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Abstract In mammals, the suprachiasmatic nucleus of the hypothalamus is the master circadian pacemaker that synchronizes the clocks in the central nervous system and periphery, thus orchestrating rhythms throughout the body. However, little is known about how so many cellular clocks within and across brain circuits can be effectively synchronized. In this work, we investigated the implication of two possible pathways: (i) astrocytes-mediated synchronization and (ii) neuronal paracrine factors-mediated synchronization. By taking advantage of a lab-on-a-chip microfluidic device developed in our laboratory, here we report that both pathways are involved. We found the paracrine factors-mediated synchronization of molecular clocks is diffusion-limited and, in our device, effective only in case of a short distance between neuronal populations. Interestingly, interconnecting astrocytes define an active signaling channel that can synchronize molecular clocks of neuronal populations also at longer distances. At mechanism level, we found that astrocytes-mediated synchronization involves both GABA and glutamate, while neuronal paracrine factors-mediated synchronization occurs through GABA signaling. These findings identify a previously unknown role of astrocytes as active cells that might distribute long-range signals to synchronize the brain clocks, thus further strengthening the importance of reciprocal interactions between glial and neuronal cells in the context of circadian circuitry.

Exhaled nitric oxide (NO) production, upregulated by inflammatory cytokines and mediators in central and peripheral airways, can be easily and non-invasively detected in exhaled air in asthma and other respiratory conditions as a promising tool for disease monitoring. The American Thoracic Society and European Respiratory Society released recommendations that standardize the measurement of the fractional exhaled NO (FeNO). In asthma, increased FeNO reflects eosinophilic-mediated inflammatory pathways and, as a biomarker of T2 inflammation can be used to identify asthma T2 phenotype. In this setting its measurement has shown to be an important tool especially in the diagnostic process, in the assessment and evaluation of poor adherence or predicting positive response to inhaled corticosteroids treatment, in phenotyping severe asthma patients and as a biomarker to predict the response to biologic treatments. The discovery of the role of NO in the pathogenesis of different diseases affecting the airways and the possibility to estimate the predominant site of increased NO production has provided new insight on its regulatory role in the airways, making it suitable for a potential extended use in clinical practice for different pulmonary diseases, even though its role remains less clear than in asthma. Monitoring FeNO in pulmonary obstructive lung diseases including chronic bronchitis and emphysema, interstitial lung diseases, obstructive sleep apnea and other pulmonary diseases is still under debate but has opened up a window to the role NO may play in the management of these diseases. The use of FeNO is reliable, cost effective and recommendable in both adults and children, and should be implemented in the management of patients with asthma and other respiratory conditions.

Background: Inflammation plays a pivotal role in the pathophysiology of asthma. Free light chains (FLC) can cause inflammation by mast cell antigen-activation. Serum immunoglobulin (Ig) FLC κ, but not λ, were shown elevated in adult males with asthma. We sought to investigate if serum Ig FLC concentrations are affected by asthma severity and their relationships with inflammatory outcomes.Methods: By using immunoassays, we measured serum κ and λ Ig FLCs in 24 severe persistent asthma patients, 15 patients with moderate persistent asthma, 15 steroid-naïve mild persistent asthma patients and 20 healthy control subjects in a cross-sectional observational study. Total and specific serum IgE concentrations, fractional exhaled nitric oxide (FENO), lung function, peripheral blood eosinophils and neutrophils, and C reactive protein (CRP) were also measured.Results: Serum κ FLC concentrations were elevated in severe asthma patients compared mild asthma patients (p < 0.05) and healthy subjects (p < 0.05). Serum λ FLCs were higher in severe asthma patients than in healthy subjects (p < 0.05) and correlated with blood eosinophil counts (percentage, κ: r = 0.51, p = 2.9678−6; λ: r = 0.42, p = 1.7377−4; absolute values, κ: r = 0.45, p = 6.1284−5; λ: r = 0.38, p = 7.8261−4), but not with total or specific serum IgE. In severe asthma patients, serum Ig FLC correlated with serum CRP (κ: r = 0.33; p = 0.003; λ: r = 0.38, p = 8.8305−4) and blood neutrophil cell counts (percentage, κ: r = 0.31; p = 0.008; λ: r = 0.29, p = 0.01; absolute values, κ: r = 0.40; p = 3.9176−4; λ: r = 0.40, p = 4.5479−4), were elevated in subjects with blood eosinophilia (≥300 cells/µL) (n = 13) compared with non-eosinophilic subjects (n = 10) (κ: 19.2 ± 1.2 mg/L versus 12.1 ± 1.3 mg/L, p < 0.001; λ: 27.2 ± 2.6 mg/L versus 16.8 ± 2.5 mg/L, p < 0.01), but were similar in atopic (n = 15) versus nonatopic subjects (n = 9) (κ: p = 0.20; λ: p = 0.80). Serum FLC were negatively correlated with lung function tests, including forced expiratory volume in one second (FEV1) (κ: r = −0.33; p = 0.0034; λ: r = −0.33; p = 0.0035), and FEV1/forced vital capacity ratio (κ: r = −0.33; p = 0.0034; λ: r = −0.33; p = 0.0036).Conclusion: Serum Ig FLCs are elevated in severe asthma adults and might represent new surrogate markers of inflammation. The pathophysiological implications of these findings require further research. This study was approved by the ethics committee of the University Hospital Agostino Gemelli Foundation and Catholic University of the Sacred Heart (approval number P/1034/CE2012).

