A murine model to study vasoreactivity and intravascular flow in lung isograft microvessels.

Intravital microscopy of orthotopic lung tissue is technically demanding, especially for repeated investigations. Therefore, we have established a novel approach, which allows non-invasive repetitive in vivo microscopy of ectopic lung tissue in dorsal skinfold chambers. Syngeneic subpleural peripheral lung tissue and autologous endometrium (control) were transplanted onto the striated muscle within dorsal skinfold chambers of C57BL/6 mice. Grafts were analysed by intravital fluorescence microscopy over 14 days. Angiogenesis occurred in the grafts on day 3, as indicated by sinusoidal microvessels on the grafts' edges with very slow blood flow, perifocal oedema, and haemorrhage. By day 10, lung transplants were completely revascularized, exhibited a dense network of microvessels with irregular diameters, chaotic angioarchitecture, and high blood flow. Compared to lung tissue, endometrial grafts contained a structured, glomerulus-like vessel architecture with lower blood flow. Despite missing ventilation, hypoxic vasoconstriction of the lung tissue arterioles occurred. In contrast, endometrium tissue arterioles dilated during hypoxia and constricted in hyperoxia. This demonstrates that ectopic lung grafts keep their ability for organ-specific hypoxic vasoconstriction. These findings indicate that our approach is suitable for repetitive in vivo pulmonary microcirculation analyses. The high blood flow and hypoxia-induced vasoconstriction in lung grafts suggest a physiological intrinsic vasoregulation independent of the recipient tissue.

Sequential Inhalational Tobramycin-Colistin-Combination in CF-Patients with Chronic P. Aeruginosa Colonization - an Observational Study.

BACKGROUND/AIMS: In cystic fibrosis (CF), chronic microbial lung infections are difficult to treat and cause morbidity and increased mortality. METHODS: In a multicentre, open-label, exploratory, non-interventional study, inhaled tobramycin (300 mg twice daily) and colistin (1 million I.U. twice daily) were sequentially combined with the aim to investigate the effect on 41 CF patients with chronic P. aeruginosa infections for six months (mean age 24 ± 10.8y). RESULTS: Six patients had adverse events that were assessed as being related to treatment. Mucus production and coughing both decreased in 39%, whereas FEV1 absolute and relative to baseline increased by 4.9% and 9.1%, respectively (p = 0.004) in 29 patients, who were definitely treated sequentially. Efficacy of the therapy was rated 'excellent' or 'good' by the physicians in 80.5% of the patients. CONCLUSIONS: The results indicate that treatment with inhaled antibiotics, sequentially combined, was very well tolerated by most patients and may have a beneficial effect, even if transitory on lung function and respiratory symptoms.

Long-Term Pulmonal Therapy of Cystic Fibrosis-Patients with Amitriptyline.

BACKGROUND/AIMS: Several recent clinical studies revealed an accumulation of ceramide in bronchial epithelial cells of patients with cystic fibrosis (CF). Degradation of ceramide concentrations in lungs of CF patients employing the functional acid sphingomyelinase inhibitor amitriptyline revealed a benefit in lung function, weight and exacerbation rates. METHODS: To test for a beneficial effect of amitriptyline in vivo, we performed two phase II randomised, double-blind, placebo-controlled studies. CF patients were treated with 25 mg amitriptyline twice daily, i.e. a total dose of 50 mg/d. After those two studies part of the patients used amitriptyline in an off-lable-use for routine treatment. These patients were observed after one, two and three years after continuous use of amitriptyline and were matched with those patients who were not treated. These patients were used as a control group. RESULTS: After one year of treatment, forced expiratory volume in 1 sec predicted (FEV1) increased significantly by 7.6±7.0%, p=<0.001, and weight increased by 2.1±2.3kg, p=<0.001 in the amitriptyline population (n=20), whereas FEV1 decreased significantly in the control group by 1.8±3.3%, p=0.010, and weight increased by 1.1±2.7kg, p=0.010 (n=14). After two years of treatment, FEV1 increased significantly by 5.6±10.3%, p=0.009, and weight increased by 3.6±2.9kg, p=<0.001 in the amitriptyline population (n=12). In contrast, FEV1 decreased in the control group by 2.1±3.7%, p=0.051 and weight increased by only 0.4±2.9kg, p=0.31 (n=10). After three years of treatment, FEV1 increased significantly by 7.7±8%, p=0.050, and weight increased by 7.3±3.8kg, p=0.016, in the amitriptyline population (n=5), whereas FEV1 decreased in the control group by 1.0±1.3%, p=0.075 and weight increased by 0.4±1.5kg, p=0.29 (n=5). CONCLUSION: Amitriptyline significantly increases FEV1, reduces ceramide in lung cells and increases weight of CF patients.

Gaseous nitric oxide to treat antibiotic resistant bacterial and fungal lung infections in patients with cystic fibrosis: a phase I clinical study.

BACKGROUND: Individuals with cystic fibrosis (CF) receive antibiotics continuously throughout their entire life which leads to drug resistant microbial lung infections which are difficult to treat. Nitric oxide (NO) gas possesses antimicrobial activity against a wide variety of microorganisms in vitro, in vivo in animal models and a phase I study in healthy adults showed administration of intermittent 160 ppm NO to be safe. METHODS: We assessed feasibility and safety of inhaled NO in eight CF patients who received 160 ppm NO for 30 min, three times daily for 2 periods of 5 days. RESULTS: The NO treatment was safe and in none of the patients were serious drug-related adverse events observed which caused termination of the study. The intention-to-treat analysis revealed a significant mean reduction of the colony forming units of all bacteria and all fungi, while mean forced expiratory volume 1 s % predicted (FEV1) relative to baseline increased 17.3 ± 8.9 % (P = 0.012). CONCLUSIONS: NO treatment may improve the therapy of chronic microbial lung infections in CF patients, particularly concerning pathogens with intrinsic or acquired resistance to antibiotics.

