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Background: Anti-DFS70 antibodies, corresponding to the dense fine speckled antinuclear antibody (ANA) pattern in HEp-2 substrates, have been observed in chronic inflammatory conditions, cancer and in healthy individuals but in only a small percentage of patients with connective tissue diseases (CTD). Objectives: The study was aimed to investigate the possible role of Anti-DFS70 antibodies to distinguish CTD associated interstitial lung disease (CTD-ILD) from idiopathic interstitial pneumonia (IIP) and to explore potential correlations between anti-DFS70 antibodies and clinical parameters. Methods: Serum samples were collected from 49 healthy controls (HC), 35 scleroderma-ILD (SSc-ILD) patients as negative controls for anti-DFS70 antibody, and 260 patients with the initial diagnosis IIP including 100 nonspecific interstitial pneumonia (NSIP) and 160 idiopathic pulmonary fibrosis (IPF) patients. ANA pattern was identified by indirect immunofluorescence on HEp-2 cells and anti-DFS70 antibodies were measured in serum by ELISA. Results: Serum anti-DFS70 antibodies were less frequently seen in ILD and SSc-ILD patients compared to HCs. Thirty-seven patients (34 initial idiopathic NSIP and 3 initial IPF patients) developed CTD during 24 months of follow-up, most of them combined with ANA positivity and anti-DFS70 antibody negativity. Anti-DFS70 antibody positivity was not significantly different between CTD-ILD and idiopathic ILD. Conclusions: The frequency of serum anti-DFS70 antibody is markedly decreased in patients with ILDs. Anti-DFS70 antibodies may be useful to predict CTD development in ILD patients.

Background and objective Pulmonary alveolar proteinosis (PAP) is a rare disease characterized by alveolar filling. YKL-40, a chitinase-like protein produced by macrophages and epithelial cells, is increased in patients with interstitial lung diseases. We aimed to evaluate the role of YKL-40 as a biomarker for PAP. Methods A total of 34 patients with autoimmune PAP and 50 healthy controls were studied. YKL-40 was measured by ELISA in serum and bronchoalveolar lavage fluid (BALF). Chitinase coding gene polymorphisms (CHI3L1−329 and −131) were detected by PCR and pyrosequencing. Correlations between serum YKL-40 levels and disease outcome were analysed. Results Baseline serum and BALF levels of YKL-40 were higher in PAP patients than in controls (286 ± 27 ng/mL vs 42 ± 4 ng/mL, P

Hypersensitivity pneumonitis (HP) is a complex syndrome caused by the inhalation of a variety of antigens in susceptible and sensitized individuals. These antigens are found in the environment, mostly derived from bird proteins and fungi. The prevalence and incidence of HP vary widely depending on the intensity of exposure, the geographical area and the local climate. Immunopathologically, HP is characterized by an exaggerated humoral and cellular immune response affecting the small airways and lung parenchyma. A complex interplay of genetic, host and environmental factors underlies the development and progression of HP. HP can be classified into acute, chronic non-fibrotic and chronic fibrotic forms. Acute HP results from intermittent, high-level exposure to the inducing antigen, usually within a few hours of exposure, whereas chronic HP mostly originates from long-term, low-level exposure (usually to birds or moulds in the home), is not easy to define in terms of time, and may occur within weeks, months or even years of exposure. Some patients with fibrotic HP may evolve to a progressive phenotype, even with complete exposure avoidance. Diagnosis is based on an accurate exposure history, clinical presentation, characteristic high-resolution CT findings, specific IgG antibodies to the offending antigen, bronchoalveolar lavage and pathological features. Complete antigen avoidance is the mainstay of treatment. The pharmacotherapy of chronic HP consists of immunosuppressive drugs such as corticosteroids, with antifibrotic therapy being a potential therapy for patients with progressive disease.

Family with sequence similarity 13, member A (FAM13A) variants have been associated with susceptibility to chronic lung diseases. A recent genome-wide association study has shown an association between a polymorphism in FAM13A rs2609255 and idiopathic interstitial pneumonias in a Caucasian population. However, the relationship between rs2609255 polymorphism and prognosis in idiopathic interstitial pneumonias has not been investigated.Sixty-five patients with idiopathic pulmonary fibrosis (IPF) and 310 Japanese healthy volunteers were enrolled in this study. Genomic DNA was extracted from all subjects. rs2609255 was genotyped by a commercially available assay. The correlations between rs2609255 polymorphism and survival and the occurrence of acute exacerbation were evaluated.The frequency of the minor G allele was significantly higher in IPF patients (59.2%) than in controls (41.9%; OR = 1.78, 95% CI; 1.29-2.44, p 0.001). The rs2609255 major T allele was associated with lower diffusing capacity of carbon monoxide values and higher composite physiologic index after adjustment for age, sex and smoking (β = -7.20, p = 0.005 and β = 5.59, p = 0.009, respectively). In the Kaplan-Meier analysis, the T allele carriers showed a significantly increased mortality compared to the non-carriers (p 0.05). In the multivariate Cox-proportional hazards analysis, the T allele of rs2609255 was independently associated with poor survival (hazard ratio, 5.37; p = 0.031; 95% confidence interval, 1.16-24.82).FAM13A gene polymorphism showed a significant association with the susceptibility to IPF, with severity of lung function impairment and with poor prognosis.

