Targeted eicosanoids lipidomics of exhaled breath condensate in healthy subjects

Background

Exhaled breath condensate collection is a non-invasive method of sampling the respiratory tract that can be repeated several times in a wide range of clinical settings. Quantitation of non-volatile compounds in the condensate requires highly sensitive analytical methods, e.g. mass spectrometry.

Objective

To validate cross-platform measurements of eicosanoids using high performance liquid chromatography or gas chromatography coupled with mass spectrometry in exhaled breath condensate sampled from 58 healthy individuals.

Methods

Twenty different eicosanoid compounds, representing major arachidonic acid lipoxygenation and cyclooxygenation pathways were measured using a stable isotope dilution method. We applied a free palmitic acid concentration as a surrogate marker for the condensate dilution factor.

Results

Eicosanoids concentrations in the condensates were consistent with their content in other biological fluids. Prostaglandin E₂ was the most abundant mediator, represented by its stable metabolite tetranor-PGEM. Prostaglandin D₂ products were at low concentration, while hydroxyacids derived from lipoxygenation were abundant. 5-HETE was elevated in current tobacco smokers. Leukotriene B₄ has the highest concentration of all 5-LO products. 15-LO analogues of cysteinyl leukotrienes–eoxins were detectable and metabolized to eoxin E₄. Two main vascular prostanoids: prostacyclin and thromboxane B₂ were present as metabolites. A marker for non-enzymatic lipid peroxidation, 8-iso-PGF_2α isoprostane was increased in smokers.

Conclusion

Presented targeted lipidomics analysis of exhaled breath condensate in healthy subjects justifies its application to investigation of inflammatory lung diseases. Measurements of non-volatile mediators of inflammation in the condensates might characterize disease-specific pathological mechanisms and responses to treatment.     

Adipocytokine Profile,Cytokine Levels and Foxp3 Expression in Multiple Sclerosis: a Possible Link to Susceptibility and Clinical Course of Disease

Background

Adipocytokines may be involved in multiple sclerosis (MS) as well as other autoimmune and inflammatory-related diseases. This study aims to compare levels of resistin, visfatin and leptin in three subgroups of MS patients with healthy subjects and also to study their relationship with Foxp3 expression and levels of several pro-inflammatory mediators such as interleukine-1 β(IL-1 β),tumor necrosis factor-α (TNF-α) and human sensitive C-reactive protein (hs-CRP).

Methods

A total of 391 subjects including 200 healthy controls and 191 MS patients were recruited for this case-control study. Circulating adipocytokines and inflammatory mediators were measured using immunoassay methods. Foxp3 gene expression in peripheral blood mononuclear cells (PBMC) was determined by quantitative real-time PCR. Fat tissue mass was evaluated by using dual energy X-ray absorptiometery (DEXA).

Results

A significant difference was observed in levels of inflammatory mediators, adipocytokines, Foxp3 gene expression and adipose tissue mass between MS patients and healthy controls. All adipocytokines were positively correlated with levels of inflammatory mediators and negatively correlated with Foxp3 expression in MS patients. In controls, there were positive correlations between circulating leptin and resistin with TNF-α and IL-1β in subgroup analysis, the highest levels of TNF-α, IL-1β, hs-CRP, resistin and leptin were observed in primary progressive-MS (PP-MS) patients. Also, expression of Foxp3 and levels of visfatin in relapsing remitting-MS(RR-MS) patients were higher compared with the other subgroups.

Conclusions

Our findings suggest the potential role of adipocytokines in pathogenesis and severity of MS. Notably, the relationship of adipocytokines levels with inflammatory cytokines as well as clinical features of MS could be considerable in translational medicine and biomarker studies.     

Distinct differences in serum eicosanoids in healthy,enteritis and colorectal cancer individuals

Background

Eicosanoids as inflammatory mediators take part in the regulation of disease progression. However, the application of serum eicosanoid in disease progression identification was still uncertain.

