Airway microbiome and host inflammatory response in bronchiectasis

Non-cystic fibrosis bronchiectasis (hereby referred to as bronchiectasis) is a chronic respiratory disease characterized by irreversible dilation of bronchi in the context of chronic syndrome mainly represented by daily cough, sputum production and frequent pulmonary exacerbations. Bronchiectasis pathophysiology is recognised in the development of a vicious circle of infection, inflammation, mucous clearance and pulmonary damage. Although microbial community and inflammation have a very important role in respiratory diseases, few is known in bronchiectasis. The aim of this PhD project was to evaluate sputum microbiome and its interaction with the local host inflammatory response in adults with bronchiectasis during their stable state. Several studies were conducted to explore this research question. In the second chapter, we report the methodological selection of techniques for both microbiome and active neutrophil elastase (aNE) analysis in sputum. The third chapter is divided into three different sections. In the first, we identified two groups of adult bronchiectasis patients with different microbiome diversity levels. The low microbiome diversity group was found to be enriched in Pseudomonas, Staphylococcus and Streptococcus. Multivariate analysis identified FEV1%predict.<50, radiology and primary ciliary dyskinesia (PCD) to be independently associated with low microbiome diversity. The following section represents a preliminary study focused on bronchiectasis aetiologies, more specifically PCD and immunodeficiency. The study concerning PCD did not identify a clear association between aetiology, microbiome and inflammation comparing a group of matched PCD with idiopathic or post infective bronchiectasis, although, a trend could be observed.Subsequently, immunodeficiency was comparedto idiopathic bronchiectasis. These two groupsidentified, primary immunodeficiency and idiopathic differed in terms of alpha diversity and quantification of H. influenzae which was higher in the immunodeficiency group. Chapter fourfocuses on neutrophilic inflammation looking at the association of aNE in bronchiectasis with microbiome analysis. We found evidences of low microbiome diversity and P. aeruginosa identification in patients with high levels of aNE. The following study focused on active Cathepsin G (Cat-G) which is a putative biomarker for bronchiectasis. Cat-G was associated with disease severity, radiological severity, quality of life and chronic infection. It was also associated with low microbiome diversity and P. aeruginosa molecular detection. Finally, Cat-G and aNE resulted to be similar in predicting bronchiectasis severity (bronchiectasis severity index -BSI), severe exacerbation and chronic infection in bronchiectasis patients during stable state. Finally, in chapter five, we analyse microbiome and inflammation among adult bronchiectasis patients with chronic P. aeruginosa infection. Association network analysis identified differences in terms of number of interactions of Pseudomonas with other genera and interactions between cytokines and microbial effectors in patients with different exacerbations/year. We hope that this thesis may be a step forward in better understanding the role of both microbiome and inflammation in bronchiectasis that may lead to unravelling of endotypes and the identification of therapeutic targets. This will finally result in advances in a precise medicine approach for bronchiectasis patients

Comparison of different sets of immunological tests to identify treatable immunodeficiencies in adult bronchiectasis patients

Immunological tests; BronchiectasisPruebas inmunologicas; BronquiectasiasProves immunològiques; BronquiectasiesBackground The reported prevalence of immunodeficiencies in bronchiectasis patients is variable depending on the frequency and extent of immunological tests performed. European Respiratory Society guidelines recommend a minimum bundle of tests. Broadening the spectrum of immunological tests could increase the number of patients diagnosed with an immunodeficiency and those who could receive specific therapy. The primary objective of the present study was to assess the performance of different sets of immunological tests in diagnosing any, primary, secondary or treatable immunodeficiencies in adults with bronchiectasis. Methods An observational, cross-sectional study was conducted at the Bronchiectasis Program of the Policlinico University Hospital in Milan, Italy, from September 2016 to June 2019. Adult outpatients with a clinical and radiological diagnosis of bronchiectasis underwent the same immunological screening during the first visit when clinically stable consisting of: complete blood count; immunoglobulin (Ig) subclass tests for IgA, IgG, IgM and IgG; total IgE; lymphocyte subsets; and HIV antibodies. The primary endpoint was the prevalence of patients with any immunodeficiencies using five different sets of immunological tests. Results A total of 401 bronchiectasis patients underwent the immunological screening. A significantly different prevalence of bronchiectasis patients diagnosed with any, primary or secondary immunodeficiencies was found across different bundles. 44.6% of bronchiectasis patients had a diagnosis of immunodeficiency when IgG subclasses and lymphocyte subsets were added to the minimum bundle suggested by the guidelines. Conclusion A four-fold increase in the diagnosis of immunodeficiencies can be found in adults with bronchiectasis when IgG subclasses and lymphocyte subsets are added to the bundle of tests recommended by guidelines

