Characterization of NKG2-A/-C, Kir and CD57 on NK Cells Stimulated with pp65 and IE-1 Antigens in Patients Awaiting Lung Transplant.

INTRODUCTION: Cytomegalovirus (CMV) is the leading opportunistic infection in lung transplant (LTx) recipients. CMV is associated with graft failure and decreased survival. Recently, new antiviral therapies have been proposed. The present study aimed to investigate NK and T cell subsets of patients awaiting LTx. We analyzed the cellular populations between reactive and non-reactive QuantiFERON (QF) CMV patients for the prediction of immunological response to infection. METHODS: Seventeen pre-LTx patients and 15 healthy controls (HC) have been enrolled. QF and IFN-γ ELISA assay detections were applied. NK cell subsets and T cell and proliferation assay were detected before and after stimulation with pp-65 and IE-1 CMV antigens after stratification as QF+ and QF-. Furthermore, we quantified the serum concentrations of NK- and T-related cytokines by bead-based multiplex analysis. RESULTS: CD56brCD16lowNKG2A+KIR+ resulted in the best discriminatory cellular subsets between pre-LTx and HC. Discrepancies emerged between serology and QF assay. Better proliferative capability emerged from patients who were QF+, in particular in CD8 and CD25-activated cells. CD56brCD16low, adaptive/memory-like NK and CD8Teff were highly increased only in QF+ patients. CONCLUSIONS: QF more than serology is useful in the detection of patients able to respond to viral infection. This study provides new insights in terms of immunological responses to CMV in pre-LTX patients, particularly in NK and T cells biology.

Peripheral and alveolar biomarkers of fibrotic hypersensitivity pneumonitis.

Fibrotic hypersensitivity pneumonitis (fHP) is a frequently misdiagnosed fibrosing interstitial pneumonia, which often remains undiagnosed due to the lack of uniformity of diagnostic criteria. Its features are similar to those of other ILDs, especially idiopathic pulmonary fibrosis (IPF), and biomarkers with potential clinical value have been proposed. We reviewed the recent literature on serum and BAL biomarkers, focusing on their clinical role in the diagnosis and management of fHP. We searched Medline/Pubmed results from 2005 until April 2020. The manuscripts of interest selected by our search were limited in number and proposed different clinical biomarkers in serum (IgG antibodies, macrophage inflammatory proteins-1, epithelial cell proteins) and BAL (lymphocytes, T-cell mediators). This is the first review to summarize all the serum and BAL biomarkers for fHP proposed in the literature. This review summarized the main biomarkers investigated in fibrotic hypersensitivity pneumonitis because an urgent aim of subsequent research will be to validate and standardize them for diagnostic purposes.

The Role of Galectins in Chronic Lung Allograft Dysfunction.

BACKGROUND: Galectins are proteins that bind ß-galactosides such as N-acetyllactosamine present in N-linked and O-linked glycoproteins and that seem to be implicated in inflammatory and immune responses as well as fibrotic mechanisms. This preliminary study investigated serum galectins as clinical biomarkers in lung transplant patients with chronic lung allograft dysfunction (CLAD), phenotype bronchiolitis obliterans syndrome (BOS). MATERIALS AND METHODS: Nineteen lung transplant patients [median age (IQR), 55 (45-62) years; 53% males] were enrolled in the study. Peripheral blood concentrations of galectins-1, 3 and 9 were determined with commercial ELISA kits. RESULTS: Galectin-1 concentrations were higher in BOS than in stable LTX patients (p = 0.0394). In logistic regression analysis, testing BOS group as dependent variable with Gal-1 and 3 as independent variables, area under the receiver operating characteristics (AUROC) curve was 98.9% (NPV 90% and PPV 88.9%, p = 0.0003). With the stable LTX group as dependent variable and Gal-1, 3 and 9 as independent variables, AUROC was 92.6% (NPV 100% and PPV 90%, p = 0.0023). In stable patients were observed an inverse correlation of Gal-3 with DLCO% and KCO%, and between Gal-9 and KCO%. CONCLUSION: Galectins-1, 3 and 9 are possible clinical biomarkers in lung transplant patients with diagnostic and prognostic meaning. These molecules may be directly implicated in the pathological mechanisms of BOS. The hypothesis that they could be new therapeutic targets in BOS patients is intriguing and also worth exploring.

Regulatory and Effector Cell Disequilibrium in Patients with Acute Cellular Rejection and Chronic Lung Allograft Dysfunction after Lung Transplantation: Comparison of Peripheral and Alveolar Distribution.

