Modulation, microbiota and inflammation in the adult CF gut: A prospective study.

BACKGROUND: Cystic Fibrosis (CF) has prominent gastrointestinal and pancreatic manifestations. The aim of this study was to determine the effect of Cystic fibrosis transmembrane conductance regulator (CFTR) modulation on, gastrointestinal inflammation, pancreatic function and gut microbiota composition in people with cystic fibrosis (CF) and the G551D-CFTR mutation. METHODS: Fourteen adult patients with the G551D-CFTR mutation were assessed clinically at baseline and for up to 1 year after treatment with ivacaftor. The change in gut inflammatory markers (calprotectin and lactoferrin), exocrine pancreatic status and gut microbiota composition and structure were assessed in stool samples. RESULTS: There was no significant change in faecal calprotectin nor lactoferrin in patients with treatment while all patients remained severely pancreatic insufficient. There was no significant change in gut microbiota diversity and richness following treatment. CONCLUSION: There was no significant change in gut inflammation after partial restoration of CFTR function with ivacaftor, suggesting that excess gut inflammation in CF is multi-factorial in aetiology. In this adult cohort, exocrine pancreatic function was irreversibly lost. Longer term follow-up may reveal more dynamic changes in the gut microbiota and possible restoration of CFTR function.

Recurrent asthma exacerbations: co-existing asthma and common variable immunodeficiency.

Common variable immunodeficiency is characterized by impaired B-cell differentiation and defective immunoglobulin production manifesting as recurrent respiratory tract infections. While the condition can masquerade as asthma, late diagnosis of CVID in known asthmatic is rarely reported.We present the case of a 43-year-old lady with recurrent episodes of wheeze, cough, sinusitis and multiple lower respiratory tract infections. Transiently responsive to antibiotics and steroids. These episodes had been occurring for many years and she had a longstanding clinical diagnosis of asthma.As part of her work up for recurrent respiratory tract infections a CT thorax was performed and demonstrated bronchiectasis. Further tests including Immunoglobulin levels revealed critically low IgG, IgM, and IgA levels. Immunoglobulin replacement therapy was commenced with a reduction in exacerbation frequency and severity, and objective improvement of asthma control. Subsequent lung function tests demonstrated reversible airflow limitation (obstructive lung function with 13% reversibility in FEV1 post-bronchodilator) consistent with asthma.Our case illustrates the importance of searching for alternate and co-existent diagnoses in patients diagnosed with asthma who are unresponsive to conventional therapy. We believe that serum immunoglobulin measurement should form a component of such a workup.

A pilot study demonstrating the altered gut microbiota functionality in stable adults with Cystic Fibrosis.

Cystic Fibrosis (CF) and its treatment result in an altered gut microbiota composition compared to non-CF controls. However, the impact of this on gut microbiota functionality has not been extensively characterised. Our aim was to conduct a proof-of-principle study to investigate if measurable changes in gut microbiota functionality occur in adult CF patients compared to controls. Metagenomic DNA was extracted from faecal samples from six CF patients and six non-CF controls and shotgun metagenomic sequencing was performed on the MiSeq platform. Metabolomic analysis using gas chromatography-mass spectrometry was conducted on faecal water. The gut microbiota of the CF group was significantly different compared to the non-CF controls, with significantly increased Firmicutes and decreased Bacteroidetes. Functionality was altered, with higher pathway abundances and gene families involved in lipid (e.g. PWY 6284 unsaturated fatty acid biosynthesis (p = 0.016)) and xenobiotic metabolism (e.g. PWY-5430 meta-cleavage pathway of aromatic compounds (p = 0.004)) in CF patients compared to the controls. Significant differences in metabolites occurred between the two groups. This proof-of-principle study demonstrates that measurable changes in gut microbiota functionality occur in CF patients compared to controls. Larger studies are thus needed to interrogate this further.

Clostridium difficile carriage in adult cystic fibrosis (CF); implications for patients with CF and the potential for transmission of nosocomial infection.