Sarcoidosis may have more than a single causative agent, including infectious and non-infectious agents. Among the potential infectious causes of sarcoidosis, Mycobacterium tuberculosis and Propionibacterium acnes are the most likely microorganisms. Potential latent infection by both microorganisms complicates the findings of molecular and immunologic studies. Immune responses to potential infectious agents of sarcoidosis should be considered together with the microorganisms detected in sarcoid granulomas, because immunologic reactivities to infectious agents reflect current and past infection, including latent infection unrelated to the cause of the granuloma formation. Histopathologic data more readily support P. acnes as a cause of sarcoidosis compared with M. tuberculosis, suggesting that normally symbiotic P. acnes leads to granuloma formation in some predisposed individuals with Th1 hypersensitivity against intracellular proliferation of latent P. acnes, which may be triggered by certain host or drug-induced conditions. Detection of bacterial nucleic acids in granulomas does not necessarily indicate co-localization of the bacterial proteins in the granulomas. In the histopathologic diagnosis of sarcoidosis, M. tuberculosis-associated and P. acnes-associated sarcoidosis will possibly be differentiated in some patients by immunohistochemistry with appropriate antibodies that specifically react with mycobacterial and propionibacterial antigens, respectively, for each etiology-based diagnosis and potential antimicrobial intervention against sarcoidosis.

Background: Genetic variants of TOLLIP and MUC5B have been reported to be associated with development and/or prognosis of idiopathic pulmonary fibrosis (IPF). Real-life experiences on the application of SNPs in the clinical management of IPF are lacking. This study was conducted to investigate the association of MUC5B and TOLLIP SNPs with disease course and outcome in a single-centre cohort. Patients and Methods: 50 IPF patients (M 43 and F 8, age 66±10 y) and 50 healthy controls (HC) (M 37, F 13, age42±2 y) were genotyped for SNPs within TOLLIP (rs3750920 and rs5743890) and MUC5B (rs35705950). Diagnosis of IPF was made according to the ATS/ERS guideline 2011. Genotype frequency was compared between IPF and HC subjects. Correlation of SNPs genotypes with survival, acute exacerbation or disease progression (a decline of ≥ 10% in FVC) was investigated. Results: TOLLIP SNPs genotype distribution did not differ between IPF and HC (p=0.6). MUC 5B T minor allele was more frequent in IPF subjects than in HC (67% vs 16% p Conclusion: While the minor allele T in MUC5B is associated with IPF, TOLLIP gene variants appear to correlate with disease progression, therefore being of potential utility to stratify IPF patients.

Introduction: We have previously shown that the GAP stage was useful for predicting an acute exacerbation of IPF (AE-IPF). However, the discriminative ability between GAP stages I and II was insufficient. Aims: To investigate whether a modified GAP score which includes KL-6 and the ethnicity has a better accuracy for predicting the risk of AE-IPF. Methods: A total of 131 patients with IPF (75 German and 56 Japanese) were studied. Baseline serum KL-6 levels >1,300 U/mL and Results: The original GAP score at baseline was 4.5±0.2 in the German and 4.3±0.2 in the Japanese cohorts (p=0.50). The modified GAP score at baseline was 4.8±0.2 in the German and 6.9±0.3 in the Japanese cohorts (p Conclusions: The modified GAP score/stage which includes baseline serum KL-6 level and Japanese ethnicity demonstrated a higher accuracy for predicting AE-IPF than the original GAP stage during the first 3 years of follow-up.

Granulomatous lung diseases are a heterogeneous group of disorders that have a wide spectrum of pathologies with variable clinical manifestations and outcomes. Precise clinical evaluation, laboratory testing, pulmonary function testing, radiological imaging including high-resolution computed tomography and often histopathological assessment contribute to make a confident diagnosis of granulomatous lung diseases. Differential diagnosis is challenging, and includes both infectious (mycobacteria and fungi) and noninfectious lung diseases (sarcoidosis, necrotising sarcoid granulomatosis, hypersensitivity pneumonitis, hot tub lung, berylliosis, granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis, rheumatoid nodules, talc granulomatosis, Langerhans cell histiocytosis and bronchocentric granulomatosis). Bronchoalveolar lavage, endobronchial ultrasound-guided transbronchial needle aspiration, transbronchial cryobiopsy, positron emission tomography and genetic evaluation are potential candidates to improve the diagnostic accuracy for granulomatous lung diseases. As granuloma alone is a nonspecific histopathological finding, the multidisciplinary approach is important for a confident diagnosis.

Hypersensitivity pneumonitis (HP) is a complex syndrome caused by the inhalation of environmental antigens. Chronic HP may mimic other fibrotic lung diseases, such as idiopathic pulmonary fibrosis. Recognition of the antigen is important for diagnosis; avoidance of further exposure is critical for treatment. Fibrosis on biopsy or high-resolution computed tomography is a predictor of increased mortality. Additional research is needed to understand why the disease develops only in a minority of exposed individuals and why cases of chronic HP may progress without further antigen exposure.

Summary Background Pulmonary alveolar proteinosis (PAP) is a rare syndrome characterized by the intra-alveolar accumulation of surfactant lipids and proteins. The aim of the study is to describe the epidemiologic, clinical, physiologic, and laboratory features of PAP in a large single-center cohort of patients with PAP. Study population Over 30 years, 70 patients with PAP were managed at our institution, 64 with primary and 6 with secondary PAP. Results The mean age at diagnosis was 43 years with a male to female ratio of 1.3. BAL was the most commonly applied diagnostic method, performed in 83% of cases. A history of smoking was seen in 79%, and of dust exposure in 54%, most commonly to aluminum, silica and sawdust. GM-CSF autoantibody correlated with clinical outcome and KL-6 with diffusing capacity. The number of whole lung lavages (WLL) necessary to reach remission was higher in current smokers. Conclusions This study shows that the use of BAL for the diagnosis of PAP can reduce the need of histological confirmation. A history of dust or fume inhalation is strongly associated with PAP, also with the autoimmune form. Smoking seems to influence the response to treatment, increasing the number of WLL necessary to reach remission.