Methods

Serum from 52 healthy volunteers, 34 enteritis patients and 55 colorectal cancer (CRC) patients were collected. Ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was used to analyze the change of serum eicosanoids.

Results

Of 158 eicosanoids, we found that lower levels of anti-inflammatory eicosanoids 13-HOTrE, 9-HOTrE, DHA, 11-HETE and 12-HHT were observed in enteritis and CRC group compared with healthy group, meanwhile the content of 5-iPF2α-VI as oxidative stress mediator in enteritis and CRC group was greater than that in healthy groups. Moreover, 9-HODE, 13-HODE, 12,13-diHOME, 8-HETE and 15-HETE were dramatically decrease in CRC group compared with non-CRC group. Additionally, the change of 5-, 12- and 15-HETE content in serum sample was associated with progression from healthy to enteritis, finally to CRC. No significant difference between serum eicosanoids and the expression of CerbB-2 and Ki67 were observed.

Conclusion

Serum eicosanoids might be used as a possible biomarker for identifying among health, enteritis and CRC.

     

Inhibiting AKT Phosphorylation Employing Non-Cytotoxic Anthraquinones Ameliorates TH2 Mediated Allergic Airways Disease and Rhinovirus Exacerbation

Background

Severe asthma is associated with T helper (T_H) 2 and 17 cell activation, airway neutrophilia and phosphoinositide-3-kinase (PI3K) activation. Asthma exacerbations are commonly caused by rhinovirus (RV) and also associated with PI3K-driven inflammation. Anthraquinone derivatives have been shown to reduce PI3K-mediated AKT phosphorylation in-vitro.

Objective

To determine the anti-inflammatory potential of anthraquinones in-vivo.

Methods

BALB/c mice were sensitized and challenged with crude house dust mite extract to induce allergic airways disease and treated with mitoxantrone and a novel non-cytotoxic anthraquinone derivative. Allergic mice were also infected with RV1B to induce an exacerbation.

Results

Anthraquinone treatment reduced AKT phosphorylation, hypoxia-inducible factor-1α and vascular endothelial growth factor expression, and ameliorated allergen- and RV-induced airways hyprereactivity, neutrophilic and eosinophilic inflammation, cytokine/chemokine expression, mucus hypersecretion, and expression of T_H2 proteins in the airways. Anthraquinones also boosted type 1 interferon responses and limited RV replication in the lung.

Conclusion

Non-cytotoxic anthraquinone derivatives may be of therapeutic benefit for the treatment of severe and RV-induced asthma by blocking pro-inflammatory pathways regulated by PI3K/AKT.     

Alterations of Plasma Lysophosphatidylcholine Species in Obesity and Weight Loss

Background

Obesity and related diseases of the metabolic syndrome contribute to the major health problems in industrialized countries. Alterations in the metabolism of lipid classes and lipid species may significantly be involved in these metabolic overload diseases. However, little is known about specific lipid species in this syndrome and existing data are contradictive.

Methods

In this study, we quantified plasma lipid species by electrospray ionization tandem mass spectrometry (ESI-MS/MS) in obese subjects before and after 3 month weight loss as well as in a control group.

Results

The comparison of obese subjects with control subjects before weight loss revealed significantly lower lysophosphatidylcholine (LPC) concentrations in obesity. LPC concentrations did not significantly increase during the observed period in the weight loss group. Analysis of LPC species revealed a decrease of most species in obesity and negative correlations with C-reactive protein (CRP) and body mass index (BMI). Correlating BMI ratio before and after weight loss with the ratio of total LPC and individual LPC species revealed significant negative relationships of LPC ratios with BMI ratio.

Conclusions

Our findings contribute to the contradictive discussion of the role of LPC in obesity and related chronic inflammation strongly supporting pre-existing data in the literature that show a decrease of LPC species in plasma of obese and a potentially anti-inflammatory role in these subjects.     

