Elevated leukotriene B4 and 8-isoprostane in exhaled breath condensate from preterm-born infants.

BACKGROUND: Inflammation and oxidative stress play a key role in the development of bronchopulmonary dysplasia (BPD), possibly contributing to persistent respiratory morbidity after preterm birth. We aimed to assess if inflammatory markers were elevated in exhaled breath condensate (EBC) of infants born very prematurely (< 32 weeks gestation) at 12-16 corrected months of age, and if increased levels were associated with BPD diagnosis and respiratory morbidity. METHODS: EBC samples and respiratory questionnaires were collected from 15 term-born infants and 33 preterm-born infants, 12 with a neonatal BPD diagnosis. EBC samples were analysed for leukotriene B4 (inflammation) and 8-isoprostane (oxidative stress) concentrations using enzyme-linked immune-assays. Differences between groups were analysed by Kruskal-Wallis Test with post-hoc comparisons, independent samples t-test or Mann-Whitney U test depending on normality of the data. RESULTS: Leukotriene B4 and 8-isoprostane levels were elevated in exhaled breath condensate of preterm-born infants compared to those born at term (mean difference [95% CI]; 1.52 [0.45, 2.59], p = 0.02; 0.77 [0.52, 1.02], p < 0.001, respectively). Leukotriene B4 and 8-isoprostane levels were independent of BPD diagnosis and respiratory morbidity over the first year of life. CONCLUSIONS: Infants born very prematurely exhibit elevated markers of airway neutrophilic inflammation and oxidative stress beyond the first year of life, regardless of a neonatal diagnosis of chronic lung disease or respiratory morbidity during infancy. These findings may have implications for future lung health. TRIAL REGISTRATION: N/A.

Metabolic profiling of chronic obstructive pulmonary disease model rats and the interventional effects of HuaTanJiangQi decoction using UHPLC-Q-TOF/MSE.

The occurrence of chronic obstructive pulmonary disease (COPD) will lead to physiological and pathological variations and endogenous metabolic disorders. A traditional Chinese medicine formula, HuaTanJiangQi decoction (HTJQ), exhibits an unambiguous therapeutic effect on COPD in China. Nevertheless, the mechanism of its therapeutic effect on COPD is not clear. With this purpose, pulmonary function, histopathological and the inflammatory factors in bronchoalveolar lavage fluid (BALF) in rats model of COPD were investigated. Then, ultra high-performance liquid chromatography quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) analysis and multivariate statistical analysis were used to further reveal the mechanism of HTJQ therapeutic effect on COPD via metabolomics study. The results showed that the characteristics of lung tissues were significantly reversed, the concentration of LTB4 and LTC4 were gradually decreased, and the lung function began to recover after HTJQ treatment. These typical indicators of COPD in HTJQ intervention group were reversed similar to the control group, suggested that HTJQ has a therapeutic effect on COPD. Moreover, 32 dysregulated metabolites, including Thromboxane a2, Sphingosine 1-phosphate, PC(18:2(9Z,12Z)/18:1(11Z)), Leukotriene B4, Glutathione, Arachidonic acid, Sphingosylphosphocholine acid, N-Acetyl-leukotriene e4, Lysopc(18:1(11Z)), L-Cysteine, and Guanosine diphosphate. All the altered metabolites were associated with the onset and development of COPD, and involved in glycerophospholipid metabolism, sphingolipid metabolism, glutathione metabolism, and arachidonic acid metabolism, which were significantly changed in rats model with COPD. Generally, these findings provide a systematic view of metabolic changes linked to the onset and development of COPD, also indicated that HTJQ could provide satisfactory therapeutic effects on COPD and metabolomics study can be utilized to further understand the molecular mechanisms.

