Are the enzymatic methods currently being used to measure bronchoalveolar lavage bile salt levels fit for purpose?

BACKGROUND: Microaspiration after gastroesophageal reflux has been implicated in the chronic loss of allograft function in lung transplant patients. Bronchoalveolar lavage fluid (BALF) assessment for pepsin and bile salts is a common method to document reflux and aspiration. Clinically used methods for bile salt analysis include tandem mass spectrometry and diagnostic enzymatic kits designed to measure bile salts in serum. In clinical research, the enzymatic kits have been commonly used for BALF assays in lung transplant recipients, with reports of detection limits of 0.2 µmol/liter, and the levels used to inform clinical decisions. This study assessed the sensitivity of detection by 2 enzymatic assay kits compared with tandem mass spectrometry. METHODS: These 2 kits were used to measure (1) the absorbance changes for 0 to 50 µmol/liter bile salts, (2) levels in gastric juice (10-10,010 µmol/liter), and (3) bile salt levels of 40 BALF samples that were also measured using tandem mass spectrometry (0.01-1.19 µmol/liter). Measurements of pH/impedance were done in 14 of 15 patients. RESULTS: Neither kit had detection limits as low as claimed in previous BALF studies. The kits could be made more sensitive with a longer incubation time, (5 µmol/liter). All patients had detectable lavage bile acids using mass spectroscopy, 71% had pathologic distal gastroesophageal reflux, and 43% had pathologic proximal reflux. CONCLUSIONS: The enzymatic kits are not sensitive enough for use in situations where bile salt levels are much below 5 µmol/liter, which is the case in BALF. In addition, reports in the literature of levels significantly below 5 µmol/liter need reassessing. Tandem mass spectrometry with a lower limit of detection of 0.01 µmol/liter should be the method of choice.

The critical role of TAK1 in accentuated epithelial to mesenchymal transition in obliterative bronchiolitis after lung transplantation.

Therapies to limit or reverse fibrosis have proven unsuccessful, highlighting the need for a greater understanding of basic mechanisms that drive fibrosis and, in particular, the link between fibrosis and inflammation. It has been shown that pro-fibrotic transforming growth factor ß1 (TGF-ß1)-driven epithelial-to-mesenchymal transition (EMT) can be accentuated by tumor necrosis factor α (TNF-α). TGF-ß-activated kinase 1 (TAK1) is activated by both TGF-ß1 and TNF-α, activating both nuclear factor kappa-light-chain-enhancer of activated B cells and mitogen-activated protein kinase signaling pathways. In this study, we evaluated the potential for TAK1 to modulate the synergistic effect between TGF-ß1 and TNF-α in driving EMT. Co-stimulation with TGF-ß1 and TNF-α induced an accentuated and extended phosphorylation of TAK1 compared to either alone. TAK1 signaled downstream via nuclear factor kappa-light-chain-enhancer of activated B cells, and Jun N-terminal kinase-2, but independent of Jun N-terminal kinase-1, extracellular signal-regulated kinase-1/2, or p38 mitogen-activated protein kinase signaling to drive EMT in bronchial epithelial cells. Blocking either TAK1 or Jun N-terminal kinase-2 inhibited EMT. TAK1 phosphorylation was increased in the airway epithelium of patients with fibrotic airway disease. These data identify factors leading to and affected by accentuated and extended TAK1 phosphorylations potential novel therapeutic targets in inflammation-driven fibrotic diseases.

Is CFTR-delF508 really absent from the apical membrane of the airway epithelium?