Clostridium difficile is an anaerobic Gram-positive, spore-forming, toxin-producing bacillus transmitted among humans through the faecal-oral route. Despite increasing carriage rates and the presence of C. difficile toxin in stool, patients with CF rarely appear to develop typical manifestations of C. difficile infection (CDI). In this study, we examined the carriage, toxin production, ribotype distribution and antibiotic susceptibility of C. difficile in a cohort of 60 adult patients with CF who were pre-lung transplant. C. difficile was detected in 50% (30/60) of patients with CF by culturing for the bacteria. C. difficile toxin was detected in 63% (19/30) of C. difficile-positive stool samples. All toxin-positive stool samples contained toxigenic C. difficile strains harbouring toxin genes, tcdA and tcdB. Despite the presence of C. difficile and its toxin in patient stool, no acute gastrointestinal symptoms were reported. Ribotyping of C. difficile strains revealed 16 distinct ribotypes (RT), 11 of which are known to be disease-causing including the hyper-virulent RT078. Additionally, strains RT002, RT014, and RT015, which are common in non-CF nosocomial infection were described. All strains were susceptible to vancomycin, metronidazole, fusidic acid and rifampicin. No correlation was observed between carriage of C. difficile or any characteristics of isolated strains and any recorded clinical parameters or treatment received. We demonstrate a high prevalence of hypervirulent, toxigenic strains of C. difficile in asymptomatic patients with CF. This highlights the potential role of asymptomatic patients with CF in nosocomial transmission of C. difficile.

A 76 year old female diagnosed with cystic fibrosis.

The diagnosis of Cystic Fibrosis (CF) requires a high clinical suspicion in patients presenting at all ages. Early recognition permits referral to a specialist centre and may reduce the morbidity and mortality associated with CF. We report the case of the oldest patient in Ireland diagnosed with CF at 76 years of age and highlight the clinical features of her presentation.

MYCN repression of Lifeguard/FAIM2 enhances neuroblastoma aggressiveness.

Neuroblastoma (NBL) is the most common solid tumor in infants and accounts for 15% of all pediatric cancer deaths. Several risk factors predict NBL outcome: age at the time of diagnosis, stage, chromosome alterations and MYCN (V-Myc Avian Myelocytomatosis Viral Oncogene Neuroblastoma-Derived Homolog) amplification, which characterizes the subset of the most aggressive NBLs with an overall survival below 30%. MYCN-amplified tumors develop exceptional chemoresistance and metastatic capacity. These properties have been linked to defects in the apoptotic machinery, either by silencing components of the extrinsic apoptotic pathway (e.g. caspase-8) or by overexpression of antiapoptotic regulators (e.g. Bcl-2, Mcl-1 or FLIP). Very little is known on the implication of death receptors and their antagonists in NBL. In this work, the expression levels of several death receptor antagonists were analyzed in multiple human NBL data sets. We report that Lifeguard (LFG/FAIM2 (Fas apoptosis inhibitory molecule 2)/NMP35) is downregulated in the most aggressive and undifferentiated tumors. Intringuingly, although LFG has been initially characterized as an antiapoptotic protein, we have found a new association with NBL differentiation. Moreover, LFG repression resulted in reduced cell adhesion, increased sphere growth and enhanced migration, thus conferring a higher metastatic capacity to NBL cells. Furthermore, LFG expression was found to be directly repressed by MYCN at the transcriptional level. Our data, which support a new functional role for a hitherto undiscovered MYCN target, provide a new link between MYCN overexpression and increased NBL metastatic properties.

Inhaled versus nebulised tobramycin: a real world comparison in adult cystic fibrosis (CF).

BACKGROUND: There are no published data on real-life clinical experience comparing inhaled antibiotic therapy via new rapid delivery systems with nebulised antibiotic therapy in CF. This real world study compares safety, effectiveness and tolerability using tobramycin inhaled powder (TIP) versus tobramycin inhaled solution (TIS). METHODS: Adult patients with CF commencing TIP (n=78) completed a questionnaire assessing safety, efficacy, tolerability, patient-satisfaction and self-reported adherence to TIS at baseline and during 12 months of TIP therapy. FEV1% predicted and exacerbation rate were recorded at each visit. RESULTS: There was a significant improvement in adherence scores, with a significant decrease in the number of intravenous antibiotic courses received during 12 months of TIP compared with the preceding 12 months using TIS. 94% of patients who had previously used TIS preferred TIP therapy over TIS. CONCLUSIONS: Inhaled powder tobramycin in CF is associated with improved adherence, tolerability and decreased exacerbation rates compared to nebulised treatment in real-life practice.

T cell-mediated induction of thymic stromal lymphopoietin in differentiated human primary bronchial epithelial cells.