Root canal contamination or exposure to lipopolysaccharide differentially modulate prostaglandin E 2 and leukotriene B 4 signaling in apical periodontitis

Abstract Purpose To evaluate the kinetics of apical periodontitis development in vivo , induced either by contamination of the root canals by microorganisms from the oral cavity or by inoculation of bacterial lipopolysaccharide (LPS) and the regulation of major enzymes and receptors involved in the arachidonic acid metabolism. Methodology Apical periodontitis was induced in C57BL6 mice (n=96), by root canal exposure to oral cavity (n=48 teeth) or inoculation of LPS (10 µL of a suspension of 0.1 µg/µL) from E. coli into the root canals (n= 48 teeth). Healthy teeth were used as control (n=48 teeth). After 7, 14, 21 and 28 days the animals were euthanized and tissues removed for histopathological and qRT-PCR analyses. Histological analysis data were analyzed using two-way ANOVA followed by Sidak's test, and qRT-PCR data using two-way ANOVA followed by Tukey's test (α=0.05). Results Contamination by microorganisms led to the development of apical periodontitis, characterized by the recruitment of inflammatory cells and bone tissue resorption, whereas inoculation of LPS induced inflammatory cells recruitment without bone resorption. Both stimuli induced mRNA expression for cyclooxygenase-2 and 5-lipoxygenase enzymes. Expression of prostaglandin E 2 and leukotriene B 4 cell surface receptors were more stimulated by LPS. Regarding nuclear peroxisome proliferator-activated receptors (PPAR), oral contamination induced the synthesis of mRNA for PPARδ, differently from inoculation of LPS, that induced PPARα and PPARγ expression. Conclusions Contamination of the root canals by microorganisms from oral cavity induced the development of apical periodontitis differently than by inoculation with LPS, characterized by less bone loss than the first model. Regardless of the model used, it was found a local increase in the synthesis of mRNA for the enzymes 5-lipoxygenase and cyclooxygenase-2 of the arachidonic acid metabolism, as well as in the surface and nuclear receptors for the lipid mediators prostaglandin E2 and leukotriene B4.

Leukotriene B4 in equine asthma syndrome: what do we know so far? / Leucotrieno B4 na síndrome asmática equina: o que sabemos até o momento?

The term "equine asthma syndrome" (EAS) was recently proposed due to the resemblance of the equine disease to human asthma. Leukotrienes cause constriction of the bronchi, especially in the lower airways and increase mucus secretion in the respiratory system. Leukotriene B4 (LTB4) has been discovered as a strong chemotactic factor, which plays a role in neutrophil migration. The immunologic background of EAS remains not fully elucidated despite many studies on the pathogenesis. This study aimed to evaluate the LTB4 concentration in the bronchoalveolar lavage fluid (BALF) of horses with and without pulmonary inflammatory disease. Thirty-five mixed breed horses were studied and LTB4 was determined by using specific ELISA Kit. The horses were grouped by 2 different criteria for statistical analysis of data: 1) according to the values for BALF citology and 2) according to the detection of LTB4 in BALF. There was significant difference of effect of age on the LTB4 detection in equine BALF. Younger animals were the majority where it was possible to detect LTB4 values in LBA. In conclusion, there was an effect of age on the detection of LTB4 in equine BALF, where LTB4 levels were more easily detected in younger animals than older animals and the results of this study raise the possibility of considering future studies with the objective of establishing the real role and the best moment to detect LTB4 in BALF of the equine asthma syndrome.(AU)

Recentemente, o termo "síndrome da asma equina" (SAE) foi proposto devido à semelhança da doença equina à asma humana. Os leucotrienos causam constrição dos brônquios, especialmente nas vias aéreas posteriores e aumentam a secreção de muco no sistema respiratório. O leucotrieno B4 (LTB4) foi descoberto como um forte fator quimiotático, que desempenha um papel na migração de neutrófilos. O fundo imunológico do SAE permanece não completamente elucidado apesar de muitos estudos sobre a patogênese. Este estudo teve como objetivo avaliar a concentração de LTB4 no lavado broncoalveolar (LBA) de equinos com e sem doença inflamatória pulmonar. Trinta e cinco cavalos de raças mistas foram estudados e o LTB4 foi determinado usando o kit ELISA específico. Os animais foram agrupados por dois critérios diferentes para análise estatística dos dados: 1) de acordo com os valores para citologia do LBA e 2) de acordo com a detecção do LTB4 no LBA. Houve diferença significativa do efeito da idade na detecção do LTB4 no LBA equino. Os animais mais jovens foram a maioria onde foi possível detectar os valores de LTB4 no LBA. Em conclusão, houve um efeito da idade na detecção de LTB4 em LBA equino, onde os níveis de LTB4 foram mais facilmente detectados em animais jovens do que em animais mais velhos e foi possível detectar a concentração de LTB4 no LBA equino e os resultados deste estudo levantam a possibilidade de considerar futuros estudos com o objetivo de estabelecer o real papel e o melhor momento para detectar LTB4 no LBA da síndrome asmática equina.(AU)

Exhaled breath condensate hydrogen peroxide, pH and leukotriene B4 are associated with lower airway inflammation and airway cytology in the horse.

