Correcting for tissue nitrogen excretion in multiple breath washout measurements.

Nitrogen excreted from body tissues impacts the calculation of multiple breath nitrogen washout (MBWN2) outcomes. The aim of this study was to determine the effect of tissue N2 on MBWN2 outcomes in both healthy subjects and patients with CF and to assess whether it is possible to correct for tissue N2. The contribution of tissue N2 to MBWN2 outcomes was estimated by comparing MBWN2-derived functional residual capacity (FRCN2) to FRC measured by body plethysmography (FRCpleth) and by comparing MBW outcome measures derived from MBWN2 and sulfur hexafluoride MBW (MBWSF6). Compared to plethysmography and MBWSF6, MBWN2 overestimated FRC and lung clearance index (LCI). Application of mathematical tissue N2 corrections reduced FRCN2 values closer to FRCpleth in health and reduced LCIN2 in both health and CF, but did not explain all of the differences observed between N2-dependent and -independent techniques. Use of earlier washout cut-offs could reduce the influence of tissue N2. Applying tissue N2 corrections to LCIN2 measurements did not significantly affect the interpretation of treatment effects reported in a previously published interventional trial. While tissue N2 excretion likely has an impact on MBWN2 outcomes, better understanding of the nature of this phenomenon is required before routine correction can be implemented into current MBWN2 protocols

Surfactant protein a in cystic fibrosis: supratrimeric structure and pulmonary outcome.

The state of oligomerization of surfactant associated protein-A (SP-A) monomers differs between individuals. This likely affects SP-A's functional properties and could thereby influence clinical status in patients with lung diseases. In this study we focus on SP-A structure in cystic fibrosis (CF) compared to both healthy subjects and disease controls. SP-A composition and function were assessed in both bronchoalveolar lavage (BAL) fluid and serum of 46 CF patients with mild disease, 25 patients with chronic bronchitis and 22 healthy subjects by gel chromatography and a functional agglutination assay. Relation of SP-A agglutination ability to disease severity of the subjects was explored. SP-A was present in seven major oligomeric forms with the majority of SP-A being structurally organized as complex oligomeric forms. More complex oligomeric forms were associated with better SP-A function with regard to its agglutination ability. These forms were more frequently observed in BAL than in serum, but there were no differences between disease groups. In CF patients, more complex forms of SP-A were associated with better lung function. Organizational structure of SP-A affects its functional activity and is linked to disease severity in CF

Upper and lower airway cultures in children with cystic fibrosis: Do not neglect the upper airways

AbstractBackgroundAirways of cystic fibrosis (CF) patients are colonised with bacteria early in life. We aimed to analyse differences between results of simultaneously taken upper airway (UAW) and lower airway (LAW) cultures, to describe clinical characteristics of patients with positive versus negative cultures and to follow up the patients with P. aeruginosa positive UAW cultures.MethodsBacteriological and clinical data from 157 children were collected during annual check up. The number of positive UAW and LAW cultures and correspondence between these results and clinical characteristics were analysed.ResultsPositive LAW and UAW cultures were found in 79.6% and 43.9% of patients respectively (p<0.001). Patients with positive LAW cultures were significantly older (11.9 vs. 9.8years, p<0.05) and had more LAW symptoms (73.6% vs. 46.7%, p<0.05), especially when P. aeruginosa was found. Patients with positive UAW cultures (especially S. aureus) had more nasal discharge (50.7% vs. 25.0%, p<0.001). In 65% of patients with positive UAW and negative LAW culture for P. aeruginosa the next LAW became P. aeruginosa positive.ConclusionUAW cultures and LAW cultures differ in children with CF and there are differences in clinical characteristics between patients with positive versus negative culture results. P. aeruginosa positive UAW cultures appeared to precede positive LAW cultures in a substantial part of patients, suggesting some kind of cross-infection between the UAW and LAW

Effect of Arginase Inhibition on Pulmonary L-Arginine Metabolism in Murine Pseudomonas Pneumonia

