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.

BACKGROUND:THIS is the first time that a bronchoalveolar lavage (BAL) neutrophil-to-lymphocyte ratio (NL-ratio) has been demonstrated in sarcoidosis and chronic hypersensitivity pneumonitis (cHP) than in idiopathic pulmonary fibrosis (IPF) patients.METHODS:Consecutive BAL samples from the 167 interstitial lung disease (ILD) patients were retrospectively enrolled in the study and clustered into three diagnostic categories: IPF, cHP and sarcoidosis.RESULTS:NL-ratio which proved higher in IPF (mean±SD, 2.1±3.8) than sarcoidosis (mean±SD 0.7±1.9; P<1E-04) and cHP patients (mean±SD 1.6±3.1; P=7.7E-03). ROC curve analysis to discriminate between Sarcoidosis and other ILDs showed an area under the curve (AUC) of 83.7%, (56% sensitivity and 96% specificity) while IPF and the other ILD were discriminated with AUC of 73% using a NL-ratio threshold value of 0.48 (73% sensitivity and 63% specificity). Interestingly, the NL-ratio was significantly correlated with other prognostic parameters: it was inversely correlated with forced vital capacity (FVC) (r=-0.3; P=2.5E-02) and forced expiration volume in 1 second (FEV1) (r=-0.3; P=2E-02) percentages and directly correlated with composite pulmonary index (CPI) score (r=0.3; P=3.2E-02). A decision-tree statistical algorithm was applied.CONCLUSIONS:This is the first time that a lower NL-ratio has been demonstrated in sarcoidosis and cHP than in IPF patients. The present preliminary report indicates a relationship between BAL NL-ratio and lung function parameters in patients with IPF: this ratio may help to optimize management of IPF patients and to improve follow-up and outcome.

Background Broncho-alveolar lavage (BAL) is a safe diagnostic procedure, useful for differentiating fibrotic lung disorders and for excluding malignancy and infection. A recent multicenter study demonstrated a new, relatively sensitive, and specific index called Bronchoalveolar Cytology Threshold (BCT), useful for distinguishing healthy individuals from patients with lung diseases. Objectives In our study, BCT was applied for the first time to the analysis of interstitial lung diseases (ILDs), investigating its potential for differential diagnosis. Combinations of BAL cells that improve diagnostic accuracy for ILDs were studied and are proposed. Methods A retrospective analysis of BAL samples was performed. We considered more than 1000 BAL samples from patients investigated for ILD, performed at Siena University Hospital. The samples enrolled for the study included 468 patients: 413 with and 55 without ILD. BAL was performed for diagnostic purposes in line with international guidelines. BCT were calculated according to available literature. Results Among ILDs, patients with fibrotic hypersensitivity pneumonitis, idiopathic pulmonary fibrosis (IPF) and sarcoidosis showed significantly lower BCTs than unclassified ILD. Asbestosis patients showed significantly lower BCTs than nonspecific interstitial pneumonia (NSIP), cryptogenic organizing pneumonia (COP), connective tissue disease related ILD (CTD-ILD), sarcoidosis and unclassified ILD patients. COP patients showed significantly higher BCT than IPF, f-HP and sarcoidosis. Moreover, COP patients were best distinguished by BCT. Conclusion The analysis of BAL features is currently included in the diagnostic algorithm of ILDs. BAL cell patterns and BCT index can provide useful information for distinguishing ILDs, reducing the need for invasive procedures. Integrated approaches to BAL analysis can improve the interpretation of results without further cost or loss of time.

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.

The house dust mite (HDM) is globally ubiquitous in human habitats. Thirty-two allergens for Dermatophagoides farinae and 21 for Dermatophagoides pteronyssinus have been detected so far. The present minireview summarizes information about the role of Der p 1 as a key coordinator of the HDM-induced allergic response and reports on a series of Italian patients who are allergic to HDMs. We studied the specific IgE profiles in a population of patients with allergic asthma and rhinitis screened for specific immunotherapy (SIT) for HDM allergies, with the aim of obtaining insights into the pathogenic role of Der p1. Patients co-sensitized to other airborne allergens showed a higher prevalence of asthma (9/12 (75%) vs. 2/7 (29%); p < 0.05) than did HDM mono-sensitized patients. The latter group showed higher Der p1 concentrations than that of the co-sensitized group (p = 0.0360), and a direct correlation between Der p1 and Der p2 (r = 0.93; p = 0.0003) was observed. In conclusion, our study offers insights into the role of Der p1 in a population of patients with allergic rhinitis and asthma who were candidates for SIT. Interestingly, Der p1 positivity was associated with bronchial asthma and co-sensitization.

The original version of this article unfortunately contained a mistake. First and last names of the authors were interchanged.

