Proliferative activity of antigen-specific CD154+ T cells against bacterial and fungal respiratory pathogens in cystic fibrosis decreases after initiation of highly effective CFTR modulator therapy.

Background: Together with impaired mucociliary clearance, lung disease in cystic fibrosis (CF) is driven by dysregulation of innate and adaptive immunity caused by dysfunctional CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), leading to airway infection and hyperinflamma-tion. The highly effective CFTR modulator therapy (HEMT) elexacaftor/tezacaftor/ivacaftor (ETI) generates substantial improvements in clinical outcomes of people with CF (pwCF) by restoration of CFTR activity. Aberrant immune responses of lymphocytes due to CFTR dysfunction has been described in the past, but not the effects of CFTR restoration by HEMT on these cells. We aimed to examine the effect of ETI on the proliferative activity of antigen-specific CD154 (+) T cells against bacterial and fungal species relevant in CF and on total IgG and IgE as markers of B cell adaptive immunity. Methods: We performed ex vivo analyses of Ki-67 expression in antigen-specific CD154 (+) T cells against Pseudomonas aeruginosa, Staphylococcus aureus, Aspergillus fumigatus, Scedosporium apiospermum and Candida albicans from 21 pwCF by cytometric assay based on antigen-reactive T cell enrichment (ARTE), and analysis of total serum IgE and IgG before and after initiation of ETI. Results: Mean Ki-67 expression in antigen-specific CD154 (+) T cells against P. aeruginosa, A. fumigatus, S. apiospermum and C. albicans, but not S. aureus, mean total serum IgG and mean total serum IgE decreased significantly after initiation of ETI. No correlation was found to change in sputum microbiology of the examined pathogens. Mean BMI and FEV1 increased significantly. Conclusion: HEMT is associated with decreased antigen-specific CD154 (+) T cell proliferation activity in our cohort, independent of findings in sputum microbiology of the examined pathogens. Together with the observed clinical improvement and the decrease in total IgE and IgG, this indicates effects due to CFTR restoration on CD154 (+) T cells by ETI and a reduction of B cell activation with subsequent lower immunoglobulin synthesis under HEMT therapy. These results endorse earlier evidence of CFTR dysfunction in T and B cells leading directly to aberrant immune responses with hyperinflammation.

A prospective phase IIA multicenter double-blinded randomized placebo-controlled clinical trial evaluating the efficacy and safety of inhaled Tobramycin in patients with ventilator-associated pneumonia (iToVAP).

OBJECTIVE: Treatment of ventilated pneumonia is often unsuccessful, even when patients are treated according to current guidelines. Therefore, we aimed to investigate the efficacy of the adjunctive inhaled Tobramycin in patients with pneumonia caused by Gram-negative pathogens in addition to the standard systemic treatment. DESIGN: Prospective, multicenter, double-blinded, randomized, placebo-controlled clinical trial. SETTING: 26 patients in medical and surgical ICUs. PATIENTS: Patients with ventilator-associated pneumonia caused by Gram-negative pathogens. MEASUREMENT AND MAIN RESULTS: Fourteen patients were assigned to the Tobramycin Inhal group and 12 patients to the control group. The microbiological eradication of the Gram-negative pathogens was significantly higher in the intervention group than in the control group (p < 0.001). The probability of eradication was 100% in the intervention group [95% Confidence Interval: 0.78-1.0] and 25% in the control group [95% CI: 0.09-0.53]. The increased eradication frequency was not associated with increased patient survival. CONCLUSION: Inhaled aerosolized Tobramycin demonstrated clinically meaningful efficacy in patients with Gram-negative ventilator-associated pneumonia. The probability of eradication in the intervention group was 100%. However, the successful eradication was not associated with a reduction in systemic anti-infective therapy, a shorter ICU stay, or even a survival benefit. In the presence of multidrug-resistant Gram-negative pathogens that are sensitive only to colistin and/or aminoglycosides, supplemental inhaled therapy with nebulizers suitable for this purpose should be considered in addition to systemic antibiotic therapy.