BACKGROUND: Understanding where mutant CFTR is localised in airway epithelia is essential in guiding the best therapeutic approach to correct the dysfunction of the CFTR protein. The widely held paradigm is that CF patients harbouring the commonest mutation, CFTR-delF508, trap CFTR within the endoplasmic reticulum and target it for degradation. However there are conflicting reports concerning expression and localisation of CFTR-delF508 in lung tissue. To attempt to resolve this fundamental issue we developed a novel approach to measure CFTR-delF508 in the lower airways of patients who have undergone lung transplantation for advanced CF. By sampling CF and non-CF epithelium simultaneously from the same individual, confounding factors of different airway microenvironments which may have influenced previous observations can be overcome. METHODS: Epithelia sampled by bronchial brushing above (CF) and below (non-CF) the bronchial anastomosis were stained for CFTR and the localisation and level of expression assessed (n = 12). RESULTS: There was no significant difference in the proportion of tall columnar cells showing CFTR immunostaining as a discrete band at the apical membrane in cells harbouring the CFTR-delF508 mutation compared to non-CF cells (p = 0.21, n = 12). However, the amount of CFTR expressed at the apical surface was reduced by ∼50% in CF cells compared to non-CF cells (p = 0.04, n = 5). CONCLUSIONS: Our novel observation challenges the prevailing paradigm that CFTR is essentially absent from the apical membrane of respiratory cells harbouring the CFTR-delF508 mutation. Moreover, it raises the possibility that the new generation of CFTR potentiators may offer a realistic therapeutic option for CF patients.

Ceramide is increased in the lower airway epithelium of people with advanced cystic fibrosis lung disease.

RATIONALE: Ceramide accumulates in the airway epithelium of mice deficient in cystic fibrosis transmembrane conductance regulator, resulting in susceptibility to Pseudomonas aeruginosa infection and inflammation. OBJECTIVES: To investigate quantitatively ceramide levels in the lower airway of people with cystic fibrosis compared with pulmonary hypertension, emphysema, and lung donors. METHODS: Immunohistochemistry was performed on the lower airway epithelium of explanted lungs (eight cystic fibrosis, emphysema, and pulmonary hypertension, respectively) and eight donor lungs using ceramide, neutrophil elastase, and myeloperoxidase antibodies. High-performance liquid chromatography-mass spectrometry was performed on tissue from five lungs with cystic fibrosis and five with pulmonary hypertension. MEASUREMENTS AND MAIN RESULTS: Staining for ceramide was significantly increased in the lower airway epithelium of people with cystic fibrosis (median, 14.11%) compared with pulmonary hypertension (3.03%; P = 0.0009); unused lung donors (3.44%; P = 0.0009); and emphysema (5.06%; P = 0.01). Ceramide staining was increased in emphysematous lungs compared with pulmonary hypertension (P = 0.0135) and unused donors (P = 0.0009). The number of neutrophil elastase- and myeloperoxidase-positive cells in the airway was positively correlated with the percentage of epithelium staining for ceramide (P = 0.001). Ceramide staining was significantly increased in lungs colonized with Pseudomonas aeruginosa (10.1%) compared with those not colonized (3.14%; P = 0.0106). Significantly raised levels of ceramides C16:0, C18:0, and C20:0 were detected by mass spectrometry in lungs with cystic fibrosis compared with pulmonary hypertension. Differences in C22:0 were not significant. CONCLUSIONS: Immunoreactive ceramide is increased in the lower airway epithelium of people with advanced cystic fibrosis. Detected by mass-spectrometry ceramide species C16:0, C18:0, and C20:0 but not C22:0 are increased.

Primary bronchial epithelial cell culture from explanted cystic fibrosis lungs.

Lung disease is responsible for more than 95% of morbidity and mortality in cystic fibrosis. The exact pathogenesis of cystic fibrosis lung disease remains poorly understood. Experimental models are therefore vital for use in research. Animal models and immortalized cell lines both have inherent limitations. Explanted lungs removed from people with cystic fibrosis at the time of transplantation represent a potentially valuable but technically and logistically challenging source of primary cystic fibrosis bronchial epithelial cells. In this study, pieces of segmental bronchus from explanted lungs were treated with patient-specific antimicrobials prior to isolation of bronchial epithelial cells. Cultured cells were characterized by their morphology under light microscopy, cytokeratin and hematoxylin-eosin staining, and electrophysiological profile. Primary bronchial epithelial cells were successfully cultured from 15 of 22 patients attempted. The cells exhibited typical epithelial morphology, staining for cytokeratin, lack of responsiveness to forskolin treatment, and remained viable after storage in liquid nitrogen. Seven unsuccessful cultures failed due to early infection with bacteria known to colonize the airways pretransplant. The results show that primary bronchial epithelial cell culture is possible from explanted cystic fibrosis lungs. This provides an important cellular model to elucidate the pathogenic mechanisms in cystic fibrosis lung disease and to investigate potential therapeutic targets.

