Microarray analysis of primary epithelial and fibroblast cells in chronic rhinosinusitis without nasal polyps.

BACKGROUND: Chronic rhinosinusitis is an inflammatory condition with an as yet unknown pathophysiology. We aimed to detect clusters of differentially regulated genes in the epithelial and fibroblast cells of patients with Chronic Rhinosinusitis without nasal polyposis (CRSsNP) and healthy controls. METHODOLOGY: Carefully phenotyped CRSsNP and healthy control participants were recruited. Primary cultures of isolated epithelial and fibroblast cells were established. Whole transcriptome analysis of the cells was performed using microarrays and replicated with quantitative RT-PCR and immunohistochemistry. RESULTS: Fibroblast cells from CRSsNP patients showed a significant upregulation (more than 2x) of the transcription factor NFE2L3 when compared to healthy controls by microarray with multiple hypothesis testing correction, qRT-PCR and immunohistochemistry. CONCLUSIONS: Here we have utilized microarray analysis to search for differentially expressed genes in isolated patient derived epithelial and fibroblast cells. The transcription factor NFE2L3 has been shown to be upregulated in fibroblast cells consistent with increasing evidence that fibroblasts play a key role in tissue specific inflammation within the paranasal sinuses.

Pseudomonas aeruginosa Induced Airway Epithelial Injury Drives Fibroblast Activation: A Mechanism in Chronic Lung Allograft Dysfunction.

Bacterial infections after lung transplantation cause airway epithelial injury and are associated with an increased risk of developing bronchiolitis obliterans syndrome. The damaged epithelium is a source of alarmins that activate the innate immune system, yet their ability to activate fibroblasts in the development of bronchiolitis obliterans syndrome has not been evaluated. Two epithelial alarmins were measured longitudinally in bronchoalveolar lavages from lung transplant recipients who developed bronchiolitis obliterans syndrome and were compared to stable controls. In addition, conditioned media from human airway epithelial cells infected with Pseudomonas aeruginosa was applied to lung fibroblasts and inflammatory responses were determined. Interleukin-1 alpha (IL-1α) was increased in bronchoalveolar lavage of lung transplant recipients growing P. aeruginosa (11.5 [5.4-21.8] vs. 2.8 [0.9-9.4] pg/mL, p < 0.01) and was significantly elevated within 3 months of developing bronchiolitis obliterans syndrome (8.3 [1.4-25.1] vs. 3.6 [0.6-17.1] pg/mL, p < 0.01), whereas high mobility group protein B1 remained unchanged. IL-1α positively correlated with elevated bronchoalveolar lavage IL-8 levels (r(2)  = 0.6095, p < 0.0001) and neutrophil percentage (r(2)  = 0.25, p = 0.01). Conditioned media from P. aeruginosa infected epithelial cells induced a potent pro-inflammatory phenotype in fibroblasts via an IL-1α/IL-1R-dependent signaling pathway. In conclusion, we propose that IL-1α may be a novel therapeutic target to limit Pseudomonas associated allograft injury after lung transplantation.

Macrophages are critical to the maintenance of IL-13-dependent lung inflammation and fibrosis.

The roles of macrophages in type 2-driven inflammation and fibrosis remain unclear. Here, using CD11b-diphtheria toxin receptor (DTR) transgenic mice and three models of interleukin 13 (IL-13)-dependent inflammation, fibrosis, and immunity, we show that CD11b(+) F4/80(+) Ly6C(+) macrophages are required for the maintenance of type 2 immunity within affected tissues but not secondary lymphoid organs. Direct depletion of macrophages during the maintenance or resolution phases of secondary Schistosoma mansoni egg-induced granuloma formation caused a profound decrease in inflammation, fibrosis, and type 2 gene expression. Additional studies with CD11c-DTR and CD11b/CD11c-DTR double-transgenic mice suggested that macrophages but not dendritic cells were critical. Mechanistically, macrophage depletion impaired effector CD4(+) T helper type 2 (Th2) cell homing and activation within the inflamed lung. Depletion of CD11b(+) F4/80(+) Ly6C(+) macrophages similarly reduced house dust mite-induced allergic lung inflammation and suppressed IL-13-dependent immunity to the nematode parasite Nippostrongylus brasiliensis. Consequently, therapeutic strategies targeting macrophages offer a novel approach to ameliorate established type 2 inflammatory diseases.

