteau, L.; Pepin, M.; Pardon, P.; Le Pape, A. )

    1990-10-01

    The recruitment of polymorphonuclear leucocytes (PMNs) during the development of experimental pyogranulomas induced by Corynebacterium pseudotuberculosis was followed in nine male lambs by scintigraphic examination. Autologous blood PMNs were labelled with 99m-technetium or 111-indium and were re-injected intravenously into infected lambs. The functional properties of the labelled cells were monitored (1) in vitro by measuring their phagocytic and bactericidal activity against C. pseudotuberculosis and their chemotaxis under agarose, and (2) in vivo by following scintigraphically their capacity to accumulate in an inflammatory focus induced by intradermal injection of latex beads coated with Salmonella abortus equi lipopolysaccharide. Following inoculation of corynebacteria into the right ear of lambs, radioactive foci were observed to be localized in the right ear and in the draining lymph nodes during the 4 days following inoculation. Histopathological examination performed 32 h after inoculation confirmed the intense accumulation of PMNs at these sites. With the exception of one animal, which presented visible foci in the neck 14 days postinoculation, no radioactive foci were observed during the later phases of experimental infection, despite the presence of multiple pyogranulomas which were confirmed by bacteriological examination after necropsy of the lambs. Histopathological examination of these lesions revealed layers of fibroblasts, lymphocytes, and macrophages surrounding a necrotic centre. The results of these studies suggest that the contribution of PMNs during the chronic phase of inflammation is considerably reduced in comparison with the acute inflammatory phase of the infectious process.

  8. Leukocyte Recruitment in Inflammation: Basic Concepts and New Mechanistic Insights Based on New Models and Microscopic Imaging Technologies

    PubMed Central

    Leick, Marion; Azcutia, Veronica; Newton, Gail; Luscinskas, Francis W.

    2014-01-01

    The immune cell system is a critical component of host defense. Recruitment of immune cells to sites of infection, immune reaction, or injury is complex and involves coordinated adhesive interactions between the leukocyte and the endothelial cell monolayer that lines blood vessels. This article will review basic mechanisms in the recruitment of leukocytes to tissues, and then selectively review new concepts that are emerging based on advances in live cell imaging microscopy and mouse strains. These emerging concepts are altering the conventional paradigms of inflammatory leukocyte recruitment established in the early 1990’s. Indeed, recent publications have identified previously unrecognized contributions from pericytes and interstitial leukocytes and their secreted products that guide leukocytes to their targets. It is also notable that investigators have begun to design organs on a chip. Recent reports indicate that this avenue of research holds much promise. PMID:24562377

  9. Opsonic activity of anti-flagellar serum against Clostridium chauvoei by mouse polymorphonuclear leucocytes.

    PubMed

    Tamura, Y; Tanaka, M

    1987-05-01

    The role of anti-flagellar serum against Clostridium chauvoei in phagocytosis by mouse polymorphonuclear leucocytes was examined. Anti-flagellar serum markedly increased phagocytic rate against the flagellated strain Okinawa but not against a non-flagellated mutant (NFM) derived from the same strain, while anti-NFM serum increased the phagocytic rate against both strains. These results indicate that anti-flagellar serum exerts its protective effect by opsonic activity.

  10. 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

  11. 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

  12. 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.

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

    PubMed

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

    2016-08-05

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Schaefer, Antje; Hordijk, Peter L

    2015-07-01

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

  16. [Effect of proteflazid on TLRs expression by mononuclear leukocytes of peripheral blood and epithelial cells of mucous membranes and skin in patients with herpes-associated erythema multiforme and erythema annulare centrifugum].

