Imbalance of Lymphocyte Subsets and CD45RA-Expressing Cells in Intrathoracic Lymph Nodes, Alveolar Compartment and Bloodstream of Pulmonary Sarcoidosis Patients.

Sarcoidosis is a systemic granulomatous disease mainly affecting the lungs and hilomediastinal lymph nodes. It is characterized by non-caseating epithelioid cell granulomas in lymph nodes and lungs. Our study aimed to evaluate and compare T, B and NK cell subsets in the alveolar compartment, lymph nodes and the bloodstream simultaneously in the same patients to elucidate the immune responses associated with the development and progression of sarcoidosis. A secondary aim was to evaluate the distribution of CD45RA-expressing cells in the different anatomical compartments. Patients suspected to have sarcoidosis and who underwent bronchoscopy with bronchoalveolar lavage (BAL), lung-draining lymph node (LLN) biopsy by EBUS-TBNA and peripheral blood (PB) sampling were included in the study. They were monitored at the Regional Referral Centre of Siena University Hospital and the Respiratory Diseases Unit of Perugia Hospital. Multicolour flow cytometry analysis through FASCLyric was performed to assess T, B and NK cell subsets. Thirty-two patients (median age (IQR) 57 (52-58) years) were consecutively and prospectively enrolled. Machine learning analysis created a model which selected CD56dim16bright, CD8, Tfc, Th17, Th12, Tfh17, Tfh2, TcemRA, ThemRA, T naïve, Tc naïve, Breg, CD1d+CD5+, Th-reg, Tfh, Th1 and CD4 cells with an accuracy of 0.9500 (kappa 0.8750). Comparative analysis found 18 cell populations that differed significantly between the three anatomical compartments. The bloodstream was enriched in ThemRA (p = 0.0416), Tfh2 (p = 0.0189), Tfh17 (p = 0.0257), Th2 (p = 0.0212), Th17 (p = 0.0177), Th-naïve (p = 0.0368), CD56dimCD16bright (p < 0.0001), CD8 (p = 0.0319), TcemRA (p < 0.0001) and Tfc cells (p = 0.0004) compared with the alveolar compartment, while Th-reg were lower in PB than BAL (p = 0.0329). The alveolar compartment was enriched in Breg (p = 0.0249) and CD1d+CD5+ (p = 0.0013) with respect to LLN samples and PB. Conversely, Tfh (p = 0.0470), Th1 (p = 0.0322), CD4 (p = 0.0486) and Tc-naïve (p = 0.0009) were more abundant in LLN than in BAL and PB. It has been speculated that changes in the relative contents of PB cells could be related to changes in production and to the selective redistribution of PB cells to granulomatous foci. This study further supports the fact that sarcoidosis is multisystemic in nature. However, the low level of immune cells in peripheral blood of patients with sarcoidosis is concerning. A re-expression of CD45RA on CD4+ and CD8+ cells could result in a reduction in peripheral immune activity. Thus, changes in the spectrum of the bloodstream may reflect both pathogenic and compensatory processes.

PD1, CTLA4 and TIGIT Expression on T and NK Cells in Granulomatous Diseases: Sarcoidosis and ANCA-Associated Vasculitis.

Sarcoidosis is a granulomatous diseases affecting the lungs. Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a histologically granulomatous B-mediated disorder characterized by activated T cells. The expression of immune checkpoint (IC) molecules (PD1, CTLA4, TIGIT) on T- and NK-cells negatively regulate the T-cell immune function. The present study aimed to explore the peripheral distribution of IC molecules to better elucidate their peripheral tolerance failure, which might reflect the development of diseases. Patients referred to Respiratory Diseases and Rheumatology Unit of Siena University Hospital were prospectively and consecutively enrolled. Healthy subjects were also enrolled as a control group. Multicolor flow cytometric analysis was performed to detect IC molecules in the peripheral blood of patients. Twenty-three patients were consecutively and prospectively enrolled in the study: 11 patients had an AAV diagnosis and 12 had sarcoidosis. CD4+PD1+ cells were higher in sarcoidosis and GPA than in HC (p = 0.0250 and p = 0.0253, respectively). CD56+CTLA4+ were higher in sarcoidosis than GPA, MPA and HC (p = 0.0085, p = 0.0042 and p = 0.0004, respectively). CTLA4+NK cells clustered for 100% of sarcoidosis patients according to decision tree analysis, while PD1+CD4 and CD8 cells for clustered for 100% of GPA patients. Our analyses showed substantial differences between sarcoidosis and AAV, further confirming the immunological peculiarity of this disease. Despite these advances, the pathogenesis remains incompletely understood, indicating an urgent need for further research to reveal the distinct immunological events in this process, with the hope to open up new therapeutic avenues and, if possible, to develop preventive measures.