BACKGROUND: Exhaled breath condensate (EBC) analysis is a noninvasive method to assess the lower respiratory tract. In human subjects, EBC hydrogen peroxide (H2 O2 ), pH and leukotriene B4 (LTB4 ) are useful for detection and monitoring of inflammatory lung diseases, including asthma. OBJECTIVES: To determine associations between EBC biomarkers and cytological and endoscopic definitions of lower airway inflammation (LAI) while controlling for sampling and environmental variables. STUDY DESIGN: Prospective, cross-sectional study. METHODS: Clinical, endoscopic and airway cytological findings from 47 horses were compared with EBC pH and concentrations of H2 O2 and LTB4 by univariate and multivariable analyses. Dichotomous (presence/absence of airway inflammation) and continuous outcome variables (differential cell counts in tracheal aspirate and bronchoalveolar lavage fluid, BALF) were evaluated and potential effects of collection and methodological factors were included. RESULTS: EBC pH and H2 O2 concentrations were higher in horses with LAI and both were positively associated with the percentage of neutrophils in BALF (P<0.05). Mast cell percentage in BALF was negatively associated with EBC pH, and BALF eosinophil percentage was positively associated with EBC LTB4 (P<0.05). Ambient temperature, relative humidity and assay methodology significantly impacted some analytes. MAIN LIMITATIONS: LAI is challenging to categorise due to a variety of clinical and cytological phenotypes. Although the study was designed to overcome this limitation, numbers of horses were small in some categories. CONCLUSIONS: EBC pH and H2 O2 concentrations are altered by airway inflammation, suggesting a role for these biomarkers in the diagnosis and monitoring of airway disease. Environmental and methodological factors can influence these biomarkers and should be considered in the interpretation of results.

Erythromycin attenuates metalloprotease/anti-metalloprotease imbalance in cigarette smoke-induced emphysema in rats via the mitogen-activated protein kinase/nuclear factor-κB activation pathway.

The present study investigated whether erythromycin (ERY) reduces cigarette smoke (CS)-induced emphysema in rats and aimed to determine the anti-inflammatory effect of ERY, which may identify potential treatments for chronic obstructive pulmonary disease. Furthermore, the current study focused on the potential effects on the imbalance between matrix metalloprotease (MMP) and anti-MMP activity, the phosphorylation of mitogen-activated protein kinases (MAPKs) and the nuclear factor­κB (NF­κB) signaling pathway. Wistar rats were divided into the following three groups (n=12 each): control (ERY vehicle only, without any CS exposure), CS (animals were exposed to CS for 12 weeks) and CS + ERY (animals were exposed to CS for 12 weeks and received 100 mg/kg/day ERY). The recruitment of inflammatory cells into the bronchoalveolar lavage fluid (BALF) and the histopathology of lung tissue from all groups was evaluated to grade the severity of the emphysema. The expression of MMP­2, MMP­9 and tissue inhibitor of metalloproteinase­1 was evaluated by immunohistochemistry and western blotting. The activation of MAPKs, NF­κB and inhibitor of NF­κB (IκBα), in lung tissues was examined by western blotting. Treatment with ERY resulted in fewer inflammatory cells and cytokines in the BALF, and fewer emphysema­associated changes in the lungs compared with control. The stimulus of CS promoted the phosphorylation of extracellular signal­regulated kinase (ERK)1/2 and p38, but not c­Jun NH2­terminal kinase, thereby inducing the activation of the ERK/MAPK signaling pathway in rats. Furthermore, CS exposure increased the expression of NF-κB and decreased the expression of IκBα. The levels of phosphorylated ERK1/2 and p38 were significantly reduced in rats with CS­induced emphysema when treated with ERY compared with the CS group. The results of the present study therefore indicate that oral administration of ERY may suppress CS­induced emphysema by regulating inflammatory cytokines and the MMP/anti-MMP imbalance via the MAPK/NF-κB pathway.

