Effects of outdoor air pollutants on platelet activation in people with type 2 diabetes.

Exposure to air pollution is associated with increased morbidity and mortality from cardiovascular disease. We hypothesized that increases in exposure to ambient air pollution are associated with platelet activation and formation of circulating tissue factor-expressing microparticles. We studied 19 subjects with type 2 diabetes, without clinical evidence of cardiovascular disease, who had previously participated in a human clinical study of exposure to ultrafine particles (UFP). Blood was obtained for measurements of platelet activation following an overnight stay in the Clinical Research Center, prior to each of their two pre-exposure visits. Air pollution and meteorological data, including UFP counts, were analyzed for the 5 days prior to the subjects' arrival at the Clinical Research Center. Contrary to expectations, increases in UFP were associated with decreases in surface expression of platelet activation markers. The number of platelet-leukocyte conjugates decreased by -80 (95% confidence interval (CI) -123 to -37, p = 0.001) on the first lag day (20-44 h prior to the blood draw) and by -85 (CI -139 to -31, p = 0.005) on combined lag days 1 to 5, per interquartile range (IQR) increase in UFP particle number (2482). However, levels of soluble CD40L increased 104 (CI 3 to 205, p = 0.04) pg/ml per IQR increase in UFP on lag day 1, a finding consistent with prior platelet activation. We speculate that, in people with diabetes, exposure to UFP activates circulating platelets within hours of exposure, followed by an increase in soluble CD40L and a rebound reduction in circulating platelet surface markers.

The ratio of arginine to dimethylarginines is reduced and predicts outcomes in patients with severe sepsis.

OBJECTIVES: Arginine deficiency may contribute to microvascular dysfunction, but previous studies suggest that arginine supplementation may be harmful in sepsis. Systemic arginine availability can be estimated by measuring the ratio of arginine to its endogenous inhibitors, asymmetric and symmetric dimethylarginine. We hypothesized that the arginine-to-dimethylarginine ratio is reduced in patients with severe sepsis and associated with severity of illness and outcomes. DESIGN: Case-control and prospective cohort study. SETTING: Medical and surgical intensive care units of an academic medical center. PATIENTS AND SUBJECTS: One hundred nine severe sepsis and 50 control subjects. MEASUREMENTS AND MAIN RESULTS: Plasma and urine were obtained in control subjects and within 48 hrs of diagnosis in severe sepsis patients. The arginine-to-dimethylarginine ratio was higher in control subjects vs. sepsis patients (median, 95; interquartile range, 85-114; vs. median, 34; interquartile range, 24-48; p < .001) and in hospital survivors vs. nonsurvivors (median, 39; interquartile range, 26-52; vs. median, 27; interquartile range, 19-32; p = .004). The arginine-to-dimethylarginine ratio was correlated with Acute Physiology and Chronic Health Evaluation II score (Spearman's correlation coefficient [ρ] = - 0.40; p < .001) and organ-failure free days (ρ = 0.30; p = .001). A declining arginine-to-dimethylarginine ratio was independently associated with hospital mortality (odds ratio, 1.63 per quartile; 95% confidence interval, 1.00-2.65; p = .048) and risk of death over the course of 6 months (hazard ratio, 1.41 per quartile; 95% confidence interval, 1.01-1.98; p = .043). The arginine-to-dimethylarginine ratio was correlated with the urinary nitrate-to-creatinine ratio (ρ = 0.46; p < .001). CONCLUSIONS: The arginine-to-dimethylarginine ratio is associated with severe sepsis, severity of illness, and clinical outcomes. The arginine-to-dimethylarginine ratio may be a useful biomarker, and interventions designed to augment systemic arginine availability in severe sepsis may still be worthy of investigation.

Vascular effects of ultrafine particles in persons with type 2 diabetes.

BACKGROUND: Diabetes confers an increased risk for cardiovascular effects of airborne particles. OBJECTIVE: We hypothesized that inhalation of elemental carbon ultrafine particles (UFP) would activate blood platelets and vascular endothelium in people with type 2 diabetes. METHODS: In a randomized, double-blind, crossover trial, 19 subjects with type 2 diabetes inhaled filtered air or 50 µg/m³ elemental carbon UFP (count median diameter, 32 nm) by mouthpiece for 2 hr at rest. We repeatedly measured markers of vascular activation, coagulation, and systemic inflammation before and after exposure. RESULTS: Compared with air, particle exposure increased platelet expression of CD40 ligand (CD40L) and the number of platelet-leukocyte conjugates 3.5 hr after exposure. Soluble CD40L decreased with UFP exposure. Plasma von Willebrand factor increased immediately after exposure. There were no effects of particles on plasma tissue factor, coagulation factors VII or IX, or D-dimer. CONCLUSIONS: Inhalation of elemental carbon UFP for 2-hr transiently activated platelets, and possibly the vascular endothelium, in people with type 2 diabetes.