Background: Angiotensin-converting enzyme (ACE) is an acid glycoprotein that converts angiotensin I into angiotensin II. It is produced mainly by activated alveolar macrophages and it resulted elevated in sarcoidosis patients. ACE is the only biomarker mentioned in WASOG international guidelines for the diagnosis and follow-up of sarcoidosis patients but its sensitivity and specificity are low. This study aimed to analyze serial measurements of ACE levels in sarcoidosis patients stratified according to concomitant ACE-inhibitor therapies (ACEIs). Subjects and methods: 136 serum samples from sarcoidosis patients were retrospectively enrolled in the study. Serial ACE concentrations were measured once year for each patient. Population were divided according to radiogical stages and ACEIs. Results: ACE concentrations resulted higher in non-ACEIs than ACEIs group (p<E-04). This result was confirmed also stratifying population according to radiological stages particularly in stage 3 (p=2E-03) or stage 2 of the disease (p<1E-04). Considering ACEIs, serum ACE levels proved to be higher in sarcoidosis patients treated with zofenopril than in those treated with perindopril (p=2E-02), enalapril (p=2E-03) or ramipril (p=2E-04). Patients treated with ACEIs showed a progressive reduction in ACE levels to five years of follow-up (p=1.3E-02) and the zofenopril group recorded the highest ACE levels (p<1E-04). Conclusions: This retrospective study investigated changes in ACE levels in patients with sarcoidosis treated or not treated with ACEIs. Considering the overall low sensitivity and specificity of this biomarker, we suggest systematically investigating medical drugs prescribed for patients with sarcoidosis, in order to optimize the interpretation of ACE in clinical management.

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.

Chronic hypersensitivity pneumonitis (cHP) is a fibrotic interstitial lung disease (ILD) resulting from inhalation of different organic substances and chemical compounds determining an inflammatory and immunological response in sensitized individuals. KL-6, a human mucin protein expressed by type 2 pneumocytes, has been proposed as a prognostic biomarker of cHP. Assessment of usefulness KL-6 in ILD has been investigated primarily in Asiatic population. The aim of this study was to evaluate clinical utility of KL-6 in serum and bronchoalveolar lavage (BAL). In this study, we retrospectively analysed clinical, radiological and immunological data of a cohort of 42 patients affected by cHP: KL-6 concentrations were collected from serum and BAL. KL-6 clinical value was assessed through the analysis of association between KL-6 concentrations and clinical, functional, immunological and radiological features. KL-6 serum concentration results increased in 28/34 patients (82%). A positive direct correlation was observed between KL-6 concentrations in BAL and serum (r = 0.62, p < 0.05). In our study population we found that patients with extensive presence of ground glass opacities and centrilobular nodules at high-resolution computed tomography (HRCT) showed the highest concentrations of KL-6 in BAL and a predominantly CD3+ CD8+ BAL lymphocytosis. BAL lymphocytosis and KL-6 concentrations showed a direct correlation. BAL KL-6, a result of alveolar damage, caused in cHP by CD3+ CD8+ mediated flogosis and suggested by radiological evidence of ground-glass opacities and centrilobular nodules, can be considered a useful biomarker to assess, along with BAL cellular analysis and HRCT findings, disease activity.

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 Al 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 Al, 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 Al, 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 Al, 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.

At the beginning of our studies on proteomics we compared serum protein profiles in interstitial lung diseases and we found increased expression of certain acute phase proteins, such as serum amyloid A (SAA), in sarcoidosis serum samples. An increased expression of SAA1 was detected by a proteomic approach (two-dimensional electrophoresis) in serum of sarcoidosis patients, therefore we decided to validate this finding evaluating its concentrations by a quantitative assay (ELISA) and to further investigate SAA patterns in patients with sarcoidosis. Our population consisted of 48 patients affected by sarcoidosis and 17 healthy controls. Patients were divided according to phenotype activity score SCAC. Proteomic analysis of SAA in serum revealed that the protein resolved in 2 different spots with isoelectric point/molecular weight of 5.69/11000 KDa and 6.15/11000 Kda. SAA1 spots were never observed in samples of healthy subjects but only in sarcoidosis patients. ELISA assay reported significantly higher concentrations of SAA in patients than controls (p=0.002). Protein concentrations inversely correlated with FEV1 percentages in sarcoidosis patients and were significantly higher in SCAC group 6 than in SCAC group 4 (p Interestingly the molecular weight of the two isoforms of SAA identified in our study corresponded to two unidentified serum proteins, proposed as sarcoidosis biomarker, in a previous proteomic study performed by Bons et al. In conclusion SAA could be a useful biomarker of sarcoidosis since its levels are significantly higher in patients than controls, especially in those with lung function impairment and requiring prolonged steroid treatment.