SummaryBackgroundTNF receptors (TNFR1 and TNFR2) and Fas belong to the system of apoptosis-signalling receptor molecules and may play a role in the pathogenesis of interstitial lung disease. Patients with cryptogenic organizing pneumonia (COP) usually respond well to corticosteroids, in contrast to those with idiopathic pulmonary fibrosis (IPF). This may be due to the different pathogenesis.MethodsThe expression of TNFR1, TNFR2 and Fas on bronchoalveolar lavage (BAL) macrophages and lymphocytes was analysed in 9 patients with COP, 10 with IPF and 12 controls. The production of soluble TNFR1, 2 and TNF-α by alveolar macrophages was measured by ELISA.ResultsTNFR1 and Fas expression on alveolar macrophages was significantly higher in COP than in controls and IPF. The expression of TNFR2 on alveolar macrophages was also increased in COP compared to controls. The expression of TNFR2 and Fas on lymphocytes was significantly higher in COP than in IPF and controls. In addition, the expression of TNFR1, TNFR2 and Fas on BAL cells correlated positively with BAL lymphocytes (p

Advances have been made in minimally invasive diagnostic procedures in sarcoidosis, including bronchoalveolar lavage (BAL), endobronchial ultrasonography-guided transbronchial needle aspiration (EBUS-TBNA), and positron emission tomography (PET). Several independent groups found almost identical predictive values of the CD4:CD8 ratio in BAL for the diagnosis of sarcoidosis. A CD4:CD8 ratio greater than 3.5 shows a high specificity of 93 to 96% for sarcoidosis, but the sensitivity is low (53 to 59%). EBUS-TBNA is a safe and useful tool for diagnosing sarcoidosis stage I and II with a sensitivity of 83 to 93% and a specificity of 100%. Novel imaging techniques have been explored, such as PET using L-[3- (18)F] fluoro-alpha-methyltyrosine ( (18)F-F MT), which is more specific for malignancy than (18)F-fluorodeoxyglucose ( (18)F-FDG)-PET. The combined modality of FMT-PET with FDG-PET could successfully discriminate sarcoidosis from malignancy. These recent developments including novel biopsy procedures and novel imaging techniques could be of value to diagnosing sarcoidosis.

Introduction:We report a survey of mesothelioma survival rates with insights into the survival benefit because of pemetrexed. We also studied a potential link between specific single nucleotide polymorphisms of transcobalamin II (TCII) gene and susceptibility to both asbestos and pemetrexed.Methods:Clinical and occupational data from 287 consecutive mesothelioma patients were collected from the north-east region of France (1989–2007). Blood or paired tumoral and normal samples were collected from the last 210 French patients to study the TCII single nucleotide polymorphisms at the codon 259 (quantitative polymerase chain reaction). Results were compared with those obtained from a group of 263 French control healthy subjects and to a group of 91 German mesothelioma patients. Patients' characteristics and genotypes results were statistically analyzed for significant correlations.Results:The mean overall patient's survival was 18.19 ± 21.07 months. Pemetrexed increased the patients' survival by 50% (21.81 versus 16.99 months). The TCII allele Proline (Pro) was overrepresented into the mesothelioma cohort when compared with the controls (35 versus 19.77%). This also concerned German patients. The alleles Pro and Proline Arginine (ProArg) were more frequent among patients exposed to asbestos (p = 0.005, p < 0.001, respectively). The allele ProArg was associated with the longest survival while under pemetrexed (p = 0.007). No difference was found in the genotypes of patients untreated with pemetrexed.Conclusions:Pemetrexed treatment is related to a survival increase in mesothelioma patients. The allele Pro seems overrepresented in mesothelioma patients. Those having the allele ProArg present a better outcome under pemetrexed.

Backgrounds: Japanese patients with idiopathic pulmonary fibrosis (IPF) appear to experience acute exacerbation (AE-IPF) more frequently than other ethnicities. Aims: To investigate pathogenetic gene polymorphisms in developing AE-IPF. Methods: A total of 139 patients with IPF (86 German and 53 Japanese) were enrolled. The correlation between ethnicity, polymorphisms in potential pathogenetic genes ( MUC1, MUC5B, ELMOD2, HER2, IL8, TERT, Caveolin, EGF, HMOX1, ICAM1, VEGFA, DPP9, ATP11A, OBFC1, DSP, FAM13A1, TOLLIP) and the incidence of AE-IPF was evaluated. Results: Six (7%) German and 17 (32%) Japanese patients experienced AE-IPF during the first 3 years of follow-up. In the univariate analysis, Japanese ethnicity, lower vital capacity, non-use of triple therapy (prednisone, azathioprine and N-acetylcysteine), the polymorphisms in MUC1, TERT, DPP9 and ATP11A gene were risk factors for AE-IPF. In the multivariate analysis, A/A polymorphisms in TERT gene and A/A polymorphisms in MUC1 gene were independent risk factors for AE-IPF (HR=53.6, p=0.0089; HR=8.2, p=0.042, respectively). The C statistic for TERT and MUC1 gene polymorphisms in addition to covariates including Japanese ethnicity was 0.896 (p TERT and MUC1 gene polymorphisms in addition to covariates was 0.914 (p Conclusions: TERT and MUC1 gene polymorphisms appear to be risk factors for AE-IPF.