Azithromycin attenuates effects of lipopolysaccharide on lung allograft bronchial epithelial cells.

BACKGROUND: The bronchial epithelium is a source of mediators that may play a role in the airway inflammation and remodeling of post-transplant obliterative bronchiolitis (OB). Traditional strategies have failed to have an impact on OB. Recent studies have suggested a role for azithromycin in managing the condition. In this study we aimed to determine the effect of azithromycin on LPS-mediated epithelial release of factors relevant to airway neutrophilia and remodeling in a unique population of primary bronchial epithelial cells (PBECs) derived from stable lung allografts. METHODS: PBECs were established from bronchial brushings of stable lung transplant recipients and treated with lipopolysaccharide (LPS, 0.1, 1 and 10 microg/ml) for 48 hours. Interleukin-8 (IL-8), granulocyte macrophage colony-stimulating factor (GM-CSF) and vascular endothelial growth factor (VEGF) protein levels were measured by Luminex analyzer. PBECs were then incubated with LPS and azithromycin, and protein levels were again determined. RESULTS: LPS caused a significant increase in IL-8 and GM-CSF at concentrations of 1 and 10 microg/ml, with no effect on VEGF release. Azithromycin caused a significant decrease in the LPS-stimulated IL-8 and GM-CSF release. CONCLUSIONS: LPS upregulates release of IL-8 and GM-CSF from PBECs derived from stable lung allografts. Sub-microbicidal concentrations of azithromycin attenuate this and may, therefore, alleviate infection-driven neutrophilic airway inflammation and remodeling in the allograft airway.

Simvastatin attenuates release of neutrophilic and remodeling factors from primary bronchial epithelial cells derived from stable lung transplant recipients.

Obliterative bronchiolitis (OB), the major cause of chronic lung allograft dysfunction, is characterized by airway neutrophilia, inflammation, and remodeling, with progressive fibroproliferation and obliteration of small airways that ultimately leads to patient death. Statins have potential anti-inflammatory effects and have been demonstrated to confer a survival advantage in lung transplant patients. We postulated that the beneficial effects of simvastatin in lung transplantation are in part due to inhibition of the epithelial production of key mediators of neutrophil chemotaxis, inflammation, and airway remodeling. Our objective was to assess the effect of simvastatin on a unique population of primary bronchial epithelial cells (PBECs) derived from stable lung allografts, with specific reference to airway neutrophilia and remodeling. PBEC cultures were stimulated with IL-17 or transforming growth factor (TGF)-beta, with and without simvastatin. Supernatant levels of factors critical to driving airway neutrophilia and remodeling were measured. IL-17 upregulated IL-8, IL-6, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), and VEGF, whereas TGF-beta increased IL-6, GM-CSF, matrix metalloproteinase (MMP)-2, and MMP-9. Simvastatin attenuated effects of both IL-17 and TGF-beta. We have demonstrated the ability of simvastatin to attenuate release of airway neutrophilic and remodeling mediators and to inhibit their upregulation by TGF-beta and IL-17. These data illustrate the potential of simvastatin to alleviate neutrophilic airway inflammation and remodeling in the transplanted lung and may have additional relevance to other neutrophilic airway conditions, such as chronic obstructive pulmonary disease.

Pepsin, a biomarker of gastric aspiration in lung allografts: a putative association with rejection.

