Nancy Elkins

Xanthine oxidoreductase promotes the inflammatory state of mononuclear phagocytes through effects on chemokine expression, peroxisome proliferator-activated receptor-gamma sumoylation, and HIF-1 alpha

The protective effects of pharmacological inhibitors of xanthine oxidoreductase (XOR) have implicated XOR in many inflammatory diseases. Nonetheless, the role played by XOR during inflammation is poorly understood. We previously observed that inhibition of XOR within the inflammatory mononuclear phagocytes (MNP) prevented neutrophil recruitment during adoptive transfer demonstrating the role of XOR in MNP-mediated neutrophil recruitment. To further explore the role of XOR in the inflammatory state of MNP, we studied MNP isolated from inflammatory lungs combined with analyses of MNP cell lines. We demonstrated that XOR activity was increased in inflammatory MNP following insufflation of Th-1 cytokines in vivo and that activity was specifically increased by MNP differentiation. Inhibition of XOR reduced levels of CINC-1 secreted by MNP. Expression of peroxisome proliferator-activated receptor γ (PPARγ) in purified rat lung MNP and MNP cell lines reflected both the presence of PPARγ isoforms and PPARγ SUMOylation, and XOR inhibitors increased levels of SUMO-PPARγ in MNP cell lines. Both ectopic overexpression of XOR cDNA and uric acid supplementation reduced SUMO-PPARγ in MNP cells. Levels of the M2 markers CD36, CD206, and arginase-1 were modulated by uric acid and oxonic acid, whereas siRNA to SUMO-1 or PIAS-1 also reduced arginase-1 in RAW264.7 cells. We also observed that HIF-1α was increased by XOR inhibitors in inflammatory MNP and in MNP cell lines. These data demonstrate that XOR promotes the inflammatory state of MNP through effects on chemokine expression, PPARγ SUMOylation, and HIF-1α and suggest that strategies for inhibiting XOR may be valuable in modulating lung inflammatory disorders.

Hyperpolarized 83Kr MRI of lungs

Hyperpolarized (hp) (83)Kr (spin I=9/2) is a promising gas-phase contrast agent that displays sensitivity to the surface chemistry, surface-to-volume ratio, and surface temperature of the surrounding environment. This proof-of-principle study demonstrates the feasibility of ex vivo hp (83)Kr magnetic resonance imaging (MRI) of lungs using natural abundance krypton gas (11.5% (83)Kr) and excised, but otherwise intact, rat lungs located within a custom designed ventilation chamber. Experiments comparing the (83)Kr MR signal intensity from lungs to that arising from a balloon with no internal structure inflated to the same volume with krypton gas mixture suggest that most of the observed signal originated from the alveoli and not merely the conducting airways. The (83)Kr longitudinal relaxation times in the rat lungs ranged from 0.7 to 3.7s but were reproducible for a given lung. Although the source of these variations was not explored in this work, hp (83)Kr T(1) differences may ultimately lead to a novel form of MRI contrast in lungs. The currently obtained 1200-fold signal enhancement for hp (83)Kr at 9.4T field strength is found to be 180 times below the theoretical upper limit.