Background: Sarcoidosis is a multisystem disease characterized by noncaseating granuloma formation, mostly affecting the lung. A variant in the TNFRSF1A region has been found to codify a protein capable of TNF antagonism. This variant is also associated with the development of multiple sclerosis but is absent in autoimmune diseases highly responsive to TNF-alpha treatment. Aim: To search for the presence of TNFRSF1A SNP in sarcoidosis patients and its potential correlation with disease phenotype. Patients and method: 79 patients with histologically proven lung sarcoidosis (49 male, 30 female) and 50 healthy controls (HC) were studied. Radiological stage and organ involvement were assessed. DNA was extracted from peripheral blood and rs1800693 SNP was determined by PCR. Genotyping was performed by pyrosequencing. Correlation with stage and clinical profile of sarcoidosis was investigated. Results: The alleles of rs1800693 SNP were in Hardy-Weinberg equilibrium in the studied subjects. The frequency of A/A genotype was 27%, A/G 54% and G/G 19% in sarcoidosis patients, similar to HC. The mean duration of disease was 4.9±3 years. No correlations were seen between genotype and age at onset, gender, serum ACE, organ involvement or use of corticosteroids. In patients with the G/G genotype, DL CO at diagnosis was significantly lower than in those with the A/G or A/A genotype (59±19 vs 73±11 and 79±11% pred, p=0.019) and duration of disease tended to be longer (6.1 vs 5.2 and 4.3 years, respectively). Conclusion: The presence of the G allele of the rs1800693 SNP in sarcoidosis can be related to more severe lung functional impairment in sarcoidosis.

Background: Nintedanib, a tyrosine kinase inhibitor approved for the treatment of idiopathic pulmonary fibrosis (IPF), exhibits antifibrotic and antiinflammatory activity. Th1 cytokines with IFN-γ inducing capacity have been shown to inhibit angiogenesis through up-regulation of ELR-CXC chemokines in mice. The aim of the study was to investigate the effect of nintedanib on the production of angiogenic/ angiostatic cytokines by AMs in ILD. Methods: AMs obtained by bronchoalveolar lavage fluid (BALF) from 9 ILD patients (5 IPF and 4 idiopathic nonspecific interstitial pneumonias (iNSIP)) were cultured with/without lipopolysaccharide (LPS). The effects of nintedanib at different concentrations (0, 0.1, 0.3, 1.0 μmol/L) on the production of Th1 cytokines (IL-12p40 and IL-18), angiogenic chemokines (IL-8, SDF-1α), and an angiostatic chemokine (MIG) were measured in the cell culture supernatants by Luminex bead based assay. Results: The spontaneous release of MIG by AMs was higher in IPF than in iNSIP (454.3±167.6 vs. 73.8±29.1 pg/mL/10 6 AMs, p 6 AMs, p Conclusions: Nintedanib seems to reduce the release of IL-12p40, IL-18 and MIG by AMs from patients with IPF and iNSIP in a similar way.

The cause(s) of sarcoidosis is unknown.Mycobacteriumspp. are suspected in Europe andPropionibacteriumspp. are suspected in Japan. The present international collaboration evaluated the possible etiological links between sarcoidosis and the suspected bacterial species. Formalin-fixed and paraffin-embedded sections of biopsy samples of lymph nodes, one from each of 108 patients with sarcoidosis and 65 patients with tuberculosis, together with 86 control samples, were collected from two institutes in Japan and three institutes in Italy, Germany, and England. Genomes ofPropionibacterium acnes,Propionibacterium granulosum,Mycobacterium tuberculosis,Mycobacterium aviumsubsp.paratuberculosis, andEscherichia coli(as the control) were counted by quantitative real-time PCR. EitherP. acnesorP. granulosumwas found in all but two of the sarcoid samples.M. aviumsubsp.paratuberculosiswas found in no sarcoid sample.M. tuberculosiswas found in 0 to 9% of the sarcoid samples but in 65 to 100% of the tuberculosis samples. In sarcoid lymph nodes, the total numbers of genomes ofP. acnesorP. granulosumwere far more than those ofM. tuberculosis. P. acnesorP. granulosumwas found in 0 to 60% of the tuberculosis and control samples, but the total numbers of genomes ofP. acnesorP. granulosumin such samples were less than those in sarcoid samples.Propionibacteriumspp. are more likely thanMycobacteriaspp. to be involved in the etiology of sarcoidosis, not only in Japanese but also in European patients with sarcoidosis.

Pulmonary alveolar proteinosis (PAP) is characterized by alveolar accumulation of surfactant lipoproteins. Proteasomes are involved in the nonlysosomal protein degradation. We hypothesize that enzymatically active proteasome is increased in the alveolar space of PAP.31 PAP patients (29 with primary, 2 with secondary form), 14 disease controls (10 with COPD and 4 with emphysema) and 18 healthy controls were studied. 20S Proteasome was measured by ELISA in bronchoalveolar lavage fluid (BALF) and in serum. Enzyme activity of extracellular proteasome (pkat/mg) was measured through fluorogenic substrate cleavage.Proteasome concentration in BAL was higher in PAP patients than in disease controls or healthy subjects (566±420 vs 53±27 vs 60±42ng/ml, respectively, p0.0001 for both). Serum proteasome levels were higher in PAP patients than in healthy controls (825±712 vs 405±176ng/ml, p=0.018). PAP patients with active disease had higher serum levels than those who achieved remission (1317±1176 vs 439±422ng/ml, p=0.008). Proteasomal enzyme activity was increased in BAL of PAP patients (p0.05).The 20S proteasome is increased and active in BAL of patients with PAP. Extracellular proteasome may contribute to the alveolar degradation of accumulated proteins in PAP.

