Metabolomic analysis of breath volatile organic compounds reveals unique breathprints in children with inflammatory bowel disease: a pilot study.

BACKGROUND: Breath testing is becoming an important diagnostic method to evaluate many disease states. In the light of rising healthcare costs, is important to develop a simple non-invasive tool to potentially identify paediatric patients who need endoscopy for suspected inflammatory bowel disease (IBD). AIM: To analyse exhaled volatile organic compounds (VOCs) and investigate the presence of a unique breath patterns to differentiate paediatric patients with (IBD) from healthy controls. METHODS: A cross-sectional, single-centre study included paediatric IBD patients and healthy controls (age range, 5-21 years). The diagnosis of IBD was confirmed by endoscopic, histological and radiographic data. Exhaled breath was collected and analysed using a selective ion flow tube mass spectroscopy (SIFT-MS) to identify new markers or patterns of IBD. RESULTS: One hundred and seventeen patients (62 with IBD and 55 healthy controls) were included in the study. Linear discriminant analysis and principle component analysis of mass scanning ion peak data demonstrated 21 pre-selected VOCs correctly classify patients with IBD or as healthy controls; P < 0.0001. Multivariable logistic regression analysis further showed three specific VOCs (1-octene, 1-decene, (E)-2-nonene) had excellent accuracy for predicting the presence of IBD with an area under the curve (AUC) of 0.96 (95% CI: 0.93-0.99). No significant difference in VOCs was found between patients with Crohn's disease or ulcerative colitis, and no significant correlation was seen with disease activity. CONCLUSION: These pilot data support the hypothesis that a unique breathprint potentially exists for paediatric IBD in the exhaled metabolome.

Biomarkers in exhaled breath condensate: a review of collection, processing and analysis.

Exhaled breath condensate (EBC) is a potential rich source for countless biomarkers that can provide valuable information about respiratory as well as systemic diseases. EBC has been studied in a variety of diseases including allergic rhinitis, asthma, chronic obstructive lung disease, cystic fibrosis, lung cancer, and obstructive sleep apnea syndrome. Although numerous biomarkers have been discovered and studied in EBC, the methods of collection and biomarker detection have not been fully standardized. While leaving standardization methods up to individual labs for the present time is optimal for the continued discovery of new biomarkers in EBC, this decreases the reproducibility and generalizability of the findings. In this review we will discuss specific biomarkers studied in specific diseases as well as some of the related technical issues including collection, processing and analysis.

Exhaled breath condensate: methodological recommendations and unresolved questions.

Collection of exhaled breath condensate (EBC) is a noninvasive method for obtaining samples from the lungs. EBC contains large number of mediators including adenosine, ammonia, hydrogen peroxide, isoprostanes, leukotrienes, nitrogen oxides, peptides and cytokines. Concentrations of these mediators are influenced by lung diseases and modulated by therapeutic interventions. Similarly EBC pH also changes in respiratory diseases. The aim of the American Thoracic Society/European Respiratory Society Task Force on EBC was to identify the important methodological issues surrounding EBC collection and assay, to provide recommendations for the measurements and to highlight areas where further research is required. Based on the currently available evidence and the consensus of the expert panel for EBC collection, the following general recommendations were put together for oral sample collection: collect during tidal breathing using a noseclip and a saliva trap; define cooling temperature and collection time (10 min is generally sufficient to obtain 1-2 mL of sample and well tolerated by patients); use inert material for condenser; do not use resistor and do not use filter between the subject and the condenser. These are only general recommendations and certain circumstances may dictate variation from them. Important areas for future research involve: ascertaining mechanisms and site of exhaled breath condensate particle formation; determination of dilution markers; improving reproducibility; employment of EBC in longitudinal studies; and determining the utility of exhaled breath condensate measures for the management of individual patients. These studies are required before recommending this technique for use in clinical practice.

HLA-DP-unrestricted TNF-alpha release in beryllium-stimulated peripheral blood mononuclear cells.