Renalase Protects against Contrast-Induced Nephropathy in Sprague-Dawley Rats

Background

Contrast-induced nephropathy (CIN) is the third leading cause of hospital-acquired acute renal failure. Oxidative stress, apoptosis and inflammation play crucial roles in CIN. Renalase is a newly discovered monoamine oxidase from the kidney. We hypothesize that renalase could protect against CIN through anti-oxidation, anti-inflammation and anti-apoptosis pathways.

Methods

We tested our hypothesis in vivo with a rat model of Ioversol-induced CIN and in vitro. Sprague-Dawley rats were divided into 4 groups (n = 6 per group): control group, Ioversol group (rats subjected to Ioversol-induced CIN), Ioversol plus vehicle group (CIN rats pretreated with vehicle) and Ioversol plus renalase group (CIN rats pretreated with 2 mg/kg recombinant renalase). HK2 cells were treated with Ioversol or H₂O₂.

Results

The results showed that pretreatment with renalase attenuated the deterioration of renal function, tubular necrosis, oxidative stress, apoptosis and inflammation (P<0.05). Furthermore, renalase protected HK2 cells against the cytotoxicity of Ioversol and suppressed Caspase-3 activity, oxidative stress and apoptosis induced by H₂O₂.

Conclusion

Recombinant renalase protected CIN in rats through anti-oxidation, anti-apoptosis and anti-inflammation mechanisms.     

Individual Variation in Lipidomic Profiles of Healthy Subjects in Response to Omega-3 Fatty Acids

Introduction

Conflicting findings in both interventional and observational studies have resulted in a lack of consensus on the benefits of ω3 fatty acids in reducing disease risk. This may be due to individual variability in response. We used a multi-platform lipidomic approach to investigate both the consistent and inconsistent responses of individuals comprehensively to a defined ω3 intervention.

Methods

The lipidomic profile including fatty acids, lipid classes, lipoprotein distribution, and oxylipins was examined multi- and uni-variately in 12 healthy subjects pre vs. post six weeks of ω3 fatty acids (1.9 g/d eicosapentaenoic acid [EPA] and 1.5 g/d docosahexaenoic acid [DHA]).

Results

Total lipidomic and oxylipin profiles were significantly different pre vs. post treatment across all subjects (p=0.00007 and p=0.00002 respectively). There was a strong correlation between oxylipin profiles and EPA and DHA incorporated into different lipid classes (r²=0.93). However, strikingly divergent responses among individuals were also observed. Both ω3 and ω6 fatty acid metabolites displayed a large degree of variation among the subjects. For example, in half of the subjects, two arachidonic acid cyclooxygenase products, prostaglandin E₂ (PGE₂) and thromboxane B2 (TXB₂), and a lipoxygenase product, 12-hydroxyeicosatetraenoic acid (12-HETE) significantly decreased post intervention, whereas in the other half they either did not change or increased. The EPA lipoxygenase metabolite 12-hydroxyeicosapentaenoic acid (12-HEPE) varied among subjects from an 82% decrease to a 5,000% increase.

Conclusions

Our results show that certain defined responses to ω3 fatty acid intervention were consistent across all subjects. However, there was also a high degree of inter-individual variability in certain aspects of lipid metabolism. This lipidomic based phenotyping approach demonstrated that individual responsiveness to ω3 fatty acids is highly variable and measurable, and could be used as a means to assess the effectiveness of ω3 interventions in modifying disease risk and determining metabolic phenotype.     

Pharmacodynamic Consequences of Administration of VLA-4 Antagonist CDP323 to Multiple Sclerosis Subjects: A Randomized,Double-Blind Phase 1/2 Study

Background

Lymphocyte inhibition by antagonism of α4 integrins is a validated therapeutic approach for relapsing multiple sclerosis (RMS).

Objective

Investigate the effect of CDP323, an oral α₄-integrin inhibitor, on lymphocyte biomarkers in RMS.

Methods

Seventy-one RMS subjects aged 18–65 years with Expanded Disability Status Scale scores ≤6.5 were randomized to 28-day treatment with CDP323 100 mg twice daily (bid), 500 mg bid, 1000 mg once daily (qd), 1000 mg bid, or placebo.