BackgroundPhosphorylation of diacylglycerol by diacylglycerol-kinases represents a major inhibitory event constraining T cell activation upon antigen engagement. Efficient TCR signalling requires the inhibition of the alpha isoform of diacylglycerol kinase, DGKα, by an unidentified signalling pathway triggered by the protein adaptor SAP. We previously demonstrated that, in SAP absence, excessive DGKα activity makes the T cells resistant to restimulation-induced cell death (RICD), an apoptotic program counteracting excessive T cell clonal expansion.ResultsHerein, we report that the Wiskott-Aldrich syndrome protein (WASp) inhibits DGKα through a specific interaction of the DGKα recoverin homology domain with the WH1 domain of WASp. Indeed, WASp is necessary and sufficient for DGKα inhibition, and this WASp function is independent of ARP2/3 activity. The adaptor protein NCK-1 and the small G protein CDC42 connect WASp-mediated DGKα inhibition to SAP and the TCR signalosome. In primary human T cells, this new signalling pathway is necessary for a full response in terms of IL-2 production, while minimally affecting TCR signalling and restimulation-induced cell death. Conversely, in T cells made resistant to RICD by SAP silencing, the enhanced DAG signalling due to DGKα inhibition is sufficient to restore apoptosis sensitivity.ConclusionWe discover a novel signalling pathway where, upon strong TCR activation, the complex between WASp and DGKα blocks DGKα activity, allowing a full cytokine response.

Bronchial asthma is a chronic inflammatory condition with increasing prevalence worldwide that may present as heterogeneous phenotypes defined by the T2-mediated pattern of airway inflammation T2-high and T2-low asthma. Severe refractory asthma includes a subset of asthmatic patients who fail to control their disease despite maximal therapy and represent a group of patients needing marked resource utilization and hence may be eligible to add-on biological therapies. Among the new biologics, we focused our attention on two monoclonal antibodies: dupilumab, exerting a dual blockade of cytokine (interleukin (IL)-4 and IL-13) signaling; and tezepelumab, acting at a higher level preventing the binding of thymic stromal lymphopoietin (TSLP) to its receptor, thus blocking TSLP, IL-25, and IL-33 signaling, hence modulating airway T2 immune responses. With their different mechanisms of action, these two biologics represent important options to provide an enhanced personalized treatment regimen. Several clinical trials have been conducted testing the efficacy and safety of dupilumab in severe refractory asthmatic patients showing improvements in lung function, asthma control, and reducing exacerbations. Similar results were reported with tezepelumab that, differently from dupilumab, acts irrespectively on eosinophilic or non-eosinophilic phenotype. In this review, we provide an overview of the most important highlights regarding dupilumab and tezepelumab characteristics and mechanism of action with a critical review of the principal results of clinical (Phase II and III) studies concluded and those still in progress.