How to Process Sputum Samples and Extract Bacterial DNA for Microbiota Analysis

Different steps and conditions for DNA extraction for microbiota analysis in sputum have been reported in the literature. We aimed at testing both dithiothreitol (DTT) and enzymatic treatments of sputum samples and identifying the most suitable DNA extraction technique for the microbiota analysis of sputum. Sputum treatments with and without DTT were compared in terms of their median levels and the coefficient of variation between replicates of both DNA extraction yield and real-time PCR for the 16S rRNA gene. Treatments with and without lysozyme and lysostaphin were compared in terms of their median levels of real-time PCR for S. aureus. Two enzyme-based and three beads-based techniques for DNA extraction were compared in terms of their DNA extraction yield, real-time PCR for the 16S rRNA gene and microbiota analysis. DTT treatment decreased the coefficient of variation between replicates of both DNA extraction yield and real-time PCR. Lysostaphin (either 0.18 or 0.36 mg/mL) and lysozyme treatments increased S. aureus detection. One enzyme-based kit offered the highest DNA yield and 16S rRNA gene real-time PCR with no significant differences in terms of alpha-diversity indexes. A condition using both DTT and lysostaphin/lysozyme treatments along with an enzymatic kit seems to be preferred for the microbiota analysis of sputum samples

Asthma Control Test and Bronchial Challenge with Exercise in Pediatric Asthma

Background: Poor asthma control can lead to exercise-induced bronchoconstriction (EIB), but the relationship between subjective disease control and EIB is unclear. No studies have compared asthma control test (ACT) scores of children with those of their parents regarding EIB. We assessed whether ACT scores predict the occurrence of EIB in two age groups. We also evaluated ACT scores and objective measures as explanatory variables for airway response to exercise. Methods: Patients (71 aged <12 years, 93 aged 6512 years) and their parents completed an ACT questionnaire separately. Current therapy, skin prick testing and spirometry at baseline and after exercise were assessed. EIB was defined as a fall in FEV1 of at least 12% from baseline. Sensitivity and specificity for cut-off values of ACT scores predictive of EIB were plotted, and the Area Under Curve (AUC) was described. Results: Atopy and current therapy were similarly frequent. EIB was observed in 23.9% of children aged <12 years and in 33.3% aged 6512 years. EIB occurrence in subjects previously scored as having full control (25), partial control (20\u201024) and no control (<20) varied according to the age group and responder. Percentages of EIB cases increased as ACT scores decreased in children aged 6512 years alone (child ACT scores, 25: 21.9%, 20-24: 31.1%, <20: 62.5%, p=0.017). Plots for ACT scores as predictors of EIB yielded low non\u2010significant AUC values in children aged <12 years; by contrast, moderate AUC values emerged in children aged 6512 years (child: 0.67, p=0.007; parent: 0.69, p=0.002). Sensitivity of ACT scores below 20 as a predictor of EIB was low in older children (child: 32.3%, parent: 22.6%), whereas specificity was high (child: 90.3%, parent: 93.5%). Multiple regression analysis with percent fall in FEV1 as dependent variable included FEV1/FVC%, ACT child score and gender in the prediction model ( r=0.42, p=0.000). Conclusion: ACT scores are a more effective means of excluding than confirming EIB in asthmatic patients aged 6512 years; their predictive value decreases in younger patients. ACT scores together with lung function may help to predict airway response to exercise. New tools for pediatric asthma assessment may optimize this association

Treatment of Biofilm Communities: An Update on New Tools from the Nanosized World

Traditionally regarded as single cell organisms, bacteria naturally and preferentially build multicellular communities that enable them to react efficiently to external stimuli in a coordinated fashion and with extremely effective outcomes. These communities are bacterial biofilms, where single cells or microcolonies are embedded in self-built Extracellular Polymeric Substance (EPS), composed of different macromolecules, e.g., polysaccharides, proteins, lipids, and extracellular DNA (eDNA). Despite being the most common form in nature and having many biotechnologically useful applications, biofilm is often regarded as a life-threatening form of bacterial infection. Since this form of bacterial life is intrinsically more resistant to antibiotic treatment and antimicrobial resistance is reaching alarming levels, we will focus our attention on how nanotechnology made new tools available to the medical community for the prevention and treatment of these infections. After a brief excursus on biofilm formation and its main characteristics, different types of nanomaterials developed to prevent or counteract these multicellular forms of bacterial infection will be described. A comparison of different classifications adopted for nanodrugs and a final discussion of challenges and future perspectives are also presente