RATIONALE: Human lung transplantation is a therapeutic option for selected patients with advanced cardiopulmonary disease, but long-term survival is limited by chronic rejection. Persistent acute rejection and gastric aspiration have been implicated as risk factors but there is little or no evidence to date that they are associated. OBJECTIVES: We have tested the hypothesis that pepsin, a marker of gastric aspiration, is present in lung transplant recipients, and that high levels are associated with biopsy-diagnosed acute rejection and/or bronchiolitis obliterans syndrome. METHODS: Levels of bronchoalveolar lavage (BAL) pepsin were measured by ELISA in 36 lung transplant recipients, 4 normal volunteers, and 17 subjects with unexplained chronic cough. MEASUREMENTS AND MAIN RESULTS: Our primary finding was that, compared with control subjects, BAL pepsin levels were elevated in stable lung transplant recipients, subjects with acute rejection, and subjects with bronchiolitis obliterans syndrome. Our secondary finding was that the highest levels were found in recipients with acute vascular rejection grade > or = A2 (median, 11.2; range, 5.4 - 51.7 ng/ml; normal median, 1.1; range, 0-2.3 ng/ml; p = 0.004). CONCLUSIONS: We have shown that elevated levels of pepsin, a biomarker of gastric aspiration, are consistently identified in the BAL of lung allografts. The highest levels were seen in patients with > or = grade A2 acute rejection. This provides further evidence supporting the possible role of aspiration in the development of overall allograft injury.

Anxiety in rheumatoid arthritis.

OBJECTIVE: To examine the level of anxiety experienced by individuals with rheumatoid arthritis (RA). METHODS: Data from 2 previous studies were used to compare the level of anxiety (measured by the State-Trait Anxiety Inventory) in the following 4 subgroups: a general RA sample, a general osteoarthritis sample, a sample with both RA and major depression, and a normative sample of age-equivalent, working adults. Canonical correlations were used to examine associations between measures of anxiety and measures of both stress and depression. The relationship between anxiety and duration of RA was also explored. RESULTS: The general RA sample had state anxiety levels that were comparable to the normative sample, although trait anxiety levels were significantly higher (P < 0.001). In addition, individuals with RA who also met criteria for depression exhibited significantly higher levels of both state anxiety (P < 0.0001) and trait anxiety (P < 0.0001) than was observed in the normative sample. Canonical correlations revealed that measures of anxiety were correlated with both measures of depression (r = 0.83) and measures of stress (r = 0.50). Anxiety was not found to be significantly related to RA disease duration. CONCLUSION: These findings demonstrated that individuals with RA, especially if concomitantly depressed, tend to exhibit levels of anxiety that are generally higher than a normative group of age-equivalent, working adults. The substantial canonical correlations between anxiety and both depression and stress revealed that anxiety shares variance with these more frequently studied variables in RA. However, anxiety was not found to be related to RA disease duration.

Management of depression in rheumatoid arthritis: a combined pharmacologic and cognitive-behavioral approach.

OBJECTIVE: To examine the effectiveness of cognitive-behavioral and pharmacologic treatment of depression in rheumatoid arthritis (RA). METHODS: Subjects (n = 54) with confirmed diagnoses of both major depression and RA were randomly assigned to 1 of 3 groups: 1) cognitive-behavioral/pharmacologic group (CB-PHARM), 2) attention-control/pharmacologic group, or 3) pharmacologic control group. Measures of depression, psychosocial status, health status, pain, and disease activity were collected at baseline, posttreatment (10 weeks), 6-month followup, and 15-month followup. Data were analyzed to compare the treatment effectiveness of the groups; data also were aggregated to examine the effects of antidepressive medication over time. Lastly, a no-treatment control group was defined from a cohort of persons who declined participation. RESULTS: Baseline comparisons on demographic and dependent measures revealed a need to assess covariates on age and education; baseline scores on dependent measures also were entered as covariates. Analyses of covariance revealed no statistically significant group differences at postintervention, 6-month followup, or 15-month followup, except higher state and trait anxiety scores for the CB-PHARM group at the 15-month followup. In the longitudinal analyses of the effects of antidepressive medication, significant improvement in psychological status and health status were found at posttreatment, 6-month followup, and 15-month followup, but no significant improvements were shown for pain or disease activity. In addition, the comparison of the aggregated pharmacologic group with a no-treatment group revealed a statistically significant benefit for the 3 groups that received the antidepressive medication. CONCLUSION: In persons with RA, cognitive-behavioral approaches to the management of depression were not found to be additive to antidepressant medication alone, but antidepressant intervention was superior to no treatment.