A crucial pathogenetic role of serum amyloid A (SAA) in granulomatous inflammation of sarcoidosis has recently been reported. In this study we analyzed SAA expression in detail, starting from proteomic analysis of serum of sarcoidosis patients. We also used the faster ELISA method that enabled us to examine a greater number of samples. Serum concentrations of SAA were significantly higher in sarcoidosis patients than controls (p<0.001), inversely correlated with FEV(1) and significantly higher in patients with subacute onset requiring prolonged and multiple steroid treatments (class 6 SCAC) than in patients with subacute onset not requiring therapy (class 4 SCAC) (p<0.001). Our results suggest that serum amyloid A could be a suitable marker of sarcoidosis: its serum concentrations are significantly higher in sarcoidosis patients than controls, the protein is only expressed in gels of sarcoidosis patients and not in healthy subjects, and the SAA1 isoforms could match the unidentified biomarker of sarcoidosis reported in a previous proteomic study by another group. The effectiveness of SAA as a clinical biomarker of sarcoidosis should now be investigated in a large prospective study.

BACKGROUND Sarcoidosis is a systemic granulomatous disease characterised by T-helper cell/macrophage alveolitis. Activated macrophages release mediators, such as cytokines, chemokines, oxygen radicals, and enzymes. In a previous paper we found higher levels of chitotriosidase, a macrophage derived enzyme, in serum of patients with sarcoidosis than in controls. Serum chitotriosidase levels were correlated with sarcoidosis radiological stages. Human chitotriosidase is involved in the pathogenesis of many lysosomal storage disorders and is selectively expressed in chronically activated tissue macrophages. METHODS In the present study we determined chitotriosidase concentrations in bronchoalveolar lavage of patients with newly diagnosed pulmonary sarcoidosis (divided into two groups according to clinical parameters) and of controls with an ELISA test. RESULTS Significantly different chitotriosidase concentrations were found in BAL of patients than controls, especially in patients with progressing disease. CONCLUSION Chitotriosidase but not angiotensin converting enzyme concentrations correlated with sarcoidosis radiological stages, and also with the degree of lung infiltrate seen by CT-scan, suggesting that the former enzyme (detected locally and sistemically) may play a role in the pathogenesis of the disease. Further studies with a greater number of patients are needed to confirm this hypothesis and to determine whether chitotriosidase may be a marker of the severity of sarcoidosis.

Background: Human chitotriosidase is a chitinase selectively expressed by activated macrophages. An increase in chitotriosidase activity was previously described by us in the serum and bronchoalveolar lavage of sarcoidosis patients. Objective: The aim of the present study was to analyze serum chitotriosidase activity in a larger number of sarcoidosis patients to verify the reported increase with respect to controls and to compare serum chitotriosidase levels in patients with sarcoidosis and tuberculosis, two granulomatous disorders of different etiology. Methods: Chitotriosidase activity was measured in the serum of 96 sarcoidosis patients, 15 pulmonary tuberculosis patients and 30 healthy controls. Results: We found significantly higher serum chitotriosidase activity in sarcoidosis patients than controls (p < 0.01) and in sarcoidosis patients than tuberculosis patients (p < 0.01), confirming a striking elevation of chitotriosidase activity (>10 times greater than normal) in pulmonary sarcoidosis patients. This is the first time that chitotriosidase activity has been analyzed in the serum of patients with pulmonary tuberculosis; it was found to be significantly lower than in sarcoidosis patients and not significantly greater than in controls. Conclusion: Although the mechanisms leading to the increase in chitotriosidase activity in sarcoidosis are still unknown, this enzyme may be specifically involved in the pathogenesis of the disease. Further studies with a greater number of patients are needed to confirm these results and to determine whether chitotriosidase could be a marker with diagnostic or prognostic value in sarcoidosis.

Background: In previous papers, we found significantly higher activity of chitotriosidase, a macrophage derived enzyme, in serum and BAL of patients with sarcoidosis, especially in those with progressing disease and lung involvement, than in controls. Locally and systemically produced chitotriosidase activity was correlated with radiological stage and also with degree of lung infiltration, suggesting that this enzyme may play a rote in the pathogenesis of sarcoidosis and may be used as a marker of disease severity.Aim: To analyse chitotriosidase activity in serum and bronchoalveolar lavage of patients with idiopathic pulmonary fibrosis and pulmonary fibrosis associated with systemic sclerosis and to compare it with chitotriosidase activity in controls and sarcoidosis patients.Methods: Chitotriosidase activity was determined by a fluorometric assay.Results: The results showed that serum chitotriosidase activity was only elevated in sarcoidosis patients; in patients with idiopathic pulmonary fibrosis and pulmonary fibrosis associated with systemic sclerosis it was in the normal range. On the contrary, in BAL of sarcoidosis and idiopathic pulmonary fibrosis patients the activity was significantly higher than in controls.Conclusion: Serum chitotriosidase is a potential marker of sarcoidosis severity; it increases in sarcoidosis in relation to radiological stage and degree of lung infiltration. The increase in chitotriosidase activity in BAL of sarcoidosis and idiopathic pulmonary fibrosis patients suggests that the enzyme could be involved in fibrogenesis in diffuse lung diseases. Further research is needed to understand the role of chitotriosidase in the pathogenesis of sarcoidosis and its involvement in fibrotic remodelling in certain diffuse lung diseases. (C) 2007 Elsevier Ltd. All rights reserved.