Antibiotic Therapy for Pulmonary Exacerbations in Cystic Fibrosis-A Single-Centre Prospective Observational Study.

People with cystic fibrosis experience bronchopulmonary exacerbations, leading to lung damage, lung function decline, increased mortality, and a poor health-related quality of life. To date, there are still open questions regarding the rationale for antibiotic use and the optimal duration of antibiotic therapy. This prospective single-center study (DRKS00012924) analyzes exacerbation treatment over 28 days in 96 pediatric and adult people with cystic fibrosis who started oral and/or intravenous antibiotic therapy in an inpatient or outpatient setting after clinician diagnosis of bronchopulmonary exacerbation. Biomarkers of exacerbation were examined in terms of their ability to predict response to treatment and the need for antibiotic therapy. The mean duration of antibiotic therapy was 14 days. Inpatient treatment was associated with a poorer health status, but no significant difference was found in the modified Fuchs exacerbation score between inpatients and outpatients. A significant increase of in-hospital FEV1, home spirometry FEV1, and body-mass index and a significant decrease of the modified Fuchs symptom score, C-reactive protein, and 8 out of the 12 domain scores of the revised cystic fibrosis questionnaire were demonstrated after 28 days. However, a trend towards a FEV1 decline in the inpatient group on day 28 could be demonstrated, while FEV1 was maintained in the outpatient group. Correlation analyses of changes between baseline and day 28 show a strong positive correlation between home spirometry and in-hospital FEV1, strong negative correlations between FEV1 and the modified Fuchs exacerbation score and between FEV1 and C-reactive protein, and a moderately negative correlation between FEV1 and the three domains of the revised cystic fibrosis questionnaire. Responders and non-responders to antibiotic therapy were defined in terms of FEV1 improvement after therapy. A higher baseline C-reactive protein, a greater decrease in C-reactive protein, a higher baseline modified Fuchs exacerbation score, and a greater decrease in the score after 28 days could be found in the responder group, while other baseline and follow-up parameters like FEV1 showed no significant differences. Our data show that the modified Fuchs exacerbation score is applicable in a clinical setting and can detect acute exacerbations regardless of health status. Home spirometry is a useful tool for outpatient exacerbation management. A change in C-reactive protein and a modified Fuchs score change are suitable follow-up markers of exacerbation due to their strong correlation with FEV1. Further studies are needed to assess which patients would benefit from a longer duration of antibiotic therapy. C-reactive protein at exacerbation onset and C-reactive protein decline during and after therapy better predict antibiotic therapy success than FEV1 at therapy onset, while the modified Fuchs score indicates exacerbation regardless of the need for antibiotic therapy, suggesting that antibiotic therapy is only part of exacerbation management.

Antigen specificity and cross-reactivity drive functionally diverse anti-Aspergillus fumigatus T cell responses in cystic fibrosis.

BACKGROUNDThe fungus Aspergillus fumigatus causes a variety of clinical phenotypes in patients with cystic fibrosis (pwCF). Th cells orchestrate immune responses against fungi, but the types of A. fumigatus-specific Th cells in pwCF and their contribution to protective immunity or inflammation remain poorly characterized.METHODSWe used antigen-reactive T cell enrichment (ARTE) to investigate fungus-reactive Th cells in peripheral blood of pwCF and healthy controls.RESULTSWe show that clonally expanded, high-avidity A. fumigatus-specific effector Th cells, which were absent in healthy donors, developed in pwCF. Individual patients were characterized by distinct Th1-, Th2-, or Th17-dominated responses that remained stable over several years. These different Th subsets target different A. fumigatus proteins, indicating that differential antigen uptake and presentation directs Th cell subset development. Patients with allergic bronchopulmonary aspergillosis (ABPA) are characterized by high frequencies of Th2 cells that cross-recognize various filamentous fungi.CONCLUSIONOur data highlight the development of heterogenous Th responses targeting different protein fractions of a single fungal pathogen and identify the development of multispecies cross-reactive Th2 cells as a potential risk factor for ABPA.FUNDINGGerman Research Foundation (DFG), under Germany's Excellence Strategy (EXC 2167-390884018 "Precision Medicine in Chronic Inflammation" and EXC 2051-390713860 "Balance of the Microverse"); Oskar Helene Heim Stiftung; Christiane Herzog Stiftung; Mukoviszidose Institut gGmb; German Cystic Fibrosis Association Mukoviszidose e.V; German Federal Ministry of Education and Science (BMBF) InfectControl 2020 Projects AnDiPath (BMBF 03ZZ0838A+B).

