Acute pain and its management.
Explore the source record for details and available documents.
Acute Disease↗
Biomedical subjects
B A Walker
Publications and source records attributed to B A Walker.
The use of flow cytometry to measure neutrophil function.
Neutrophils are important professional phagocytic cells that provide the host with a first line of defense against acute bacterial and fungal diseases and recurrent, severe or unusual infections are associated with inherited defects of neutrophil function. Furthermore, abundant evidence links inappropriate neutrophil-mediated tissue damage to the pathogenesis of conditions such as acute respiratory distress syndrome, septicemia with multiorgan failure, ischemia-reperfusion injury and rheumatoid arthritis. Flow cytometry has been increasingly used to evaluate the functional capabilities of neutrophils. In this review, we discuss the use of flow cytometry to assess neutrophil functional responses including calcium mobilization, F-actin assembly, adhesion, aggregation, degranulation, phagocytosis and reactive oxygen species (ROS) production. The use of flow cytometry to identify neutrophil priming is also discussed. The advantage of flow cytometry is that the majority of neutrophil functions can be measured using a small volume of whole blood that reduces artifactual changes in function caused by purification procedures. The advent of numerous new fluorochromes and multiparametric analysis allows the simultaneous measurement of several neutrophil functions in the same population of cells. Flow cytometric analysis provides a rapid screen for abnormalities of neutrophil function and reflects more accurately their behavior in vivo.
Animals↗
The chicken B locus is a minimal essential major histocompatibility complex.
Here we report the sequence of the region that determines rapid allograft rejection in chickens, the chicken major histocompatibility complex (MHC). This 92-kilobase region of the B locus contains only 19 genes, making the chicken MHC roughly 20-fold smaller than the human MHC. Virtually all the genes have counterparts in the human MHC, defining a minimal essential set of MHC genes conserved over 200 million years of divergence between birds and mammals. They are organized differently, with the class III region genes located outside the class II and class I region genes. The absence of proteasome genes is unexpected and might explain unusual peptide-binding specificities of chicken class I molecules. The presence of putative natural killer receptor gene(s) is unprecedented and might explain the importance of the B locus in the response to the herpes virus responsible for Marek's diseases. The small size and simplicity of the chicken MHC allows co-evolution of genes as haplotypes over considerable periods of time, and makes it possible to study the striking MHC-determined pathogen-specific disease resistance at the molecular level.
Amino Acid Sequence↗
Vitamin D binding protein variants and the risk of COPD.
Although the development of chronic obstructive pulmonary disease (COPD) in smokers shows genetic susceptibility, only alpha1-antitrypsin deficiency has been identified as a definite genetic risk factor. There have been three previous studies in which associations between Gc-globulin phenotypes and COPD have been investigated. Although some data suggest an association, the results were inconclusive. Because smoking is the major risk factor for COPD, it may have been a confounding factor in previous studies. We have investigated Gc-globulin genotypic frequencies among 75 COPD patients and 64 nonobstructed controls. Both groups had significant smoking histories: pack-years (mean +/- SD) of 52 +/- 30 and 48 +/- 27, respectively. The results show that homozygosity for the Gc2 allele is protective against COPD (OR = 0.17, 95% CI = 0.03 to 0.83). There were no differences between genotypes for lung elastic recoil values or for the level of upstream airway resistance. Gc-globulin can enhance complement (C5a)-mediated neutrophil chemotaxis. Because neutrophils play a role in parenchymal destruction and airway inflammation, we examined whether Gc-globulin's ability to enhance neutrophil chemotaxis varied with genotype. We found no difference among genotypes with respect to neutrophil chemotaxis suggesting that the protective effect of the Gc2 allele is mediated through a different mechanism.
Airway Resistance↗
Adenosine A2a receptor activation delays apoptosis in human neutrophils.