A dual inhibitor of cyclooxygenase and 5-lipoxygenase protects against kainic acid-induced brain injury.

Systemic administration of kainic acid causes inflammation and apoptosis in the brain, resulting in neuronal loss. Dual cyclooxygenase/5-lipoxygenase (COX/5-LOX) inhibitors could represent a possible neuroprotective approach in preventing glutamate excitotoxicity. Consequently, we investigated the effects of a dual inhibitor of COX/5-LOX following intraperitoneal administration of kainic acid (KA, 10 mg/kg) in rats. Animals were randomized to receive either the dual inhibitor of COX/5-LOX (flavocoxid, 20 mg/kg i.p.) or its vehicle (1 ml/kg i.p.) 30 min after KA administration. Sham brain injury rats were used as controls. We evaluated protein expression of phosphorylated extracellular signal-regulated kinase (p-ERK1/2) and tumor necrosis factor alpha (TNF-α) as well as levels of malondialdehyde (MDA), prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) in the hippocampus. Animals were also observed for monitoring behavioral changes according to Racine Scale. Finally, histological analysis and brain edema evaluation were carried out. Treatment with the dual inhibitor of COX/5-LOX decreased protein expression of p-ERK1/2 and TNF-α in hippocampus, markedly reduced MDA, LTB4 and PGE2 hippocampal levels, and also ameliorated brain edema. Histological analysis showed a reduction in cell damage in rats treated with the dual inhibitor of COX/5-LOX, particularly in hippocampal subregion CA3c. Moreover, flavocoxid significantly improved behavioral signs following kainic acid administration. Our results suggest that dual inhibition of COX/5-LOX by flavocoxid has neuroprotective effects during kainic acid-induced excitotoxicity.

[Determining asthma treatment in children by monitoring fractional exhaled nitric oxide, sputum eosinophils and leukotriene B4]. / Monitorización del tratamiento en el asma bronquial mediante determinación de fracción exhalada de óxido nítrico, eosinófilos y leucotrieno B4 en esputo inducido de población infantil.

Sputum eosinophils and exhaled fractional nitric oxide (FENO) are markers of airway inflammation in asthma. Cytokines, cysteinyl-leukotrienes and leukotriene B4 (LTB4) are responsible for this inflammation. The aim of this study is to determine the usefulness of these markers in monitoring asthma treatment in children. FENO, sputum eosinophils, and LTB4 in induced sputum were performed in 10 children (9-15 years old). These determinations were repeated four months later, after the beginning or an increase in the treatment. FENO values tended to decrease (P=.15), pulmonary function tended to improve (P=.10), and sputum eosinophils decreased (P=.003) compared to the first determination. There were no differences in LTB4 concentrations (P=.88). Sputum eosinophils seem to be more precise than FENO in the monitoring of inflammation in asthmatic children.

Synthesis of leukotrienes in porcine uteri with endometritis induced by infection with Escherichia coli.

Leukotrienes (LTs) are lipid mediators that play a significant role in the inflammatory process. Their production in inflamed uteri is not fully understood. The present experiment aimed to determine LTB4 and LTC4 amounts, 5-lipooxygenase (5-LO), LTA4 hydrolase (LTAH) and LTC4 synthase (LTCS) mRNA levels and protein expression in inflamed porcine uteri. On Day 3 of the oestrous cycle (Day 0 of the study), either Escherichia coli suspension or saline were infused into uterine horns. Collection of uterine tissues and washings took place eight or sixteen days later. In gilts suffering from endometritis increased LTB4 and LTC4 levels in the endometrium and washings and 5-LO mRNA levels in the myometrium on Days 8 and 16, 5-LO protein levels in the endometrium and myometrium on Day 8, LTAH mRNA and protein levels in the endometrium and myometrium on Days 8 and 16, respectively. Although LTCS mRNA and protein expression in the myometrium and LTCS protein expression in the endometrium were enhanced on Day 16 after Escherichia coli inoculation, LTCS mRNA levels decreased on Day 8 in both tissues. Our study shows the upregulation of LT production in inflamed porcine uteri, which suggests the importance of these factors to the process of uterine inflammation.

Inflammatory mediators in saliva associated with arterial stiffness and subclinical atherosclerosis.