IL-1α released from damaged epithelial cells is sufficient and essential to trigger inflammatory responses in human lung fibroblasts.

Activation of the innate immune system plays a key role in exacerbations of chronic lung disease, yet the potential role of lung fibroblasts in innate immunity and the identity of epithelial danger signals (alarmins) that may contribute to this process are unclear. The objective of the study was to identify lung epithelial-derived alarmins released during endoplasmic reticulum stress (ER stress) and oxidative stress and evaluate their potential to induce innate immune responses in lung fibroblasts. We found that treatment of primary human lung fibroblasts (PHLFs) with conditioned media from damaged lung epithelial cells significantly upregulated interleukin IL-6, IL-8, monocyte chemotactic protein-1, and granulocyte macrophage colony-stimulating factor expression (P<0.05). This effect was reduced with anti-IL-1α or IL-1Ra but not anti-IL-1ß antibody. Costimulation with a Toll-like receptor 3 ligand, polyinosinic-polycytidylic acid (poly I:C), significantly accentuated the IL-1α-induced inflammatory phenotype in PHLFs, and this effect was blocked with inhibitor of nuclear factor kappa-B kinase subunit beta and TGFß-activated kinase-1 inhibitors. Finally, Il1r1-/- and Il1a-/- mice exhibit reduced bronchoalveolar lavage (BAL) neutrophilia and collagen deposition in response to bleomycin treatment. We conclude that IL-1α plays a pivotal role in triggering proinflammatory responses in fibroblasts and this process is accentuated in the presence of double-stranded RNA. This mechanism may be important in the repeated cycles of injury and exacerbation in chronic lung disease.

TNFα from classically activated macrophages accentuates epithelial to mesenchymal transition in obliterative bronchiolitis.

Bronchiolitis obliterans syndrome is characterized by fibrotic obliteration of small airways which severely impairs graft function and survival after lung transplantation. Bronchial epithelial cells from the transplanted lung can undergo epithelial to mesenchymal transition and this can be accentuated by activated macrophages. Macrophages demonstrate significant plasticity and change phenotype in response to their microenvironment. In this study we aimed to identify secretory products from macrophages that might be therapeutic targets for limiting the inflammatory accentuation of epithelial to mesenchymal transition in bronchiolitis obliterans syndrome. TNFα, IL-1ß and IL-8 are elevated in bronchoalveolar lavage from lung transplant patients prior to diagnosis of bronchiolitis obliterans syndrome. Classically activated macrophages secrete more TNFα and IL-1ß than alternatively activated macrophages and dramatically accentuate TGF-ß1-driven epithelial to mesenchymal transition in bronchial epithelial cells isolated from lung transplant patients. Blocking TNFα, but not IL-1ß, inhibits the accentuation of epithelial to mesenchymal transition. In a pilot unblinded therapeutic intervention in five patients with progressive bronchiolitis obliterans syndrome, anti-TNFα treatment improved forced expiratory volume in 1 second and 6-min walk distances in four patients. Our data identify TNFα as a potential new therapeutic target in bronchiolitis obliterans syndrome deserving of a randomized placebo controlled clinical trial.

Volatile biomarkers of Pseudomonas aeruginosa in cystic fibrosis and noncystic fibrosis bronchiectasis.