Genetic diversification of persistent Mycobacterium abscessus within cystic fibrosis patients.

Mycobacterium (M.) abscessus infections in Cystic Fibrosis (CF) patients cause a deterioration of lung function. Treatment of these multidrug-resistant pathogens is associated with severe side-effects, while frequently unsuccessful. Insight on M. abscessus genomic evolvement during chronic lung infection would be beneficial for improving treatment strategies. A longitudinal study enrolling 42 CF patients was performed at a CF center in Berlin, Germany, to elaborate phylogeny and genomic diversification of in-patient M. abscessus. Eleven of the 42 CF patients were infected with M. abscessus. Five of these 11 patients were infected with global human-transmissible M. abscessus cluster strains. Phylogenetic analysis of 88 genomes from isolates of the 11 patients excluded occurrence of M. abscessus transmission among members of the study group. Genome sequencing and variant analysis of 30 isolates from 11 serial respiratory samples collected over 4.5 years from a chronically infected patient demonstrated accumulation of gene mutations. In total, 53 genes exhibiting non-synonymous variations were identified. Enrichment analysis emphasized genes involved in synthesis of glycopeptidolipids, genes from the embABC (arabinosyltransferase) operon, betA (glucose-methanol-choline oxidoreductase) and choD (cholesterol oxidase). Genetic diversity evolved in a variety of virulence- and resistance-associated genes. The strategy of M. abscessus populations in chronic lung infection is not clonal expansion of dominant variants, but to sustain simultaneously a wide range of genetic variants facilitating adaptation of the population to changing living conditions in the lung. Genomic diversification during chronic infection requires increased attention when new control strategies against M. abscessus infections are explored.

Clinical impact of levofloxacin inhalation solution in cystic fibrosis patients in a real-world setting.

BACKGROUND: Levofloxacin inhalation solution is the most recently approved inhaled antibiotic in Europe and Canada for adult cystic fibrosis patients. Its efficacy and safety have been assessed in randomized controlled trials. Our aim was to evaluate real life experience and outcomes in our treatment centre. METHODS: We evaluated the efficacy of inhaled levofloxacin solution in 86 patients with cystic fibrosis in terms of the following outcome parameters: changes in %-predicted forced expiratory volume in one second (FEV1), body-mass index (BMI), and exacerbation rate. We conducted an intraindividual analysis of patients who received levofloxacin inhalation solution twice daily 240 mg for at least 4 weeks. RESULTS: Change in FEV1% predicted for the treatment period was +2.27% (p=0.0027) after 4 weeks. There was no change in BMI for overall group, but exacerbation rate compared to one year before initiation of inhaled levofloxacin decreased significantly (p=0.0024) after 1 year of treatment (3.23 ± 1.39 versus 2.71 ± 1.58). CONCLUSIONS: In patients with cystic fibrosis, inhaled levofloxacin solution has the potential to improve FEV1 and to reduce the number of bronchopulmonary exacerbations.

Risk factors for cystic fibrosis arthropathy: Data from the German cystic fibrosis registry.