OBJECTIVE: Whereas circulating levels of C-reactive protein (CRP) have been associated with, for example, arterial stiffness, subclinical atherosclerosis and metabolic syndrome, other inflammatory biomarkers with potential interest for these conditions may not be measurable systemically. The predictive value of salivary biomarkers in these contexts has remained largely unexplored. The aim of the present study was to establish the association of different salivary biomarkers of inflammation with subclinical cardiovascular disease. METHODS: Two hundred and fifty-nine individuals were included in the study. Saliva and plasma samples were collected, and each individual underwent carotid ultrasound and measures of pulse wave velocity and blood pressure. Medical history of previous cardiovascular disease, current medications and smoking were collected by questionnaire. RESULTS: Salivary levels of CRP, leukotriene B4 (LTB4), prostaglandin E2 (PGE2), matrix metalloproteinase 9 (MMP-9), creatinine and lysozyme were measured. Salivary levels of CRP were significantly correlated with plasma levels (r = 0.73, P < 0.0001). In an age-adjusted and sex-adjusted analysis, salivary CRP was significantly and positively correlated with mean arterial blood pressure, pulse pressure, pulse wave velocity, BMI, metabolic syndrome, waist-to-hip ratio and intima-media thickness. Increasing age and sex-adjusted salivary CRP tertiles were in addition associated with carotid plaques. In a multivariate analysis, CRP and MMP-9 were associated with intima-media thickness, LTB4 and PGE2 with arterial stiffness, and lysozyme with hypertension. CONCLUSION: Saliva may represent an alternative mean for evaluation of cardiovascular risk.

Effect of allergen sensitization on external root resorption.

In orthodontic tooth movement (OTM), we should be concerned about external root resorption (ERR) as an undesirable iatrogenic problem, but its mechanisms are not fully understood. Since our previous epidemiologic studies found that patients with allergic diseases showed higher rates of ERR during orthodontic treatment, we explored the possible effect of allergic sensitization on ERR. In ovalbumin (OVA)-sensitized Brown-Norway rats, the amounts of ERR and OTM were greater than those in animals subjected to orthodontic force alone. The expression levels of RANKL and pro-inflammatory cytokines were increased in the periodontal tissues of sensitized rats with OTM, compared with control rats. Furthermore, leukotriene B4 (LTB4), a potent lipid mediator of allergic inflammation, and enzymes of the 5-lipoxygenase pathway, the biosynthetic pathway of leukotrienes, were also up-regulated. We found that low doses of aspirin suppressed ERR in allergen-sensitized rats, as well as the expressions of RANKL, pro-inflammatory cytokines, and LTB4. The present findings indicate that allergen sensitization has adverse effects on ERR under OTM, and that aspirin is a potential therapeutic agent for combating ERR.

Comparison of different non-invasive methods for detection of allergic asthma.

Non-invasive methods to assess inflammation of lower airways are induced sputum (IS), exhaled nitric oxide (eNO), and exhaled breath condensate (EBC). Here we focused on the assessment of airway inflammation with a panel of non-invasive methods in health care workers (HCWs) with suspected latex allergy with and without current allergic respiratory symptoms about 10 years after the latex ban in German health care facilities. Seventy-seven non-smoking subjects were examined by skin prick test and specific IgE measurements, eNO, IS, and EBC. Sensitivity, specificity, and positive and negative predicted values for relevant biomarkers were calculated using current asthma symptoms as the gold standard. Twenty-nine subjects (38%) reported ongoing asthmatic symptoms (AS). In these subjects the EBC concentrations of nitrogen oxides (NO(x); p=0.027) and leukotriene B(4) (p=0.025) were significantly higher than in subjects without AS. In addition, in the subjects with AS the numbers of eosinophils (p=0.015) and the concentrations of IL-5 (p= 0.021) in IS samples were significantly higher than in the subjects without AS. A good correlation between several inflammatory markers in IS was detected. The maximum Youden Index was reached for IS total eosinophils ≥3.5·10(4) with a test efficiency of 0.72. In conclusion, non-invasive inflammatory monitoring with EBC and IS may assist the diagnosis of allergic asthma. Self-reported current asthmatic symptoms were reflected by eosinophilic inflammation and the best parameter to support the asthma diagnosis is a total number of eosinophils ≥3.5·10(4) in IS.