AIMS: The purpose of this study was to determine whether volatile organic compounds specific to Pseudomonas aeruginosa could be detected in clinical sputum specimens. METHODS AND RESULTS: Patients were recruited from specialist bronchiectasis and cystic fibrosis clinics. The gold standard for diagnosing Ps. aeruginosa infection was a positive sputum culture. About 72 sputum headspace samples taken from patients at risk of or known to have prior Ps. aeruginosa infection were analysed by solid phase micro-extraction mass spectrometry. 2-nonanone was a marker in Ps. aeruginosa in sputum headspace gas with sensitivity of 72% and specificity of 88%. A combination of volatile compounds, a sputum library of 17 compounds with 2-nonanone, increased sensitivity in the detection of Ps. aeruginosa to 91% with specificity of 88%. CONCLUSIONS: In contrast to the 48-hour turnaround for classical microbiological culture, these results were available within 1-2 h. These data demonstrate the potential for rapid and accurate diagnosis of Ps. aeruginosa infection from sputum samples. SIGNIFICANCE AND IMPACT OF THE STUDY: 2-Nonanone is a compound requiring further study in the exhaled breath as it may improve diagnostic of Ps. aeruginosa infection when combined with other reported volatile markers.

Raised interleukin-17 is immunolocalised to neutrophils in cystic fibrosis lung disease.

Interleukin (IL)-17 is pivotal in orchestrating the activity of neutrophils. Neutrophilic inflammation is the dominant pathology in cystic fibrosis (CF) lung disease. We investigated IL-17 protein expression in the lower airway in CF, its cellular immunolocalisation and the effects of IL-17 on CF primary bronchial epithelial cells. Immunohistochemistry was performed on explanted CF lungs and compared with the non-suppurative condition pulmonary hypertension (PH). Airway lavages and epithelial cultures were generated from explanted CF lungs. Immunoreactivity for IL-17 was significantly increased in the lower airway epithelium in CF (median 14.1%) compared with PH (2.95%, p=0.0001). The number of cells staining positive for IL-17 in the lower airway mucosa was also increased (64 cells·mm(-1) compared with 9 cells·mm(-1) basement membrane, p=0.0005) and included both neutrophils in addition to mononuclear cells. IL-17 was detectable in airway lavages from explanted CF lungs. Treatment of epithelial cultures with IL-17 increased production of IL-8, IL-6 and granulocyte macrophage colony-stimulating factor. In conclusion, immunoreactive IL-17 is raised in the lower airway of people with CF and localises to both neutrophils and mononuclear cells. IL-17 increases production of pro-neutrophilic mediators by CF epithelial cells, suggesting potential for a positive feedback element in airway inflammation.

Inflammation and epithelial to mesenchymal transition in lung transplant recipients: role in dysregulated epithelial wound repair.

Epithelial to mesenchymal transition (EMT) has been implicated in the pathogenesis of obliterative bronchiolitis (OB) after lung transplant. Although TNF-alpha accentuates TGF-beta1 driven EMT in primary human bronchial epithelial cells (PBECs), we hypothesized that other acute pro-inflammatory cytokines elevated in the airways of patients with OB may also accentuate EMT and contribute to dysregulated epithelial wound repair. PBECs from lung transplant recipients were stimulated with TGF-beta1+/-IL-1beta, IL-8, TNF-alpha or activated macrophages in co-culture and EMT assessed. The quality and rate of wound closure in a standardized model of lung epithelial injury was assessed in response to above stimuli. Co-treatment with TGF-beta1+TNF-alpha or IL-1beta significantly accentuates phenotypic and some functional features of EMT compared to TGF-beta1 alone. Co-treatment with TGF-beta1+TNF-alpha or IL-1beta accelerates epithelial wound closure however the quality of repair is highly dysregulated. Co-treatment with TGF-beta1+IL-8 has no significant effect on EMT or the speed or quality of wound healing. Activated macrophages dramatically accentuate TGF-beta1-driven EMT and cause dysregulated wound repair. Crosstalk between macrophage-derived acute inflammation in the airway and elevated TGF-beta1 may favor dysregulated airway epithelial repair and fibrosis in the lung allograft via EMT.

Epithelial to mesenchymal transition (EMT) and airway remodelling after human lung transplantation.