    PubMed

    Sorokina, E V; Akhmatova, N K; Skhodova, S A

    2014-01-01

    The article reports survey data on 23 patients with erythemas, including 19 patients with herpes-associated erythema multiforme (HAEM) and 4 patients with Darier's erythema annulare centrifugum (DEAC). Patients in the initial state (baseline) and after two weeks of therapy with proteflazid were characterized by measuring the levels of Toll-like receptor (TLR) expression in peripheral blood mononuclear cells (PBMC) and in epithelial cells of the throat and the skin. The TLR expression in PBMC and skin was assessed by flow cytometry with monoclonal antibodies (ICA) (Caltag Laboratories, USA; Hycult Biotech, Netherlands) against relevant antigens. In addition, patients were also characterized by the content of subpopulations of lymphocytes expressing surface markers CD3, CD4, CD8, CD16, CD21, CD23, CD72, CD25, and HLA-DR in the peripheral blood, which was measured by flow cytometry. The therapy with proteflazid in patients with both HAEM and DEAC led to normalization of the level of both T-cell and B-cell immunity, which was manifested by an increase in the total number of lymphocytes, CD3+, CD4+, CD21+, and CD72+. Measurements of the dynamics of TLR expression in the course of immunotherapy showed an increase in the number of TLR 2, 3, 4, 7, 8, and 9 in PBMC (which was especially pronounced for TLR2) and in epithelium of the pharyngeal mucosa and skin (increased expression of TLR3, 7, and 9).

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

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

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

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

    PubMed

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

    2014-06-21

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

  19. Expansion of polymorphonuclear myeloid-derived suppressor cells in patients with end-stage renal disease may lead to infectious complications.

    PubMed

    Xing, Yan-Fang; Cai, Rui-Ming; Lin, Qu; Ye, Qing-Jian; Ren, Jian-Hua; Yin, Liang-Hong; Li, Xing

    2017-02-16

    Myeloid-derived suppressor cells (MDSCs) are recently identified immune suppressive cells in multiple chronic inflammations. Here, we investigated MDSCs in patients with end-stage renal disease (ESRD) and their clinical significance in these patients and healthy individuals (49 each). Polymorphonuclear and mononuclear MDSCs were investigated by flow cytometry. Patients with ESRD before hemodialysis presented a significantly higher level of polymorphonuclear MDSCs. Depletion of polymorphonuclear-MDSCs resolved T cell IFN-γ responses. By co-culture, T cell proliferation and the production of IFN-γ were abrogated by the addition of polymorphonuclear MDSCs in a dose-dependent manner. Both of these effects were reversed by a reactive oxygen species inhibitor. The levels of reactive oxygen species were higher in polymorphonuclear MDSCs derived from patients with ESRD than from normal individuals. The mRNA level of NOX2, the key protein complex responsible for reactive oxygen species production, was higher in ESRD-related polymorphonuclear MDSCs. The phospho-STAT3 level, a key activator of MDSCs, was higher in ESRD-related polymorphonuclear MDSCs. Finally, the polymorphonuclear MDSC level before and after hemodialysis was positively related to infectious diseases. Patients with ESRD were dichotomized into 2 groups by the amount of polymorphonuclear MDSCs. Patients with high levels of polymorphonuclear MDSCs presented with a higher incidence of infectious events. Thus, polymorphonuclear MDSCs were elevated in ESRD patients with strong immune-suppressive capability through a phospho-STAT3/reactive oxygen species pathway. Hence, polymorphonuclear MDSCs might increase the risk of infectious complications.

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

    PubMed

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

    2006-04-15

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

  1. 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

    The main objective of this study was to analyse the role and mode of action of the mast cell mediator histamine in leukocyte-endothelium interactions in small venules in vivo. For this purpose, we used a histological approach (combined with intravital microscopy) that allows studies of rapid mediator-induced venular leukocyte accumulation, reflecting leukocyte rolling, in the undisturbed microcirculation of the rat mesentery where rolling is normally absent. We first examined the relative importance of histamine and 5-hydroxytryptamine (5-HT) in acute mast cell-dependent leukocyte recruitment. The mast cell secretagogue compound 48/80 (i.p. for 15 min) induced a marked venular accumulation of polymorphonuclear leukocytes (PMNL) which was almost abolished by combined histamine1 (H1)- and histamine2 (H2)-receptor blockade. In contrast, the 5-HT-receptor antagonist methysergide was inactive in this regard. Moreover, exogenous 5-HT was less active than exogenous histamine in evoking venular PMNL accumulation (histamine response dose-dependent; 5-HT response bell shaped). Prostaglandin D2 did not cause PMNL accumulation. The venular PMNL response to exogenous histamine peaked between 15 min and 1 h, was still significantly elevated at 2 h, and then returned to prechallenge values after 3 h. At all time points, the histamine-induced PMNL accumulation was nearly abolished by i.v. treatment with the polysaccharide fucoidin (which blocks rolling but not firm adhesion per se), suggesting that the PMNL response to histamine was due to rolling rather than firm adhesion over the entire 3 h period. At no time point did histamine trigger accumulation of mononuclear leukocytes (MNL). To examine the role of histamine-receptors in the histamine-induced PMNL accumulation (i.e. rolling), the animals were pretreated with diphenhydramine (H1-receptor antagonist), cimetidine, or ranitidine (H2-receptor antagonists). Diphenhydramine alone inhibited the venular PMNL response to