Adenosine has both pro- and anti-inflammatory effects on neutrophils. Exposure of cultured neutrophils to 2-chloroadenosine or 5'-N-ethylcarboxamidoadenosine (NECA) decreased apoptosis after 16 h, with half-maximal responses for NECA and 2-chloroadenosine of 7.1 +/- 7.7 and 59.0 +/- 32.0 nM, respectively. Adenosine receptor agonists exhibited a rank order of potency for decreasing apoptosis of 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) > NECA > or = 2-chloro-N6-cyclopentyladenosine >> 2-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide, which is consistent with the affinity order profile established for human A2a receptors. The reduction in apoptosis in cultured neutrophils at 16 h by CGS 21680 was due to a delay in apoptosis. The addition of CGS 21680 (100 nM) increased the half-life for the appearance of apoptosis from 10.9 +/- 3.1 to 21.0 +/- 1.0 h. Addition of the non-xanthine phosphodiesterase inhibitor 4-(3-butoxy-4-methoxybenzyl)-2-imidazalidinone (Ro-20-1724; 1 microM) enhanced the effects of CGS 21680 at all agonist concentrations. PGE1 (10 microM), PGE2 (0.1-10 microM), and dibutyryl cAMP (5-500 microM) all decreased apoptosis in cultured neutrophils. The enhancement of the effect of adenosine by a phosphodiesterase inhibitor and the similar actions of PGE2, PGE1, and dibutyryl cAMP suggest that this decrease in apoptosis may be mediated by a cAMP-dependent pathway.
Adenosine↗
Polymorphonuclear leukocytes L-selectin expression decreases as they age in circulation.
We recently reported that L-selectin expression increases on circulating polymorphonuclear leukocytes (PMN) during active bone marrow release, which suggests that older cells in the circulation have lower levels of L-selectin than those recently released from the bone marrow. The present study was designed to test the hypothesis that L-selectin expression reduces on PMN as they age in the circulation. In vitro studies using flow cytometry showed that PMN L-selectin decreased to 14.6 +/- 2.3% of baseline during a 24-h incubation at 37 degrees C. To test this hypothesis in vivo, rabbit PMN, labeled in vivo with 5'-bromo-2-deoxyuridine (PMN-brdU) in donor animals, were infused into recipient rabbits as whole blood (n = 5) and followed over 24 h in the circulation with a double immunolabeling technique. These results showed that the fraction of the L-selectin-negative PMN-BrdU in the circulation increased with time (P < 0.001), and nearly all of the PMNBrdU in the circulation were L-selectin negative after 24 h. Removal of L-selectin from the surface of PMN-BrdU with chymotrypsin before infusion did not change their rate of removal from the circulation (half-life or t 1/2 262 vs. 296 min, P = NS). We conclude that there is a continuous loss of L-selectin from PMN during their life span in the circulation, which supports the hypothesis that L-selectin expression decreases on PMN as they age.
Animals↗
Apoptosis in circulating PMN: increased susceptibility in L-selectin-deficient PMN.
Previous work from our laboratory has shown that polymorphonuclear leukocytes (PMN) lose L-selectin as they age in the circulation. The present study was designed to examine the relationship between PMN age and susceptibility to apoptosis in the circulation using L-selectin as a marker of PMN age in rabbits. L-selectin-deficient leukocytes were separated from a mixed population of PMN in leukocyte-rich plasma using magnetic beads. Apoptosis was measured both with both morphological criteria and by determining the level of DNA fragmentation. The L-selectin-deficient cells separated in vitro showed morphological features of apoptosis (P < 0.01) and had higher levels of DNA fragmentation (P < 0.01) than the mixed population of PMN from which they were obtained. To determine if aging had a similar effect in vivo, PMN were labeled in the bone marrow with 5'-bromo-2'-deoxyuridine (BrdU) and L-selectin levels (immunocytochemistry) and DNA fragmentation (sandwich enzyme-linked immunosorbent assay) were measured in BrdU-labeled PMN in peripheral blood. The results showed that the peak release of BrdU-labeled PMN from the bone marrow into peripheral blood was associated with high levels of L-selectin expression, and these PMN had the lowest levels of DNA fragmentation. These results confirm that the level of L-selectin expression can be used as a marker of cell age and extend this observation by showing that aging in the circulation is associated with an increased susceptibility to apoptosis.
Animals↗
Effect of mechanical deformation on structure and function of polymorphonuclear leukocytes.