BACKGROUND: Aberrant epithelial repair is a key event in the airway remodelling which characterises obliterative bronchiolitis (OB) in the transplanted lung. The potential for airway epithelium from lung transplant recipients to undergo epithelial to mesenchymal cell transition (EMT) was assessed in culture and in vivo in lung allograft tissue. METHODS: Change in epithelial and mesenchymal marker expression was assessed after stimulation with transforming growth factor beta(1) (TGF-beta(1)) alone or in combination with tumour necrosis factor alpha (TNFalpha) and compared with untreated controls. The ability of cells to deposit extracellular matrix, secrete matrix metalloproteinases (MMPs) and invade collagen was investigated. Immunolocalisation of epithelial and mesenchymal markers was compared in airway tissue from stable recipients and those with OB. RESULTS: Untreated cells maintained epithelial morphology and phenotype. TGF-beta(1) reduced expression of epithelial markers, increased expression of vimentin and fibronectin, promoted collagen I and fibronectin deposition and increased MMP-9 production. Co-treatment with TNFalpha dramatically accentuated phenotypic and some functional features of EMT. Airway epithelial biopsies from recipients with OB demonstrated significantly increased staining for mesenchymal markers and significantly reduced E-cadherin staining compared with stable recipients. CONCLUSIONS: These observations demonstrate the ability of human airway epithelium to undergo EMT and suggest this phenomenon may be a potential link between inflammatory injury and TGF-beta(1)-driven airway remodelling in the development of OB.

Pseudomembranous colitis in four patients with cystic fibrosis following lung transplantation.

In the present study, 4 patients with cystic fibrosis undergoing lung transplantation (from a total of 137) who developed fulminant pseudomembranous colitis are described. Initial presentation was variable and the mortality rate was 50% despite urgent colectomy. In one case the presenting abdominal distension was thought to be due to meconium ileus equivalent. It is concluded that Clostridium difficile colitis may be a difficult diagnosis in patients with cystic fibrosis and follows a fulminant course after lung transplantation.

Airway epithelial cell senescence in the lung allograft.

Chronic lung allograft dysfunction, manifesting as bronchiolitis obliterans syndrome (BOS), is characterized by airway epithelial injury, impaired epithelial regeneration and subsequent airway remodeling. Increased cellular senescence has been reported in renal and liver allografts affected by chronic allograft dysfunction but the significance of cellular senescence in the airway epithelium of the transplanted lung is unknown. Thirty-four lung transplant recipients, 20 with stable graft function and 14 with BOS, underwent transbronchial lung biopsy and histochemical studies for senescence markers in small airways. Compared to nontransplant control lung tissue (n = 9), lung allografts demonstrate significantly increased airway epithelial staining for senescence-associated beta galactosidase (SA beta-gal) (p = 0.0215), p16(ink4a) (p = 0.0002) and p21(waf1/cip) (p = 0.0138) but there was no difference in expression of these markers between stable and BOS affected recipients (p > 0.05). This preliminary cross-sectional study demonstrates that cellular senescence occurs with increased frequency in the airway epithelium of the lung allograft but does not establish any association between airway epithelial senescence and BOS. A prospective longitudinal study is required to better address any potential causal association between airway epithelial senescence in stable allograft recipients and the subsequent development of BOS.

Outcomes of lung transplantation for cystic fibrosis in a large UK cohort.

BACKGROUND: Lung transplantation is an important option to treat patients with advanced cystic fibrosis (CF) lung disease. The outcomes of a large UK cohort of CF lung transplantation recipients is reported. METHODS: Retrospective review of case notes and transplantation databases. RESULTS: 176 patients with CF underwent lung transplantation at our centre. The majority (168) had bilateral sequential lung transplantation. Median age at transplantation was 26 years. Diabetes was common pretransplantation (40%). Polymicrobial infection was common in individual recipients. A diverse range of pathogens were encountered, including the Burkholderia cepacia complex (BCC). The bronchial anastomotic complication rate was 2%. Pulmonary function (forced expiratory volume in 1 s % predicted) improved from a pretransplantation median of 0.8 l (21% predicted) to 2.95 l (78% predicted) at 1 year following transplantation. We noted an acute rejection rate of 41% within the first month. Our survival values were 82% survival at 1 year, 70% at 3 years, 62% at 5 years and 51% at 10 years. Patients with BCC infection had poorer outcomes and represented the majority of those who had a septic death. Data are presented on those free from these infections. Bronchiolitis obliterans syndrome (BOS) and sepsis were common causes of death. Freedom from BOS was 74% at 5 years and 38% at 10 years. Biochemical evidence of renal dysfunction was common although renal replacement was infrequently required (<5%). CONCLUSION: Lung transplantation is an important therapeutic option in patients with CF even in those with more complex microbiology. Good functional outcomes are noted although transplantation associated morbidities accrue with time.