Gene expression profile in circulating mononuclear cells after exposure to ultrafine carbon particles.

CONTEXT: Exposure to particulate matter (PM) is associated with systemic health effects, but the cellular and molecular mechanisms are unclear. OBJECTIVE: We hypothesized that, if circulating mononuclear cells play an important role in mediating systemic effects of PM, they would show gene expression changes following exposure. MATERIALS AND METHODS: Peripheral blood samples were collected before (0 h) and at 24 h from healthy subjects exposed to filtered air (FA) and ultrafine carbon particles (UFPs, 50 microg/m(3)) for 2 h in a previous study (n = 3 each). RNA from mononuclear cell fraction (> 85% lymphocytes) was extracted, amplified and hybridized to Affymetrix HU133 plus 2 microarrays. Selected genes were confirmed in five additional subjects from the same study. RESULTS: We identified 1713 genes (UFP 24 h vs. FA 0 and 24 h, P < 0.05, false discovery rate of 0.01). The top 10 upregulated genes (fold) were CDKN1C (1.86), ZNF12 (1.83), SRGAP2 (1.82), FYB (1.79), LSM14B (1.79), CD93 (1.76), NCSTN (1.70), DUSP6 (1.69), TACC1 (1.68), and H2AFY (1.68). Upregulation of CDKN1C and SRGAP2 was confirmed by real-time-PCR. We entered 1020 genes with a ratio >1.1 or <-1.1 into the Ingenuity Pathway Analysis and identified pathways related to inflammation, tissue growth and host defense against environmental insults, such as, insulin growth factor 1 signaling, insulin receptor signaling and NF-E2-related factor-2-mediated oxidative stress response pathway. DISCUSSION AND CONCLUSIONS: Two-hour exposures to UFP produced gene expression changes in circulating mononuclear cells. These gene changes provide biologically plausible links to PM-induced systemic health effects, especially those in the cardiovascular system and glucose metabolism.

Artery-to-vein differences in nitric oxide metabolites are diminished in sepsis.

OBJECTIVE: Nitric oxide deficiency may contribute to microvascular dysfunction in sepsis. Current physiologic paradigms contend that nitrite and/or S-nitrosohemoglobin mediate intravascular delivery of nitric oxide. These nitric oxide metabolites are purportedly consumed during hemoglobin deoxygenation, producing nitric oxide and coupling intravascular nitric oxide delivery with metabolic demand. Systemic nitrite and S-nitrosohemoglobin consumption can be assessed by comparing their concentrations in arterial vs. venous blood. We hypothesized that arterial vs. venous differences in nitrite and S-nitrosohemoglobin are diminished in sepsis and associated with mortality. DESIGN: Case-control and prospective cohort study. SETTING: Adult intensive care units of an academic medical center. PATIENTS AND SUBJECTS: Eighty-seven critically ill septic patients and 52 control subjects. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Nitrite and S-nitrosohemoglobin were measured using tri-iodide-based reductive chemiluminescence. In control subjects, arterial plasma, whole blood, and red blood cell nitrite levels were higher than the corresponding venous levels. In contrast, S-nitrosohemoglobin was higher in venous compared to arterial blood. In septic patients, arterial vs. venous red blood cell nitrite and S-nitrosohemoglobin differences were absent. Furthermore, the plasma nitrite arterial vs. venous difference was absent in nonsurvivors. CONCLUSIONS: In health, nitrite levels are higher in arterial vs. venous blood (suggesting systemic nitrite consumption), whereas S-nitrosohemoglobin levels are higher in venous vs. arterial blood (suggesting systemic S-nitrosohemoglobin production). These arterial vs. venous differences are diminished in sepsis, and diminished arterial vs. venous plasma nitrite differences are associated with mortality. These data suggest pathologic disruption of systemic nitrite utilization in sepsis.

Changes in fluorescence intensity of selected leukocyte surface markers following fixation.

BACKGROUND: Immunophenotyping of blood leukocytes often involves fixation with paraformaldehyde prior to cytometry analysis. However, the influence of cell type and marker specificity on the stability of fluorescence intensity after fixation has not been well studied. METHODS: Human whole blood was stained using a panel of fluorescein isothiocyanate-labeled antibodies to surface markers. Unfixed and fixed samples were analyzed by flow cytometry at 0, 2, 4, 6, 24, 48, and 96 h after staining. Fluorescence measurements were converted to molecules of equivalent soluble fluorochrome for comparison. RESULTS: Fixation caused a significant decrease in both forward and side scatter at 48 h which required gating adjustments to achieve resolution of cell populations. The autofluorescence increased progressively in fixed samples (ninefold at 96 h for monocytes). Variable decreases in marker-associated fluorescence became apparent after correction for autofluorescence. The magnitude of the decrease at 96 h varied with cell type and marker, from 5% for CD32 on monocytes to 39% for CD16 on neutrophils. CONCLUSION: The change in fluorescence intensity following staining and fixation of leukocytes varies with cell type and surface marker. Fluorescence stability should be determined for each cell type and marker used, and the confounding effects of fixation on cell autofluorescence should be considered.