Mepolizumab Effectiveness and Allergic Status in Real Life.

BACKGROUND: It is not clear whether mepolizumab is differently effective in allergic and nonallergic severe eosinophilic asthmatics (SEA) in real life. OBJECTIVE: We tested mepolizumab effectiveness in allergic/nonallergic SEA in real life. A strict criterion to identify the 2 phenotypes was used. METHOD: We retrospectively considered 134 consecutive patients divided into allergic, with a positivity to at least 1 allergen to prick tests and/or IgE values ≥100 UI/mL (severe allergic eosinophilic asthma [SAEA]; n: 97-72.4%), and nonallergic, with no prick test results and normal IgE levels <100 UI/mL (severe nonallergic eosinophilic asthma [SNAEA]; n: 37-27.6%). They had taken mepolizumab for at least 6 months. RESULTS: After 10.9 ± 3.7 months, improvements in FEV1%, FEF25-75%, exacerbation numbers, blood eosinophil (BE) counts, fractional exhaled nitric oxide (FENO) (ppb), percentages of patients that stopped/reduced short-acting ß2-agonists (SABAs) or oral corticosteroid (OC), observed after treatment, were similar in both groups. Only Asthma Control Test (ACT) increases were higher in SNAEA (8 [5-9]) than in SAEA (5 [2.5-8.5]; p = 0.016). However, no differences were found after treatment in percentages of subjects with ACT ≥20, as well as with FEV1 >80%, FEF25-75 >65%, exacerbations ≤2, BE <300 cells/µL, and FENO <25 ppb between SAEA and SNAEA. Besides, no significant relationships were found, comparing SNAEA with SAEA, for FEV1% (ß = -0.110; p = 0.266), FEF25-75% (ß = -0.228; p = 0.06), BE counts (ß = -0.012; p = 0.918), FENO (ß = 0.234; p = 0.085), ACT (ß = 0.046; p = 0.660), and exacerbations (ß = -0.070; p = 0.437). No different associations between lung function and SNAEA occurrence when compared to SAEA condition (FEV1 >80%: OR = 1.04 [95% CI: 0.43-2.55], p = 0.923; FEF25-75 >65%: OR = 0.41 [95% CI: 0.08-2.03], p = 0.272) were detected. Neither all other parameters, such as ACT >20 (OR = 0.73 [95% CI: 0.32-1.63], p = 0.440), presence of exacerbations (OR = 1.35 [95% CI: 0.55-3.27], p = 0.512), SABA discontinuation (OR = 1.16 [95% CI: 0.40-3.39], p = 0.790), and OC cessation/reduction (OR = 3.44 [95% CI: 0.40-29.27], p = 0.258), were differently associated with 1 or the other phenotype. CONCLUSION: Mepolizumab can be considered as a valid therapeutic choice for either allergic or nonallergic SEA in real life.

Mepolizumab effectiveness on small airway obstruction, corticosteroid sparing and maintenance therapy step-down in real life.