Acute and chronic hypokalemia sensitize the isolated heart to hypoxic injury

We examined the effects of acute and/or chronic hypokalemia on responses to 30 min of hypoxia and recovery in the isolated, perfused heart model. We found that both acute hypokalemia and chronic hypokalemia impaired contractility [expressed as maximum slope of pressure increase over time (dP/d t): 501 ± 49 and 529 ± 48 vs. 1,302 ± 118 mmHg/s, P < 0.01] and recovery of ATP concentrations (determined with31P NMR spectroscopy: 30 ± 6 and 40 ± 10 vs. 67 ± 5% initial, P < 0.05) at 30 min of recovery. Moreover, the combination of acute hypokalemia and chronic hypokalemia had additive effects (dP/d t 166 ± 15 mmHg/s and ATP 21 ± 7% initial, both P < 0.01). We also measured cytosolic calcium with surface fluorescence spectroscopy after indo 1 loading. Acute hypokalemia and acute hypokalemia + chronic hypokalemia increased cytosolic calcium (averaged throughout the cardiac cycle) during and after hypoxia (390- to 460-nm ratio at 30 min of recovery: 0.46 ± 0.07 and 0.65 ± 0.07 vs. 0.18 ± 0.03, P < 0.01), whereas control and chronic hypokalemia hearts had only small changes with hypoxia and recovery. Finally, when we examined mitochondria isolated from hearts perfused under experimental conditions, we found that chronic hypokalemia-alone mitochondria and chronic hypokalemia + acute hypokalemia mitochondria had marked impairment of state 3 respiration compared with control hearts (52 ± 13 and 50 ± 9 vs. 128 ± 10 natm ⋅ min-1 ⋅ mg protein-1 with succinate as substrate, P < 0.01), whereas acute hypokalemia mitochondria demonstrated only subtle changes. These data suggest that both acute hypokalemia and chronic hypokalemia impair cardiac responses to hypoxia. The mechanism may involve impairment of calcium metabolism, but cytosolic calcium alterations do not explain all of the metabolic and functional effects of acute hypokalemia and chronic hypokalemia in the setting of hypoxia.We examined the effects of acute and/or chronic hypokalemia on responses to 30 min of hypoxia and recovery in the isolated, perfused heart model. We found that both acute hypokalemia and chronic hypokalemia impaired contractility [expressed as maximum slope of pressure increase over time (dP/dt): 501 +/- 49 and 529 +/- 48 vs. 1,302 +/- 118 mmHg/s, P < 0.01] and recovery of ATP concentrations (determined with 31P NMR spectroscopy: 30 +/- 6 and 40 +/- 10 vs. 67 +/- 5% initial, P < 0.05) at 30 min of recovery. Moreover, the combination of acute hypokalemia and chronic hypokalemia had additive effects (dP/dt 166 +/- 15 mmHg/s and ATP 21 +/- 7% initial, both P < 0.01). We also measured cytosolic calcium with surface fluorescence spectroscopy after indo 1 loading. Acute hypokalemia and acute hypokalemia + chronic hypokalemia increased cytosolic calcium (averaged throughout the cardiac cycle) during and after hypoxia (390- to 460-nm ratio at 30 min of recovery: 0.46 +/- 0.07 and 0.65 +/- 0.07 vs. 0.18 +/- 0.03, P < 0.01), whereas control and chronic hypokalemia hearts had only small changes with hypoxia and recovery. Finally, when we examined mitochondria isolated from hearts perfused under experimental conditions, we found that chronic hypokalemia-alone mitochondria and chronic hypokalemia + acute hypokalemia mitochondria had marked impairment of state 3 respiration compared with control hearts (52 +/- 13 and 50 +/- 9 vs. 128 +/- 10 natm.min-1.mg protein-1 with succinate as substrate, P < 0.01), whereas acute hypokalemia mitochondria demonstrated only subtle changes. These data suggest that both acute hypokalemia and chronic hypokalemia impair cardiac responses to hypoxia. The mechanism may involve impairment of calcium metabolism, but cytosolic calcium alterations do not explain all of the metabolic and functional effects of acute hypokalemia and chronic hypokalemia in the setting of hypoxia.

Effect of ergothioneine on acute lung injury and inflammation in cytokine insufflated rats

OBJECTIVE The Acute Respiratory Distress Syndrome (ARDS), the most severe form of Acute Lung Injury (ALI), is a highly-fatal, diffuse non-cardiogenic edematous lung disorder. The pathogenesis of ARDS is unknown but lung inflammation and lung oxidative stress are likely contributing factors. Since no specific pharmacologic intervention exists for ARDS, our objective was to determine the effect of treatment with ergothioneine-a safe agent with multiple anti-inflammatory and antioxidant properties on the development of lung injury and inflammation in rats insufflated with cytokines found in lung lavages of ARDS patients. METHOD Sprague-Dawley rats (3-10/group) were given 15 mg/kg or 150 mg/kg l-ergothioneine intravenously 1h before or 18 h after cytokine (IL-1 and IFNγ) insufflation. Lung injury (lavage LDH levels) and lung inflammation (lavage neutrophil numbers) were measured 24h after cytokine insufflation. RESULTS Ergothioneine pre- and post-treatment generally decreased lung injury and lung inflammation in cytokine insufflated rats. CONCLUSION Ergothioneine should be considered for additional testing as a potential therapy for treating and preventing ARDS.

Effects of fine carbonaceous particles containing high and low unpaired electron spin densities on lungs of female mice

The negative impacts on human health that accompany inhalation of atmospheric particles are documented in numerous epidemiologic studies, but the effect of specific chemical properties of the particles is generally unknown. We developed and employed technology for generating inhalable aerosols of carbonaceous air pollution particles that have specific physical and chemical properties. We find that inhaling particles with greater unpaired electron spin (free radical) densities stimulates greater lung inflammatory and oxidative stress responses. Cultured alveolar macrophages take up more particles of greater free radical content, develop mitochondrial abnormalities, and release more leukotriene B(4) (LTB(4)) than alveolar macrophages exposed to lesser free-radical-containing particles in vitro. Mice exposed to high free radical particles in vivo also develop mitochondrial abnormalities in alveolar macrophages and increased oxidative stress, which is reflected by increases in lung nitrotyrosine staining and lung lavage nitrogen oxide levels compared with those of lesser free radical density. These results provide insight for the unexplained geographic differences and have implications for fossil fuel combustion conditions and the impact of fine particles on health and disease.