BACKGROUND: The immune mechanisms occurring during acute rejection (AR) and chronic lung allograft dysfunction are a challenge for research and the balance between effector and regulatory cells has not been defined completely. In this study, we aimed to elucidate the interaction of effector cells, mainly Th17, Th1 and Th2, and regulatory cells including (CD4+CD25+CD127low/-) T reg cells and phenotypes of B regs, CD19+CD24hiCD38hi, CD19+CD24hiCD27hi and CD19+CD5+CD1d+. METHODS: Bronchoalveolar lavage cells (BAL) and peripheral blood mononuclear cells (PBMCs) from stable lung transplanted (LTx )subjects (n = 4), AR patients (n = 6) and bronchiolitis obliterans syndrome (BOS) (n = 6) were collected at the same time. Cellular subsets were detected through flow cytometry. RESULTS: A predominance of Th17 cells subtypes in the PBMCs and BAL and a depletion of Tregs, that resulted in decrease Treg/Th17 ratio, was observed in the AR group. CD19+CD24hiCD38hi Bregs resulted increased in BAL of AR patients. Th1 cells predominance and a reduction of Tregs cells was observed in BAL from AR patients. Moreover, multivariate analysis showed interdependences within studied variables revealing that effector cells and regulatory cells can effectively discriminate patients' immunological status. CONCLUSIONS: In AR, BOS and stable lung transplant, regulatory and effector cells clearly demonstrated different pathways of activation. Understanding of the balance of T cells and T and B regulatory cells can offers insights into rejection.

Serial KL-6 measurements in COVID-19 patients.

SARS-CoV2-induced direct cytopathic effects against type II pneumocytes are suspected to play a role in mediating and perpetuating lung damage. The aim of this study was to evaluate serum KL-6 behavior in COVID-19 patients to investigate its potential role in predicting clinical course. Sixty patients (median age IQR, 65 (52-69), 43 males), hospitalized for COVID-19 at Siena COVID Unit University Hospital, were prospectively enrolled. Twenty-six patients were selected (median age IQR, 63 (55-71), 16 males); all of them underwent follow-up evaluations, including clinical, radiological, functional, and serum KL-6 assessments, after 6 (t1) and 9 (t2) months from hospital discharge. At t0, KL-6 concentrations were significantly higher than those at t1 (760 (311-1218) vs. 309 (210-408) p = 0.0208) and t2 (760 (311-1218) vs 324 (279-458), p = 0.0365). At t0, KL-6 concentrations were increased in patients with fibrotic lung alterations than in non-fibrotic group (755 (370-1023) vs. 305 (225-608), p = 0.0225). Area under the receiver operating curve (AUROC) analysis showed that basal KL-6 levels showed good accuracy in discriminating patients with fibrotic sequelae radiologically documented (AUC 85%, p = 0.0404). KL-6 concentrations in patients with fibrotic involvement were significantly reduced at t1 (755 (370-1023) vs. 290 (197-521), p = 0.0366) and t2 (755 (370-1023) vs. 318 (173-435), p = 0.0490). Serum concentrations of KL-6 in hospitalized COVID-19 patients may contribute to identify severe patients requiring mechanical ventilation and to predict those who will develop pulmonary fibrotic sequelae in the follow-up.

Portable Pocket-Sized Ultrasound Scanner for the Evaluation of Lung Involvement in Coronavirus Disease 2019 Patients.

Ultrasound imaging of the lung (LUS) and associated tissues has demonstrated clinical utility in coronavirus disease 2019 (COVID-19) patients. The aim of the present study was to evaluate the possibilities of a portable pocket-sized ultrasound scanner in the evaluation of lung involvement in patients with COVID-19 pneumonia. We conducted 437 paired readings in 34 LUS evaluations of hospitalized individuals with COVID-19. The LUS scans were performed on the same day with a standard high-end ultrasound scanner (Venue GO, GE Healthcare, Chicago, IL, USA) and a pocket-sized ultrasound scanner (Butterfly iQ, Butterfly Network Inc., Guilford, CT, USA). Fourteen scans were performed on individuals with severe cases, 11 on individuals with moderate cases and nine on individuals with mild cases. No difference was observed between groups in days since onset of symptoms (23.29 ± 10.07, 22.91 ± 8.91 and 28.56 ± 11.13 d, respectively; p = 0.38). No significant differences were found between LUS scores obtained with the high-end and the portable pocket-sized ultrasound scanner. LUS scores in individuals with mild respiratory impairment were significantly lower than in those with moderate and severe cases. Our study confirms the possibilities of portable pocket-sized ultrasound imaging of the lung in COVID-19 patients. Portable pocket-sized ultrasound scanners are cheap, easy to handle and equivalent to standard scanners for non-invasive assessment of severity and dynamic observation of lung lesions in COVID-19 patients.