  2. Blood leukocytes from benznidazole-treated indeterminate chagas disease patients display an overall type-1-modulated cytokine profile upon short-term in vitro stimulation with trypanosoma cruzi antigens

    PubMed Central

    2012-01-01

    Background Benznidazole (Bz)-chemotherapy is recommended to prevent Chagas disease progression, despite its limited efficacy during chronic disease. However, the host mechanisms underlying these benefits still remain to be elucidated. Methods In this study, we have used short-term whole blood cultures to describe the cytokine profile of Bz-treated Indeterminate Chagas disease patients-(INDt) as compared to untreated patients-(IND). Results Our findings showed that IND presented increased levels of IL-10+neutrophils, IL-12+ and IL-10+monocytes and IFN-γ+NK-cells. Moreover, IND showed slight increase of IL-4+CD4+T-cells and enhanced levels of IL-10+CD8+T-cells and B-cells. Additional analysis of cytokine Low and High producers also highlighted the presence of High cytokine producers within IND, including IL-10 from CD4+ T-cells and IFN-γ from CD8+ T-cells, as compared to NI. The Bz-treatment lead to an overall cytokine down-regulation in the innate and adaptive compartments, including low levels of IL-12+ and IL-10+neutrophils and monocytes, IFN-γ+NK-cells, IL-12+, TNF-α+, IFN-γ+ and IL-5+CD4+T-cells and IL-10+B-cells, along with basal levels of cytokine-expressing CD8+T-cells in INDt as compared to IND. The in vitro antigen stimulation shifted the cytokine profile toward a type 1-modulated profile, with increased levels of IL-12+ and IL-10+ monocytes, IFN-γ+ and IL-4+NK-cells along with TNF-α+ and IFN-γ+CD8+T-cells. Analysis of Low and High cytokine producers, upon in vitro antigen stimulation, further confirm these data. Conclusion Together, our findings showed that the Bz treatment of Indeterminate Chagas’ disease patients shifts the cytokine patterns of peripheral blood monocytes, NK-cells and CD8+ T-cells towards a long-lasting Type-1-modulated profile that could be important to the maintenance of a non-deleterious immunological microenvironment. PMID:22625224

  3. 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.

