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Background: Serum mid-regional proadrenomedullin (MR-proADM) has emerged as a marker of organ failure (mainly lungs and kidneys) and poor prognosis in patients admitted to intensive care (IC); some reports also suggest it and other markers, such as Krebs von den Lungen-6 (KL-6) and interleukin-6 (IL-6), as a prognostic biomarker of COVID-19. The aim of the study was to evaluate the performance MR-proADM in hospitalized COVID-19 patients for predicting in-hospital mortality and need for non-invasive or invasive respiratory support. Methods: We enrolled 74 patients hospitalized in the COVID Unit of Siena Hospital from March to May 2020, for whom serum samples were available on admission for assay of MR-proADM, KL-6 and IL-6. Demographic data, comorbidities, medical history and clinical laboratory data on days 1–3 of admission and Simplified Acute Physiology Score and Simplified Organ Failure Assessment scores calculated at day 1 were collected retrospectively, as well as mortality and IC admission data. Results: 12 patients died in hospital (16%) and 14 patients were admitted to IC (19%). Serum concentrations of MR-proADM on admission and on day 1 were higher among non-survivors than among survivors (p = 0.015 and p = 0.045, respectively), while those on day 3 were not significantly different. Patients needing respiratory support had higher MR-proADM concentrations on admission than the others (p = 0.046), and those requiring invasive mechanical ventilation had higher MR-proADM on day 1 (p = 0.017). Serum concentrations of KL-6 and IL-6 were significantly higher in non-survivors (p = 0.03 and p = 0.004, respectively). ROC curve analysis showed that serum MR-proADM on day 1 had the best accuracy in predicting death and/or IC admission (AUC = 0.9583, p = 0.0006); the combination of all three biomarkers further improved the accuracy of prediction of death or IC admission (AUC = 0.9793; p = 0.00004). Conclusions: Our data sustain the potential of serum MR-proADM as a reliable prognostic biomarker of hospitalized COVID-19 patients and confirms the utility of the three markers in the management and risk stratification of hospitalized patients. The markers are collected mini-invasively and are quick to analyze and cost-effective.

Background: The pathogenesis of sarcoidosis involves T cells and B lymphocytes that produce autoantibodies. We compared the expression of different T and B cell subsets in sarcoidosis and three B-mediated rheumatic diseases that can affect the lungs in an attempt to identify similarities and differences that distinguish these diseases. Methods: The study included patients referred to Siena University Hospital’s respiratory disease and rheumatology units. Patients were enrolled prospectively and consecutively. Healthy volunteers were also included. Multicolor flow cytometry was performed on phenotype T and B cell subsets. Multivariate analysis was carried out to reduce the dimensionality of the data. Results: Fifteen patients had a diagnosis of sarcoidosis, fourteen idiopathic inflammatory myopathies (IIM), five granulomatosis with polyangiitis (GPA), ten microscopic polyangiitis (MPA), and seven were controls. Thirty-five T and B cell subsets were phenotyped, 15 of which were significantly different in sarcoidosis, B-mediated rheumatic disorders, and controls. Principal components analysis distinguished the four groups of patients with a total explained variance of 54.7%. A decision tree was constructed to determine which clustering variables would be most useful for distinguishing sarcoidosis, IIM, MPA, and GPA. The model showed regulatory T helper cells (Th-reg) > 5.70% in 91% of sarcoidosis patients as well as Th-reg ≤ 5.70 and Th17 > 43.27 in 100% of MPA. It also showed Th-reg ≤ 5.70, Th17 ≤ 43.27 and Tfh-reg ≥ 7.81 in 100% of GPA patients, and Th-reg ≤ 5.70, Th17 ≤ 43.27 and Tfh-reg ≤ 7.81 in 100% of IIM patients. Conclusion: The immune cell profile sheds light on similarities and differences between sarcoidosis and B-mediated rheumatic diseases. Sarcoidosis and autoimmune diseases show similar patterns of cellular immune dysregulation, suggesting a common pathogenic pathway that may provide an opportunity for further understanding autoimmunity and exploring biological therapies to treat sarcoidosis.

Objectives: Systemic sclerosis (SSc) is a disease characterized by diffuse sclerosis of skin and organs and small vessel vasculopathy. Despite it, large vessels can also be involved with ulnar artery vasculopathy, revealing as a more frequent feature of SSc. The aim of this paper is to assess the macrovascular involvement of SSc patients through an ultrasound (US) evaluation of radial and ulnar arteries. Methods: Radial and ulnar resistance indices (RIs) and peak systolic velocity (PV) (cm/s) together with clinical features of SSc patients were evaluated. Raynaud phenomenon (RP) and healthy control (HC) groups were used for comparison. Results: Forty-three SSc patients were evaluated. Twelve patients (28%) had ulnar artery occlusions (UAOs). In nine cases (75%), UAOs were bilateral. A high UAO prevalence (42%) was found in SSc patients with late nailfold-video-capillaroscopy (NVC) pattern (p = 0.0264). Patients with UAOs had digital ulcers (DUs) in 10 cases (83.3%). Radial and ulnar PVs were lower in SSc and RP patients than the HC group. Radial and ulnar RIs were higher in SSc and RP patients than the HC group. A decision tree analysis led to the classification of 70% of SSc patients with an ulnar RI > 0.82 and ulnar PV > 2.8 cm/s. The most influential variables on UAO development were interstitial lung disease (ILD) (p = 0.002) and NVC pattern (p = 0.002). A positive correlation was shown between modified Rodnan skin score (mRSS) and ILD (p = 0.283; r = 0.033), mRSS and DU (r = 0.344; p = 0.012) and DU and ILD (r = 0.303; p = 0.024). Male sex was associated with increased UAO frequency (p = 0.042). Conclusions: UAO is a peculiar feature of severe SSc present in 28% of the cases, particularly associated with the presence of ILD and late NVC pattern. In 75% of the cases, UAOs are bilateral. DUs are very frequent in patients with UAOs (83%). The RI evaluated by US could be useful to distinguish SSc from HC patients. US could be a useful tool for assessing high-risk DU development in patients.