Adiponectin and leptin levels in idiopathic pulmonary fibrosis: A new method for BAL and serum assessment.

Adipokines (APN) are mainly secreted by adipocytes, macrophages and various other cells, along with their role in the regulation and mediation of inflammatory responses. APN is almost exclusively synthesized by adipocytes and regulated by peroxisome proliferator-activated receptor γ (PPARγ) that is involved in the epithelial-mesenchymal transition, linked lung fibrosis. Leptin is involved in acute lung injury with a role in lung fibrogenesis. Little is known about the relationship between APN/leptin and idiopathic pulmonary fibrosis (IPF) and the few studies available in the literature used ELISA to detect these lipid mediators. Our study is also the first to measure adipokines by the new multiplex assay and for the first time were performed in bronchoalveolar lavage (BAL) from IPF patients. This preliminary study suggests that APN levels in serum could be useful for predicting the prognosis of IPF, as they are inversely correlated with DLco percentages and BMI. Moreover, this first analysis of APN in BAL from IPF patients by a new method demonstrated an inverse correlation between these levels and BMI values and a direct correlation with eosinophil percentages, both of which are negative prognostic factors of IPF.

Efficacy of baricitinib in treating rheumatoid arthritis: Modulatory effects on fibrotic and inflammatory biomarkers in a real-life setting.

BACKGROUND: Baricitinib is a JAK inhibitor that blocks intracellular signalling pathways of inflammatory cytokines recommended for Rheumatoid arthritis (RA) patients not responding to initial treatment. Among RA extrareticular features, interstitial lung involvement is primarly characterized by fibrotic evolution. The aim of the present study was to analyse the effects of baricitinib in a population of RA and RA-ILD patients in a real-life setting, describing any changes in lung function parameters, serum inflammatory biomarkers and fibrotic biomarkers after 6 months of treatment. MATERIALS AND METHODS: 15 patients (median (IQR) 65 (55-66); 13% males and 74% smokers) treated with baricitinib were enrolled. 4 patients (27%) were classified as RA-ILD before baricitinib therapy. Our study is the first to evaluate adipokine levels in RA patients (including a small population with RA-ILD) after six months of baricitinib treatment with a novel multiplex method. RESULTS: The modulatory effects of baricitinib on lipid mediators were associated with clinical and functional improvement, demonstrated by the significant increase in DLco and KCO percentages after six months of treatment. Baricitinib decreased the systemic inflammation by lowering expression of IL-6 and CRP and reducing ESR and serum concentrations of adiponectin. A significant reduction of KL-6 levels in RA-ILD patients after six months of baricitinib therapy reflects the stability of interstitial lung involvement in these patients. CONCLUSION: Baricitinib was demonstrated to be a safe immune modulator that reduces the concentrations biomarkers of lung fibrosis and inflammation in RA patients, including a subgroup with interstitial lung involvement.

BAL and serum multiplex lipid profiling in idiopathic pulmonary fibrosis and fibrotic hypersensitivity pneumonitis.

BACKGROUND: Differential diagnosis between IPF and fibrotic HP (fHP) can be challenging: these two ILDs share many common features but call for different therapeutic approaches. In the present study, differential lipid mediator profiles were analysed by a new method in BAL and serum from HP and IPF patients. MATERIALS AND METHODS: 76 patients were enrolled retrospectively in the study. Median age (IQR) was 67 years (51-74); 63% were males, 30 had fHP and 46 had IPF. Serum and BAL samples were collected at initial diagnosis. For quantification of serum and BAL lipid mediators was used bead-based multiplex LEGENDPlex™ analysis (Biolegend). RESULTS: Serum Apo A1 levels were significantly higher in IPF than fHP patients (p = 0.314); indeed, serum levels of CCL2 and Apo C3 were lower in HP than in IPF patients (p = 0.013 and p = 0.041, respectively). BAL concentrations of Apo A1, adipsin, Apo C3 and APN were significantly lower in IPF than in fHP patients (p < 0.0001, p < 0.0001, p = 0.007 and p = 0.023, respectively). In the logistic regression, IPF was tested as dependent variable. Serum levels of Apo A1, CCL2 and Apo C3 were tested as independent variables and ROC curve analysis of model performance showed AUC 93% (p < 0.0001); on the other hand, BAL concentrations of Apo A1, adipsin, Apo C3 and APN showed AUC 81% (p < 0.0001). DISCUSSION: Lipid biomarkers evaluated in BAL in our study confirm the hypothesis that fHP and IPF have different lung fibrosis phenotypes. The former is a post-inflammatory cell-regulated ILD and the second is more related to tissue remodeling and repair.