  4. 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

  5. Myeloperoxidase deficient polymorphonuclear leucocytes in leukaemia and allied disorders.

    PubMed

    Bendix-Hansen, K

    1988-12-01

    This thesis is a survey of nine previously published articles on MPO deficient PMN. The incidences in leukaemia and allied disorders of the presence of this abnormal subpopulation of mature neutrophils and the relationship to clinical course in AML, susceptibility to infections in AML, FAB classification in AML and MDS, cytogenetically defined aberrations in MDS and morphometrical characteristics were investigated. The aims of the studies were to examine the diagnostic as well as the prognostic value of the parameter, to examine the usefulness of the parameter as an predictive indicator of CR and relapse in AML and to examine the concept that MPO deficient PMN may originate from leukaemic precursors. MPO deficient PMN were found to occur in a minor number (less than 4% of the total number of PMN) in normal humans and the incidences of an abnormal number (greater than 4%) were found to be about 40% in AML (I, II, III, IV, VIII), 60% in CML (I, VII), 30% in MPD other than CML (VII) and 30% in MDS (V). The highest incidences in AML were found in the FAB subtypes possessing the most myeloid differentiation potential i.e. FAB M2 and FAB M4 (IV). In ALL, CLL, HCL, Hodgkin's disease, anaemia not related to leukaemia and leukaemoid reactions the incidences all were 0% (I, unpublished data). The abnormal MPO deficient PMN subpopulation, if present, disappeared when CR was achieved and reappeared when relapse eventually was developed (II, VIII). In both situations serial determinations showed that the change occurred before the usual routine blood examinations predicted CR and relapse; several days and several months prior, respectively (VIII). The probability of obtaining CR was lower in the AML patients with the abnormal subpopulation and the risk of developing relapse higher than in AML patients without the anomaly (II, VIII). These differences were not statistically significant, however. AML patients, showing an increased number of MPO deficient PMN, revealed a

  6. 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.

  7. Preparation of surface-modified poly(butylene terephthalate) nonwovens and their application as leukocyte removal filters.

    PubMed

    Kim, Eun Jin; Yeo, Gwu-Dong; Pai, Chaul-Min; Kang, Inn-Kyu

    2009-08-01

    Blood transfusion-related adverse reactions have been reported to be caused by leukocytes in blood products. It is now generally accepted that it would be highly desirable to reduce leukocytes level as low as possible. In this study, melt-blown poly(butylene terephthalate) nonwoven (PBT-NW) was treated with a hydroxyapatite (HA) surface-modification method for removal of leukocytes from blood components. Acrylic acid was graft-polymerized onto the surface of the PBT-NW after oxygen plasma glow discharge treatment. The PBT-NW surface was covered with a thin layer of HA produced by immersing the polymer surface in an aqueous solution containing high concentrations of PO(4) (3-) and Ca(2+) after graft-polymerization of acrylic acid, which provided the nucleus for HA crystallization. The surface was characterized using water contact angles, attenuated total reflection-Fourier transform infrared (ATR-FT-IR), and electron spectroscopy for chemical analysis. When filtration was performed with a unit of red blood cell concentrates, HA-deposited PBT-NW (PBT-HA) removed 98.5% of the leukocytes and recovered 99.5% of the erythrocytes, suggesting that HA-deposited PBT-NW is a very promising blood filter for selective removal of leukocytes.

  8. Effects of intra-mammary bacterial infection with coagulase negative staphylococci and stage of lactation on shedding of epithelial cells and infiltration of leukocytes into milk: comparison among cows, goats and sheep.

    PubMed

    Leitner, Gabriel; Merin, Uzi; Krifucks, Oleg; Blum, Shlomo; Rivas, Ariel L; Silanikove, Nissim

    2012-06-30

    The effects of mammary gland bacterial infection and stage of lactation on leukocyte infiltration into the mammary gland were compared among cows, goats and sheep. Animals were at two stages of lactation: mid or late. In mid-lactation animals, bacterial-free glands and coagulase negative Staphylococcus (CNS)-infected glands were compared. In late lactation only uninfected glands were studied. Of mid-lactation bacteria-free animals, goats had the highest number of leukocytes and % polymorphonuclears (PMNs), whereas sheep had the lowest and leukocytes number in cows were intermediate between sheep and goats. Based on %PMN, two cell clusters were found in sheep, which overlapped with the parallel cell clusters of cows and goats, but with a slightly higher number of leukocytes in each cell cluster. At late lactation, goats had higher values for %PMN and leukocyte numbers in comparison to cows, which had a similar cellular profile to sheep. The cellular immune response to CNS infection was similar for the three animal species, although the number of cells was different, while the basal cell level at mid-lactation and especially at the end of lactation was species specific.