Berylliosis is a granulomatous disorder of the lung caused by inhalation of beryllium (Be) and dominated by the accumulation of CD4+ T-helper (Th)1 memory T-cells proliferating in response to Be in the lower respiratory tract. Two gene markers have been associated with susceptibility to berylliosis: 1) the human leucocyte antigen (HLA)-DP gene whose allelic variants, carrying glutamate in position 69 of the beta-chain (HLA-DPGlu69), can bind Be directly and present it to interferon (IFN)-gamma releasing Th1 T-cell clones from patients with berylliosis; and 2) the cytokine gene tumour necrosis factor (TNF)-alpha which has been shown to increase berylliosis risk independent of HLA-DPGlu69. In order to determine whether TNF-alpha release was triggered by Th1 T-cell activation by Be stimulation in the context of HLA-DPGlu69 molecules, the proliferation of BeSO4-stimulated blood mononuclear cells and the release of IFN-gamma, TNF-alpha, RANTES (regulated on activation normal T-cell expressed and secreted), granulocyte-macrophage colony-stimulating factor, interleukin (IL)-4, IL-6, IL-8, IL-10 and IL-12 by BeSO4-stimulated blood mononuclear cells was quantified in 11 individuals with berylliosis using an anti-HLA-DP antibody as a probe for HLA-DP restricted T-cell activation. While proliferation and IFN-gamma release were completely abrogated by HLA-DP inhibition (inhibition with anti-HLA-DP monoclonal antibody (mAb): 88+/-16 and 77+/-16%, respectively; anti-HLA-DR: 29+/-38 and 14+/-10%, respectively), the release of TNF-alpha was not (inhibition with anti-HLA-DP mAb: 8.9+/-7.8%). No other cytokine was detected at significant levels. Moreover, Be was able to induce TNF-alpha production in healthy control subjects not exposed to Be in the absence of T-cell proliferation and IFN-gamma production. In conclusion, these data suggest that the tumour necrosis factor-alpha response of mononuclear cells is independent of the activation of beryllium-specific human leucocyte anitgen-DP restricted T-cells, which is consistent with the finding that the tumour necrosis factorA2 and the human leucocyte anitgen-DPGlu69 genetic markers are independently interacting in increasing berylliosis risk.

Alterations in exhaled gas profile during allergen-induced asthmatic response.

The source of exhaled carbon monoxide (CO) and the relationship to airway inflammation are not clear. If CO is produced by the inflamed airway, we hypothesized that inflammation induced by allergen challenge would increase exhaled CO of atopic asthmatics. Eight atopic asthmatics underwent whole lung allergen challenge. CO, nitric oxide (NO), oxygen, and carbon dioxide (CO(2)) were measured simultaneously in exhaled breath which was collected into Mylar balloons before (baseline), immediately after, and at subsequent times after allergen. NO was higher in asthmatics than control subjects at baseline, increased further in seven of the eight asthmatics after allergen, and was inversely correlated to specific conductance. In contrast, exhaled CO of asthmatics was not higher than that of control individuals at baseline, decreased immediately after allergen, and returned to baseline levels during the late asthmatic response. Thus, allergen-induced airway inflammation did not lead to increased exhaled CO in asthma.

Major histocompatibility locus genetic markers of beryllium sensitization and disease.