Direct hemoperfusion with polymyxin B-immobilized fiber (PMX-DHP) might be beneficial for treating acute exacerbation (AE) of interstitial pneumonia (IP). Venovenous extracorporeal membranous oxygenation (VV-ECMO) is an emerging tool to avoid ventilator-induced lung injury. This is a report presenting the first three patients with AE of IP treated with a combined therapy of PMX-DHP and VV-ECMO.Patient 1 was a 68-year-old male with acute interstitial pneumonia, patient 2 a 67-year-old male with AE of idiopathic pulmonary fibrosis, and patient 3 a 61-year-old female with AE of collagen vascular disease-associated interstitial pneumonia. All patients were severely hypoxemic and required mechanical ventilation. A combined therapy using PMX-DHP and VV-ECMO was initiated with support of intravenous corticosteroids and antibiotics. Radiological findings, oxygenation and laboratory findings markedly improved and all patients survived without severe complications.A combined therapy of PMX-DHP and VV-ECMO might be a therapeutic option for AE of IP.

Tumor necrosis factor-alpha (TNF-alpha) is an important proinflammatory cytokine. Recently, pentoxifylline (POF) has been shown to suppress the synthesis of TNF-alpha from lipopolysaccharide (LPS)-stimulated human monocytes in cell cultures and in vivo. The aim of this study was to investigate whether POF-induced suppression of TNF-alpha secretion affects peripheral blood monocytes (PBM) and alveolar macrophages (AM) equally, and whether POF is able to suppress the spontaneous TNF-alpha production from AM in pulmonary sarcoidosis in vitro. In seven patients without interstitial lung disease we studied the effect of POF on LPS-stimulated PBM and AM cultured for 24 h. In six patients with sarcoidosis we investigated the effect of POF on the enhanced spontaneous TNF-alpha production by AM in vitro. POF induced a dose-dependent suppression of the LPS-stimulated TNF-alpha production which was not different for PBM and AM, respectively. In sarcoidosis, POF inhibited the spontaneous TNF-alpha production of AM at 0.1 mM by 91% and at 1 mM by 98%. In conclusion, POF inhibits LPS-induced TNF-alpha production from PBM and AM to a similar extent and can also inhibit the exaggerated spontaneous TNF-alpha production from AM in sarcoidosis in vitro. This may be the basis for further clinical trials to evaluate POF as an immunotherapeutic agent in sarcoidosis.

Alveolar macrophages (AM) originate from blood monocytes and, during the maturation process, undergo functional and morphological changes which are also reflected in their phenotypic pattern. Among the macrophage membrane antigens, adhesion molecules of the integrin family are particularly important for effector functions and cell-cell interactions. The aim of this study was to analyse the membrane expression of selected integrins by AM recovered from bronchoalveolar lavage (BAL) as compared to their precursors, peripheral blood monocytes (PBM). The cells were stained using a sensitive immunoperoxidase assay with 10 different monoclonal antibodies. The data showed a higher expression by AM than PBM of all but one of the studied adhesion molecules. The only exception was CD11b (Mac-1, CR3) which showed a higher expression in PBM than in AM. Several molecules, for example, CD49d (VLA-4), CD51 (vitronectin receptor), and CD54 (intercellular adhesion molecule-1, ICAM-1) were found to be upregulated by AM in patients with a lymphocytic pattern of BAL. In contrast, the phenotype of PBM does not show any changes in these patients. In conclusion, we have demonstrated differences in the expression of integrins between AM and PBM which can be partially responsible for some of their functional differences.

Bronchiolitis obliterans organizing pneumonia (BOOP) is a distinct clinicopathological entity histologically characterized by intra-alveolar granulation tissue and absence of extensive fibrotic lesions. Effective macrolide treatment of BOOP has been reported anecdotally. This study aimed to investigate whether alveolar macrophages (AMs) produce aberrant proinflammatory cytokines in BOOP and whether this can be inhibited by clarithromycin (CAM) or azithromycin (AZM).AMs collected by bronchoalveolar lavage (BAL) from 6 BOOP patients and 8 non-ILD controls were cultured for 24h in the presence or absence of CAM, AZM, lipopolysaccharide (LPS), or dexamethasone (DEX). Tumor necrosis factor alpha (TNF-α), soluble TNF receptor 1 (sTNFR1), sTNFR2, interleukin 1beta (IL-1β), IL-6, IL-8, IL-10, interferon gamma inducible protein 10 (IP-10) and CC chemokine ligand 18 (CCL18) were measured in the culture supernatant by ELISA.The spontaneous and LPS-stimulated production of all investigated cytokines by AMs was significantly increased in BOOP compared to controls. CAM and AZM induced a dose-dependent suppression of spontaneous TNF-α, sTNFR2, IL-6, IL-8 and CCL18 production (p0.05). CAM also inhibited the IL-1β production. CAM and AZM significantly and dose-dependently attenuated the LPS-stimulated production of sTNFR1, sTNFR2, IL-8 and CCL18 (p0.05). CAM also inhibited the LPS-stimulated TNF-α, IL-1β, IL-6 and IL-10 production.AMs from BOOP patients produce abundant proinflammatory cytokines which may be pivotal in the disease pathogenesis. Macrolides inhibit this cytokine production, CAM more efficiently than AZM.

YKL-40, a chitinase-like protein mainly secreted by macrophages, neutrophils and epithelial cells, is increased in patients with idiopathic interstitial pneumonia and sarcoidosis. We aimed to investigate the role of YKL-40 as a biomarker in hypersensitivity pneumonitis (HP).72 HP patients, 100 interstitial lung disease (ILD) controls and 60 healthy controls were studied. YKL-40 was measured by ELISA in serum and bronchoalveolar lavage fluid (BALF) at baseline and follow-up. The relationship between YKL-40 levels, clinical variables and disease outcome was evaluated.Baseline serum YKL-40 levels were significantly higher in HP patients than in healthy controls (p−1, the baseline serum YKL-40 level predicted disease progression (hazard ratio 6.567; p−1was associated with mortality (hazard ratio 9.989; pSerum YKL-40 may be a useful prognostic biomarker in HP patients.