Pseudomembranous colitis in four patients with cystic fibrosis following lung transplantation.

Pseudomembranous colitis is an uncommon complication in patients with cystic fibrosis, despite the use of multiple high-dose antibiotic regimens and the frequency of hospital admissions. Four patients from a total of 137 patients with cystic fibrosis undergoing lung transplantation are described who developed fulminant pseudomembranous colitis. Initial presentation was variable and the mortality rate was 50% despite urgent colectomy. In one case the presenting abdominal distension was thought to be due to meconium ileus equivalent. It is concluded that Clostridium difficile colitis may be a difficult diagnosis in patients with cystic fibrosis and follows a fulminant course after lung transplantation.

Formation of Neu/ErbB2-induced mammary tumors is unaffected by loss of ErbB4.

The four members of the ErbB family of receptor tyrosine kinases are involved in development and tumorigenesis of the mammary gland. Whereas the epidermal growth factor receptor, ErbB2 and ErbB3 are positively associated with various cancers, clinical studies of ErbB4 in breast cancer are contradictory. Results from tissue culture analyses and some clinical studies suggested that ErbB4 is either a tumor suppressor or is a negative regulator of ErbB2-driven tumors. Neu-Cre-ErbB4(flox/null) mice in which ErbB4 was inactivated by Cre-lox-mediated recombination in the mammary gland developed MMTV-Neu-driven mammary tumors with a similar latency period to mice with one or two wild-type ErbB4 alleles. Moreover, there was no difference in the histologies of tumors that developed, nor in the propensity to form lung metastases. Taken together these results suggest that ErbB4 is not a potent, highly penetrant tumor suppressor, nor is it a factor in Neu-mediated tumorigenesis in this model.

Phenotype of airway epithelial cells suggests epithelial to mesenchymal cell transition in clinically stable lung transplant recipients.

BACKGROUND: Obliterative bronchiolitis in chronic rejection of lung allografts is characterised by airway epithelial damage and fibrosis. The process whereby normal epithelium is lost and replaced by fibroblastic scar tissue is poorly understood, but recent findings suggest that epithelial cells can become fibroblasts through epithelial-mesenchymal transition (EMT). It is hypothesised that EMT occurs in lung allografts and plays a potential role in airway remodelling. METHODS: Sixteen stable lung transplant recipients underwent bronchoscopy with bronchoalveolar lavage (BAL), endobronchial biopsies, and bronchial brushings. Biopsy sections were stained for the fibroblast marker S100A4. Brushings were cultured on collagen, stained with anti-S100A4, and examined for further EMT markers including matrix metalloproteinase (MMP) zymographic activity and epithelial invasion through collagen coated filters. RESULTS: A median 15% (0-48%) of the biopsy epithelium stained for S100A4 in stable lung transplant recipients and MMP-7 co-localisation was observed. In non-stimulated epithelial cultures from lung allografts, S100A4 staining was identified with MMP-2 and MMP-9 production and zymographic activity. MMP total protein and activity was increased following stimulation with transforming growth factor (TGF)-beta1. Non-stimulated transplant epithelial cells were invasive and penetration of collagen coated filters increased following TGF-beta1 stimulation. CONCLUSIONS: This study provides evidence of EMT markers in lung allografts of patients without loss of lung function. The EMT process may represent a final common pathway following injury in more common diseases characterised by airway remodelling.

Objective assessment of criteria for selection of donor lungs suitable for transplantation.