BACKGROUND: Epidemiology and potential risk factors for cystic fibrosis arthropathy (CFA) were studied in a relevant cystic fibrosis (CF) patient cohort. METHODS: Cohort study of patients included in the German CF registry in 2016-2017. Descriptive analysis, exploratory tests and multivariable logistic regression were used to assess prevalence of CFA and associated potential risk factors for adult patients with/without chronic Pseudomonas aeruginosa infection. RESULTS: 6069 CF patients aged from 0 to 78 years were analysed. CFA was observed in 4.9% of the patients. Prevalence was significantly higher in adult patients (8.4%) compared to patients <18 years (0.7%; p<0.0001). Logistic regression analyses in adult patients (n=3319) showed that CFA was significantly associated with increasing age (OR=1.04; 95% CI: 1.02-1.05; p<0.0001), female gender (OR=2.10; 95%CI:1.52-2.90; p<0.0001), number of hospitalizations (OR=1.24; 95%CI:1.12-1.36; p<0.0001), chronic P. aeruginosa infection (OR=1.83; 95%CI:1.28-2.61; p=0.0009), CF-related diabetes (OR=1.69; 95%CI:1.23-2.33; p=0.0013), pancreatic insufficiency (OR=2.39; 95%CI:1.28-4.46; p=0.0060) and sinusitis/polyps (OR=1.91; 95%CI:1.39-2.62; p<0.0001). In a subgroup analysis of adults without chronic P. aeruginosa infection (n=1550) CFA was also significantly associated with increasing age, female gender, increasing number of hospitalizations, pancreatic insufficiency as well as sinusitis/polyps; antimycotic treatment associated only in this subgroup while association with CF-related diabetes was not significant. CONCLUSION: CFA is a frequent and clinically relevant co-morbidity particularly in adult CF patients. CFA is significantly more common in patients with chronic P. aeruginosa colonization but associations with other indicators for a more severe disease course were identified regardless of P. aeruginosa colonization status.

Urban Life as Risk Factor for Aspergillosis.

Aspergillus fumigatus (Af) frequently colonizes the airways of patients with cystic fibrosis (CF) and can cause severe diseases, such as allergic bronchopulmonary aspergillosis, Af bronchitis or even Af pneumonia. However, risk factors, including environmental factors, for acquiring Af in the respiratory tract of patients with CF are rarely studied and described. The aim of this study was to investigate whether urban or rural life could affect colonization with Af in the respiratory tract of patients with CF. Due to privacy policy, registry data are usually not linked to patients´ home addresses. It is therefore very difficult to analyze the influence of the patient´s residential environment. This prospective questionnaire survey was carried out in 31 German CF centers in 2018. Only completed surveys, including a clearly assigned type of residential area were included. Statistical analysis was performed by chi-squared test and logistic regression models. A total of 1016 questionnaires were analyzed (Patients` age: 23 ± 13; 0-88 years; female gender: n=492; 48%). The majority of patients with CF live in large cities (n =314; 30.9%) or urban districts (n=461; 45.4%). Prevalence of 30.2% was found for Af, within the 12 months of investigation period. Af colonization was significantly associated with urban life (p=0.004). Urban live should be considered as possible new risk factor for colonization with Af in the respiratory tract of patients with CF. These new results may raise the awareness of the influence of environmental factors on patient outcomes and should be included in patient guidance and preventive measures.

Frequent Pet Contact as Risk Factor for Allergic Bronchopulmonary Aspergillosis in Cystic Fibrosis.

Aspergillus fumigatus (Af) frequently colonizes the respiratory tract of patients with cystic fibrosis (CF). Af is associated with loss of pulmonary function and allergic bronchopulmonary aspergillosis (ABPA), a hypersensitivity fungal lung disease. Environmental factors have impact on CF patients' lung function variation. The aim of this nationwide questionnaire survey was to investigate the amount of CF patients with frequent pet contact including pet species and to examine the potential impact of frequent pet contact on the occurrence of Af colonization and ABPA diagnosis in these patients. The survey was carried out in 31 German CF centers in 2018. A total of 1232 who completed the surveys were included, and statistical analysis was performed by chi-squared test. Within the study cohort 49.8% of subjects (n = 614; CF patients < 18years: 49.4%, n = 234; ≥ 18years: 50.1%, n = 380) reported frequent contact to pets, of which 60.7% reported frequent contact to dogs, 42.3% to cats and other animals. Of those with frequent pet contact, 71.8% (n = 441) had contact to one pet or more pets from the same family. Af colonization was not significantly associated with frequent pet contact. ABPA diagnosis was documented in 16.7% (n = 206) of all included CF patients and was significantly associated with frequent pet contact (18.9%, n = 116, p = 0.042), confirming previous single center examinations. Particularly, patients with frequent contact to dogs showed an increased ABPA prevalence of 21.3%. Frequent pet contact might be a risk factor for ABPA. CF patients who are sensitized to Af should be informed about the increased risk to develop an ABPA by frequent pet contact. Patients with recurrent onset of ABPA should be evaluated in terms of frequent pet contact.