Leukotrienes B4, C4, D4 and E4 in the exhaled breath condensate (EBC), blood and urine in patients with pneumoconiosis.

Leukotrienes (LTs) are involved in the pathogenesis of lung fibrosis and were increased in exhaled breath condensate (EBC) of the patients with pneumoconiosis. However the possible influence of extra-pulmonary disorders on the EBC markers is not known. Therefore in parallel with EBC, LTs' levels in the plasma and urine were measured in patients with pneumoconiosis (45 × asbestos exposure, 37 × silica exposure) and in 27 controls. Individual LTs B4, C4, D4 and E4 were measured by liquid chromatography - electrospray ionization - tandem mass spectrometry (LC-ESI-MS/MS). In EBC, LT D4 and LT E4 were increased in both groups of patients (p<0.001 and p<0.05), comparing with the controls. Both LT B4 and cysteinyl LTs were elevated in asbestos-exposed subjects (p<0.05). Asbestosis with more severe radiological signs (s1/s2-t3/u2) and lung functions impairment has shown higher cysteinyl LTs and LT C4 in the EBC (p<0.05) than mild asbestosis (s1/s0-s1/s1). In addition, in the subjects with asbestosis, cysteinyl LTs in EBC correlated with TLC (-0.313, p<0.05) and TLCO/Hb (-0.307, p<0.05), and LT C4 with TLC (-0.358, p<0.05). In pneumoconioses, EBC appears the most useful from the 3 fluids studied.

Effects of herpes simplex virus type 1 infection on immune functions of human neutrophils.

BACKGROUND AND OBJECTIVE: Herpesviruses may play roles in the development of periodontal diseases. This study analyzed the effects of herpes simplex virus type 1 (HSV-1) infection on neutrophil function. The effects of lipopolysaccharide (LPS) from the periodontal pathogen, Porphyromonas gingivalis, during HSV-1 infection were also determined. MATERIAL AND METHODS: Purified HSV-1 was pretreated with buffer containing no serum, with HSV-1 immunoglobulin G (IgG)-positive serum (HSV-1 antiserum) or with control serum. Neutrophils were mock-infected or infected with the pretreated HSV-1. Viral binding and phagosome formation were detected using immunostaining. Intracellular reactive oxygen species (ROS) were determined using 2',7'-dichlorofluorescin diacetate and fluorometry. Leukotriene B(4) (LTB(4)) and interleukin-8 (IL-8) were detected using enzyme immunoassays. Release of matrix metalloproteinase-9 (MMP-9) was examined using gelatin zymography. Phosphorylation of Akt/glycogen synthase kinase-3 (GSK-3) was determined using western blotting. RESULTS: HSV-1 bound directly to neutrophils and enhanced the release of MMP-9. HSV-1 immune complexes, formed in the HSV-1 antiserum, bound neutrophils and induced the formation of early phagosome more effectively than did HSV-1 alone. The relative levels of ROS and phosphorylation of Akt/GSK-3 were increased significantly in neutrophils after infection with HSV-1 immune complexes. Infection with HSV-1 and HSV-1 immune complexes also stimulated the production of inflammatory mediators, LTB(4) and IL-8. Moreover, LPS enhanced the HSV-1-stimulatory production of IL-8. CONCLUSION: This study demonstrated differences in neutrophils infected with HSV-1 alone or with HSV-1 immune complexes, suggesting that opsonization of HSV-1 might enhance its effects on neutrophils. The in vitro findings suggest that HSV-1 infection may induce the inflammatory response and affect periodontal health.

Monocyte chemoattractant protein 1 regulates pulmonary host defense via neutrophil recruitment during Escherichia coli infection.