Results

Relative to placebo, all dosages of CDP323 significantly decreased the capacity of lymphocytes to bind vascular adhesion molecule-1 (VCAM-1) and the expression of α₄-integrin on VCAM-1–binding cells. All but the 100-mg bid dosage significantly increased total lymphocytes and naive B cells, memory B cells, and T cells in peripheral blood compared with placebo, and the dose-response relationship was shown to be linear. Marked increases were also observed in natural killer cells and hematopoietic progenitor cells, but only with the 500-mg bid and 1000-mg bid dosages. There were no significant changes in monocytes. The number of samples for regulator and inflammatory T cells was too small to draw any definitive conclusions.

Conclusions

CDP323 at daily doses of 1000 or 2000 mg induced significant increases in total lymphocyte count and suppressed VCAM-1 binding by reducing unbound very late antigen-4 expression on lymphocytes.

Trial Registration

ClinicalTrials.gov NCT00726648.     

Association between Traffic-Related Air Pollution,Subclinical Inflammation and Impaired Glucose Metabolism: Results from the SALIA Study

Background

Environmental and lifestyle factors regulate the expression and release of immune mediators. It has been hypothesised that ambient air pollution may be such an external factor and that the association between air pollution and impaired glucose metabolism may be attributable to inflammatory processes. Therefore, we assessed the associations between air pollution, circulating immune mediators and impaired glucose metabolism.

Methods

We analysed concentrations of 14 pro- and anti-inflammatory immune mediators as well as fasting glucose and insulin levels in plasma of 363 women from the Study on the influence of Air pollution on Lung function, Inflammation and Aging (SALIA, Germany). Exposure data for a group of pollutants such as nitrogen oxides (NO₂, NO_x) and different fractions of particulate matter were available for the participants'' residences. We calculated the association between the pollutants and impaired glucose metabolism by multiple regression models.

Results

The study participants had a mean age of 74.1 (SD 2.6) years and 48% showed impaired glucose metabolism based on impaired fasting glucose or previously diagnosed type 2 diabetes. Only long-term exposure NO₂ and NO_x concentrations showed positive associations (NO₂: OR 1.465, 95% CI 1.049-2.046, NO_x: OR 1.409, 95% CI 1.010-1.967) per increased interquartile range of NO₂ (14.65 µg/m³) or NO_x (43.16 µg/m³), respectively, but statistical significance was lost after correction for multiple comparisons. Additional adjustment for circulating immune mediators or the use of anti-inflammatory medication had hardly any impact on the observed ORs.

Conclusions

Our results suggest that exposure to nitrogen oxides may contribute to impaired glucose metabolism, but the associations did not reach statistical significance so that further studies with larger sample sizes are required to substantiate our findings. Our data do not preclude a role of inflammatory mechanisms in adipose or other tissues which may not be reflected by immune mediators in plasma.     

Allergic asthmatics show divergent lipid mediator profiles from healthy controls both at baseline and following birch pollen provocation

Background

Asthma is a respiratory tract disorder characterized by airway hyper-reactivity and chronic inflammation. Allergic asthma is associated with the production of allergen-specific IgE and expansion of allergen-specific T-cell populations. Progression of allergic inflammation is driven by T-helper type 2 (Th2) mediators and is associated with alterations in the levels of lipid mediators.

Objectives

Responses of the respiratory system to birch allergen provocation in allergic asthmatics were investigated. Eicosanoids and other oxylipins were quantified in the bronchoalveolar lumen to provide a measure of shifts in lipid mediators associated with allergen challenge in allergic asthmatics.

Methods

Eighty-seven lipid mediators representing the cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 (CYP) metabolic pathways were screened via LC-MS/MS following off-line extraction of bronchoalveolar lavage fluid (BALF). Multivariate statistics using OPLS were employed to interrogate acquired oxylipin data in combination with immunological markers.