Hypersensitivity to beryllium (Be) is found in 1-16% of exposed workers undergoing immunological screening for beryllium disease using the beryllium lymphocyte proliferation test (BeLPT). However, only approximately 50% of BeLPT-positive workers present with lung granulomas (i.e. berylliosis). As berylliosis is associated with the human leukocyte antigen (HLA)-DP supratypic marker DPGlu69, the authors asked whether this marker is differentially associated with disease presentation. A population of 639 workers from a beryllium factory undergoing BeLPT screening was evaluated in a nested case-control study for the prevalence of HLA-DPGlu69, the HLA-DPB1, HLA-DQ and HLA-DR alleles and of the biallelic tumour necrosis factor (TNF)-alpha polymorphism TNF-alpha-308 in 23 individuals presenting as "sensitized" (i.e. BeLPT-positive without lung granulomas) and in 22 presenting as "diseased" (i.e. BeLPT-positive with granulomas in the lung biopsy). The HLA-DPGlu69 marker was associated with "disease" (odds ratio (OR) 3.7, p=0.016, 95% confidence interval (CI) 1.4-10.0), whilst the high TNF-alpha production-related TNF-alpha-308*2 marker was associated with both a positive BeLPT (OR 7.8, corrected p<0.0001, 95% CI 3.2-19.1) with no difference between "sensitization" and "disease". Furthermore, the HLA-DRArg74 marker was associated with "sensitization" without disease (OR 3.96, p=0.005, 95%, CI 1.5-10.1). The data indicate that tumour necrosis factor-alpha, human leukocyte antigen-DR and human leukocyte antigen-DP markers play different roles in beryllium sensitization and granuloma formation in beryllium-exposed workers.

High levels of exhaled nitric oxide (NO) and NO synthase III expression in lesional smooth muscle in lymphangioleiomyomatosis.

Smooth-muscle proliferation is the hallmark of lymphangioleiomyomatosis (LAM). Although little is known about the pathogenesis of LAM, nitric oxide (NO) is a key regulator of smooth-muscle proliferation. NO is linked to the pathogenesis of other lung diseases such as asthma, in part by the finding of higher-than-normal levels of exhaled NO. If NO were involved in the abnormal smooth-muscle proliferation in LAM, we reasoned that exhaled NO from individuals with LAM would also differ from that of healthy control subjects. To evaluate this hypothesis, we studied exhaled NO in individuals with LAM in comparison with healthy and asthmatic women using a chemiluminescent NO analyzer. Women with LAM had higher exhaled NO than did healthy women but lower than asthmatic women (NO [parts per billion] median (25 to 75%): LAM 8 [7 to 15] [n = 28], control 6 [5 to 8] [n = 21], asthma 14 [8 to 25] [n = 22]; Kruskal-Wallis P < 0.001). Immunohistochemical studies on formalin-fixed, paraffin-embedded sections of surgical and autopsy material from lungs of individuals with LAM showed diffuse NO synthase III (NOSIII) expression in the lesional smooth muscle of LAM similar to that in the vascular endothelium. NOSIII expression was limited to the vascular endothelium and bronchial smooth muscle in healthy control lungs. The increased NO and the presence of NOSIII expression in lesional smooth muscle warrants further study into the potential role for NO in the pathogenesis of LAM.

A 65-year-old factory worker with dyspnea on exertion and a normal chest x-ray.

Chronic beryllium disease is an occupationally acquired granulomatous lung disease similar to sarcoidosis. It is caused by exposure to beryllium in genetically susceptible persons. It should be suspected in patients with beryllium exposure who present with pulmonary symptoms or have a positive screening blood beryllium-specific lymphocyte proliferation test. The diagnosis is confirmed by the finding of granulomas on transbronchial biopsy in the appropriate clinical and epidemiologic setting. Although there is no cure, treatment with corticosteroids is usually beneficial. In view of the potential side effects, treatment is reserved for patients with symptoms or a decline in pulmonary function.

Molecular mechanisms of increased nitric oxide (NO) in asthma: evidence for transcriptional and post-translational regulation of NO synthesis.