Neutrophil accumulation is a critical event to clear bacteria. Since uncontrolled neutrophil recruitment can cause severe lung damage, understanding neutrophil trafficking mechanisms is important to attenuate neutrophil-mediated damage. While monocyte chemoattractant protein 1 (MCP-1) is known to be a monocyte chemoattractant, its role in pulmonary neutrophil-mediated host defense against Gram-negative bacterial infection is not understood. We hypothesized that MCP-1/chemokine (C-C motif) ligand 2 is important for neutrophil-mediated host defense. Reduced bacterial clearance in the lungs was observed in MCP-1(-/-) mice following Escherichia coli infection. Neutrophil influx, along with cytokines/chemokines, leukotriene B(4) (LTB(4)), and vascular cell adhesion molecule 1 levels in the lungs, was reduced in MCP-1(-/-) mice after infection. E. coli-induced activation of NF-κB and mitogen-activated protein kinases in the lung was also reduced in MCP-1(-/-) mice. Administration of intratracheal recombinant MCP-1 (rMCP-1) to MCP-1(-/-) mice induced pulmonary neutrophil influx and cytokine/chemokine responses in the presence or absence of E. coli infection. Our in vitro migration experiment demonstrates MCP-1-mediated neutrophil chemotaxis. Notably, chemokine receptor 2 is expressed on lung and blood neutrophils, which are increased upon E. coli infection. Furthermore, our findings show that neutrophil depletion impairs E. coli clearance and that exogenous rMCP-1 after infection improves bacterial clearance in the lungs. Overall, these new findings demonstrate that E. coli-induced MCP-1 causes neutrophil recruitment directly via chemotaxis as well as indirectly via modulation of keratinocyte cell-derived chemokine, macrophage inflammatory protein 2, and LTB(4).

The effects of dietary fish oil on inflammation, fibrosis and oxidative stress associated with obstructive renal injury in rats.

SCOPE: We examined whether dietary supplementation with fish oil modulates inflammation, fibrosis and oxidative stress following obstructive renal injury. METHODS AND RESULTS: Three groups of Sprague-Dawley rats (n=16 per group) were fed for 4 wk on normal rat chow (oleic acid), chow containing fish oil (33 g eicosapentaenoic acid and 26 g docosahexaenoic acid per kg diet), or chow containing safflower oil (60 g linoleic acid per kg diet). All diets contained 7% fat. After 4 wk, the rats were further subdivided into four smaller groups (n=4 per group). Unilateral ureteral obstruction was induced in three groups (for 4, 7 and 14 days). The fourth group for each diet did not undergo surgery, and was sacrificed as controls at 14 days. When rats were sacrificed, plasma and portions of the kidneys were removed and frozen; other portions of kidney tissue were fixed and prepared for histology. Compared with normal chow and safflower oil, fish oil attenuated collagen deposition, macrophage infiltration, TGF-ß expression, apoptosis, and tissue levels of arachidonic acid, MIP-1α, IL-1ß, MCP-1 and leukotriene B(4). Compared with normal chow, fish oil increased the expression of HO-1 protein in kidney tissue. CONCLUSIONS: Fish oil intake reduced inflammation, fibrosis and oxidative stress following obstructive renal injury.

A phase II randomized placebo-controlled trial of omega-3 fatty acids for the treatment of acute lung injury.

OBJECTIVES: Administration of eicosapentaenoic acid and docosahexanoic acid, omega-3 fatty acids in fish oil, has been associated with improved patient outcomes in acute lung injury when studied in a commercial enteral formula. However, fish oil has not been tested independently in acute lung injury. We therefore sought to determine whether enteral fish oil alone would reduce pulmonary and systemic inflammation in patients with acute lung injury. DESIGN: Phase II randomized controlled trial. SETTING: Five North American medical centers. PATIENTS: Mechanically ventilated patients with acute lung injury ≥18 yrs of age. INTERVENTIONS: Subjects were randomized to receive enteral fish oil (9.75 g eicosapentaenoic acid and 6.75 g docosahexanoic acid daily) or saline placebo for up to 14 days. MEASUREMENTS AND MAIN RESULTS: Bronchoalveolar lavage fluid and blood were collected at baseline (day 0), day 4 ± 1, and day 8 ± 1. The primary end point was bronchoalveolar lavage fluid interleukin-8 levels. Forty-one participants received fish oil and 49 received placebo. Enteral fish oil administration was associated with increased serum eicosapentaenoic acid concentration (p < .0001). However, there was no significant difference in the change in bronchoalveolar lavage fluid interleukin-8 from baseline to day 4 (p = .37) or day 8 (p = .55) between treatment arms. There were no appreciable improvements in other bronchoalveolar lavage fluid or plasma biomarkers in the fish oil group compared with the control group. Similarly, organ failure score, ventilator-free days, intensive care unit-free days, and 60-day mortality did not differ between the groups. CONCLUSIONS: Fish oil did not reduce biomarkers of pulmonary or systemic inflammation in patients with acute lung injury, and the results do not support the conduct of a larger clinical trial in this population with this agent. This experimental approach is feasible for proof-of-concept studies evaluating new treatments for acute lung injury.