Results

Thirty-two oxylipins were quantified, with baseline asthmatics possessing a different oxylipin profile relative to healthy individuals that became more distinct following allergen provocation. The most prominent differences included 15-LOX-derived ω-3 and ω-6 oxylipins. Shared-and-Unique-Structures (SUS)-plot modeling showed a correlation (R² = 0.7) between OPLS models for baseline asthmatics (R²Y[cum] = 0.87, Q²[cum] = 0.51) and allergen-provoked asthmatics (R²Y[cum] = 0.95, Q²[cum] = 0.73), with the majority of quantified lipid mediators and cytokines contributing equally to both groups. Unique structures for allergen provocation included leukotrienes (LTB₄ and 6-trans-LTB₄), CYP-derivatives of linoleic acid (epoxides/diols), and IL-10.

Conclusions

Differences in asthmatic relative to healthy profiles suggest a role for 15-LOX products of both ω-6 and ω-3 origin in allergic inflammation. Prominent differences at baseline levels indicate that non-symptomatic asthmatics are subject to an underlying inflammatory condition not observed with other traditional mediators. Results suggest that oxylipin profiling may provide a sensitive means of characterizing low-level inflammation and that even individuals with mild disease display distinct phenotypic profiles, which may have clinical ramifications for disease.     

Co-Culture of Human Bronchial Fibroblasts and CD4+ T Cells Increases Th17 Cytokine Signature

Background

Airway inflammation is an important characteristic of asthma and has been associated with airway remodelling and bronchial hyperreactivity. The mucosal microenvironment composed of structural cells and highly specialised extracellular matrix is able to amplify and promote inflammation. This microenvironment leads to the development and maintenance of a specific adaptive response characterized by Th2 and Th17. Bronchial fibroblasts produce multiple mediators that may play a role in maintaining and amplifying this response in asthma.

Objective

To investigate the role of bronchial fibroblasts obtained from asthmatic subjects and healthy controls in regulating Th17 response by creating a local micro-environment that promotes this response in the airways.

Methods

Human bronchial fibroblasts and CD4⁺T cells were isolated from atopic asthmatics and non-atopic healthy controls. CD4⁺T were co-cultured with bronchial fibroblasts of asthmatic subjects and healthy controls. RORc gene expression was detected by qPCR. Phosphorylated STAT-3 and RORγt were evaluated by western blots. Th17 phenotype was measured by flow cytometry. IL-22, IL17, IL-6 TGF-β and IL1-β were assessed by qPCR and ELISA.

Results

Co-culture of CD4⁺T cells with bronchial fibroblasts significantly stimulated RORc expression and induced a significant increase in Th17 cells as characterized by the percentage of IL-17⁺/CCR6⁺ staining in asthmatic conditions. IL-17 and IL-22 were increased in both normal and asthmatic conditions with a significantly higher amount in asthmatics compared to controls. IL-6, IL-1β, TGF-β and IL-23 were significantly elevated in fibroblasts from asthmatic subjects upon co-culture with CD4⁺T cells. IL-23 stimulates IL-6 and IL-1β expression by bronchial fibroblasts.

Conclusion

Interaction between bronchial fibroblasts and T cells seems to promote specifically Th17 cells profile in asthma. These results suggest that cellular interaction particularly between T cells and fibroblasts may play a pivotal role in the regulation of the inflammatory response in asthma.     

Enhanced Expression of Cystathionine β-Synthase and Cystathionine γ-Lyase During Acute Cholecystitis-Induced Gallbladder Inflammation

Background

Hydrogen sulfide (H₂S) has recently been shown to play an important role in the digestive system, but the role of endogenous H₂S produced locally in the gallbladder is unknown. The aim of this study was to investigate whether gallbladder possesses the enzymatic machinery to synthesize H₂S, and whether H₂S synthesis is changed in gallbladder inflammation during acute acalculous cholecystitis (AC).