Rationale: Infection of the lung with Pseudomonas aeruginosa results in upregulation of nitric oxide synthases (NOS) and arginase expression, and both enzymes compete for L-arginine as substrate. Nitric oxide (NO) production may be regulated by arginase as it controls L-arginine availability for NOS. We here studied the effect of systemic arginase inhibition on pulmonary L-arginine metabolism in Pseudomonas pneumonia in the mouse. Methods: Mice (C57BL/6, 8–10 weeks old, female) underwent direct tracheal instillation of Pseudomonas (PAO-1)-coated agar beads and were treated by repeated intra-peritoneal injections of the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) or PBS until lungs were harvested on day 3 of the infection. L-arginine metabolites were quantified using liquid chromatography-tandem mass spectrometry, NO metabolites nitrate and nitrite by Griess reagent and cytokines by ELISA. Results: NO metabolite concentrations (48.5±2.9 vs. 10.9±2.3 µM, p<0.0001), as well as L-ornithine (29.6±1.7 vs 2.3±0.4 µM, p<0.0001), the product of arginase activity, were increased in Pseudomonas infected lungs compared to naïve controls. Concentrations of the NOS inhibitor asymmetric dimethylarginine (ADMA) were also increased (0.44±0.02 vs. 0.16±0.01 µM, p<0.0001). Arginase inhibition in the infected animals resulted in a significant decrease in L-ornithine (14.6±1.6 µM, p<0.0001) but increase in L-arginine concentration (p<0.001), L-arginine/ADMA ratio (p<0.001), L-arginine availability for NOS (p<0.001), and NO metabolite concentrations (67.3±5.7 µM, p<0.05). Arginase inhibitor treatment also resulted in an increase in NO metabolite levels in animals following intratracheal injection of LPS (p = 0.015). Arginase inhibition was not associated with an increase in inflammatory markers (IFN-γ, IL-1β, IL-6, MIP-2, KC or TNF-α) in lung. Concentrations of the L-ornithine-dependent polyamines putrescine, spermidine and spermine were increased in Pseudomonas infected lungs (p<0.001, respectively) but were unaffected by ABH treatment. Conclusions: Systemic arginase inhibition with ABH during Pseudomonas pneumonia in mice results in an increase in pulmonary NO formation but no pro-inflammatory effect

Infant lung function tests as endpoints in the ISIS multicenter clinical trial in cystic fibrosis

BACKGROUND: The Infant Study of Inhaled Saline (ISIS) in CF was the first multicenter clinical trial to utilize infant pulmonary function tests (iPFTs) as an endpoint. METHODS: Secondary analysis of ISIS data was conducted in order to assess feasibility of iPFT measures and their associations with respiratory symptoms. Standard deviations were calculated to aid in power calculations for future clinical trials. RESULTS: Seventy-three participants enrolled, 70 returned for the final visit; 62 (89%) and 45 (64%) had acceptable paired functional residual capacity (FRC) and raised volume measurements, respectively. Mean baseline FEV0.5, FEF75 and FRC z-scores were 0.3 (SD: 1.2), -0.2 (SD: 2.0), and 1.8 (SD: 2.0). CONCLUSIONS: iPFTs are not appropriate primary endpoints for multicenter clinical trials due to challenges of obtaining acceptable data and near-normal average raised volume measurements. Raised volume measures have potential to serve as secondary endpoints in future clinical CF trials

T helper cell subsets specific for pseudomonas aeruginosa in healthy individuals and patients with cystic fibrosis

Background: We set out to determine the magnitude of antigen-specific memory T helper cell responses to Pseudomonas aeruginosa in healthy humans and patients with cystic fibrosis. Methods: Peripheral blood human memory CD4+ T cells were co-cultured with dendritic cells that had been infected with different strains of Pseudomonas aeruginosa. The T helper response was determined by measuring proliferation, immunoassay of cytokine output, and immunostaining of intracellular cytokines. Results: Healthy individuals and patients with cystic fibrosis had robust antigen-specific memory CD4+ T cell responses to Pseudomonas aeruginosa that not only contained a Th1 and Th17 component but also Th22 cells. In contrast to previous descriptions of human Th22 cells, these Pseudomonal-specific Th22 cells lacked the skin homing markers CCR4 or CCR10, although were CCR6+. Healthy individuals and patients with cystic fibrosis had similar levels of Th22 cells, but the patient group had significantly fewer Th17 cells in peripheral blood. Conclusions: Th22 cells specific to Pseudomonas aeruginosa are induced in both healthy individuals and patients with cystic fibrosis. Along with Th17 cells, they may play an important role in the pulmonary response to this microbe in patients with cystic fibrosis and other conditions