Evidence supporting increased nitric oxide (NO) in asthma is substantial, although the cellular and molecular mechanisms leading to increased NO are not known. Here, we provide a clear picture of the events regulating NO synthesis in the human asthmatic airway in vivo. We show that human airway epithelium has abundant expression of NO synthase II (NOSII) due to continuous transcriptional activation of the gene in vivo. Individuals with asthma have higher than normal NO concentrations and increased NOSII mRNA and protein due to transcriptional regulation through activation of Stat1. NOSII mRNA expression decreases in asthmatics receiving inhaled corticosteroid, treatment effective in reducing inflammation in asthmatic airways. In addition to transcriptional mechanisms, post-translational events contribute to increased NO synthesis. Specifically, high output production of NO is fueled by a previously unsuspected increase in the NOS substrate, l -arginine, in airway epithelial cells of asthmatic individuals. Finally, nitration of proteins in airway epithelium provide evidence of functional consequences of increased NO. In conclusion, these studies define multiple mechanisms that function coordinately to support high level NO synthesis in the asthmatic airway. These findings represent a crucial cornerstone for future therapeutic strategies aimed at regulating NO synthesis in asthma.

Eosinophils generate brominating oxidants in allergen-induced asthma.

Eosinophils promote tissue injury and contribute to the pathogenesis of allergen-triggered diseases like asthma, but the chemical basis of damage to eosinophil targets is unknown. We now demonstrate that eosinophil activation in vivo results in oxidative damage of proteins through bromination of tyrosine residues, a heretofore unrecognized pathway for covalent modification of biologic targets in human tissues. Mass spectrometric studies demonstrated that 3-bromotyrosine serves as a specific "molecular fingerprint" for proteins modified through the eosinophil peroxidase-H(2)O(2) system in the presence of plasma levels of halides. We applied a localized allergen challenge to model the effects of eosinophils and brominating oxidants in human lung injury. Endobronchial biopsy specimens from allergen-challenged lung segments of asthmatic, but not healthy control, subjects demonstrated significant enrichments in eosinophils and eosinophil peroxidase. Baseline levels of 3-bromotyrosine in bronchoalveolar lavage (BAL) proteins from mildly allergic asthmatic individuals were modestly but not statistically significantly elevated over those in control subjects. After exposure to segmental allergen challenge, lung segments of asthmatics, but not healthy control subjects, exhibited a >10-fold increase in BAL 3-bromotyrosine content, but only two- to threefold increases in 3-chlorotyrosine, a specific oxidation product formed by neutrophil- and monocyte-derived myeloperoxidase. These results identify reactive brominating species produced by eosinophils as a distinct class of oxidants formed in vivo. They also reveal eosinophil peroxidase as a potential therapeutic target for allergen-triggered inflammatory tissue injury in humans.

Nitric oxide regulation of asthmatic airway inflammation with segmental allergen challenge.

BACKGROUND: Despite evidence of increased nitric oxide (NO) in asthmatic compared with healthy individuals, the role of NO in airway inflammation is unclear. OBJECTIVE: The purpose of the study was to determine the in vivo effects of localized allergen challenge on airway NO levels and transcription factor activation. METHODS: In this study localized allergen challenge was used as a model of asthmatic exacerbation to determine the relationship of NO to airway inflammation. RESULTS: With allergen challenge, asthmatic patients had a rise in airway NO levels, whereas NO levels in healthy controls did not change. The increased NO in asthma with allergen challenge compared with healthy control subjects was associated with an increase in inflammatory cytokines (GM-CSF and macrophage inflammatory protein-1) in epithelial lining fluid and eosinophilic infiltrate in bronchoalveolar lavage fluid (BAL) and biopsy specimens. To investigate the mechanisms of cytokine gene expression, activation of the transcription factors activator protein-1 and nuclear factor-kappaB (NF-kappaB) in cells from BAL were evaluated. Activator protein-1 was not activated before or after local allergen challenge. In contrast, NF-kappaB activation was less in BAL cells from asthmatic patients with increased NO in comparison with controls. CONCLUSION: Our studies are the first to suggest an inverse correlation between NF-kappaB and airway NO in a localized segmental allergen challenge model in allergic asthmatic patients. The current study demonstrates that activation of the inflammatory response (eg, cytokines, cellular infiltrate) in allergic asthmatic patients is temporally associated with increased airway NO. We propose that NO that is up-regulated by cytokines is part of an autoregulatory feedback loop (ie, allergen challenge stimulates inflammatory cytokine production, which in turn stimulates NO production, and NO down-regulates cytokine production).