Methods

Adult male guinea pigs underwent either a sham operation or common bile duct ligation (CBDL). One, two, or three days after CBDL, the animals were sacrificed separately. Hematoxylin and eosin-stained slides of gallbladder samples were scored for inflammation. H₂S production rate in gallbladder tissue from each group was determined; immunohistochemistry and western blotting were used to determine expression levels of the H₂S-producing enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) in gallbladder.

Results

There was a progressive inflammatory response after CBDL. Immunohistochemistry analysis showed that CBS and CSE were expressed in the gallbladder epithelium, muscular layer, and blood vessels and that the expression increased progressively with increasing inflammation following CBDL. The expression of CBS protein as well as the H₂S-production rate was significantly increased in the animals that underwent CBDL, compared to those that underwent the sham operation.

Conclusions

Both CBS and CSE are expressed in gallbladder tissues. The expression of these enzymes, as well as H₂S synthesis, was up-regulated in the context of inflammation during AC.     

Depth-Dependent Differences in Community Structure of the Human Colonic Microbiota in Health

Objective

The aims of this study were to develop techniques for spatial microbial assessment in humans and to establish colonic luminal and mucosal spatial ecology, encompassing longitudinal and cross-sectional axes.

Design

A microbiological protected specimen brush was used in conjunction with a biopsy forceps to sample the colon in nine healthy volunteers undergoing colonoscopy. Terminal Restriction Fragment Length Polymorphism analysis was used to determine the major variables in the spatial organization of the colonic microbiota.

Results

Protected Specimen Brush sampling retrieved region-specific, uncontaminated samples that were enriched for bacterial DNA and depleted in human DNA when compared to biopsy samples. Terminal Restriction Fragment Length Polymorphism analysis revealed a segmentation of bacterial communities between the luminal brush and biopsy-associated ecological niches with little variability across the longitudinal axis of the colon and reduced diversity in brush samples.

Conclusion

These results support the concept of a microbiota with little longitudinal variability but with some degree of segregation between luminal and mucosal communities.     

Endoplasmic Reticulum Stress,Unfolded Protein Response and Altered T Cell Differentiation in Necrotizing Enterocolitis

Background

Endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) play important roles in chronic intestinal inflammation. Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in preterm infants and is characterized by acute intestinal inflammation and necrosis. The objective of the study is to investigate the role of ER stress and the UPR in NEC patients.

Methods

Ileal tissues from NEC and control patients were obtained during surgical resection and/or at stoma closure. Splicing of XBP1 was detected using PCR, and gene expression was quantified using qPCR and Western blot.

Results

Splicing of XBP1 was only detected in a subset of acute NEC (A-NEC) patients, and not in NEC patients who had undergone reanastomosis (R-NEC). The other ER stress and the UPR pathways, PERK and ATF6, were not activated in NEC patients. A-NEC patients showing XBP1 splicing (A-NEC-XBP1s) had increased mucosal expression of GRP78, CHOP, IL6 and IL8. Similar results were obtained by inducing ER stress and the UPR in vitro. A-NEC-XBP1s patients showed altered T cell differentiation indicated by decreased mucosal expression of RORC, IL17A and FOXP3. A-NEC-XBP1s patients additionally showed more severe morphological damage and a worse surgical outcome. Compared with A-NEC patients, R-NEC patients showed lower mucosal IL6 and IL8 expression and higher mucosal FOXP3 expression.

Conclusions

XBP1 splicing, ER stress and the UPR in NEC are associated with increased IL6 and IL8 expression levels, altered T cell differentiation and severe epithelial injury.     

Low Levels of Blood Lipids Are Associated with Etiology and Lethal Outcome in Acute Liver Failure

Background/Aims

Emerging data links different aspects of lipid metabolism to liver regeneration. In patients with acute liver failure (ALF), low levels of lipids may correlate with disease severity. Thus, we determined whether there is an etiology-specific link between lipid levels in patients suffering from ALF and aimed to investigate an effect of lipid levels on the prognosis of ALF.