-Organic Lewis Acid Catalysis- Vinylogous Mukaiyama Aldol Reactions And New Catalysts

This thesis delineates the development of efficient disulfonimide-catalyzed enantioselective vinylogous Mukaiyama aldol reactions and their extensions towards double vinlyogous versions. The disulfonimides proved to be very general, tolerating aromatic and aliphatic aldehydes as electrophiles. Focusing on open chain nucleophiles, we investigated differently substituted crotonates and sorbates. The unprecedented double vinylogous Mukaiyama aldol reactions, additions of six carbon atoms to carbonyl compounds, offered a rapid access to eight-membered ring lactones (ζ-lactones). Our endeavors towards the improvement of the disulfonimide-catalysts, led us to the exploitation of cooperative effects. This strategy found its successful embodiment in diarylmethanol-substituted hydroxy-disulfonimides, catalysts bearing a tertiary alcohol moiety in the substituents. With these catalysts we were able to efficiently convert very unreactive electrophiles in Mukaiyama aldol processes. Based on mechanistic studies, indicating a Lewis acid mechanism as operative, we presume that hydroxy-disulfonimides are Brønsted acid assisted Lewis acids. First results with these catalysts allowed for catalyst loadings unprecedented in metal-free catalysis (as low as 0.0001 mol%)

Antibiotic strategies for eradicating Pseudomonas aeruginosa in people with cystic fibrosis

Background: Respiratory tract infection with Pseudomonas aeruginosa occurs inmost people with cystic fibrosis. Once chronic infection is established, Pseudomonas aeruginosa is virtually impossible to eradicate and is associated with increased mortality and morbidity. Early infection may be easier to eradicate. This is an update of a Cochrane review first published in 2003, and previously updated in 2006 and 2009. Objectives: To determine whether antibiotic treatment of early Pseudomonas aeruginosa infection in children and adults with cystic fibrosis eradicates the organism, delays the onset of chronic infection, and results in clinical improvement. To evaluate whether there is evidence that a particular antibiotic strategy is superior to or more cost-effective than other strategies and to compare the adverse effects of different antibiotic strategies (including respiratory infection with other micro-organisms). Search methods: We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Trials Register comprising references identified from comprehensive electronic database searches and handsearches of relevant journals and abstract books of conference proceedings. Most recent search: 08 September 2014. Selection criteria: We included randomised controlled trials of people with cystic fibrosis, in whom Pseudomonas aeruginosa had recently been isolated from respiratory secretions. We compared combinations of inhaled, oral or intravenous antibiotics with placebo, usual treatment or other combinations of inhaled, oral or intravenous antibiotics. We excluded non-randomised trials, cross-over trials, and those utilising historical controls. Data collection and analysis: Both authors independently selected trials, assessed risk of bias and extracted data. Main results: The search identified 49 trials; seven trials (744 participants) with a duration between 28 days and 27 months were eligible for inclusion. Three of the trials are over 10 years old and their results may be less applicable today given the changes in standard treatment. Some of the trials had low numbers of participants and most had relatively short follow-up periods; however, there was generally a low risk of bias from missing data. In most trials it was difficult to blind participants and clinicians to treatment given the interventions and comparators used. Two trials were supported by the manufacturers of the antibiotic used. Evidence from two trials (38 participants) at the two-month time-point showed treatment of early Pseudomonas aeruginosa infection with inhaled tobramycin results in microbiological eradication of the organism from respiratory secretions more often than placebo, odds ratio 0.15 (95% confidence interval 0.03 to 0.65) and data from one of these trials, with longer follow up, suggested that this effect may persist for up to 12 months. One randomised controlled trial (26 participants) compared oral ciprofloxacin and nebulised colistin versus usual treatment. Results after two years suggested treatment of early infection results in microbiological eradication of Pseudomonas aeruginosa more often than no anti-pseudomonal treatment, odds ratio 0.12 (95% confidence interval 0.02 to 0.79). One trial comparing 28 days to 56 days treatment with nebulised tobramycin solution for inhalation in 88 participants showed that both treatments were effective and well-tolerated, with no notable additional improvement with longer over shorter duration of therapy. However, this trial was not powered to detect non- inferiority or equivalence. A trial of oral ciprofloxacin with inhaled colistin versus nebulised tobramycin solution for inhalation alone (223 participants) failed to show a difference between the two strategies, although it was underpowered to show this. A further trial of inhaled colistin with oral ciprofloxacin versus nebulised tobramycin solution for inhalation with oral ciprofloxacin also showed no superiority of the former, with increased isolation of Stenotrophomonas maltophilia in both groups. A recent, large trial in 306 children aged between one and 12 years compared cycled nebulised tobramycin solution for inhalation to culture-based therapy and also ciprofloxacin to placebo. The primary analysis showed no difference in time to pulmonary exacerbation or proportion of Pseudomonas aeruginosa positive cultures. An analysis performed in this review (not adjusted for age) showed fewer participants in the cycled therapy group with one or more isolates of Pseudomonas aeruginosa, odds ratio 0.51 (95% CI 0.31 to 0.28). Authors’ conclusions: We found that nebulised antibiotics, alone or in combination with oral antibiotics, were better than no treatment for early infection with Pseudomonas aeruginosa. Eradication may be sustained for up to two years. There is insufficient evidence to determine whether antibiotic strategies for the eradication of early Pseudomonas aeruginosa decrease mortality or morbidity, improve quality of life, or are associated with adverse effects compared to placebo or standard treatment. Four trials of two active treatments have failed to show differences in rates of eradication of Pseudomonas aeruginosa. There have been no published randomised controlled trials that investigate the efficacy of intravenous antibiotics to eradicate Pseudomonas aeruginosa in cystic fibrosis. Overall, there is still insufficient evidence from this review to state which antibiotic strategy should be used for the eradication of early Pseudomonas aeruginosa infection in cystic fibrosis