  9. Insulin Resistance in PCOS Patients Enhances Oxidative Stress and Leukocyte Adhesion: Role of Myeloperoxidase

    PubMed Central

    Victor, Victor M.; Rovira-Llopis, Susana; Bañuls, Celia; Diaz-Morales, Noelia; Martinez de Marañon, Arantxa; Rios-Navarro, Cesar; Alvarez, Angeles; Gomez, Marcelino; Rocha, Milagros; Hernández-Mijares, Antonio

    2016-01-01

    Cardiovascular diseases and oxidative stress are related to polycystic ovary syndrome (PCOS) and insulin resistance (IR). We have evaluated the relationship between myeloperoxidase (MPO) and leukocyte activation in PCOS patients according to homeostatic model assessment of IR (HOMA-IR), and have explored a possible correlation between these factors and endocrine and inflammatory parameters. This was a prospective controlled study conducted in an academic medical center. The study population consisted of 101 PCOS subjects and 105 control subjects. We divided PCOS subjects into PCOS non-IR (HOMA-IR<2.5) and PCOS IR (HOMA-IR>2.5). Metabolic and anthropometric parameters, total and mitochondrial reactive oxygen species (ROS) production, MPO levels, interactions between human umbilical vein endothelial cells and leukocytes, adhesion molecules (E-selectin, ICAM-1 and VCAM-1) and proinflammatory cytokines (IL-6 and TNF-α) were evaluated. Oxidative stress was observed in PCOS patients, in whom there was an increase in total and mitochondrial ROS production and MPO levels. Enhanced rolling flux and adhesion, and a decrease in polymorphonuclear cell rolling velocity were also detected in PCOS subjects. Increases in IL-6 and TNF-α and adhesion molecules (E-selectin, ICAM-1 and VCAM-1) were also observed, particularly in the PCOS IR group, providing evidence that inflammation and oxidative stress are related in PCOS patients. HOMA-IR was positively correlated with hsCRP (p<0.001, r = 0.304), ROS production (p<0.01, r = 0.593), leukocyte rolling flux (p<0.05, r = 0.446), E-selectin (p<0.01, r = 0.436) and IL-6 (p<0.001, r = 0.443). The results show an increase in the rate of ROS and MPO levels in PCOS patients in general, and particularly in those with IR. Inflammation in PCOS induces leukocyte-endothelium interactions and a simultaneous increase in IL-6, TNF-α, E-selectin, ICAM-1 and VCAM-1. These conditions are aggravated by the presence of IR. PMID:27007571

  10. Insulin Resistance in PCOS Patients Enhances Oxidative Stress and Leukocyte Adhesion: Role of Myeloperoxidase.

    PubMed

    Victor, Victor M; Rovira-Llopis, Susana; Bañuls, Celia; Diaz-Morales, Noelia; Martinez de Marañon, Arantxa; Rios-Navarro, Cesar; Alvarez, Angeles; Gomez, Marcelino; Rocha, Milagros; Hernández-Mijares, Antonio

    2016-01-01

    Cardiovascular diseases and oxidative stress are related to polycystic ovary syndrome (PCOS) and insulin resistance (IR). We have evaluated the relationship between myeloperoxidase (MPO) and leukocyte activation in PCOS patients according to homeostatic model assessment of IR (HOMA-IR), and have explored a possible correlation between these factors and endocrine and inflammatory parameters. This was a prospective controlled study conducted in an academic medical center. The study population consisted of 101 PCOS subjects and 105 control subjects. We divided PCOS subjects into PCOS non-IR (HOMA-IR<2.5) and PCOS IR (HOMA-IR>2.5). Metabolic and anthropometric parameters, total and mitochondrial reactive oxygen species (ROS) production, MPO levels, interactions between human umbilical vein endothelial cells and leukocytes, adhesion molecules (E-selectin, ICAM-1 and VCAM-1) and proinflammatory cytokines (IL-6 and TNF-α) were evaluated. Oxidative stress was observed in PCOS patients, in whom there was an increase in total and mitochondrial ROS production and MPO levels. Enhanced rolling flux and adhesion, and a decrease in polymorphonuclear cell rolling velocity were also detected in PCOS subjects. Increases in IL-6 and TNF-α and adhesion molecules (E-selectin, ICAM-1 and VCAM-1) were also observed, particularly in the PCOS IR group, providing evidence that inflammation and oxidative stress are related in PCOS patients. HOMA-IR was positively correlated with hsCRP (p<0.001, r = 0.304), ROS production (p<0.01, r = 0.593), leukocyte rolling flux (p<0.05, r = 0.446), E-selectin (p<0.01, r = 0.436) and IL-6 (p<0.001, r = 0.443). The results show an increase in the rate of ROS and MPO levels in PCOS patients in general, and particularly in those with IR. Inflammation in PCOS induces leukocyte-endothelium interactions and a simultaneous increase in IL-6, TNF-α, E-selectin, ICAM-1 and VCAM-1. These conditions are aggravated by the presence of IR.