Abstract Background Work‐related lower airway symptoms (WR‐LAS), rhinitis (WRR), and asthma (WRA) are very common among bakers, due to airborne exposure to wheat flour and multigrain. Limited data is available regarding fractional exhaled nitric oxide (FeNO) in bakers in relation to respiratory burden and occupational sensitization in a real‐life situation. Objective To analyze FeNO levels in relation to WRR, WR‐LAS, and WRA with regard to allergic sensitization to occupational allergen in bakers. Methods Cross‐sectional, observational study of 174 bakers employed in traditional small bakeries in the Verona District. Subjects did FeNO measurements, spirometry, methacholine challenge, and skin prick test to common inhalant aeroallergens and bakeries occupational allergens. Results FeNO levels were higher in subjects sensitized to occupational allergens compared with bakers not sensitized to occupational allergens (22.8 ppb (18.9, 27.6) vs. 12.0 ppb (9.9, 14.5), p

Sleep-related breathing disorders (SBDs) are characterized by abnormal respiration during sleep. Obstructive sleep apnea (OSA), a common SBD increasingly recognized by physicians, is characterized by recurrent episodes of partial or complete closure of the upper airway resulting in disturbed breathing during sleep. OSA syndrome (OSAS) is associated with decreased patients' quality of life (QoL) and the presence of significant comorbidities, such as daytime sleepiness. Similarly to what seen for OSAS, the prevalence of asthma has been steadily rising in recent years. Interestingly, severe asthma (SA) patients are also affected by poor sleep quality—often attributed to nocturnal worsening of their asthma—and increased daytime sleepiness and snoring compared to the general population. The fact that such symptoms are also found in OSAS, and that these two conditions share common risk factors, such as obesity, rhinitis, and gastroesophageal reflux, has led many to postulate an association between these two conditions. Specifically, it has been proposed a bidirectional correlation between SA and OSAS, with a mutual negative effect in term of disease severity. According to this model, OSAS not only acts as an independent risk factor of asthma exacerbations, but its co-existence can also worsen asthma symptoms, and the same is true for asthma with respect to OSAS. In this comprehensive review, we summarize past and present studies on the interrelationship between OSAS and SA, from endo-phenotype to clinical aspects, highlighting possible implications for clinical practice and future research directions.

Cough variant asthma (CVA), a common asthma phenotype characterized by nonproductive cough and bronchial hyperreactivity (BHR), is usually detected by bronchial provocation tests (BPTs) which are time-consuming, expensive, and unsafe. The primary study objective was to provide proof of concept for the use of fractional exhaled nitric oxide (FENO), eosinophil count percentage in induced sputum (sEOS%), forced expiratory flow between 25 and 75% of forced vital capacity (FEF25–75%) % predicted value, and FEF25–75% z-scores as surrogate markers predicting BHR in young adults with suspected CVA; the secondary objective was to compare the diagnostic performance of the various techniques. Three hundred and ten subjects (median age 24 years) were included in a cross-sectional study. Subjects were characterized as BHR positive (POS) (n = 147) or BHR negative (NEG) (n = 163) according to methacholine BPT. Classification accuracies were expressed as areas under the receiver operator characteristic curves (AUC). Compared with BHR NEG, FEF25–75% % predicted value and FEF25–75% z-scores were lower in the BHR POS group (p < 0.001), whereas FENO (p < 0.001) and sEOS% were higher (p < 0.001). AUC values for detecting BHR were as follows: FENO, 0.98 (SD = 0.02); sEOS%, 0.98 (SD = 0.02); FEF25–75% % pred, 0.93 (SD = 0.05); FEF25–75% z scores, 0.92 (SD = 0.05). Optimal cutoff values (OCV) for BHR prediction were as follows: FENO, 32.7 ppb (sensitivity = 0.93, specificity = 0.96), sEOS%, 3.80% (sensitivity = 0.94, specificity = 0.94), FEF25–75% % predicted value, 80.0% (sensitivity = 0.90, specificity = 0.87), and FEF25–75% z-score, −0.87 (sensitivity = 0.89, specificity = 0.87). Non-invasive/semi-invasive airway inflammatory or small airway functional measures might be used as surrogate markers predicting BHR in young adults with suspected CVA.