The present studies were designed to test the hypothesis that mechanical deformation of polymorphonuclear leukocytes (PMN) leads to functional changes that might influence their transit in the pulmonary capillaries. Human leukocytes were passed through 5- or 3-micron-pore polycarbonate filters under controlled conditions. Morphometric analysis showed that the majority of PMN were deformed and that this deformation persisted longer after filtration through 3-micron filters than through 5-micron filters (P < 0.05) but did not result in the cytoskeletal polarization characteristic of migrating cells. Flow cytometric studies of the filtered PMN showed that there was a transient increase in the cytosolic free Ca2+ concentration after both 3- and 5-micron filtration (P < 0.01) with an increase in F-actin content after 3-micron filtration (P < 0.05). Although L-selectin expression on PMN was not changed by either 5- or 3-micron filtration, CD18 and CD11b were increased by 3-micron filtration (P < 0.05). Priming of the PMN with N-formyl-methionyl-leucyl-phenylalanine (0.5 nM) before filtration resulted in an increase of CD11b by both 5 (P < 0.05)- and 3-micron (P < 0.01) filtration. Neither 5- nor 3-micron filtration induced hydrogen peroxide production. We conclude that mechanical deformation of PMN, similar to what occurs in the pulmonary microvessels, induces both structural and functional changes in the cells, which might influence their passage through the pulmonary capillary bed.
Actins↗
Adenosine A3 receptor expression and function in eosinophils.
The A3 adenosine receptor is widely expressed in human tissues with the most abundant expression in the lung and liver, but the predominant cellular localization and functions of this receptor in humans are unknown. Since adenosine influences the activation of circulating and resident inflammatory cells within the lung and leads to exaggerated airway narrowing in individuals with inflammatory airway disorders, we hypothesized that A3 receptor gene expression is localized to inflammatory cells and that gene expression is upregulated in airway inflammation. Lung and airway tissue were obtained at thoracotomy from nonsmoking subjects and subjects with inflammatory airway disorders associated with tobacco smoke or asthma. In situ hybridization identified A3 receptors in mesenchymal cells and eosinophils within the lamina propria of the airways and the adventitia of blood vessels, but not in mast cells. A3 receptor transcripts were highly expressed in peripheral blood eosinophils purified from atopic donors (6.36 +/- 0.60 pg/microg total RNA) in comparison with neutrophils (0.26 +/- 0.06 pg/microg) or mononuclear cells (0.9 +/- 0.15 pg/microg). Mean A3 receptor transcript abundance was greater in lung tissue from subjects with airway inflammation (0.33 +/- 0.04 pg/microg total RNA) than in normal lung (0.24 +/- 0.03 pg/microg total RNA, P = 0.035). The A3 receptor agonist N6-(4-amino-3-iodobenzyl)adenosine dose-dependently inhibited platelet activating factor-induced eosinophil chemotaxis to a maximum of 41%. This inhibitory effect was completely abolished by addition of the A3 receptor selective antagonist 3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl-1-propylxanthine. We conclude that A3 receptors are primarily expressed on eosinophils in human lung, where they mediate inhibition of eosinophil chemotaxis. Specific A3 receptor ligands may be useful agents in the treatment of eosinophil-dependent diseases such as asthma and rhinitis.
Adenosine↗
Effects of adenosine on guinea pig pulmonary eosinophils.
Extracellular adenosine has pharmacological activity on a wide variety of cell types and may play an important role as an inflammatory modulator with both pro- and anti-inflammatory activities. These studies examine the effects of adenosine on guinea pig pulmonary eosinophils. Adenosine alone did not directly induce superoxide (O2-) production. Pretreatment with adenosine primed the O2- response of guinea pig pulmonary eosinophils following the addition of 1 or 10 microM platelet-activating factor (PAF). Priming was seen at adenosine concentrations greater than 1 microM and was maximal at 100 microM. At this maximal dose, adenosine priming increased the O2- response to 1 microM and 10 microM PAF by 86% and 51%, respectively. Priming by adenosine was not seen when ionomycin or phorbol myristate acid (PMA) were used as agonists. In fura-2 loaded eosinophils, the addition of 100 microM adenosine resulted in a small but significant rise in intracellular calcium of 54.4 +/- 9.2 nM above baseline. In contrast, similar adenosine concentrations had no effect on cytosolic calcium levels in guinea pig neutrophils. These data demonstrate a pro-inflammatory role for adenosine in elicited guinea pig pulmonary eosinophils.
Adenosine↗
Polymorphonuclear leucocyte traffic in lung inflammation.
Explore the source record for details and available documents.
Adult↗
Effect of chronic antigen and beta 2 agonist exposure on airway remodeling in guinea pigs.