  11. Induced expression and functional effects of aquaporin-1 in human leukocytes in sepsis

    PubMed Central

    2013-01-01

    Introduction Gene expression profiling was performed via DNA microarrays in leukocytes from critically ill trauma patients nonseptic upon admission to the ICU, who subsequently developed either sepsis (n = 2) or severe sepsis and acute respiratory distress syndrome (n = 3). By comparing our results with published expression profiling studies in animal models of sepsis and lung injury, we found aquaporin-1 to be differentially expressed across all studies. Our aim was to determine how the water channel aquaporin-1 is involved in regulating the immune response in critically ill patients during infection acquired in the ICU. Methods Following the results of the initial genetic screening study, we prospectively followed aquaporin-1 leukocyte expression patterns in patients with ICU-acquired sepsis who subsequently developed septic shock (n = 16) versus critically ill patients who were discharged without developing sepsis (n = 13). We additionally determined aquaporin-1 expression upon lipopolysaccharide (LPS) exposure and explored functional effects of aquaporin-1 induction in polymorphonuclear granulocytes (PMNs). Results Leukocyte aquaporin-1 expression was induced at the onset of sepsis (median 1.71-fold increase; interquartile range: 0.99 to 2.42, P = 0.012 from baseline) and was further increased upon septic shock (median 3.00-fold increase; interquartile range: 1.20 to 5.40, P = 0.023 from sepsis, Wilcoxon signed-rank test); no difference was observed between baseline and discharge in patients who did not develop sepsis. Stimulation of PMNs by LPS led to increased expression of aquaporin-1 in vitro, which could be abrogated by the NF-κB inhibitor EF-24. PMN hypotonic challenge resulted in a transient increase of the relative cell volume, which returned to baseline after 600 seconds, while incubation in the presence of LPS resulted in persistently increased cell volume. The latter could be abolished by blocking aquaporin-1 with mercury and restored by incubation

  12. Response of laying hens to feeding low-protein amino acid-supplemented diets under high ambient temperature: performance, egg quality, leukocyte profile, blood lipids, and excreta pH.

    PubMed

    Torki, Mehran; Mohebbifar, Ahmad; Ghasemi, Hossein Ali; Zardast, Afshin

    2015-05-01

    An experiment was conducted to determine whether, by using a low-protein amino acid-supplemented diet, the health status, stress response, and excreta quality could be improved without affecting the productive performance of heat-stressed laying hens. The requirements for egg production, egg mass, and feed conversion ratio were also estimated using second-order equations and broken-line regression. A total of 150 Lohmann Selected Leghorn (LSL-Lite) hens were divided randomly into five groups of 30 with five replicates of six hens. The hens were raised for an 8-week period (52 to 60 weeks) in wire cages situated in high ambient temperature in an open-sided housing system. The five experimental diets (ME; 2,720 kcal/kg) varied according to five crude protein (CP) levels: normal-CP diet (control, 16.5 % CP) and low-CP diets containing 15.0, 13.5, 12.0, or 10.5 % CP. All experimental diets were supplemented with crystalline amino acids at the levels sufficient to meet their requirements. The results showed that under high temperature conditions, all productive performance and egg quality parameters in the birds fed with 15.0, 13.5, and 12.0 % CP diets were similar to those of birds fed with control diet (16.5 % CP), whereas feeding 10.5 % CP diet significantly decreased egg production and egg mass. Estimations of requirements were of 13.93 and 12.77 % CP for egg production, 14.62 and 13.22 % CP for egg mass, and 12.93 and 12.26 % CP for feed conversion ratio using quadratic and broken-line models, respectively. Egg yolk color index, blood triglyceride level, and excreta acidity were also significantly higher in birds fed with 12.0 and 10.5 % CP diets compared with those of control birds. The heterophil to lymphocyte ratio, as a stress indicator, was significantly decreased by 15.0, 13.5, and 12 % CP diets. On the basis of our findings, reducing dietary CP from 16.5 to 12.0 % and supplementing the diets with the essential amino acids showed merit for improving the