Estimating long-term treatment effects in observational data: A comparison of the performance of different methods under real-world uncertainty.

In the presence of time-dependent confounding, there are several methods available to estimate treatment effects. With correctly specified models and appropriate structural assumptions, any of these methods could provide consistent effect estimates, but with real-world data, all models will be misspecified and it is difficult to know if assumptions are violated. In this paper, we investigate five methods: inverse probability weighting of marginal structural models, history-adjusted marginal structural models, sequential conditional mean models, g-computation formula, and g-estimation of structural nested models. This work is motivated by an investigation of the effects of treatments in cystic fibrosis using the UK Cystic Fibrosis Registry data focussing on two outcomes: lung function (continuous outcome) and annual number of days receiving intravenous antibiotics (count outcome). We identified five features of this data that may affect the performance of the methods: misspecification of the causal null, long-term treatment effects, effect modification by time-varying covariates, misspecification of the direction of causal pathways, and censoring. In simulation studies, under ideal settings, all five methods provide consistent estimates of the treatment effect with little difference between methods. However, all methods performed poorly under some settings, highlighting the importance of using appropriate methods based on the data available. Furthermore, with the count outcome, the issue of non-collapsibility makes comparison between methods delivering marginal and conditional effects difficult. In many situations, we would recommend using more than one of the available methods for analysis, as if the effect estimates are very different, this would indicate potential issues with the analyses

Calcifediol-loaded liposomes for local treatment of pulmonary bacterial infections.

The influence of vitamin D3 and its metabolites calcifediol (25(OH)D) and calcitriol on immune regulation and inflammation is well described, and raises the question of potential benefit against bacterial infections. In the current study, 25(OH)D was encapsulated in liposomes to enable aerosolisation, and tested for the ability to prevent pulmonary infection by Pseudomonas aeruginosa. Prepared 25(OH)D-loaded liposomes were nanosized and monodisperse, with a negative surface charge and a 25(OH)D entrapment efficiency of approximately 23%. Jet nebulisation of liposomes was seen to yield an aerosol suitable for tracheo-bronchial deposition. Interestingly, 25(OH)D in either liposomes or ethanolic solution had no effect on the release of the proinflammatory cytokine KC from Pseudomonas-infected murine epithelial cells (LA-4); treatment of infected, human bronchial 16-HBE cells with 25(OH)D liposomes however resulted in a significant reduction in bacterial survival. Together with the importance of selecting an application-appropriate in vitro model, the current study illustrates the feasibility and practicality of employing liposomes as a means to achieve 25(OH)D lung deposition. 25(OH)D-loaded liposomes further demonstrated promising effects regarding prevention of Pseudomonas infection in human bronchial epithelial cells