Clinical manifestations of hypersensitivity pneumonitis may closely mimic other interstitial lung diseases, and the disease onset is usually insidious. High-resolution computed tomography and bronchoalveolar lavage are the sensitive and characteristic diagnostic tests for hypersensitivity pneumonitis. The relevant antigen to hypersensitivity pneumonitis cannot be identified in up to 20% to 30% of patients. Clinicians should be aware that hypersensitivity pneumonitis must be considered in all cases of interstitial lung disease, and a detailed environmental exposure history is mandatory.

Sarcoidosis likely results from the exposure of a genetically susceptible subject to an environmental agent, possibly an infectious one. Mycobacterial and propionibacterial organisms are the most commonly implicated potential etiologic agents. Propionibacterium acnes is the only microorganism, however, found in sarcoid lesions by bacterial culture. To evaluate the pathogenic role of this indigenous bacterium, we screened for the bacterium in sarcoid and non-sarcoid tissues using immunohistochemical methods with novel P. acnes-specific monoclonal antibodies that react with cell-membrane-bound lipoteichoic acid (PAB antibody) and ribosome-bound trigger-factor protein (TIG antibody). We examined formalin-fixed and paraffin-embedded samples of lungs and lymph nodes from 196 patients with sarcoidosis, and corresponding control samples from 275 patients with non-sarcoidosis diseases. The samples were mostly from Japanese patients, with 64 lymph node samples from German patients. Immunohistochemistry with PAB antibody revealed small round bodies within sarcoid granulomas in 20/27 (74%) video-assisted thoracic surgery lung samples, 24/50 (48%) transbronchial lung biopsy samples, 71/81 (88%) Japanese lymph node samples, and 34/38 (89%) German lymph node samples. PAB antibody did not react with non-sarcoid granulomas in any of the 45 tuberculosis samples or the 34 samples with sarcoid reaction. In nongranulomatous areas, small round bodies detected by PAB antibody were found in alveolar macrophages of lungs and paracortical macrophages of lymph nodes from many sarcoid and some non-sarcoid patients. Large-spheroidal acid-fast bodies, Hamazaki-Wesenberg bodies, which were found in 50% of sarcoid and 15% of non-sarcoid lymph node samples, reacted with both PAB and TIG antibodies. Electron microscopy revealed that these Hamazaki-Wesenberg bodies had a single bacterial structure and lacked a cell wall with occasional protrusions from the body. The high frequency and specificity of P. acnes, detected by PAB antibody within sarcoid granulomas, indicates that this indigenous bacterium might be the cause of granuloma formation in many sarcoid patients. © 2012 USCAP, Inc. All rights reserved.

SummaryBackgroundKL-6 is a high-molecular-weight glycoprotein classified as human Mucin-1 (MUC1). KL-6 has been reported to be a sensitive biomarker for interstitial lung diseases (ILDs) in the Japanese population. It is also known that polymorphisms in the MUC1 gene affect serum levels of KL-6. This study was conducted to evaluate serum levels of KL-6 and MUC1 polymorphisms in both German and Japanese populations.MethodsSerum levels of KL-6 were measured in 267 patients with ILDs (152 German and 115 Japanese) and 186 healthy subjects (HS) (76 German and 110 Japanese). In addition, rs4072037 single nucleotide polymorphisms (SNPs) were genotyped by polymerase chain reaction. The optimal cutoff values for discriminating patients with ILDs from HS was determined by receiver operating characteristic analysis based on ethnicity and rs4072037 genotypes.ResultsThe serum KL-6 levels in patients with ILDs were significantly higher compared with HS in both the German and the Japanese cohorts (both p

Whole lung lavage (WLL) is the standard treatment for pulmonary alveolar proteinosis (PAP). This study aimed to provide data about the kinetics of the protein wash-out, to identify factors influencing the protein concentration in the recovered fluid and to assess the efficacy of a modified lavage technique. Samples of 180 WLLs from 42 adult PAP patients were collected. 110 WLLs were performed according to the classical technique. In 70 WLLs, repeated manual ventilation was applied during the procedure. Spectrophotometry was used to measure the protein concentration in the recovered fluid. The initial protein concentration in the recovered fluid was 460 mg · dL(-1), the final concentration was 26 mg · dL(-1) and the total amount of removed proteins during a lavage was 17.5 g. A history of dust exposure was associated with a higher residual protein concentration in the recovered fluid (p=0.000013). The amount of removed proteins correlated inversely with the diffusing capacity of the lung for carbon monoxide (p=0.001) and oxygen tension (p=0.004). The modified technique removed a greater amount of proteins than the classical technique and prolonged the time to relapse (p=0.011). Exposure to dust seems to influence the kinetics of the protein wash-out. Applying manual ventilation during the procedure can enhance the efficacy of WLL.

Adhesion molecules are involved in the recruitment of leucocytes to sites of inflammation. In this study, we determined the expression of several adhesion molecules on isolated human alveolar type II pneumonocytes. Type II pneumocytes were isolated from 10 normal lung specimens, by enzymatic digestion with dispase, followed by metrizamide gradient centrifugation and panning on immunoglobulin G (IgG)-coated plastic dishes. With the freshly isolated type II cells, immunostaining was performed using a sensitive immunoperoxidase slide technique. In all cases, 60-90% of type II cells were positive for intercellular adhesion molecule-1 (ICAM-1) (CD54). A minor portion of type II cells expressed the alpha 4 (CD49d) subunit of the beta 1-integrins, and the alpha-v (CD51) subunit of the vitronectin receptor. CD11a, CD11b, CD11c, CD18, CD49b, and CD49f failed to demonstrate any immunostaining with type II cells. In conclusion, the observation of the expression of ICAM-1 and, to a lesser degree, of some integrin subunits, may indicate that alveolar type II cells participate in local immune and inflammatory responses.