BACKGROUND: Mepolizumab (MEP) has been recently introduced to treat severe eosinophilic asthma. Trials have demonstrated a significant effectiveness in this asthma phenotype. We evaluated MEP efficacy on lung function, symptoms, asthma exacerbations, biologic markers, steroid dependence and controller treatment level in real-life. METHODS: We retrospectively analyzed 134 severe asthmatics (61 males; mean age 58.3 ± 11; mean FEV1%:72 ± 21), treated with MEP for at least 6 months (mean duration:10.9 ± 3.7 months). RESULTS: FEV1% improved significantly after MEP. Mean FEF25-75 also increased from 37.4 ± 25.4% to 47.2 ± 27.2% (p < 0.0001). Mean baseline blood eosinophil level was 712 ± 731/µL (8.4 ± 5.2%) decreasing to 151 ± 384/µL (1.6 ± 1.6%) (p < 0.0001), FENO levels decreased likewise. MEP treatment also led to a significant ACT improvement (mean pre:14.2 ± 4.4; mean post:20.5 ± 28) and exacerbations significantly fell from 3.8 ± 1.9 to 0.8 ± 1.1 (p < 0.0001). 74% of patients were steroid-dependent before MEP. 45.4% and 46.4% of them showed a suspension and dose reduction respectively (p < 0.0001). A significant number reduced also ICS doses. Only 67% of subjects used SABA as needed before MEP, falling to 20% after MEP. About 40% of patients highlighted a maintenance therapy step-down. Subjects showing an omalizumab treatment failure before MEP had a similar positive response when compared with omalizumab untreated patients. CONCLUSION: In real-life, MEP improved significantly all outcomes even small airway obstruction, suggesting its possible role also in distal lung region treatment. Furthermore, it demonstrated its high effectiveness in OC/ICS-sparing, in reducing SABA as needed and in stepping-down maintenance therapy. MEP is a valid alternative for patients with previous omalizumab treatment failure.

Monitoring of indoor bioaerosol for the detection of SARS-CoV-2 in different hospital settings.

Background: Spore Trap is an environmental detection technology, already used in the field of allergology to monitor the presence and composition of potentially inspirable airborne micronic bioparticulate. This device is potentially suitable for environmental monitoring of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in hospital, as well as in other high-risk closed environments. The aim of the present study is to investigate the accuracy of the Spore Trap system in detecting SARS-CoV-2 in indoor bioaerosol of hospital rooms. Methods: The Spore Trap was placed in hospital rooms hosting patients with documented SARS-CoV-2 infection (n = 36) or, as a negative control, in rooms where patients with documented negativity to a Real-Time Polymerase Chain Reaction molecular test for SARS-CoV-2 were admitted (n = 10). The monitoring of the bioaerosol was carried on for 24 h. Collected samples were analyzed by real-time polymerase chain reaction. Results: The estimated sensitivity of the Spore Trap device for detecting SARS-CoV-2 in an indoor environment is 69.4% (95% C.I. 54.3-84.4%), with a specificity of 100%. Conclusion: The Spore Trap technology is effective in detecting airborne SARS-CoV-2 virus with excellent specificity and high sensitivity, when compared to previous reports. The SARS-CoV-2 pandemic scenario has suggested that indoor air quality control will be a priority in future public health management and will certainly need to include an environmental bio-investigation protocol.

CD103 Expression on Regulatory and Follicular T Cells in Lymph Nodes, Bronchoalveolar Lavage Fluid and Peripheral Blood of Sarcoidosis Patients.

(1) Background: Sarcoidosis is a chronic multisystem disorder of unknown aetiology, driven by a T-cell mechanism allowing T-cell attachment and transmigration through the endothelium, and endorsed by the expression of an integrin alpha-E beta-7 (CD103). This study aimed to analyse the different distribution and compartmentalisation of CD103 expression on T cell subsets in BAL, peripheral blood mononuclear cells (PBMC) and lymph nodes (LLN) from sarcoidosis patients. (2) Patients: We consecutively and prospectively enrolled 14 sarcoidosis patients. We collected PBMC, LLN and BAL at the same time from all patients. Through flow cytometric analysis, we analysed the expression of CD103 on regulatory and follicular T cell subsets. (3) Results: All patients were in radiological Scadding stage II. The multivariate analysis found that the variables which most influenced the peripheral blood compartment were high CD8+ and low ThReg, CD8+CD103+ and Tfh cell percentages. A principal component analysis plot performed to distinguish LLN, BAL and PBMC showed that they separated on the basis of CD4+, CD4+CD103+, CD8+, CD8+CD103+, TcEffector, TcNaive, ThNaive, ThEffector, Threg, ThregCD103+, Tfh, TcfCXC5+ and CD4+CD103+/CD4+ with 65.96% of the total variance. (4) Conclusions: Our study is the first to report a link between the imbalance in circulating, alveolar and lymph node CD8+ and CD8+CD103+ T cells, ThReg, Tfh and ThNaive and the CD103+CD4+/CD4+ T cell ratio in the development of sarcoidosis. These findings shine a spotlight on the pathogenesis of sarcoidosis and may offer new predictors for diagnosis. Our study provides additional understanding for a personalised, and hopefully more effective treatment of sarcoidosis.