Methods

In this retrospective single center study, we reviewed 89 consecutive ALF patients, who met the criteria of the “Acute Liver Failure Study Group”. Patient characteristics, clinical data and laboratory parameters were individually analyzed at admission and correlated with the patients'' outcome after a four week follow up. Possible endpoints were either discharge, or death or liver transplantation.

Results

High-density lipoprotein (HDL), cholesterol and triglyceride levels were significantly lower in patients who died or required a liver transplant. HDL levels were significantly higher in patients with ALF caused by acetaminophen intoxication, compared to fulminant HBV infection or drug induced liver injury. HDL levels correlated with hepatic injury by ALT levels, and Albumin, and inversely correlated with the MELD score, INR, and bilirubin.

Conclusion

In our cohort of patients with ALF, we could show that HDL and cholesterol are suppressed. In addition novel etiology specific patterns between acteminophen and non-acteminophen induced liver failure were detected for serum lipid components. Further studies are needed to address the role of cholesterol and lipid metabolism and the according pathways in different etiologies of ALF.     

Oxygen and Ventilatory Output during Several Activities of Daily Living Performed by COPD Patients Stratified According to Disease Severity

Objectives

To measure the oxygen and ventilatory output across all COPD stages performing 18 common ADL and identify the activities that present the highest metabolic and ventilatory output as well as to compare the energy expenditure within each disease severity.

Materials and Methods

Metabolic (VO₂ and VCO₂), ventilatory (f and V_E), cardiovascular (HR) and dyspnea (Borg score) variables were assessed in one hundred COPD patients during the completion of eighteen ADL grouped into four activities domains: rest, personal care, labor activities and efforts.

Results

The activities with the highest proportional metabolic and ventilatory output (VO₂/VO₂max and VE/MVV) were walking with 2.5 Kg in each hand and walking with 5.0 Kg in one hand. Very severe patients presented the highest metabolic, ventilatory output and dyspnea than mild patients (p<0.05).

Conclusions

COPD patients present an increased proportion of energy expenditure while performing activities of daily living. The activities that developed the highest metabolic and ventilatory output are the ones associated to upper and lower limbs movements combined. Very severe patients present the highest proportional estimated metabolic and ventilatory output and dyspnea. Activities of daily living are mainly limited by COPD’s reduced ventilatory reserve.     

Imaging of an Inflammatory Injury in the Newborn Rat Brain with Photoacoustic Tomography

Background

The precise assessment of cerebral saturation changes during an inflammatory injury in the developing brain, such as seen in periventricular leukomalacia, is not well defined. This study investigated the impact of inflammation on locoregional cerebral oxygen saturation in a newborn rodent model using photoacoustic imaging.

Methods

1 mg/kg of lipopolysaccharide(LPS) diluted in saline or saline alone was injected under ultrasound guidance directly in the corpus callosum of P3 rat pups. Coronal photoacoustic images were carried out 24 h after LPS exposure. Locoregional oxygen saturation (SO₂) and resting state connectivity were assessed in the cortex and the corpus callosum. Microvasculature was then evaluated on cryosection slices by lectin histochemistry.

Results

Significant reduction of SO₂ was found in the corpus callosum; reduced SO₂ was also found in the cortex ipsilateral to the injection site. Seed-based functional connectivity analysis showed that bilateral connectivity was not affected by LPS exposure. Changes in locoregional oxygen saturation were accompanied by a significant reduction in the average length of microvessels in the left cortex but no differences were observed in the corpus callosum.

Conclusion

Inflammation in the developing brain induces marked reduction of locoregional oxygen saturation, predominantly in the white matter not explained by microvascular degeneration. The ability to examine regional saturation offers a new way to monitor injury and understand physiological disturbance non-invasively.     

Effects of N-Acetylcysteine in Ozone-Induced Chronic Obstructive Pulmonary Disease Model

Introduction

Chronic exposure to high levels of ozone induces emphysema and chronic inflammation in mice. We determined the recovery from ozone-induced injury and whether an antioxidant, N-acetylcysteine (NAC), could prevent or reverse the lung damage.