Changes in markers of epithelial permeability and inflammation in chronic smokers switching to a nonburning tobacco device (Eclipse).

Eclipse, produced by R. J. Reynolds Tobacco Company, is a potential reduced exposure product (PREP) that heats rather than burns tobacco. We hypothesized that switching to Eclipse would result in relative normalization of pulmonary epithelial permeability, airway inflammation, and blood leukocyte activation in current smokers. We assessed 10 healthy smokers (aged 21-50 years, 19+/-8 pack-years) at baseline and after 2 and 4 weeks of switching to Eclipse, for symptoms, pulmonary function, airway inflammation, lung clearance of (99m)technicium-diethylenetriaminepentaacetic acid, and blood leukocyte activation and production of reactive oxygen species. Values were compared before and after Eclipse use and with those of healthy, lifetime nonsmokers (aged 18-53 years). Compared with baseline values before switching to Eclipse, lung permeability half-time increased from 33+/-3 to 43+/-6 min (p = .017) after 2 weeks and to 44+/-7 min (p = .10) after 4 weeks of Eclipse use. Carboxyhemoglobin levels increased from 5%+/-2% to 7%+/-2% (p<.01) at 4 weeks. Compared with smoking the usual brand of cigarettes, after smoking Eclipse the percentage of natural killer cells, the expression of intercellular adhesion molecule-1 on monocytes, and the expression of CD45RO on T cells showed significant improvement. However, expression of other surface markers, notably CD23 on monocytes, became more abnormal. Production of reactive oxygen species by smokers' neutrophils and monocytes increased further with Eclipse use. We found no significant effects on pulmonary function, cells in induced sputum, or exhaled nitric oxide. Switching to Eclipse reduces alveolar epithelial injury in some smokers but may increase carboxyhemoglobin levels and oxidative stress.

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

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.

Comparing inhaled ultrafine versus fine zinc oxide particles in healthy adults: a human inhalation study.

RATIONALE: Zinc oxide is a common, biologically active constituent of particulate air pollution as well as a workplace toxin. Ultrafine particles (< 0.1 microm diameter) are believed to be more potent than an equal mass of inhaled accumulation mode particles (0.1-1.0 microm diameter). OBJECTIVES: We compared exposure-response relationships for respiratory, hematologic, and cardiovascular endpoints between ultrafine and accumulation mode zinc oxide particles. METHODS: In a human inhalation study, 12 healthy adults inhaled 500 microg/m3 of ultrafine zinc oxide, the same mass of fine zinc oxide, and filtered air while at rest for 2 hours. MEASUREMENTS AND MAIN RESULTS: Preexposure and follow-up studies of symptoms, leukocyte surface markers, hemostasis, and cardiac electrophysiology were conducted to 24 hours post-exposure. Induced sputum was sampled 24 hours after exposure. No differences were detected between any of the three exposure conditions at this level of exposure. CONCLUSIONS: Freshly generated zinc oxide in the fine or ultrafine fractions inhaled by healthy subjects at rest at a concentration of 500 microg/m3 for 2 hours is below the threshold for acute systemic effects as detected by these endpoints.

Alveolar epithelial cell-macrophage interactions affect oxygen-stimulated interleukin-8 release.

Interactions of alveolar macrophages with respiratory epithelium may play a key role in hyperoxia-induced lung inflammation. We studied the effect of cell-cell contact with epithelial cells in hyperoxia on macrophages' secretion of interleukin-8 (IL-8). A549 pulmonary epithelial cells and THP-1 monocyte/macrophage cells were cultured either singly, in contact coculture, or prevented from contact by a porous membrane, and exposed to oxygen or room air. Phorbol-12-myristate-13-acetate-(PMA)-treated THP-1 cells were exposed to the same conditions. Neither cell line cultured alone produced appreciable amounts of IL-8 in hyperoxia. Contact cocultures exposed to hyperoxia produced increased IL-8, while in the noncontact coculture it was attenuated. Both cell-cell contact and PMA increased THP-1 cell CD54 expression. Intracellular IL-8 production was increased in contact cocultured, hyperoxia exposed THP-1 cells, while the A549 sample showed no change. Increased IL-8 mRNA expression was not demonstrated in cocultured, hyperoxic exposed THP-1 cells, suggesting nontranscriptional regulation of IL-8 protein levels. Contact with epithelial cells appears to potentiate macrophage responses to hyperoxia.