A point of care neutrophil elastase activity assay identifies bronchiectasis severity, airway infection and risk of exacerbation

Introduction: Neutrophil elastase activity in sputum can identify patients at high risk of airway infection and exacerbations in bronchiectasis. Application of this biomarker in clinical practice is limited, because no point-of-care test is available. We tested whether a novel semi-quantitative lateral flow device (neutrophil elastase airway test stick - NEATstik\uae) can stratify bronchiectasis patients according to severity, airway infection and exacerbation risk. Methods: Sputum samples from 124 patients with stable bronchiectasis enrolled in the UK and Spain were tested using the NEATstik\uae, which scores neutrophil elastase concentration from 0 (&lt;8 \u3bcg\ub7mL-1 elastase activity) to 10 (maximum detectable neutrophil elastase activity). High neutrophil elastase activity was regarded as a NEATstik\uae grade &gt;6. Severity of disease, airway infection from sputum culture and exacerbations over the 12 months were recorded. An independent validation was conducted in 50 patients from Milan, Italy. Measurements and main results: Patients had a median age of 69 years and forced expiratory volume in 1 s (FEV1) 69%. High neutrophil elastase activity was associated with worse bronchiectasis severity using the bronchiectasis severity index (p=0.0007) and FEV1 ( p=0.02). A high NEATstik\uae grade was associated with a significant increase in exacerbation frequency, incident rate ratio 2.75 (95% CI 1.63-4.64, p&lt;0.001). The median time to next exacerbation for patients with a NEATstik\uae grade &gt;6 was 103 days compared to 278 days. The hazard ratio was 2.59 (95% CI 1.71-3.94, p&lt;0.001). Results were confirmed in the independent validation cohort. Conclusions: A novel lateral flow device provides assessment of neutrophil elastase activity from sputum in minutes and identifies patients at increasing risk of airway infection and future exacerbations

Comparison of different sets of immunological tests to identify treatable immunodeficiencies in adult bronchiectasis patients

BACKGROUND: The reported prevalence of immunodeficiencies in bronchiectasis patients is variable depending on the frequency and extent of immunological tests performed. European Respiratory Society guidelines recommend a minimum bundle of tests. Broadening the spectrum of immunological tests could increase the number of patients diagnosed with an immunodeficiency and those who could receive specific therapy. The primary objective of the present study was to assess the performance of different sets of immunological tests in diagnosing any, primary, secondary or treatable immunodeficiencies in adults with bronchiectasis. METHODS: An observational, cross-sectional study was conducted at the Bronchiectasis Program of the Policlinico University Hospital in Milan, Italy, from September 2016 to June 2019. Adult outpatients with a clinical and radiological diagnosis of bronchiectasis underwent the same immunological screening during the first visit when clinically stable consisting of: complete blood count; immunoglobulin (Ig) subclass tests for IgA, IgG, IgM and IgG; total IgE; lymphocyte subsets; and HIV antibodies. The primary endpoint was the prevalence of patients with any immunodeficiencies using five different sets of immunological tests. RESULTS: A total of 401 bronchiectasis patients underwent the immunological screening. A significantly different prevalence of bronchiectasis patients diagnosed with any, primary or secondary immunodeficiencies was found across different bundles. 44.6% of bronchiectasis patients had a diagnosis of immunodeficiency when IgG subclasses and lymphocyte subsets were added to the minimum bundle suggested by the guidelines. CONCLUSION: A four-fold increase in the diagnosis of immunodeficiencies can be found in adults with bronchiectasis when IgG subclasses and lymphocyte subsets are added to the bundle of tests recommended by guidelines