BACKGROUND: Donor organ shortage severely limits lung transplantation as a therapeutic option, yet many potential donor lungs are deemed unsuitable by clinical selection criteria. METHODS: Of 39 consecutive potential donor lungs, 14 were accepted and 25 excluded by clinical selection criteria. All were evaluated prospectively by clinical assessment, bronchoscopy, and bronchoalveolar lavage (BAL) to evaluate objectively the discrimination of pulmonary infection and injury. RESULTS: Accepted donors were significantly younger than those excluded (mean (SD) age 36.7 (15.3) years v 49.5 (13.2) years; p = 0.009, unpaired t test) and were more likely to have suffered traumatic brain death (50% v 20%; p = 0.07, Fisher's exact test). Oxygenation (PaO(2):FiO(2)) was higher in accepted donors than in excluded donors (median (range) 63.2 (48-82.5) kPa v 43.1 (7.7-71.7) kPa; p = 0.0001, Mann-Whitney test). Positive formal BAL culture was more frequent in accepted donors (75%) than in those excluded (43%; p = 0.1, Fisher's exact test). There was no significant difference in the percentage and concentration of neutrophils in BAL fluid between accepted and excluded donors (median (range) 37.9 (0-96.9)% and 44.6 (0-1190)x10(3)/ml v 36 (1-98.1)% and 46 (0.2-1457)x10(3)/ml), nor in the BAL fluid concentration of tumour necrosis factor-alpha (140 (0-340) pg/ml v 160 (0-760) pg/ml) or interleukin 8 (810 (33-17 600) pg/ml v 540 (0-15 110) pg/ml). CONCLUSION: Current selection criteria are poor discriminators of pulmonary injury and infection and lead to the exclusion of potentially usable donor lungs.

Reticular basement membrane thickening in airways of lung transplant recipients is not affected by inhaled corticosteroids.

INTRODUCTION: Chronic rejection is a major problem for all lung transplant programmes, which is functionally manifested by fixed airflow limitation, Bronchiolitis Obliterans Syndrome (BOS). The inclusion of a Pre-BOS category, BOS(0 approximately p), in newly revised guidelines, recognizes the potential importance of early changes. We have previously demonstrated reticular basement membrane (Rbm) thickening in clinically stable lung transplant recipients free from BOS. The present study extends this, testing the hypothesis that inhaled corticosteroid (ICS) therapy will lead to a decrease in Rbm thickness in lung transplant recipients. METHODS: A parallel group, bronchoscopic intervention study of clinically stable lung allograft recipients, free from BOS, but with evidence of airway inflammation. Following baseline assessment of Rbm thickening, subjects were randomized to 3 months of either chlorofluorocarbon-driven beclomethasone diproprionate (BDP) 400 microg b.i.d., or a formulation designed to yield at least an equivalent dose, hydrofluoroalkane-driven BDP, 200 microg b.i.d. RESULTS: Three months treatment with a moderate dose of ICS, including a formulation designed for preferential small airway deposition, had no effect on Rbm thickening (13+/-3 vs. 14+/-5 microm post-ICS). CONCLUSION: Our data would suggest that airway remodelling can occur early in lung allografts and is not affected by moderate dose ICS therapy. Longitudinal studies are required to describe the pathophysiological processes involved in BOS, and specifically to elucidate potential relationships between airway remodelling, airflow obstruction and allograft failure.

Preliminary findings of quorum signal molecules in clinically stable lung allograft recipients.