Aim We aimed to characterise a large population of coronavirus disease 2019 (COVID-19) patients with moderate-to-severe hypoxaemic acute respiratory failure (ARF) receiving continuous positive airway pressure (CPAP) outside the intensive care unit (ICU), and to ascertain whether the duration of CPAP application increased the risk of mortality for patients requiring intubation. Methods In this retrospective, multicentre cohort study, we included adult COVID-19 patients, treated with CPAP outside ICU for hypoxaemic ARF from 1 March to 15 April, 2020. We collected demographic and clinical data, including CPAP therapeutic goal, hospital length of stay and 60-day in-hospital mortality. Results The study included 537 patients with a median (interquartile range (IQR) age of 69 (60–76) years. 391 (73%) were male. According to the pre-defined CPAP therapeutic goal, 397 (74%) patients were included in the full treatment subgroup, and 140 (26%) in the do not intubate (DNI) subgroup. Median (IQR) CPAP duration was 4 (1–8) days, while hospital length of stay was 16 (9–27) days. 60-day in-hospital mortality was 34% (95% CI 0.304–0.384%) overall, and 21% (95% CI 0.169–0.249%) and 73% (95% CI 0.648–0.787%) for full treatment and DNI subgroups, respectively. In the full treatment subgroup, in-hospital mortality was 42% (95% CI 0.345–0.488%) for 180 (45%) CPAP failures requiring intubation, and 2% (95% CI 0.008–0.035%) for the remaining 217 (55%) patients who succeeded. Delaying intubation was associated with increased mortality (hazard ratio 1.093, 95% CI 1.010–1.184). Conclusions We described a large population of COVID-19 patients treated with CPAP outside ICU. Intubation delay represents a risk factor for mortality. Further investigation is needed for early identification of CPAP failures.

Background and Aims: The long-term health effects of the use of electronic cigarettes (ECs) in patients with chronic obstructive pulmonary disease (COPD) are largely unexplored. We present findings from a 5-year prospective assessment of respiratory parameters in a cohort of COPD patients who substantially reduced conventional smoking or achieved abstinence by switching to ECs. Methods: Patients were evaluated prospectively for their measurements of respiratory exacerbations, spirometric indices, quality of life using the COPD assessment tool (CAT), 6-min walk distance (6MWD), as well as conventional cigarette consumption. Baseline measurements prior to switching to EC use were compared with follow-up visits at 12-, 24-, 48- and 60-months. Age- and sex-matched COPD patients reporting to be regular smokers (not using ECs) were the reference group for the analysis. Results: Complete data were available from 39 patients. Those in the EC user group achieved a marked decline in cigarette smoking or abstinence. COPD EC users had a significant diminution in COPD exacerbations; with the mean (±SD) exacerbation rate falling from 2.3 (±0.9) at baseline to 1.1 (±1.0) at 5 years ( p

Abstract Idiopathic pulmonary fibrosis (IPF) is a fibrotic lung disease characterized by progressive loss of lung function and poor prognosis. The so-called acute exacerbation of IPF (AE-IPF) may lead to severe hypoxemia requiring mechanical ventilation in the intensive care unit (ICU). AE-IPF shares several pathophysiological features with acute respiratory distress syndrome (ARDS), a very severe condition commonly treated in this setting. A review of the literature has been conducted to underline similarities and differences in the management of patients with AE-IPF and ARDS. During AE-IPF, diffuse alveolar damage and massive loss of aeration occurs, similar to what is observed in patients with ARDS. Differently from ARDS, no studies have yet concluded on the optimal ventilatory strategy and management in AE-IPF patients admitted to the ICU. Notwithstanding, a protective ventilation strategy with low tidal volume and low driving pressure could be recommended similarly to ARDS. The beneficial effect of high levels of positive end-expiratory pressure and prone positioning has still to be elucidated in AE-IPF patients, as well as the precise role of other types of respiratory assistance (e.g., extracorporeal membrane oxygenation) or innovative therapies (e.g., polymyxin-B direct hemoperfusion). The use of systemic drugs such as steroids or immunosuppressive agents in AE-IPF is controversial and potentially associated with an increased risk of serious adverse reactions. Common pathophysiological abnormalities and similar clinical needs suggest translating to AE-IPF the lessons learned from the management of ARDS patients. Studies focused on specific therapeutic strategies during AE-IPF are warranted.