Nitric oxide blocks nuclear factor-kappaB activation in alveolar macrophages.

Nitric oxide (NO) is an important endogenous regulatory molecule implicated in both proinflammatory and antiinflammatory processes in the lung. Previously, we demonstrated that in human alveolar macrophages (AM), NO decreased inflammatory cytokine production, including that of interleukin-1beta, tumor necrosis factor-alpha and macrophage inflammatory protein-1alpha. One mechanism by which NO could regulate such diverse cytokine production is through effects on the transcription factor nuclear factor-kappaB (NF-kappaB), which controls the expression of the genes for these inflammatory cytokines and growth factors. We therefore investigated whether NO affects NF-kappaB activation in AM in vitro and in vivo. In vitro studies with AM showed that NF-kappaB activation by lipopolysaccharide (LPS) is decreased by NO in a dose-dependent manner. NO prevented an LPS-mediated decrease in the NF-kappaB inhibitory protein IkappaB-alpha. In asthma, airway NO levels are increased, whereas in primary pulmonary hypertension (PPH), airway NO levels are lower than in healthy lungs. In vivo investigations were conducted with freshly isolated AM from healthy controls, asthmatic individuals, and PPH patients. Healthy individuals had airway NO levels of 8 +/- 2 ppb (mean +/- SEM), which is associated with low NF-kappaB activation. Asthma patients with airway NO levels > 17 ppb showed minimal NF-kappaB activation, whereas asthmatic individuals with NO levels

Future of flexible bronchoscopy.

Despite some potential "threats" (Table 3), the future of bronchoscopy is likely full with ever expanding applications in clinical medicine and research (Tables 1 and 2). The role of bronchoscopy in lung cancer continues to expand and the usefulness of newer techniques needs to be established. The role of FB is also ever expanding in the immuno compromised host and critically ill patients. Future studies, however, should concentrate on patient outcome, especially survival and performance status. Therapeutic bronchoscopy has been extended to many areas and the emphasis of future research will likely continue to be at the molecular level rather than on studies of better instruments for retrieval purposes. The most exciting developments are likely to be in the field of gene therapy, which has made remarkable progress in the past decade. Gene therapy can be a powerful technology, but several hurdles must be overcome to make gene therapy a reality for treating human disease in the future. As the cost of technology continues to challenge health care delivery systems, a major challenge for the future of bronchoscopy is to evaluate new technologies and applications based on their impact on patient outcome, survival, and management of cost.

Role of bronchoscopy in massive hemoptysis.

Massive hemoptysis accounts for a minority of all patients with hemoptysis but poses a major challenge for the acute and long-term treatment. Massive hemoptysis can lead to asphyxiation and airway obstruction, shock, and exsanguination. Bronchoscopy plays an integral part in managing massive hemoptysis in diagnosis and treatment (Table 5). Specifically, bronchoscopy allows lateralization and more specific localization of bleeding that is critically important for effective management. Furthermore, acute control of bleeding can sometimes be achieved with instruments and catheters placed through the bronchoscope or by agents instilled into the airways through the bronchoscope.

Role of bronchoscopy in asthma research.