BACKGROUND: Inhaled peptide challenge has been shown to induce T cell-mediated, isolated late asthmatic reaction (LAR), characterized by recruitment of CD4(+) T cells and increased levels of thymus and activation-regulated chemokine (TARC; CCL17). Epithelial-derived thymic stromal lymphopoietin (TSLP) has been shown to modulate dendritic cell function to promote TH 2 responses via CCL17 production. OBJECTIVES: To elucidate the mechanisms involved in allergen-specific T cell-induced LAR and recruitment of CD4(+) T cells by examining the effects of T cell-derived factors on the induction of TSLP in primary bronchial epithelial cells (PBEC). METHODS: PBEC grown at air-liquid interface from healthy individuals and patients with asthma were stimulated with double-stranded RNA (dsRNA) or supernatants from activated allergen-specific T cells. TSLP was measured in PBEC culture supernatants. Neutralizing antibodies and signalling inhibitors were used to examine the mechanisms responsible for the induction of epithelial-derived TSLP. The functional activity of PBEC-derived TSLP was measured using a bioassay involving the induction of CCL17 production from monocyte-derived dendritic cells (moDC). RESULTS: Both dsRNA and allergen-specific T cells induced enhanced TSLP secretion from asthmatic PBEC compared to healthy PBEC. Activated PBEC culture supernatant induced TSLP-dependent CCL17 production from moDC in a manner related to clinical asthmatic status. IL-1ß, IL-6, and CXCL8, rather than TH 2 cytokines (IL-4/5/13), appeared to be the principle mediators of allergen-specific T cell-dependent induction of epithelial-derived TSLP, which was regulated by the MEK, MAPK, and NFκB pathways. CONCLUSION AND CLINICAL RELEVANCE: Our data reveal a novel effect of allergen-specific T cells as a positive regulator of TSLP production by epithelial cells, suggesting T cell-airway epithelium interactions that may lead to maintenance and amplification of allergic inflammation. TSLP is currently a candidate for therapeutic intervention in asthma, but the factors that drive TSLP expression (T cell-derived factors) may be equally relevant in the treatment of allergic inflammation.

Organic aerosol formation downwind from the Deepwater Horizon oil spill.

A large fraction of atmospheric aerosols are derived from organic compounds with various volatilities. A National Oceanic and Atmospheric Administration (NOAA) WP-3D research aircraft made airborne measurements of the gaseous and aerosol composition of air over the Deepwater Horizon (DWH) oil spill in the Gulf of Mexico that occurred from April to August 2010. A narrow plume of hydrocarbons was observed downwind of DWH that is attributed to the evaporation of fresh oil on the sea surface. A much wider plume with high concentrations of organic aerosol (>25 micrograms per cubic meter) was attributed to the formation of secondary organic aerosol (SOA) from unmeasured, less volatile hydrocarbons that were emitted from a wider area around DWH. These observations provide direct and compelling evidence for the importance of formation of SOA from less volatile hydrocarbons.

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.

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.

Primary airway epithelial cell culture from lung transplant recipients.

Long-term survival in lung transplantation is limited by the development of obliterative bronchiolitis, a condition characterised by inflammation, epithelial injury, fibroproliferation and obliteration of bronchioles leading to airflow obstruction. To investigate the role of the bronchial epithelium in the pathogenesis of obliterative bronchiolitis the current study aimed to establish primary bronchial epithelial cell cultures (PBEC) from lung allografts. Four to six bronchial brushings were obtained from sub-segmental bronchi of lung allografts. Cells were seeded onto collagen-coated plates and grown to confluence in bronchial epithelial growth medium. Bronchial brushings (n=33) were obtained from 27 patients. PBECs were grown to confluence from 12 out of 33 (39%) brushings. Failure to reach confluence was due to early innate infection. Bacteria were usually isolated from both bronchoalveolar lavage and culture media, but a separate population was identified in culture media only. Primary culture of bronchial epithelial cells from lung transplant recipients is feasible, despite a high rate of early, patient-derived infection. Latent infection of the allograft, identified only by bronchial brushings, may itself be a persistent stimulus for epithelial injury. This technique facilitates future mechanistic studies of airway epithelial responses in the pathogenesis of obliterative bronchiolitis.

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.

Is mechanical bowel preparation necessary in patients undergoing cystectomy and urinary diversion?

OBJECTIVE: To compare the surgical outcome in patients with or with no bowel preparation before cystectomy and ileal conduit urinary diversion, specifically assessing local and systemic complications. PATIENTS AND METHODS: All patients undergoing cystectomy and ileal conduit urinary diversion between January 1991 and December 1999 were assessed retrospectively. Twenty-two receive no bowel preparation (group 1) and were compared with 64 who had (group 2). Patients had similar demographic characteristics, stage and grade of tumour. Patients in group 2 received a standard 4-day bowel preparation and group 1 received no lavage or enemas. All patients underwent a standard iliac and obturator lymph node dissection, and cystoprostatectomy or anterior exenteration and ileal conduit urinary diversion. All patients received intraoperative metronidazole and gentamicin intravenously, and two further doses after surgery. RESULTS: Deaths after surgery were comparable in the two groups (two in group 1 and four in group 2) and the incidence of wound infection was similar (three and seven, respectively). There were no significant differences between the respective groups for fistula and anastomotic dehiscence (two and six) or sepsis (three and six). Group 2 had a higher incidence of wound dehiscence (one) than in group 1 (none). The incidence of prolonged postoperative ileus was lower in group 1 (one vs 12), as was the length of hospital stay (31.6 days vs 22.8 days). CONCLUSIONS: Bowel preparation had no advantage for the surgical outcome but it increased the length of hospital stay.

Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells.

Melanosomes and premelanosomes are lysosome-related organelles with a unique structure and cohort of resident proteins. We have positioned these organelles relative to endosomes and lysosomes in pigmented melanoma cells and melanocytes. Melanosome resident proteins Pmel17 and TRP1 localized to separate vesicular structures that were distinct from those enriched in lysosomal proteins. In immunogold-labeled ultrathin cryosections, Pmel17 was most enriched along the intralumenal striations of premelanosomes. Increased pigmentation was accompanied by a decrease in Pmel17 and by an increase in TRP1 in the limiting membrane. Both proteins were largely excluded from lysosomal compartments enriched in LAMP1 and cathepsin D. By kinetic analysis of fluid phase uptake and immunogold labeling, premelanosomal proteins segregated from endocytic markers within an unusual endosomal compartment. This compartment contained Pmel17, was accessed by BSA-gold after 15 min, was acidic, and displayed a cytoplasmic planar coat that contained clathrin. Our results indicate that premelanosomes and melanosomes represent a distinct lineage of organelles, separable from conventional endosomes and lysosomes within pigmented cells. Furthermore, they implicate an unusual clathrin-coated endosomal compartment as a site from which proteins destined for premelanosomes and lysosomes are sorted.

The clinical and economic impact of deep chest surgical site infections following coronary artery bypass graft surgery.

STUDY OBJECTIVES: To examine how deep chest surgical site infections following coronary artery bypass graft (CABG) surgery impact hospital inpatient length of stay (LOS), costs, and mortality. SETTING: A large, Midwestern community medical center. DESIGN: All CABG patients who developed deep chest infection (n = 41) were compared to a set of control subjects (n = 160) systematically selected as every tenth uninfected CABG patient. Clinical data were abstracted from patient records, and cost information was obtained from the cost accounting database of the hospital. RESULTS: Variables that significantly increased the risk of deep chest surgical site infection included obesity (odds ratio [OR], 11; p = 0. 0001), renal insufficiency (OR, 8.9; p = 0.0001), connective tissue disease (OR, 25.4; p = 0.0003), reexploration for bleeding (OR, 8.2; p = 0.0015), and the timing of antibiotic prophylaxis (> 60 min before incision; OR, 5.3; p = 0.0128). Within 1 year postoperatively, patients with deep chest surgical site infection had a mortality rate of 22%, vs 0.6% for uninfected patients (p = 0.0001). Infected patients also incurred an average of 20 additional hospital days (p = 0.0001). Univariate analysis indicated that patients who developed deep chest surgical site infection incurred $20,012 in additional costs in the first year (p = 0.0001). Infected patients who died incurred on average $60,547 more than infected patients who survived (p = 0.034). Multivariate analysis confirmed the magnitude of the estimate of the cost for deep chest surgical site infection ($18, 938; p = 0.0001). CONCLUSIONS: Deep chest surgical site infections following CABG surgery are associated with significant increases in LOS, hospitalization costs, and mortality. These results suggest the need for improved infection control measures to reduce deep chest surgical site infection rates.

Renal transplantation in patients with an augmentation cystoplasty.

OBJECTIVE: To retrospectively review our experience over a 10-year period of renal transplantation in patients with augmented bladders and thus assess the safety of this procedure. PATIENTS AND METHODS: Ten transplant recipients who had previously undergone augmentation cystoplasty were reviewed; a cadaveric kidney was transplant in each case. The donor ureter was anastomosed to the augment bladder in six patients, in three to the native ureter and in one the donor renal pelvis was anastomosed to the native ureter. RESULTS: No patients died and nine of the 10 grafts were functioning at a mean follow-up of 27 months. The mean (SD) serum creatinine level at the follow-up was 100.8 (27.25) mmol/L. Four patients had 10 episodes of urosepsis requiring hospital admission, with only one graft lost. CONCLUSION: Renal transplantation can be performed safely in patients with an augmentation cystoplasty.