BACKGROUND: Infection with bacteria such as Pseudomonas is common in lung allograft recipients, particularly during chronic rejection. Analysis of sputum samples from patients with cystic fibrosis infected with Pseudomonas aeruginosa or Burkholderia cepacia has indicated the presence of bacterial N-acylhomoserine lactones (AHLs) quorum sensing signalling molecules. AHLs not only control the expression of bacterial virulence genes but are also involved in stimulating the maturation of antibiotic resistant biofilms and host chemokine release. It was hypothesised that AHLs may be detected even in clinically stable lung transplant recipients free of clinical infection or rejection. METHODS: Three 60 ml samples of bronchoalveolar lavage (BAL) fluid were taken from nine stable lung transplant recipients 3-12 months after transplantation. Detection of AHLs was carried out on dichloromethane extracted supernatants using the bioluminescence based AHL reporter plasmid pSB1075. This responds to the presence of AHLs with long acyl chains (C10-C14), generating light. Synthetic AHLs were included as positive controls. RESULTS: Five of the nine BAL fluid supernatants exhibited AHL activity, suggesting the presence of AHLs with long N-acyl chains. There was no correlation between the levels of AHLs detected or their absence and BAL fluid microbiology or diagnosis before transplantation. CONCLUSIONS: This is the first evidence for the presence of AHL quorum sensing signals in human lung allograft recipients, even in subjects with no rejection or apparent infection. Further longitudinal follow up of these preliminary findings is required to elucidate potential links with infection, rejection, and allograft deterioration.

Crystal structure of ATP sulfurylase from the bacterial symbiont of the hydrothermal vent tubeworm Riftia pachyptila.

In sulfur chemolithotrophic bacteria, the enzyme ATP sulfurylase functions to produce ATP and inorganic sulfate from APS and inorganic pyrophosphate, which is the final step in the biological oxidation of hydrogen sulfide to sulfate. The giant tubeworm, Riftia pachyptila, which lives near hydrothermal vents on the ocean floor, harbors a sulfur chemolithotroph as an endosymbiont in its trophosome tissue. This yet-to-be-named bacterium was found to contain high levels of ATP sulfurylase that may provide a substantial fraction of the organisms ATP. We present here, the crystal structure of ATP sulfurylase from this bacterium at 1.7 A resolution. As predicted from sequence homology, the enzyme folds into distinct N-terminal and catalytic domains, but lacks the APS kinase-like C-terminal domain that is present in fungal ATP sulfurylase. The enzyme crystallizes as a dimer with one subunit in the crystallographic asymmetric unit. Many buried solvent molecules mediate subunit contacts at the interface. Despite the high concentration of sulfate needed for crystallization, no ordered sulfate was observed in the sulfate-binding pocket. The structure reveals a mobile loop positioned over the active site. This loop is in a "closed" or "down" position in the reported crystal structures of fungal ATP sulfurylases, which contained bound substrates, but it is in an "open" or "up" position in the ligand-free Riftia symbiont enzyme. Thus, closure of the loop correlates with occupancy of the active site, although the loop itself does not interact directly with bound ligands. Rather, it appears to assist in the orientation of residues that do interact with active-site ligands. Amino acid differences between the mobile loops of the enzymes from sulfate assimilators and sulfur chemolithotrophs may account for the significant kinetic differences between the two classes of ATP sulfurylase.

Inhaled corticosteroids and the treatment of lymphocytic bronchiolitis following lung transplantation.

Airway rejection after lung transplantation is recognized histologically as lymphocytic bronchiolitis (LB). We hypothesized that inhaled steroids could control LB and that changes in exhaled nitric oxide (eNO) would correlate with the development of LB and also have a role in monitoring response to treatment. A cohort of 120 lung transplant (LT) recipients attending for review and biopsy had eNO measurements, FEV1, lavage microbiology, and biopsy histology performed prospectively. Wilcoxon signed-rank test was used to assess the significance of changes in eNO and FEV1. The coefficient of reproducibility of eNO measurement in stable recipients was 2.36 ppb. Fourteen developed graft dysfunction owing to isolated LB and were treated with inhaled budesonide 800 microg twice daily. They showed significant increases in eNO at diagnosis, median (range) 10.9 ppb (4.6 to 48) ppb compared with baseline, 4.33 (1.0 to 10.76), p = 0.008, and a decrease in FEV1. After inhaled treatment, both eNO and FEV1 returned to baseline values. Seven developed acute vascular rejection (with or without LB) and were treated with oral corticosteroids; no changes in eNO occurred at diagnosis or after treatment. Serial eNO measurements provide a useful noninvasive method of identifying airway inflammation in LT recipients. Inhaled budesonide may be a useful addition to systemic immunosuppressants in controlling airway inflammation posttransplant.