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Introduction: Microbiome imbalance could be associated with the occurrence of acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF). Aims: To investigate the association between oral bacterial flora and the occurrence of AE-IPF. Methods: We have prospectively collected 142 patients with IPF, 264 healthy subjects and 31 patients with bacterial pneumonia. Baseline serum antibody levels against 17 bacteria were measured by ELISA. The correlations between these values and the occurrence of AE-IPF were evaluated. An imbalance in the number of bacteria was defined as the number of pathogenic bacteria minus that of inhibitory bacteria. Results: We have identified 7 bacteria potentially promoting AE-IPF (Porphyromonas type I, III, IV, V, Helicobacter, Campylobacter, and Tannerella) and 3 bacteria potentially inhibiting AE-IPF (Prevotella, Fusobacterium, and Aggregatibacter). The baseline number of pathogenic bacteria was increased in patients with AE-IPF compared with those without AE-IPF (4.1±2.3 vs 2.9±2.4, p=0.0049), whereas the number of inhibitory bacteria was decreased (1.1±1.1 vs 1.6±1.1, p=0.007). The imbalance in the number of bacteria was increased in patients with AE-IPF (3.0±2.2 vs 1.3±2.2, p Conclusions: The imbalance of pathogenic and inhibitory bacteria was associated with the occurrence of AE-IPF.

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.

Introduction: Microbiome imbalance could be one of the risk factors for acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF). However, little is known about the role of the oral bacterial flora in AE-IPF. Aims: To investigate the significance of oral bacterial flora as a risk factor for AE-IPF. Methods: A total of 142 patients with IPF (77 German and 65 Japanese), 264 healthy subjects (164 German and 100 Japanese) and 31 Japanese patients with bacterial pneumonia were prospectively collected. Baseline serum levels of antibodies against 16 bacteria of the oral flora were measured by ELISA. The correlations between baseline serum levels of these antibodies and the incidence of AE-IPF were evaluated. Results: Baseline serum levels of Porphyromonas type III, IV, V, and Fusobacterium antibodies were significantly higher in the Japanese IPF patients compared with the other 4 groups (Japanese healthy, Japanese pneumonia, German healthy and German IPF). Baseline serum levels of several antibacterial antibodies (Porphyromonas type I, III, IV, V, Tannerella, and Campylobacter) were positively correlated with the occurrence of AE-IPF (AUC 0.554-0.608), whereas others (Aggregatibacter, Prevotella and Fusobcterium) were negatively correlated (AUC 0.400-0.441). Among them, the ratio of Tannerella to Aggregatibacter was significantly associated with the occurrence of AE-IPF (AUC 0.520-0.747, p=0.015). In the multivariate analysis, the ratio of Tannerella to Aggregatibacter > 0.72 and Japanese ethnicity were independent risk factors for AE-IPF (hazard ratio 2.23, p=0.03; 4.48, p=0.0002). Conclusions: An imbalance in the oral bacterial flora could be a risk factor for AE-IPF.

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.

Background and objective A single nucleotide polymorphism (SNP) rs35705950 in the promoter of Mucin 5B (MUC5B) has been reported to be associated with idiopathic pulmonary fibrosis (IPF) mainly in Caucasian populations. This study was conducted to confirm the association between rs35705950 and IPF in a Japanese population. Methods Genomic DNA was extracted from blood samples in 384 Japanese and 137 German subjects, and rs35705950 was detected by commercially available genotyping assay. Results The genotype distributions of rs35705950 in Japanese patients with IPF, nonspecific interstitial pneumonia (NSIP) and healthy subjects (HS) were significantly different from those in the German counterparts (P

Background: Anti-DFS70 antibody, corresponding to the dense fine ANA speckled pattern at indirect immunofluorescence (IIF) in HEp-2 substrates, have been detected in a variety of chronic inflammatory conditions, cancer and in healthy individuals, but only in a small percentage of systemic ANA- associated rheumatic disease (SARD). In a previous study we found that anti-DFS70 positivity can be used to largely exclude SARD in patients with ILD and ANA positivity (IPAF). Aim of this study: To investigate whether serum anti-DFS70 levels correlate with clinical parameters in ILD. Patients and Methods: 260 patients with ILD (100 NSIP and 160 IPF), 49 healthy controls (HC) and 35 scleroderma-ILD (SSc-ILD) patients as negative control were studied. Serum anti-DFS70 at baseline was measured by ELISA (MBL Co., Ltd., Japan). Results: During 24 months of follow-up, 37 ILD patients (34 NSIP and 3 IPF) developed connective tissue disease (CTD). Anti-DFS70 levels in the ILD group were significantly lower than in HC (119 vs 320 U/ml, p In patients with idiopathic NSIP (iNSIP) anti-DFS70 titers did not differ from those of IPF patients or CTD-NSIP (119 vs 123 vs 101 U/ml, respectively). In iNSIP patients, anti-DFS70 titers inversely correlated with FVC (%pred.) (r=-0.320, p Conclusions: iNSIP patients with higher anti-DFS70 levels showed more impairment of lung function and gas exchange than CTD-NSIP.

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.

Die bronchoalveolare Lavage (BAL) bietet heute, neben einem hohen Sicherheitsprofil, eine Fulle von Befunden, die als diagnostische Marker eine Rolle spielen. Sie ist bei allen unklaren interstitiellen Lungenerkrankungen, auch bei solchen mit infektioser Genese, zur diagnostischen Standarduntersuchung geworden. Bei relativ spezifischen Befunden wie der pulmonalen Alveolarproteinose, Langerhans-Zell-Histiozytose, der diffusen alveolaren Blutung, diffusen malignen Infiltraten oder bei Staubexposition ist eine Lungenbiopsie meist nicht mehr erforderlich. Bei anderen Erkrankungen wie der Sarkoidose oder der exogen-allergischen Alveolitis kann bei entsprechender Klinik und radiologischem Befund das differenzialdiagnostische Spektrum stark eingeschrankt werden und auch in vielen Fallen die Diagnose ohne Biopsie gestellt werden. Bei der idiopathischen Lungenfibrose tragt die BAL zur Ausschlussdiagnostik, vor allem von Erkrankungen mit einer lymphozytaren BAL, bei.