Prospective Evaluation of Aspergillus fumigatus-Specific IgG in Patients With Cystic Fibrosis.

Background: In Cystic Fibrosis (CF), the airways are often colonized by opportunistic fungi. The most frequently detected mold is Aspergillus fumigatus (Af). Af diseases are associated with significant morbidity and mortality. The most common clinical picture caused by Af is allergic bronchopulmonary aspergillosis (ABPA), triggered by an immunological reaction against Af. Af bronchitis and invasive aspergillosis rarely occur in CF as a result of spore colonization and germination. Since pulmonary mycoses and exacerbations by other pathogens overlap in clinical, radiological, and immunological characteristics, diagnosis still remains a challenge. The search for reliable, widely available biomarkers for Af diseases is therefore still an important task today. Objectives: Af-specific IgG m3 is broadly available. Sensitivity and specificity data are contradictory and differ depending on the study population. In our prospective study on pulmonary Af diseases in CF, we determined specific IgG m3 in order to test its suitability as a biomarker for acute Af diseases and as a follow-up parameter. Methods: In this prospective single center study, 109 patients with CF were screened from 2016 to 2019 for Af-associated diseases. According to diagnostic criteria, they were divided into four groups (control, bronchitis, ABPA, pneumonia). The groups were compared with respect to the level of Af-specific IgG (ImmunoCAP Gm3). We performed a receiver operating characteristic (ROC) curve analysis to determine cut-off, sensitivity and specificity. Twenty-one patients could be enrolled for a follow-up examination. Results: Of the 109 patients, 36 were classified as acute Af-disease (Af bronchitis, ABPA, Af pneumonia). Of these, 21 patients completed follow up-screening. The median Af-specific Gm3 was higher in the acute Af-disease groups. There was a significant difference in Af-specific IgG m3 compared to the control group without acute Af-disease. Overall, there was a large interindividual distribution of Gm3. A cut-off value of 78.05 mg/L for Gm3 was calculated to discriminate controls and patients with ABPA/pneumonia with a specificity of 75% and a sensitivity of 74.6%. The follow up examination of 21 patients showed a decrease of Gm3 in most patients without statistical significance due to the small number of follow up patients. Conclusion: Af specific IgG may be a useful biomarker for acute ABPA and Af pneumonia, but not for Af bronchitis in CF. However, due to the large interindividual variability of Gm3, it should only be interpreted alongside other biomarkers. Therefore, due to its broad availability, it could be suitable as a biomarker for ABPA and Af pneumonia in CF, if the results can be supported by a larger multicenter cohort.

Human Anti-fungal Th17 Immunity and Pathology Rely on Cross-Reactivity against Candida albicans.

Th17 cells provide protection at barrier tissues but may also contribute to immune pathology. The relevance and induction mechanisms of pathologic Th17 responses in humans are poorly understood. Here, we identify the mucocutaneous pathobiont Candida albicans as the major direct inducer of human anti-fungal Th17 cells. Th17 cells directed against other fungi are induced by cross-reactivity to C. albicans. Intestinal inflammation expands total C. albicans and cross-reactive Th17 cells. Strikingly, Th17 cells cross-reactive to the airborne fungus Aspergillus fumigatus are selectively activated and expanded in patients with airway inflammation, especially during acute allergic bronchopulmonary aspergillosis. This indicates a direct link between protective intestinal Th17 responses against C. albicans and lung inflammation caused by airborne fungi. We identify heterologous immunity to a single, ubiquitous member of the microbiota as a central mechanism for systemic induction of human anti-fungal Th17 responses and as a potential risk factor for pulmonary inflammatory diseases.