Over the past 15 years, much has been learned about the presence of airway inflammation in asthma through the use of investigative bronchoscopy. It has become quite clear that inflammation is present even in mild asthma. In addition to the eosinophils, T-lymphocytes and a variety of cytokines have been identified to play a prominent role in asthmatic inflammation. The concept of delayed asthmatic response after allergen exposure and its relationship to cellular inflammation and airway hyper-reactivity has become more clearly established. Our understanding of asthmatic airway inflammation, however, is incomplete. As interesting as the database has been so far, investigative FB has not defined a unique profile for patients with asthma. Specifically, lavage or endobronchial biopsy has not identified parameters that help in the diagnosis, assessment of disease severity, prognosis, or likelihood to respond to specific therapies. Also, the exact relationship between parameters in lavage compared with mucosal biopsy and how these are related to airway hyper-reactivity and the clinical syndrome of asthma remains poorly understood. In this regard, it must be confessed that currently FB with lavage and biopsy in asthmatics needs to be considered as a research tool for specimen retrieval to help characterize and express inflammation. Although these techniques have contributed immensely to our understanding of asthma pathogenesis, presently these techniques do not have any practical role or clinical usefulness.

Biochemical reaction products of nitric oxide as quantitative markers of primary pulmonary hypertension.

Primary pulmonary hypertension (PPH) is a rare and fatal disease of unknown etiology. Inflammatory oxidant mechanisms and deficiency in nitric oxide (NO) have been implicated in the pathogenesis of pulmonary hypertension. In order to investigate abnormalities in oxidants and antioxidants in PPH, we studied intrapulmonary NO levels, biochemical reaction products of NO, and antioxidants (glutathione [GSH], glutathione peroxidase [GPx], and superoxide dismutase [SOD]) in patients with PPH (n = 8) and healthy controls (n = 8). Intrapulmonary gases and fluids were sampled at bronchoscopy. Pulmonary hypertension was determined by right-heart catheterization. NO and biochemical reaction products of NO in the lung were decreased in PPH patients in comparison with healthy controls (NO [ppb] in airway gases: control, 8 +/- 1; PPH, 2.8 +/- 0. 9; p = 0.016; and NO products [microM] in bronchoalveolar lavage fluid [BALF]: control, 3.3 +/- 1.05; PPH, 0.69 +/- 0.21; p = 0.03). However, GSH in the lungs of PPH patients was higher than in those of controls (GSH [microM] in BALF: 0.55 +/- 0.04; PPH, 0.9 +/- 0.1; p = 0.015). SOD and GPx activities were similar in the two groups (p >/= 0.50). Biochemical reaction products of NO were inversely correlated with pulmonary artery pressures (R = -0.713; p = 0.047) and with years since diagnosis of PPH (R = -0.776; p = 0.023). NO reaction products are formed through interactions between oxidants and NO, with the end products of reaction dependent upon the relative levels of the two types of molecules. The findings of the study therefore show that NO and oxidant reactions in the lung are related to the increased pulmonary artery pressures in PPH.

iNOS expression in human intestinal microvascular endothelial cells inhibits leukocyte adhesion.

Increased nitric oxide (NO) production by inducible nitric oxide synthase (iNOS) has been associated with intestinal inflammation, including human inflammatory bowel disease. However, NO can downregulate endothelial activation and leukocyte adhesion, critical steps in the inflammatory response. Using primary cultures of human intestinal microvascular endothelial cells (HIMEC), we determined the role of NO in the regulation of HIMEC activation and interaction with leukocytes. Both nonselective (NG-monomethyl-L-arginine) and specific (N-iminoethyl-L-lysine) competitive inhibitors of iNOS significantly increased binding of leukocytes by HIMEC activated with cytokines and lipopolysaccharide. Increased adhesion was reversible with the NOS substrate L-arginine and was not observed in human umbilical vein endothelial cells (HUVEC). Activation of HIMEC significantly upregulated HIMEC iNOS expression and NO production. NOS inhibitors did not augment cell adhesion molecule levels in activated HIMEC but did result in sustained increases in intracellular reactive oxygen species. In addition, antioxidant compounds reversed the effect of NOS inhibitors on HIMEC-leukocyte interaction. Taken together, these data suggest that after HIMEC activation, iNOS-derived NO is an endogenous antioxidant, downregulating leukocyte binding and potentially downregulating intestinal inflammation.