  13. Response of laying hens to feeding low-protein amino acid-supplemented diets under high ambient temperature: performance, egg quality, leukocyte profile, blood lipids, and excreta pH

    NASA Astrophysics Data System (ADS)

    Torki, Mehran; Mohebbifar, Ahmad; Ghasemi, Hossein Ali; Zardast, Afshin

    2015-05-01

    An experiment was conducted to determine whether, by using a low-protein amino acid-supplemented diet, the health status, stress response, and excreta quality could be improved without affecting the productive performance of heat-stressed laying hens. The requirements for egg production, egg mass, and feed conversion ratio were also estimated using second-order equations and broken-line regression. A total of 150 Lohmann Selected Leghorn (LSL-Lite) hens were divided randomly into five groups of 30 with five replicates of six hens. The hens were raised for an 8-week period (52 to 60 weeks) in wire cages situated in high ambient temperature in an open-sided housing system. The five experimental diets (ME; 2,720 kcal/kg) varied according to five crude protein (CP) levels: normal-CP diet (control, 16.5 % CP) and low-CP diets containing 15.0, 13.5, 12.0, or 10.5 % CP. All experimental diets were supplemented with crystalline amino acids at the levels sufficient to meet their requirements. The results showed that under high temperature conditions, all productive performance and egg quality parameters in the birds fed with 15.0, 13.5, and 12.0 % CP diets were similar to those of birds fed with control diet (16.5 % CP), whereas feeding 10.5 % CP diet significantly decreased egg production and egg mass. Estimations of requirements were of 13.93 and 12.77 % CP for egg production, 14.62 and 13.22 % CP for egg mass, and 12.93 and 12.26 % CP for feed conversion ratio using quadratic and broken-line models, respectively. Egg yolk color index, blood triglyceride level, and excreta acidity were also significantly higher in birds fed with 12.0 and 10.5 % CP diets compared with those of control birds. The heterophil to lymphocyte ratio, as a stress indicator, was significantly decreased by 15.0, 13.5, and 12 % CP diets. On the basis of our findings, reducing dietary CP from 16.5 to 12.0 % and supplementing the diets with the essential amino acids showed merit for improving the

  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. 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

  16. Hemoglobin stimulates mononuclear leukocytes to release interleukin-8 and tumor necrosis factor alpha.

    PubMed

    McFaul, S J; Bowman, P D; Villa, V M; Gutierrez-Ibanez, M J; Johnson, M; Smith, D

    1994-11-01

    Incubation of human mononuclear leukocytes (MNL) with human stroma-free hemolysate (SFH), purified adult hemoglobin Ao (HbAo), and oxidized HbAo (METHb) caused MNL to release compounds into the supernate that mediated neutrophil (polymorphonuclear leukocytes, PMN) chemotaxis and PMN adherence to human umbilical vein endothelial cells (HUVEC). Chemotaxis and PMN adherence to HUVEC were reduced significantly when supernates were preincubated with neutralizing antibodies to interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-alpha), respectively, suggesting that IL-8 and TNF-alpha played significant roles in mediating these activities. Greatest chemotactic activity was observed in supernates of MNL treated with HbAo; while greatest PMN/endothelial cell (EC) adherence activity was observed in supernates of MNL treated with METHb. Furthermore, PMN/EC adherence activity was a function of METHb content in each hemoglobin solution. PMN chemotaxis, PMN adherence to HUVEC, and cytokine release increased as a function of increasing incubation time. Chemotactic activity was detected in HbAo-treated and METHb-treated MNL supernates after incubation for 6 hours and was maximal by 10 hours. IL-8 was detected in both HbAo and METHb-MNL supernates by 4 hours. PMN/EC adherence activity was detected in HbAo-MNL supernates at 10 hours and in METHb-MNL supernates at 4 hours. TNF-alpha was detected in METHb and HbAo-MNL supernates at 4 and 12 hours, respectively. These results suggest that hemoglobin solutions stimulate MNL to release IL-8 and TNF-alpha in quantities sufficient to induce PMN chemotaxis and PMN adherence to HUVEC. This is a US government work. There are no restrictions on its use.