Immune-Checkpoint Expression on CD4, CD8 and NK Cells in Blood, Bronchoalveolar Lavage and Lymph Nodes of Sarcoidosis.

BACKGROUND: Sarcoidosis features non-necrotizing granulomas consisting mainly of activated CD4-lymphocytes. T-cell activation is regulated by immune checkpoint (IC) molecules. The present study aimed to compare IC expression on CD4, CD8 and NK cells from peripheral, alveolar and lung-draining lymph node (LLN) samples of sarcoidosis patients. METHODS: Flow-cytometry analysis was performed to detect IC molecules and a regression decision tree model was constructed to investigate potential binary classifiers for sarcoidosis diagnosis as well as for the IC distribution. RESULTS: Fourteen patients (7 females) were consecutively recruited in the study; all enrolled patients showed hilo-mediastinal lymph node enlargement and lung parenchyma involvement with chest X-rays and high resolution computed tomography. CD4+PD1+ and CD8+PD1+ were higher in bronchoalveolar lavage (BAL) than in LLN (p = 0.0159 and p = 0.0439, respectively). CD4+ T-cell immunoglobulin and ITIM domain (TIGIT)+ were higher in BAL than in peripheral blood mononuclear cells (PBMCs) (p = 0.0239), while CD8+TIGIT+ were higher in PBMC than in BAL (p = 0.0386). CD56+TIGIT+ were higher in LLN than in PBMC (p = 0.0126). The decision-tree model showed the best clustering cells of PBMC, BAL and LLN: CD56, CD4/CD8 and CD4+TIGIT+ cells. Considering patients and controls, the best subset was CD4+CTLA-4+. CONCLUSION: High expression of PD1 and TIGIT on T cells in BAL, as well as CTLA-4 and TIGIT on T cells in LLN, suggest that inhibition of these molecules could be a therapeutic strategy for avoiding the development of chronic inflammation and tissue damage in sarcoidosis patients.

Allergen induces the migration of platelets to lung tissue in allergic asthma.

RATIONALE: Platelets are essential for pulmonary leukocyte recruitment, airway hyperresponsiveness, and bronchial remodeling in animals with allergic inflammation and can be found in bronchoalveolar lavage of sensitized animals. No studies, however, have explored the direct migration of platelets to lungs. OBJECTIVES: To assess whether platelets migrate into lung parenchyma in response to inhaled allergen in ovalbumin-sensitized mice; to assess the role of the FcepsilonRI receptor in this phenomenon; and to evaluate whether platelets from patients with asthma, or from sensitized mice, undergo chemotaxis in vitro in response to relevant antigens. METHODS: Ovalbumin-sensitized wild-type (WT) mice, or FcRgamma(-/-) mice lacking the FcepsilonRIgamma, were challenged with aerosolized allergen and lungs analyzed by platelet-specific immunohistochemistry. In some experiments, mice were depleted of platelets and cross-transfused with either WT or FcRgamma(-/-) platelets to assess the role of platelet FcRgamma(-/-). Chemotaxis of platelets from patients with asthma or from sensitized mice was studied in vitro. MEASUREMENTS AND MAIN RESULTS: Histology of lungs revealed isolated platelets, migrating out of vessels and localizing underneath the airways after allergen challenge in WT but not in FcRgamma(-/-) mice. Platelets from patients with asthma and from sensitized WT mice, but not from sensitized FcRgamma(-/-) mice, migrated in vitro toward the relevant allergen or an anti-IgE. Platelets from normal mice were found to express FcepsilonRIgamma and platelet-bound IgEs were increased in sensitized mice. CONCLUSIONS: Platelets migrate extravascularly in response to a sensitizing allergen via a mechanism dependent on the interaction among allergen, allergen-specific IgE, and the FcepsilonRI, and this may allow them to participate directly in allergic tissue inflammation.

Quality assessment for recycling aggregates from construction and demolition waste: An image-based approach for particle size estimation.