We recently reported that chronic exposure to fenoterol (FEN) in guinea pigs increases in vivo and in vitro airway responsiveness to a degree similar to that induced by chronic antigen (ovalbumin [OA]) exposure. We hypothesized that these changes were due to airway inflammation and airway remodeling. To trace newly recruited granulocytes as a marker of inflammation and to detect DNA replication in resident airway wall cells, the nucleotide 5'-bromo-2'-deoxyuridine (BrdU) was administered intermittently over the six-wk period of chronic FEN and/or OA exposure. Noncartilaginous airway dimensions were measured and the area fraction of BrdU-immunoreactive and total nuclei in adventitia, smooth muscle, and epithelium was determined by immunohistochemistry and point counting. The proliferation index was defined as the ratio of the two area fractions in each wall area. The adventitial areas of FEN- and OA-treated airways were respectively 62 and 88% greater than those of control airways (p < 0.05). The inner wall areas were not increased. The smooth muscle cell and epithelial cell proliferation index was increased after OA (smooth muscle index: control, 2.7 +/- 1.1% [SEM]; OA, 23.0 +/- 3.7%; p < 0.02) but not after FEN exposure, and there was an increased number of BrdU-immunoreactive granulocytes in the adventitia and epithelium after OA but not after FEN exposure. The increased in vivo airways responsiveness produced by chronic OA or FEN exposure may be attributable to adventitial thickening and increased in vitro muscle contractility, but the cellular mechanisms underlying these and other airway wall responses are different.(ABSTRACT TRUNCATED AT 250 WORDS)
Adrenergic beta-Agonists↗
HIV testing and life insurance.
Explore the source record for details and available documents.
Female↗
Increased insulin requirement in a patient with type 1 diabetes on rifampicin.
Explore the source record for details and available documents.
Blood Glucose↗
Formyl peptide stimulates and ATP gamma S potentiates [3H]cytidine 5'-diphosphate diglyceride accumulation in human neutrophils.
Although it is evident that the chemotactic peptide FMLP activates O2-formation in neutrophils via the phosphoinositidase-mediated second messenger system, it is less clear how endogenous priming agents such as ATP and platelet activating factor potentiate FMLP action. In our study, we have examined the possible effects of the stable ATP analog adenosine 5'-O-[3-thiotriphosphate] (ATP gamma S) on cellular levels of inositol 1,4,5-trisphosphate, [Ca2+]i and diglyceride (DG), in resting and in FMLP-stimulated neutrophils. Although all three measures were increased in the presence of FMLP, only the increase in DG was enhanced by pretreatment (priming) with ATP gamma S. We also measured the accumulation of the phosphoinositide cycle intermediate cytidine 5'-diphosphate (CDP)-DG to assess possible effects of priming on phosphoinositide resynthesis. The addition of FMLP to [3H]cytidine-prelabeled neutrophils elicited an increase in the accumulation of [3H]CDP-DG that was maximally enhanced in cells that were pretreated with cytochalasin B. The stimulated accumulation of [3H]CDP-DG was completely reversed by the addition of myo-inositol. Treatment of [3H]cytidine-prelabeled neutrophils with ATP gamma S (10-100 microM) resulted in a dose-dependent synergistic increase in FMLP-stimulated [3H]CDP-DG accumulation, whereas ATP gamma S alone had no effect. The observed increases in DG and in [3H]CDP-DG, in contrast to inositol 1,4,5-trisphosphate and [Ca2+]i responses, correlates well with the ATP gamma S-priming of FMLP-induced O2-formation. A similar potentiation of FMLP-induced stimulation of CDP-DG formation was also observed with platelet-activating factor. It is proposed that the priming of FMLP responses in neutrophils proceeds via a mechanism that selectively enhances DG production through a mechanism that is independent of FMLP-induced breakdown of phosphatidylinositol bisphosphate.
Adenosine Triphosphate↗
Superoxide responses to formyl-methionyl-leucyl-phenylalanine in primed neutrophils. Role of intracellular and extracellular calcium.