According to the 2002 ATS/ERS Consensus Classification, a confident diagnosis of idiopathic pulmonary fibrosis (IPF) without surgical lung biopsy is made with consistent clinical/physiological findings and the typical features on high-resolution computed tomography (HRCT). Bronchoalveolar lavage (BAL) and/or transbronchial biopsy, one of four major criteria in the 2000 ATS/ERS IPF Statement, was no more essential in the diagnostic algorithm of 2002 ATS/ERS Consensus Classification.To evaluate the additional utility of BAL for the diagnosis of IPF.A total of 101 patients with suspected IPF on HRCT were studied. Twenty-seven patients were excluded because of lack of functional impairment (n = 20), an underlying condition causing fibrosis (n = 5), or a clinical history inconsistent with IPF (n = 2). The remaining 74 patients met all the criteria recommended in the 2002 ATS/ERS Consensus Classification for making a diagnosis in the absence of surgical biopsy. The final diagnosis was made with further examinations, including pathological analysis, in patients who showed inconsistent findings for IPF on BAL.A cut-off level of 30% for lymphocytes in BAL demonstrated a favorable discriminative power for the diagnosis of IPF. Six of the 74 patients (8%) showed a lymphocytosis of 30% or greater in BAL. Their final diagnoses were idiopathic nonspecific interstitial pneumonia (n = 3) and extrinsic allergic alveolitis (n = 3). The change in perception of the diagnosis was validated by a surgical biopsy in two cases and by subsequent outcome in four cases.BAL lymphocytosis changed the diagnostic perception in six of 74 patients who would have been misdiagnosed as having IPF without BAL.

Haem oxygenase (HO)-1 is an oxidative stress responsive protein that may be involved in the pathogenesis of interstitial lung disease. HO-1 expression in alveolar macrophages from bronchoalveolar lavage was investigated in 24 patients with idiopathic pulmonary fibrosis (IPF), 16 with sarcoidosis, 14 with hypersensitivity pneumonitis (HP) and 13 controls. Using immunocytochemistry, HO-1 expression in macrophages was scored semiquantitatively from 0-3 according to increasing intensity. The mean score of 100 macrophages was calculated. Macrophages were cultured and levels of interleukin (IL)-12 and IL-18 in the culture supernatants were measured by ELISA. The mean score of HO-1 was significantly lower in IPF (67) than in sarcoidosis (105), HP (106) or controls (106). There was no significant difference between sarcoidosis, HP and controls. The score of HO-1 correlated positively with the lymphocyte percentage in sarcoidosis and HP. Positive correlations were found between the score of HO-1 and the release of IL-12 and IL-18 by macrophages in IPF. The expression of haem oxygenase-1, a critical defender against oxidative stress, is decreased in macrophages of idiopathic pulmonary fibrosis patients compared with those with granulomatous lung disorders. This supports the hypothesis of an oxidant-antioxidant imbalance in the pathogenesis of idiopathic pulmonary fibrosis. Eur Respir J

Thalidomide exhibits diverse actions of anti-inflammation, immunomodulation and anti-angiogenesis. The efficacy of thalidomide treatment in sarcoidosis with lupus pernio is thought to be due to inhibition of tumour necrosis factor (TNF)-alpha. The mechanisms that underlie the properties of thalidomide are still unclear in interstitial lung disease. The current authors investigated the potential inhibitory effects of thalidomide at concentrations of 0.1, 0.01 and 0.001 mM on the production of transforming growth factor-beta, TNF-alpha, interleukin (IL)-1beta, IL-6, IL-8, IL-10, IL-12p70, IL-12p40 and IL-18 by alveolar macrophages from bronchoalveolar lavage in patients with sarcoidosis (n = 8), hypersensitivity pneumonitis (HP; n = 8) and idiopathic pulmonary fibrosis (IPF; n = 12). In sarcoidosis and HP patients, thalidomide induced a dose-dependent, partial suppression of lipopolysacchride (LPS)-stimulated TNF-alpha, IL-12p40 and IL-18 release. At the highest thalidomide concentration (0.1 mM), LPS-stimulated IL-8 production was also suppressed. In IPF patients, although spontaneous production of TNF-alpha, IL-12p40, IL-18 and IL-8 was lower than in sarcoidosis and HP patients, with LPS stimulation the cytokines were significantly elevated and also partially inhibited by thalidomide. In conclusion, thalidomide has the potential to improve the therapeutic regimens for sarcoidosis, hypersensitivity pneumonitis and idiopathic pulmonary fibrosis by reducing tumour necrosis factor-alpha, interleukin-12p40, interleukin-18 and interleukin-8 production.

Alveolar macrophages (AM) from smokers contain characteristic smoker's inclusion bodies within the cytoplasm as a result of ingestion of substances in the inhaled smoke. How long these smoking-related changes in the AM population can be seen after smoking cessation is largely unknown. We had the unique opportunity to investigate a 51-yr-old never-smoker after single lung transplantation (TX) for alpha 1-antitrypsin deficiency emphysema who received a donor's lung from a heavy cigarette smoker. Serial bronchoalveolar lavage (BAL) was performed in the donor's lung for transplant surveillance at defined time intervals, and the percentage of AM with characteristic smoker's inclusions was counted on slides stained with May-Grünwald-Giemsa stain. The patient had an uneventful course after TX with no major infectious complications or episodes of rejection. One month after TX the percentage of smoker's AM was 98%. BAL after 2, 5, 7, and 12 mo showed a similar high percentage. After 18 mo a first a decrease was seen, down to 78%, and after 2 yr a decrease to 59% was seen. After 3 yr, the smoker's AM had mostly disappeared, only 3% were still present. In conclusion, smoker's inclusions in AM may be detected for at least 2 yr after smoking has ceased, which is considerably longer than the estimated life span of the AM.