The size distribution of aggregates has direct and important effects on fundamental properties of construction materials such as workability, strength and durability. The size distribution of aggregates from construction and demolition waste (C&D) is one of the parameters which determine the degree of recyclability and therefore the quality of such materials. Unfortunately, standard methods like sieving or laser diffraction can be either very time consuming (sieving) or possible only in laboratory conditions (laser diffraction). As an alternative we propose and evaluate the use of image analysis to estimate the size distribution of aggregates from C&D in a fast yet accurate manner. The effectiveness of the procedure was tested on aggregates generated by an existing C&D mechanical treatment plant. Experimental comparison with manual sieving showed agreement in the range 81-85%. The proposed technique demonstrated potential for being used on on-line systems within mechanical treatment plants of C&D.

Low triiodothyronine (T3) state: a predictor of outcome in respiratory failure? Results of a clinical pilot study.

BACKGROUND AND AIM: Various low triiodothyronine (T3) states have been described in severe nonthyroidal diseases and associated with a poor prognosis in cardiovascular disease patients. We assessed thyroid function in patients with severe respiratory failure from pulmonary disorders, and needing invasive or noninvasive mechanical ventilation, in order to evaluate the prognostic value of nonthyroidal illness syndrome. METHODS: We studied 32 consecutive patients with acute or acute-on-chronic respiratory failure. Measured variables upon admission included APACHE II score, the ratio of the partial pressure of oxygen in arterial blood to the fraction of oxygen in inspired gas (PaO(2)/FiO(2)), and plasma levels of free T3 (fT3) and free thyroxine (fT4), and TSH levels. Thyroid function was further evaluated at discharge. RESULTS: Plasma levels of fT3 were below normal in 17 patients (53%). Plasma fT3 was correlated with PaO(2)/FiO(2) (P<0.001), and with APACHE II score (P=0.003). In four patients (12.5%) who died, fT3 levels were significantly lower (P=0.002) than in patients who survived. In univariate logistic regression analysis, fT3 was the only factor significantly associated with an increased risk of death (odds ratio, 64.23; 95% confidence interval, 1.78-2316.86, P=0.023). Normalization of thyroid function was observed at discharge with a significant correlation between the percent increase in both fT3 and PaO(2)/FiO(2) (P=0.015). P values were calculated using Spearman's Correlation Coefficient. CONCLUSION: Our preliminary data suggest that the low T3 state is a predictor of outcome in pulmonary patients with respiratory failure.

Treatment of acute exacerbations of chronic respiratory failure: integrated use of negative pressure ventilation and noninvasive positive pressure ventilation.

STUDY OBJECTIVES: Acute respiratory failure (ARF) can be treated with either invasive mechanical ventilation (IMV) or noninvasive mechanical ventilation (NIMV), which can spare the complications of artificial airways. To evaluate the efficacy of an integrated approach using negative pressure ventilation (NPV) with iron lung and noninvasive positive pressure ventilation (NPPV), we performed a prospective study in a group of patients admitted to our respiratory ICU (RICU) for ARF due to exacerbation of chronic respiratory failure (CRF). SETTING: RICU at "R. Silvestrini" Hospital in Perugia, Italy. PATIENTS AND METHODS: One hundred fifty-two consecutive patients were included in the study and treated with iron lung as first choice or, when contraindicated or not tolerated, with NPPV using a nasal or facial mask. After 2 h of noninvasive mechanical ventilation (NIMV), the patients were reevaluated; in case of clinical deterioration, patients receiving NPV were switched to NPPV. When NPPV as a first or second line of treatment failed the patients were intubated. MEASUREMENTS AND RESULTS: One hundred fifty-two patients received NIMV, 97 with iron lung as the first choice of treatment, and 55 with NPPV. Six patients treated with NPV were switched to NPPV during the first 2 h of treatment. Twenty-five patients required IMV. The success rate of the integrated use of NIMV (NPV plus NPPV) was 81.6%, compared to that of NPV (83.5%) and NPPV (70.5%). Twenty-one patients (13.8%) required tracheostomy; the duration of hospital stay was significantly lower in patients treated with NIMV only. Thirty patients required mechanical ventilation at home. Few severe complications were observed in patients receiving IMV. CONCLUSIONS: The integrated use of two NIMV techniques is effective in patients with acute exacerbation of CRF. In most cases intubation and tracheostomy were avoided, thus reducing the complication rate of mechanical ventilation.