BIIL 284 reduces neutrophil numbers but increases P. aeruginosa bacteremia and inflammation in mouse lungs.

BACKGROUND: A clinical study to investigate the leukotriene B(4) (LTB(4))-receptor antagonist BIIL 284 in cystic fibrosis (CF) patients was prematurely terminated due to a significantly increased risk of adverse pulmonary events. We aimed to establish the effect of BIIL284 in models of Pseudomonas aeruginosa lung infection, thereby contributing to a better understanding of what could have led to adverse pulmonary events in CF patients. METHODS: P. aeruginosa DNA in the blood of CF patients during and after acute pulmonary exacerbations and in stable patients with non-CF bronchiectasis (NCFB) and healthy individuals was assessed by PCR. The effect of BIIL 284 treatment was tested in an agar bead murine model of P. aeruginosa lung infection. Bacterial count and inflammation were evaluated in lung and other organs. RESULTS: Most CF patients (98%) and all patients with NCFB and healthy individuals had negative P. aeruginosa DNA in their blood. Similarly, the P. aeruginosa-infected mice showed bacterial counts in the lung but not in the blood or spleen. BIIL 284 treatment decreased pulmonary neutrophils and increased P. aeruginosa numbers in mouse lungs leading to significantly higher bacteremia rates and lung inflammation compared to placebo treated animals. CONCLUSIONS: Decreased airway neutrophils induced lung proliferation and severe bacteremia in a murine model of P. aeruginosa lung infection. These data suggest that caution should be taken when administering anti-inflammatory compounds to patients with bacterial infections.

Inhalation treatment with glutathione in patients with cystic fibrosis. A randomized clinical trial.

RATIONALE: Glutathione is the major antioxidant in the extracellular lining fluid of the lungs and depleted in patients with cystic fibrosis (CF). OBJECTIVES: We aimed to assess glutathione delivered by inhalation as a potential treatment for CF lung disease. METHODS: This randomized, double-blind, placebo-controlled trial evaluated inhaled glutathione in subjects with CF 8 years of age and older and FEV1 of 40-90% of predicted. Subjects were randomized to receive 646 mg glutathione in 4 ml (n = 73) or placebo (n = 80) via an investigational eFlow nebulizer every 12 hours for 6 months. MEASUREMENTS AND MAIN RESULTS: FEV1 (absolute values), both as pre-post differences (P = 0.180) and as area under the curves (P = 0.205), were the primary efficacy endpoints, and were not different between the glutathione group and the placebo group over the 6-month treatment period. Exploratory analysis showed an increase of FEV1 from baseline over placebo of 100 ml or 2.2% predicted; this was significant at 3 months, but not later. Subjects receiving glutathione had neither fewer pulmonary exacerbations, nor better scores for quality of life. Whereas increased glutathione and metabolites in sputum demonstrated significant delivery to the lungs, there was no indication of diminished oxidative stress to proteins or lipids, and no evidence for anti-inflammatory or antiproteolytic actions of glutathione supplemented to the airways. The adverse event incidence was similar between glutathione and placebo. CONCLUSIONS: Inhaled glutathione in the dose administered did not demonstrate clinically relevant improvements in lung function, pulmonary exacerbation frequency, or patient-reported outcomes. Glutathione delivery to the airways was not associated with changes in markers of oxidation, proteolysis, or inflammation. Clinical trial registered with www.clinicaltrials.gov (NCT00506688) and https://eudract.ema.europa.eu/index.html (EudraCT 2005-003870-88).

Therapy of CF-patients with amitriptyline and placebo--a randomised, double-blind, placebo-controlled phase IIb multicenter, cohort-study.

BACKGROUND/AIMS: Several recent studies revealed an accumulation of ceramide in bronchial, tracheal and intestinal epithelial cells of mice and patients with cystic fibrosis (CF). Normalization of ceramide concentrations in lungs of CF mice employing the functional acid sphingomyelinase inhibitor amitriptyline also normalized mucociliary clearance, chronic inflammation and infection susceptibility to pulmonary P. aeruginosa in these mice. METHODS: To test for a beneficial effect of amitriptyline in vivo, we performed a phase IIb randomised, double-blind, placebo-controlled study. Twenty-one CF patients were treated with 25 mg/d amitriptyline twice daily for 28 days. The placebo consisted of 19 patients and was also treated twice per day. The primary endpoint was the change in lung function in the intention-to-treat (ITT) population. Secondary endpoints were ceramide levels in epithelial cells and safety. RESULTS: After treatment, forced expiratory volume in 1 sec predicted (FEV1) increased 6.3 ± 11.5% (p=0.08) in the ITT population (36 of 40 CF patients) and 8.5 ± 10% (p=0.013) in the per protocol (PP) population (29 of 40 patients). Ceramide levels decreased in nasal epithelial cells after amitriptyline treatment. Amitriptyline had no severe and only mild and mostly transient adverse effects, i.e. xerostomia and tiredness. CONCLUSION: Amitriptyline is safe in CF-patients, increases FEV1 and reduces ceramide in lung cells of CF patients.