For superoxide (O2-) responses of human neutrophils stimulated by FMLP, experiments were designed to assess the requirement of extracellular calcium [( Ca2+]o) for priming of O2- responses by platelet-activating factor (PAF), PMA, or ionomycin. Although priming by PMA did not require [Ca2+]o, there was, as expected, a requirement for [Ca2+]o for the optimal priming effects of PAF and ionomycin. The ED50 value for [Ca2+]o in the priming function of PAF was 105 microM. The [Ca2+]o-dependent priming with ionomycin was bimodal with two ED50 values for [Ca2+]o of 90 microM and 3.2 mM. Optimal priming by PAF required at least 4-min exposure of cells to [Ca2+]o. Cells primed by PAF exhibited faster initial rates of O2-production after addition of FMLP, but the duration of O2- production was not prolonged. Whereas PAF-primed responses to FMLP are usually associated with increases in intracellular calcium [( Ca2+]i) after addition of FMLP, two conditions were found in which O2- responses to FMLP in PAF-primed cells occurred in the absence of any detectable increase in [Ca2+]i. When cells were loaded with the calcium chelator, bis-(O-aminophenoxy)-ethane-H,N,N',N'-tetraacetic acid, and then primed with PAF, normal amounts of inositol 1,4,5-trisphosphate were formed, but no increase in [Ca2+]i occurred after addition of FMLP even though the cells exhibited a fully primed O2- response; in Ca2(+)-depleted and ionomycin-permeabilized cells that were primed with PAF and then stimulated with FMLP, O2- was generated in amounts comparable to reference control (primed) cells, but there was suppressed production of inositol 1,4,5-trisphosphate and no increase in [Ca2+]i after addition of FMLP to PAF-primed cells. These data confirm the requirement of [Ca2+]o for optimal priming of neutrophils by PAF and ionomycin (but not cells primed by PMA) and indicate that, under certain conditions, generation of O2- in response to FMLP in PAF-primed neutrophils can occur independent of any increase in [Ca2+]i.
Calcium↗
Signal transduction events and Fc gamma R engagement in human neutrophils stimulated with immune complexes.
Signal transduction events have been evaluated in human neutrophils stimulated with immune complexes consisting of polyclonal rabbit antibody complexed with BSA. Immune complexes induced dose-related O2- responses, but very small increases in intracellular calcium ([Ca2+]i) levels were observed, in contrast to FMLP-stimulated cells. Measurements employing [45Ca2+] demonstrated that calcium influx and efflux in cells stimulated with immune complexes was substantially less than fluxes found in FMLP-stimulated cells. With respect to inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) formation under conditions in which the O2- responses to immune complexes or FMLP were similar, the Ins(1,4,5)P3 response to immune complexes was much smaller (by 65%) as compared to that induced by FMLP. Although pertussis toxin-treated cells showed a greatly diminished O2- response (by 89%) to FMLP, the response to immune complexes was largely resistant (only 26% reduction) to the inhibitory effects of this toxin. Antibodies to Fc gamma R indicated that engagement of Fc gamma RII and Fc gamma RIII, but not Fc gamma RI, receptors was related to the O2- response of neutrophils to immune complexes. O2- formation occurred in neutrophils incubated with Staphylococcus aureus cell walls bearing antibodies to Fc gamma RII or Fc gamma RIII. These data indicate that, in human neutrophils stimulated with immune complexes, signal transduction events involve engagement of Fc gamma RII and Fc gamma RIII. The O2- response is largely pertussis-toxin insensitive, is not associated with a significant increase in levels of [Ca2+]i, and is associated with relatively little formation of Ins(1,4,5)P3. This is in contrast to cells stimulated with FMLP in which O2- responses are largely pertussis toxin-sensitive and associated with large increases in [Ca2+]i as well as formation of Ins(1,4,5)P3. Signal transduction events involving Fc gamma R appear to be quite different from those events related to engagement of FMLP receptors.
Antibodies, Monoclonal↗
Absence of FMLP receptors on rat macrophages.
Although rat peritoneal neutrophils in the presence of cytochalasin B demonstrate superoxide (O2-) responses to the chemotactic peptide N'-formyl-met-leu-phe (FMLP), neither elicited rat peritoneal macrophages nor rat alveolar macrophages show an O2- response to FMLP (in the presence or absence of cytochalasin B), although a good O2- response to opsonized zymosan is demonstrated by both types of macrophages. Using Fura-2 loaded cells, peritoneal macrophages failed to show an increase in intracellular calcium after exposure to FMLP, f-nor-leu-phe, F-met-met-met, or F-norleu-leu-phe-norleu-lys. FMLP also failed to induce elevations in intracellular calcium in alveolar macrophages. In 3H-FMLP binding studies, the lack of responsiveness of peritoneal and alveolar macrophages was associated with the lack of FMLP receptors on these cell types, in striking contrast to the presence of functional receptors on rat neutrophils.
Animals↗