  17. COMPARISON OF TOTAL LEUKOCYTE QUANTIFICATION METHODS IN FREE-LIVING GALAPAGOS TORTOISES (CHELONOIDIS SPP.).

    PubMed

    Sheldon, Julie D; Stacy, Nicole I; Blake, Stephen; Cabrera, Fredy; Deem, Sharon L

    2016-03-01

    Reptile hematologic data provide important health information for conservation efforts of vulnerable wildlife species such as the Galapagos tortoise (Chelonoidis spp.). Given the reported discrepancies between manual leukocyte counts for nonmammalian species, two manual leukocyte quantification methods, the Natt and Herrick's (NH) and the Eopette (EO), were compared to white blood cell (WBC) estimates from blood films of 42 free-living, clinically healthy, adult female Galapagos tortoises. To investigate the effects of delay in sample processing, estimated WBC counts and leukocyte differentials were compared for blood films prepared at time of collection under field conditions (T0) to blood films prepared from samples that were stored for 18-23 hr at 4°C in the laboratory (T1). Passing-Bablok regression analysis revealed no constant or proportional error between the NH and WBC estimates (T0 and T1) with slopes of 1.1 and 0.9, respectively. However both constant and proportional errors were present between EO and WBC estimates (T0 and T1) with slopes of 3.1 and 2.7, respectively. Bland Altman plots also showed agreement between the NH and WBC estimates where the points fell within the confidence-interval limit lines and were evenly distributed about the mean. In contrast, the EO and WBC estimate comparisons showed numerous points above the upper limit line, especially at higher concentrations. WBC estimates obtained from T0 and T1 films were in agreement, whereas heterophil and monocyte percentages based on differentials were not. Cell morphology and preservation were superior in T0 blood films because thrombocytes exhibited swelling after storage, becoming difficult to differentiate from lymphocytes. In this study, the highest quality and most reliable hematologic data in Galapagos tortoises were obtained by combining immediate blood film preparation with the NH leukocyte quantification method and a confirmatory WBC estimate from the blood film.

  18. Comparison of Venous and Capillary Differential Leukocyte Counts Using a Standard Hematology Analyzer and a Novel Microfluidic Impedance Cytometer

    PubMed Central

    Harris, Scott; Spencer, Daniel; van Berkel, Cees; Morgan, Hywel

    2012-01-01

    Capillary blood sampling has been identified as a potentially suitable technique for use in diagnostic testing of the full blood count (FBC) at the point-of-care (POC), for which a recent need has been highlighted. In this study we assess the accuracy of capillary blood counts and evaluate the potential of a miniaturized cytometer developed for POC testing. Differential leukocyte counts in the normal clinical range from fingerprick (capillary) and venous blood samples were measured and compared using a standard hematology analyzer. The accuracy of our novel microfluidic impedance cytometer (MIC) was then tested by comparing same-site measurements to those obtained with the standard analyzer. The concordance between measurements of fingerprick and venous blood samples using the standard hematology analyzer was high, with no clinically relevant differences observed between the mean differential leukocyte counts. Concordance data between the MIC and the standard analyzer on same-site measurements presented significantly lower leukocyte counts determined by the MIC. This systematic undercount was consistent across the measured (normal) concentration range, suggesting that an internal correction factor could be applied. Differential leukocyte counts obtained from fingerprick samples accurately reflect those from venous blood, which confirms the potential of capillary blood sampling for POC testing of the FBC. Furthermore, the MIC device demonstrated here presents a realistic technology for the future development of FBC and related tests for use at the site of patient care. PMID:23028467

  19. 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.

  20. 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