Methods

Mice were exposed to ozone (2.5 ppm, 3 hours/12 exposures, over 6 weeks) and studied 24 hours (24h) or 6 weeks (6W) later. Nac (100 mg/kg, intraperitoneally) was administered either before each exposure (preventive) or after completion of exposure (therapeutic) for 6 weeks.

Results

After ozone exposure, there was an increase in functional residual capacity, total lung volume, and lung compliance, and a reduction in the ratio of forced expiratory volume at 25 and 50 milliseconds to forced vital capacity (FEV₂₅/FVC, FEV₅₀/FVC). Mean linear intercept (L_m) and airway hyperresponsiveness (AHR) to acetylcholine increased, and remained unchanged at 6W after cessation of exposure. Preventive NAC reduced the number of BAL macrophages and airway smooth muscle (ASM) mass. Therapeutic NAC reversed AHR, and reduced ASM mass and apoptotic cells.

Conclusion

Emphysema and lung function changes were irreversible up to 6W after cessation of ozone exposure, and were not reversed by NAC. The beneficial effects of therapeutic NAC may be restricted to the ASM.     

Rhinovirus-16 Induced Release of IP-10 and IL-8 Is Augmented by Th2 Cytokines in a Pediatric Bronchial Epithelial Cell Model

Background

In response to viral infection, bronchial epithelial cells increase inflammatory cytokine release to activate the immune response and curtail viral replication. In atopic asthma, enhanced expression of Th2 cytokines is observed and we postulated that Th2 cytokines may augment the effects of rhinovirus-induced inflammation.

Methods

Primary bronchial epithelial cell cultures from pediatric subjects were treated with Th2 cytokines for 24 h before infection with RV16. Release of IL-8, IP-10 and GM-CSF was measured by ELISA. Infection was quantified using RTqPCR and TCID₅₀. Phosphatidyl inositol 3-kinase (PI3K) and P38 mitogen activated protein kinase (MAPK) inhibitors and dexamethasone were used to investigate differences in signaling pathways.

Results

The presence of Th2 cytokines did not affect RV replication or viral titre, yet there was a synergistic increase in IP-10 release from virally infected cells in the presence of Th2 cytokines. Release of IL-8 and GM-CSF was also augmented. IP-10 release was blocked by a PI3K inhibitor and IL-8 by dexamethasone.

Conclusion

Th2 cytokines increase release of inflammatory cytokines in the presence of rhinovirus infection. This increase is independent of effects of virus replication. Inhibition of the PI3K pathway inhibits IP-10 expression.     

Association of INSIG2 Polymorphism with Overweight and LDL in Children

Background

Dyslipidemia and overweight are common issues in children. Identifying genetic markers of risk could lead to targeted interventions. A polymorphism of SNP rs7566605 near insulin-induced gene 2 (INSIG2) has been identified as a strong candidate gene for obesity, through its feedback control of lipid synthesis.

Objective

To identify polymorphisms in INSIG2 which are associated with overweight (BMI ≥ 85% for age) and dyslipidemia in children. Hypothesis: The C allele of rs7566605 would be significantly associated with BMI and LDL.

Design/Methods

We genotyped 15 SNPs in/near INSIG2 in 1,058 healthy children (53% non-Hispanic white (NHW), 37% overweight) participating in a school based study. Genotype was compared with BMI and lipid markers, adjusting for age, gender, and puberty.

Results

We found a significant association between the SNP rs12464355 and LDL in NHW children, p < 0.001. The G allele is protective (lower LDL). A different SNP was associated with overweight in NHW: rs17047757. SNP rs7566605 was not associated with overweight or lipid levels.

Conclusions

We identified novel genetic associations between INSIG2 and both overweight and LDL in NHW children. Polymorphisms in INSIG2 may be important in the development of obesity through its effects on lipid regulation.