Pentoxifylline (POF) is a well established drug with haemorrheological properties. Various evidence suggests an additional therapeutic potential in regard to inflammation and immunomodulation. Extrinsic allergic alveolitis (EAA) is a granulomatous disease which is driven by T cell and alveolar macrophage (AM) derived cytokines. To investigate the effects of POF on the production of tumor necrosis factor (TNF) alpha, interleukin (IL)-1beta, IL-6, IL-8, IL-10 and the soluble TNF receptors (sTNFR1 and sTNFR2) from AM in EAA, also in comparison with dexamethasone (DEX).AM from 9 patients with EAA were cultured for 24 h with 10% RPMI medium alone, or with lipopolysaccharide (LPS, 100 micro g/L), and with POF at concentrations of 0.01 mmol/L, 0.1 mmol/L and 1 mmol/L, or with 0.1 mmol/L DEX. Cytokines in the culture supernatants were analysed by ELISA.POF induced a dose dependent suppression of spontaneous TNFalpha and IL-10 release from AM in EAA (P0.001 and P0.05). The spontaneous production of other cytokines was unaffected by POF at all tested concentrations. DEX inhibited only the spontaneous release of TNFalpha significantly (P0.05). POF and DEX also inhibited the LPS-stimulated production of all cytokines except of IL-1beta and sTNFR1 (P0.001 or P0.01 or P0.05).Our results may be the basis for clinical trials to evaluate the role of POF as an immunotherapeutic agent in the treatment of EAA.

The Fas receptor (FasR) and the tumour necrosis factor (TNF) receptor 55/60 kDa (TNFR1) are recognized as apoptosis signalling receptors. They are known to be expressed by lymphocytes in association with immune regulation and immunological disorders. This study aimed to investigate the expression of FasR and TNFR1 by alveolar macrophages (AM) in patients with sarcoidosis. Bronchoalveolar lavage (BAL) was performed in 12 patients with active sarcoidosis and 11 control subjects. BAL cells were characterized by monoclonal antibodies using a peroxidase-antiperoxidase method. Both FasR and TNFR1 were expressed on a higher percentage of AM in sarcoidosis with respect to control subjects (mean +/-sem 40.8+/-3.1% versus 14.9+/-1.7%, p

Background: Surfactant protein A (SP-A) and SP-D are clinically established in Japan as serum biomarkers for diagnosing interstitial lung diseases (ILDs). Serum SP-D levels are affected by genetic variants. We conducted the present study to examine whether serum SP-A and/or SP-D levels in healthy subjects (HS) and patients with ILDs differ between populations with different genetic backgrounds. Methods: German subjects (n = 303; 138 patients with idiopathic interstitial pneumonias [IIPs] and 165 HS) and Japanese subjects (n = 369; 94 patients with IIPs and 275 HS) were enrolled. Serum SP-A and SP-D levels were measured using an enzyme-linked immunosorbent assay, and four single-nucleotide polymorphisms (SNPs) in the SFTPD gene were genotyped using genomic DNA extracted from blood samples. Results: In both the German and Japanese cohorts, serum SP-A and SP-D levels were significantly higher in patients with IIPs than in HS. There were no significant differences in SP-A levels between the German and Japanese cohorts; however, we found that serum SP-D levels were significantly higher in the German cohort, both in patients with IIPs and in HS (p < 0.001 and p = 0.005, respectively). Furthermore, the genotype distributions of the four SNPs in the SFTPD gene (rs721917, rs1998374, rs2243639, and rs3088308) were significantly different between German and Japanese cohorts (p < 0.001, p, This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and Arbeitsgemeinschaft zur Förderung der Pneumologie an der Ruhrlandklinik (AFPR).

Background CCL18 is a CC chemokine produced mainly by antigen-presenting cells, and is chemotactic predominantly for T-lymphocytes. CCL18 can stimulate pulmonary fibroblasts and increase the collagen production in vitro. Objectives This study aimed to compare the CCL18 levels in a variety of human biological fluids between various interstitial lung diseases (ILDs), and to reveal potential correlations with BAL cell differentials. Methods Serum and bronchoalveolar lavage fluid (BALF) samples were collected from 199 patients with idiopathic pulmonary fibrosis (IPF), idiopathic non-specific interstitial pneumonia (iNSIP), respiratory bronchiolitis interstitial lung disease/desquamative interstitial pneumonia (RB-ILD/DIP), cryptogenic organizing pneumonia (COP), hypersensitivity pneumonitis (HP) or sarcoidosis. Alveolar macrophage (AM) culture was performed in 44 patients with IPF, iNSIP, COP, HP, sarcoidosis or non-ILDs. The CCL18 levels in serum, BALF and AM culture supernatant were measured with ELISA. Results Both serum and BALF CCL18 levels in all ILDs were higher than in controls (all p < 0.005). In HP, CCL18 serum levels were the highest of all ILDs, and its BALF levels were significantly higher than in other ILDs except iNSIP. The BALF CCL18 levels markedly correlated with BAL cell differentials, especially with the percentage of BAL lymphocytes. In AM culture supernatant, the spontaneous CCL18 production was higher in HP and COP than in IPF and controls. Conclusion CCL18 levels in serum, BALF and AM culture supernatant are markedly increased in various inflammatory and fibrotic ILDs. However, the CCL18 level being highest in HP among the investigated ILDs suggests that CCL18 may be more profoundly involved in inflammatory immune responses. © 2013 Elsevier Ltd. All rights reserved.