Antimicrobial peptides, disease severity and exacerbations in bronchiectasis

Rationale: Recently a frequent exacerbator phenotype has been described in bronchiectasis, but the underlying biological mechanisms are unknown. Antimicrobial peptides (AMPs) are important in host defence against microbes but can be proinflammatory in chronic lung disease. Objectives: To determine pulmonary and systemic levels of AMP and their relationship with disease severity and future risk of exacerbations in bronchiectasis. Methods: A total of 135 adults with bronchiectasis were prospectively enrolled at three European centres. Levels of cathelicidin LL-37, lactoferrin, lysozyme and secretory leucocyte protease inhibitor (SLPI) in serum and sputum were determined at baseline by ELISA. Patients were followed up for 12 months. We examined the ability of sputum AMP to predict future exacerbation risk. Measurements and main results: AMP levels were higher in sputum than in serum, suggesting local AMP release. Patients with more severe disease at baseline had dysregulation of airway AMP. Higher LL-37 and lower SLPI levels were associated with Bronchiectasis Severity Index, lower FEV1 (forced expiratory volume in 1 s) and Pseudomonas aeruginosa infection. Low SLPI levels were also associated with the exacerbation frequency at baseline. During follow-up, higher LL-37 and lower SLPI levels were associated with a shorter time to the next exacerbation, whereas LL-37 alone predicted exacerbation frequency over the next 12 months. Conclusions: Patients with bronchiectasis showed dysregulated sputum AMP levels, characterised by elevated LL-37 and reduced SLPI levels in the frequent exacerbator phenotype. Elevated LL-37 and reduced SLPI levels are associated with Pseudomonas aeruginosa infection and can predict future risk of exacerbations in bronchiectasis

Brivaracetam as Early Add-On Treatment in Patients with Focal Seizures: A Retrospective, Multicenter, Real-World Study

Introduction In randomized controlled trials, add-on brivaracetam (BRV) reduced seizure frequency in patients with drug-resistant focal epilepsy. Most real-world research on BRV has focused on refractory epilepsy. The aim of this analysis was to assess the 12-month effectiveness and tolerability of adjunctive BRV when used as early or late adjunctive treatment in patients included in the BRIVAracetam add-on First Italian netwoRk Study (BRIVAFIRST). Methods BRIVAFIRST was a 12-month retrospective, multicenter study including adult patients prescribed adjunctive BRV. Effectiveness outcomes included the rates of sustained seizure response, sustained seizure freedom, and treatment discontinuation. Safety and tolerability outcomes included the rate of treatment discontinuation due to adverse events (AEs) and the incidence of AEs. Data were compared for patients treated with add-on BRV after 1-2 (early add-on) and &gt;= 3 (late add-on) prior antiseizure medications. Results A total of 1029 patients with focal epilepsy were included in the study, of whom 176 (17.1%) received BRV as early add-on treatment. The median daily dose of BRV at 12 months was 125 (100-200) mg in the early add-on group and 200 (100-200) in the late add-on group (p &lt; 0.001). Sustained seizure response was reached by 97/161 (60.3%) of patients in the early add-on group and 286/833 (34.3%) of patients in the late add-on group (p &lt; 0.001). Sustained seizure freedom was achieved by 51/161 (31.7%) of patients in the early add-on group and 91/833 (10.9%) of patients in the late add-on group (p &lt; 0.001). During the 1-year study period, 29 (16.5%) patients in the early add-on group and 241 (28.3%) in the late add-on group discontinued BRV (p = 0.001). Adverse events were reported by 38.7% and 28.5% (p = 0.017) of patients who received BRV as early and late add-on treatment, respectively. Conclusion Brivaracetam was effective and well tolerated both as first add-on and late adjunctive treatment in patients with focal epilepsy

The First Trimester Gravid Serum Regulates Procalcitonin Expression in Human Macrophages Skewing Their Phenotype In Vitro

Procalcitonin (PCT) is one of the best diagnostic and prognostic markers in clinical practice, widely used to evaluate the evolution of bacterial infections. Although it is mainly produced by thyroid, during sepsis almost all the peripheral tissues are involved in PCT production. Parenchymal cells have been suggested as the main source of PCT expression; however the contribution of macrophages is not clear yet. In response to environmental cues, tissue macrophages acquire distinct functional phenotypes, ranging from proinflammatory (M1) to anti-inflammatory (M2) phenotype. Macrophages at the fetal-maternal interface show immunosuppressive M2-like activities required for the maintenance of immunological homeostasis during pregnancy. This study aims to clarify the ability to synthesise PCT of fully differentiated (M0), polarized (M1/M2) macrophages and those cultured either in the presence of first trimester gravid serum (GS) or pregnancy hormones. We found out that M1 macrophages upregulate PCT expression following LPS stimulation compared to M0 and M2. The GS downregulates PCT expression in macrophages, skewing them towards an M2-like phenotype. This effect seems only partially mediated by the hormonal milieu. Our findings strengthen the key role of macrophages in counteracting inflammatory stimuli during pregnancy, suggesting PCT as a possible new marker of M1-like macrophages