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.

Background: Nintedanib is an oral multitarget tyrosine kinase inhibitor approved for the treatment of patients with idiopathic pulmonary fibrosis (IPF). Recent evidence demonstrated that nintedanib reduced functional disease progression also in subjects with non-IPF progressive fibrosing interstitial lung disease (PF-ILD). However, real-life data on the effectiveness of nintedanib in PF-ILD and familial pulmonary fibrosis (FPF) are lacking. Methods: this retrospective monocentric study enrolled 197 patients affected with IPF, PF-ILD and FPF treated with nintedanib at the Referral Centre of Siena from 2014 to 2021. Pulmonary functional tests and survival data were collected throughout the observation period for the evaluation of mortality and disease progression outcomes. Results: nintedanib treatment significantly reduced the FVC decline rate in IPF and PF-ILD subgroups, but not in FPF subjects. No significant differences were observed among the subgroups in terms of survival, which appeared to be influenced by gender and impaired lung function (FVC < 70% of predicted value). Concerning disease progression rate, a diagnosis of FPF is associated with more pronounced FVC decline despite nintedanib treatment. Conclusions: our research studies the effectiveness and safety of nintedanib in reducing functional disease progression of IPF and PF-ILD. FPF appeared to be less responsive to nintedanib, even though no differences were observed in terms of survival.

Severe eosinophilic asthma is characterized by chronic airway inflammation, oxidative stress, and elevated proinflammatory cytokines, especially IL-5. Mepolizumab and benralizumab are both humanized IgG antibodies directed against IL-5 signaling, directly acting on eosinophils count. Together with the complexity of severe asthma classification and patient selection for the targeted treatment, there is also the urgency to clarify the follow-up of therapy to identify biomarkers, in addition to eosinophils, for the optimal duration of treatment, persistence of effectiveness, and safety. To this purpose, here we performed a follow-up study using differential proteomic analysis on serum samples after 1 and 6 months of both therapies and sera from healthy patients. Statistical analysis by PCA and heatmap analyses were performed, and identified proteins were used for enrichment analysis by MetaCore software. The analysis highlighted 82 differences among all considered conditions. In particular, 30 referred to benralizumab time point (T0, T1B, T6B) and 24 to mepolizumab time point (T0, T1M, T6M) analyses. t-SNE and heatmap analyses evidence that the differential serum protein profile at 6 months of both treatments is more similar to that of the healthy subjects. Among the identified proteins, APOAI, APOC-II, and APOC-III are upregulated principally after 6 months of benralizumab treatment, plasminogen is upregulated after 6 months of both treatments and ceruloplasmin, upregulated already after 1 month of benralizumab, becoming higher after 6 months of mepolizumab. Using enrichment analysis, identified proteins were related to lipid metabolism and transport, blood coagulation, and ECM remodeling.

Background: Krebs von den Lungen-6 (KL-6) has been proposed as a disease severity marker of COVID-19. All research articles reported the KL-6 assay detected through Fujirebio reagents by Lumipulse G600/G1200 instrument. In the present study, KL-6 assay was analysed through Tosoh AIA-360 and compared with analytical results by Lumipulse G600 in a population of COVID-19 patients. Materials and methods: Sixty-four patients (median age, IQR 67 (58–76) years), all hospitalized for COVID-19 interstitial pneumonia at Siena COVID Unit. KL-6 was measured by two methods, chemiluminescence enzyme immunoassay (CLEIA) and fluorescent enzyme immunoassay (FEIA) method by Lumipulse G600 II and AIA 360 systems, respectively. Results: KL-6 concentrations evaluated by Lumipulse G600II were significantly higher in severe than those in non-severe patients (p < 0.0001) as well as evaluating by AIA360 (p < 0.0001). Receiver operating curve (ROC) curve analysis showed that KL-6 concentrations, by Lumipuse G600II, distinguished severe from non-severe COVID-19 patients with an area under the curve (AUC) of 99.8% and the best cut-off value was 448 U/mL. AUROC between severe and non-severe COVID-19 patients using T0 KL-6 concentrations by AIA360 was 97.4% and the best cut-off value was 398 U/mL. According to T0 KL-6 concentrations in COVID-19 patients, Bland–Altman difference analysis revealed a mean bias of 78 ± 174.8; while using T1 KL-6 concentrations in COVID-19 patients, Bland–Altman difference analysis revealed a mean bias of 48 ± 126 (95% limits of agreement −199–295) between the Lumipulse G600 II and the AIA360 systems. Conclusions: In conclusion, our study demonstrated that CLEIA and FEIA methods for serum KL-6 detection are comparable and reliable. KL-6 was confirmed as an easily detectable and effective biomarker to identify severe COVID-19 patients.