Sea-cod oil supplementation alters the course of Streptococcus pneumoniae infection in BALB/c mice.

The existing reports on the role of ω-3 polyunsaturated fatty acids (PUFA) in infectious diseases are contradictory. The present study was conducted to evaluate the effect of sea-cod oil on the course of respiratory tract infection by Streptococcus pneumoniae in BALB/c mice. Animals were given enteral sea-cod oil for a period of 30 and 60 days and challenged intra-tracheally with S. pneumoniae D39 serotype 2. The survival of animals and various inflammatory parameters, i.e. myeloperoxidase (MPO), malondialdehyde (MDA), nitric oxide (NO) and leukotriene B(4) in the lung homogenates, were investigated. The inflammatory cytokines levels (IL-1ß, TNF-α and IL-10) were also determined. Continuous sea-cod oil supplementation for 60 days significantly improved survival among the animals. A significant reduction in the bacterial load in the lungs of sea-cod oil-fed animals compared to the controls was observed. As the disease progressed, the reduced bacterial colonisation correlated well with the histopathological observation. This was accompanied by a decrease in the production of inflammatory mediators and cytokines in the lung homogenates. However, not even a minor difference was seen in animals given sea-cod oil supplementation for 30 days duration; therefore, long-term treatment was required to attain an effect. Sea-cod oil supplementation modulated the host immune response and, thus, protected the host from ensuing inflammatory damage due to S. pneumoniae-mediated infection.

Analysis of exhaled breath condensate in a mixed population of psittacine birds.

Collection of exhaled breath condensate (EBC) and the measurement of inflammatory markers contained therein (eg, hydrogen peroxide [H2O2], leukotriene B4 [LTB4], and pH) have been reported to be noninvasive tools for the investigation of respiratory disease in various species. In this study, the EBC of clinically healthy psittacine birds (n = 15) and psittacine birds with respiratory tract disease (n = 19) was examined, and inflammatory markers contained in the EBC were analyzed and compared. Awake birds were placed in an acrylic container from which the outflow passed through a condensation system that collected the EBC. All samples were analyzed for pH, H2O2, and LTB4. The mean values for each of these components, as well as the mean volume of the total EBC, measured from the apparently healthy birds did not differ significantly from those measured in birds with signs of respiratory tract disease. However, LTB4 in the EBC of diseased birds was higher than that of the apparently healthy birds and showed a trend toward significance. The study demonstrated the establishment of a standardized method for collecting and analyzing EBC in psittacine birds and a measurement protocol for pH, H2O2, and LTB4.

Improved sensitivity mass spectrometric detection of eicosanoids by charge reversal derivatization.

Combined liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) is a powerful method for the analysis of oxygenated metabolites of polyunsaturated fatty acids including eicosanoids. Here we describe the synthesis of a new derivatization reagent N-(4-aminomethylphenyl)pyridinium (AMPP) that can be coupled to eicosanoids via an amide linkage in quantitative yield. Conversion of the carboxylic acid of eicosanoids to a cationic AMPP amide improves sensitivity of detection by 10- to 20-fold compared to negative mode electrospray ionization detection of underivatized analytes. This charge reversal derivatization allows detection of cations rather than anions in the electrospray ionization mass spectrometer, which enhances sensitivity. Another factor is that AMPP amides undergo considerable collision-induced dissociation in the analyte portion rather than exclusively in the cationic tag portion, which allows isobaric derivatives to be distinguished by tandem mass spectrometry, and this further enhances sensitivity and specificity. This simple derivatization method allows prostaglandins, thromboxane B(2), leukotriene B(4), hydroxyeicosatetraenoic acid isomers, and arachidonic acid to be quantified in complex biological samples with limits of quantification in the 200-900 fg range. One can anticipate that the AMPP derivatization method can be extended to other carboxylic acid analytes for enhanced sensitivity detection.