A Comprehensive Evaluation of Mepolizumab Effectiveness in a Real-Life Setting.

INTRODUCTION: Interleukin-5 (IL-5) is the principal cytokine regulating eosinophil growth, differentiation, activation, and expression. It is a specific target of mepolizumab, an anti-IL-5 monoclonal antibody used in the treatment of severe eosinophilic asthma. This new drug can improve symptoms, reduce asthma exacerbations and steroid use. Few data are available on its efficacy for nasal symptoms. OBJECTIVE: To describe the all-round clinical impact of mepolizumab in a real-life setting, evaluating the efficacy and safety of the drug in severe eosinophilic asthma patients. POPULATION AND METHODS: We retrospectively collected the clinical and functional data on 27 patients (16 males) affected with severe eosinophilic asthma, diagnosed at the Siena Regional Referral Centre and monitored for 6 months. Clinical, immunological, and functional data at baseline and follow-up were entered in a database together with comorbidities, number of exacerbations, steroid treatment, multiple-flow exhaled nitric oxide, and validated questionnaires. RESULTS: A significant reduction in asthma exacerbations was observed in all patients after 6 months of the biological therapy (p = 0.0009), and 4/6 patients discontinued chronic oral steroids. A significant improvement in ACT, FEV1, SNOT22, and alveolar nitric oxide was observed after 1 month of mepolizumab (p = 0.003, p = 0.007, p = 0.047, and p = 0.019, respectively) and maintained after 6 months of treatment. After 6 months, FeNO 50 was reduced as well (p = 0.030). Mepolizumab was very well tolerated, and no major side effects were observed. CONCLUSIONS: Our study suggests that mepolizumab is effective in improving control of asthma, lung function parameters, exhaled biomarkers, and nasal symptoms in patients with severe eosinophilic asthma.

Pirfenidone in chronic lung allograft dysfunction: a single cohort study.

BACKGROUND: Chronic lung allograft dysfunction (CLAD) is still the principal long-term cause of mortality after lung transplant. Animal studies and small case series have proposed pirfenidone, a potent antifibrotic agent registered for idiopathic pulmonary fibrosis (IPF), for treatment of CLAD. The aim of this study was to evaluate the safety profile and potential efficacy of pirfenidone in patients with CLAD. METHODS: The present study concerns a cohort of nine CLAD patients treated with pirfenidone. Pulmonary function tests were performed before and after beginning treatment. Side effects were recorded and survival was analyzed. All data were retrospectively collected. RESULTS: The duration of treatment was 408.5±534.8 days. Significant side effects occurred in one case. FEV1 decline reduced from -44.5±40.7 mL/month in the 6 months before therapy to -12.8±34.3 mL/month in the following 6 months. However, data was only available for three patients (three patients died before 6 months of therapy, two patients lacked lung function parameters, one discontinued therapy and one was still in the early months of therapy). Median survival was 686 days. No significant survival differences were observed in relation to CLAD phenotype (BOS, RAS and BOS/RAS). Median survival from the start of pirfenidone therapy was 221 days. CONCLUSIONS: Our CLAD patients treated with pirfenidone showed a good safety profile, similarly to that reported for IPF patients. The drug showed potential for stabilizing decline in respiratory function. Further studies are needed in order to draw conclusions about the effectiveness of this therapy.

The effect of cigarette smoking on bronchoalveolar lavage protein profiles from patients with different interstitial lung diseases.

The proteomic approach applied to the analysis of BAL gives a panorama of the complex network of proteins of different origin and function and their modifications at alveolar level. Cigarette smoking may influence BAL protein composition and it represents the most relevant risk factor for several lung diseases. This review, for the first time, discusses the available literature regarding the effects of cigarette smoking on BAL protein composition of healthy subjects and patients affected by interstitial lung diseases (ILD). The comparison of BAL protein profiles of smokers and non-smoker healthy controls revealed alterations of proteins related to oxidative stress and protease/antiprotease imbalance (such as alpha 1 antitrypsin, alpha-1-antichymotrypsin, apolipoprotein A1, peroxiredoxin 1 and glutathione S transferase P). Smoking exposure leads to a significant dysregulation of a large number of molecular pathways involved in interstitial lung diseases and the proteomic studies applied to the study of BAL of idiopathic pulmonary fibrosis, sarcoidosis and other ILD contributed to clarify the underlying pathogenetic mechanisms facilitating ILD development and biomarker discovery.