Brief Glutamine Pretreatment Increases Alveolar Macrophage CD163/Heme Oxygenase-1/p38-MAPK Dephosphorylation Pathway and Decreases Capillary Damage but Not Neutrophil Recruitment in IL-1/LPS-Insufflated Rats.

Background Glutamine (GLN) attenuates acute lung injury (ALI) but its effect on alveolar macrophages is unknown. We hypothesized that GLN pretreatment would induce the anti-inflammatory CD163/heme oxygenase (HO)-1/p38-MAPK dephosphorylation pathway in alveolar macrophages and reduce ALI in rats insufflated with interleukin-1 (IL-1) and lipopolysaccharide (LPS). Methods Male Sprague-Dawley rats were randomized to the following groups: GLN-IL-1/LPS-, GLN+IL-1/LPS-, GLN-IL-1/LPS+, and GLN+IL-1/LPS+. GLN pretreatment was given via gavage (1g/kg L-alanyl-L-glutamine) daily for 2 days. ALI was subsequently induced by insufflating 50ng IL-1 followed by 5mg/kg E.coli LPS. After 24h, bronchoalveolar lavage (BAL) protein, lactate dehydrogenase (LDH) and neutrophil concentrations were analyzed. BAL alveolar macrophage CD163+ expression, HO-1 and p38-MAPK concentrations were measured, as well as alveolar macrophage tumor necrosis factor (TNF)-α and interleukin (IL)-10 concentrations. Histology and immunofluorescence studies were also performed. Results Following IL-1/LPS insufflation, GLN pretreated rats had significantly decreased BAL protein and LDH concentrations, but not BAL neutrophil counts, compared to non-GLN pretreated rats. The number of alveolar macrophages and the number of CD163+ macrophages were significantly increased in GLN pretreated IL-1/LPS-insufflated rats compared to non-GLN pretreated, IL-1/LPS-insufflated rats. GLN pretreatment before IL-1/LPS also significantly increased HO-1 concentrations and dephosphorylated p38-MAPK levels but not cytokine levels in alveolar macrophages. Immunofluorescence localized CD163 and HO-1 in alveolar macrophages. Conclusion Short-term GLN pretreatment activates the anti-inflammatory CD163/HO-1/p38-MAPK dephosphorylation pathway of alveolar macrophages and decreases capillary damage but not neutrophil recruitment in IL-1/LPS-insufflated rats.

Preventing the acute respiratory distress syndrome.

Laudable supportive advances have been made to improve the care of patients with the Acute Respiratory Distress Syndrome (ARDS) but no pharmacologic interventions are known to reduce the high mortality of this disorder once it is established. This commentary discusses some of the challenges that arise in preventing ARDS in at-risk individuals and the likely dependence of this approach on biomarker panels that can be done in real time.

Inhalation of two putative Gulf War toxins by mice

ABSTRACT We employed our inhalation methodology to examine whether biomarkers of inflammation and oxidative stress would be produced in mice following inhalation of aerosols containing carbonaceous particles or the vapor of pesticides prevalent during the first Gulf War. Exposure to two putative Gulf War Illness toxins, fine airborne particles and the pesticide malathion, increased biomarkers of inflammation and oxidative stress in Friend virus B (FVB) female mice. Mice inhaling particles 24 h before had increased lung lavage and plasma Leukotriene B4 (LTB4) (a biomarker of inflammation) and PGF2α (a biomarker of oxidative stress) levels, lung lavage protein and lung lavage lactic dehydrogenase (LDH) levels. These changes were a function of particle density and exposure time. Compared to particle inhalation, mice inhaling malathion 24 h before had small increase in plasma LTB4 and PGF2α levels but no increase in lung lavage LTB4, lung lavage protein, lung lavage LDH, and lung lavage alveolar macrophage (AM) levels compared to unexposed control mice. AM from particle-exposed mice contained phagocytosed particles, while AM from malathion-exposed mice showed no abnormalities. Our results indicate that inhaling particles or malathion can alter inflammatory and oxidative biomarkers in mice and raise the possibility that these toxins may have altered inflammation and oxidative stress biomarkers in Gulf War-exposed individuals.