Chronic obstructive pulmonary disease (COPD) is a common disabling disease characterized by progressive airflow obstruction. Great efforts were spent in the development of drugs able to improve symptoms, quality of life, reduce exacerbations, hospitalizations and the frequency of death of patients with COPD. The cornerstones of treatment are bronchodilator drugs of two different classes: beta agonists and muscarinic antagonists. Currently the Global initiative for COPD suggests the use of long acting beta agonists (LABAs) and long acting muscarinic antagonists (LAMAs) in combination for the majority of COPD patients, thus great interest is associated with the developing of LAMA/LABA fixed combination in the maintenance treatment of stable COPD. Many LAMA/LABA fixed dose combinations have been licensed in different countries and the clinical use of these drugs stimulated the performance of many clinical trials. The purpose of this review is a complete criticism of pharmacological and clinical aspects related to the use of LAMA/LABA single inhalers for the maintenance treatment of stable COPD, with particular mention to the most debated topics and future prospects in the field.

PKCζ and PKCι/λ form the atypical protein kinase C subgroup, characterised by a lack of regulation by calcium and the neutral lipid diacylglycerol. To better understand the regulation of these kinases, we systematically explored their interactions with various purified phospholipids using the lipid overlay assays, followed by kinase activity assays to evaluate the lipid effects on their enzymatic activity. We observed that both PKCζ and PKCι interact with phosphatidic acid and phosphatidylserine. Conversely, PKCι is unique in binding also to phosphatidylinositol-monophosphates (e.g., phosphatidylinositol 3-phosphate, 4-phosphate, and 5-phosphate). Moreover, we observed that phosphatidylinositol 4-phosphate specifically activates PKCι, while both isoforms are responsive to phosphatidic acid and phosphatidylserine. Overall, our results suggest that atypical Protein kinase C (PKC) localisation and activity are regulated by membrane lipids distinct from those involved in conventional PKCs and unveil a specific regulation of PKCι by phosphatidylinositol-monophosphates.

Maintenance pharmacological treatment for stable chronic obstructive pulmonary disease (COPD) is based on inhaled drugs, including long-acting muscarinic receptor antagonists (LAMA), long-acting β 2 -adrenoceptor agonists (LABA) and inhaled corticosteroids (ICS). Inhaled pharmacological treatment can improve patients’ daily symptoms and reduce decline of pulmonary function and acute exacerbation rate. Treatment with all three inhaled drug classes is reserved for selected, more severe, patients with COPD when symptoms are not sufficiently controlled by dual LABA/LAMA therapy and exacerbations are frequent. This review focuses on the role of single-inhaler triple therapy with once-daily fluticasone furoate/umeclidinium/vilanterol fixed-dose combination, which is in phase III clinical development for maintenance treatment of severe-to-very severe COPD. In this review, we summarize evidence providing the rationale for its use in COPD and discuss the gaps to be filled in this pharmacotherapeutic area.

The chemical environment of cells is an extremely complex and multifaceted system that includes many types of proteins, lipids, nucleic acids and various other components. With the final aim of studying these components in detail, we have developed multiband plasmonic antennas, which are suitable for highly sensitive surface enhanced Raman spectroscopy (SERS) and are activated by a wide range of excitation wavelengths. The three-dimensional hollow nanoantennas were produced on an optical resist by a secondary electron lithography approach, generated by fast ion-beam milling on the polymer and then covered with silver in order to obtain plasmonic functionalities. The optical properties of these structures have been studied through finite element analysis simulations that demonstrated the presence of broadband absorption and multiband enhancement due to the unusual geometry of the antennas. The enhancement was confirmed by SERS measurements, which showed a large enhancement of the vibrational features both in the case of resonant excitation and out-of-resonance excitation. Such characteristics indicate that these structures are potential candidates for plasmonic enhancers in multifunctional opto-electronic biosensors.