Background Hypersensitivity pneumonitis (HP) is characterized by a granulomatous inflammation and may show various forms of clinical presentation, such as the acute, subacute, and chronic forms. The T H 1-associated cytokines interleukin (IL) 12 and IL-18 and tumor necrosis factor α (TNF-α) may be involved in the pathogenesis of both the acute and chronic forms of HP. Objective To compare the release of IL-12, IL-18, and TNF-α from bronchoalveolar lavage (BAL) macrophages in these 2 forms of HP. Methods Patients underwent BAL 0 to 6 days after the last antigen exposure. Alveolar macrophages (AMs) from BAL in 6 patients with acute HP, 16 with chronic HP, and 11 controls were cultured for 24 hours. Cytokines in the culture supernatants were measured by enzyme-linked immunosorbent assay. Results The production of IL-12, IL-18, and TNF-α by AMs was increased in patients with both acute and chronic forms in either the absence or presence of lipopolysaccharide compared with controls. The levels of IL-12, IL-18, and TNF-α showed no difference between patients with acute and chronic HP. The spontaneous production of IL-12, IL-18, and TNF-α did not correlate with the CD4/CD8 ratio in BAL. The spontaneous and lipopolysaccharide-stimulated release of IL-12 showed a positive correlation with the percentage of lymphocytes ( r = .470, P = .03; r = .496, P = .02; respectively) in BAL. Conclusions This study demonstrates that an increased release of IL-12, IL-18, and TNF-α by AMs is associated with both the acute and chronic forms of HP.

This article reviews the changes in bronchoalveolar lavage (BAL) cytology and cell differentials in some of the rarer interstitial lung diseases. In a few of these diseases BAL has a diagnostic value and can replace lung biopsy. In pulmonary Langerhans cell histiocytosis the characteristic diagnostic finding is an increase in CD1 + Langerhans cells greater than 4% of total cells. The sensitivity of this cutoff value is low because only approximately 50% of patients show this elevation. In pulmonary alveolar proteinosis, the sensitivity of a diagnostic BAL is almost 100%, and the characteristic finding of milky and turbid fluid on gross examination and the characteristic findings with acellular globules that stain pink with PAS (periodic-acid-Schiff), along with abnormal foamy macrophages and a characteristic dirty background obviates the need for lung biopsy. In diffuse alveolar hemorrhage, BAL is the method of choice to diagnose the alveolar bleeding by showing free red blood cells and hemosiderin-laden, iron-positive macrophages. The underlying disorder has to be identified by history and clinical and laboratory tests. In eosinophilic lung disease, the diagnosis can be made if the BAL cell differentials show 25% or more eosinophils. In collagen vascular disease-associated lung fibrosis, the precise role of BAL in assessment and monitoring disease remains unclear. In drug-induced interstitial lung disease BAL may support a certain clinical/pathological pattern of lung involvement and is helpful for exclusion of other diseases, such as malignancies with pulmonary metastasis, heart disease with pulmonary congestion, or infections. The same is true for radiation-induced lung injury.

The diagnosis of sarcoidosis cannot be established by the presence of granuloma in a single organ, such as the skin. Therefore, the patient with potential cutaneous sarcoidosis should undergo a systemic evaluation for two reasons: to help confirm the diagnosis, and to identify other potential problems. This article reviews the recommended testing in patients with suspected or known sarcoidosis. The evaluation leads to a wide number of organs being assessed by either history, physical examination or routine laboratory investigations. The major organs routinely assessed are the lungs, the eyes, the liver and the heart.

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 and Objectives: In immunocytochemical preparations of bronchoalveolar lavage (BAL) cells from patients with extrinsic allergic alveolitis (EAA), we observed the presence of alveolar macrophages with cockade-like structures in their cytoplasm (cockade+ alveolar macrophages). These cockade+ alveolar macrophages may reflect a subpopulation of alveolar macrophages which may show a different predominance in various interstitial lung diseases. In this study we aimed to compare the frequency of cockade+ alveolar macrophages in patients with EAA (n = 14) with the results obtained in patients with sarcoidosis (n = 11), idiopathic interstitial pneumonia (IIP; n = 10) and control subjects (n = 8). We also investigated the expression of the transferrin receptor CD71 on cockade+ alveolar macrophages. Methods: In BAL fluid, the total number of cells and differential counts were determined, and immunocytologic examinations of macrophages and lymphocytes were done using monoclonal antibodies. The percentage of cockade+ alveolar macrophages was determined by counting 300 macrophages in the CD20 field of an immunocytochemical slide. Results: The percentage of cockade+ alveolar macrophages was significantly higher in the EAA group (36 ± 9%) compared to patients with sarcoidosis (12 ± 5%) or IIP (11 ± 10%) and control subjects (3 ± 1%; p < 0.001). The proportion of CD71+ alveolar macrophages was significantly lower in EAA than in the other groups (p < 0.01), and the CD71 antigen was expressed on a significantly lower proportion of cockade+ alveolar macrophages compared to cockade– alveolar macrophages in EAA (p < 0.001). Conclusion: We conclude that cockade+ alveolar macrophages could play a role in the pathogenesis and differential diagnosis EAA.

Study objectives Pentoxifylline (POF) has been shown to suppress the cytokine production from lipopolysaccharide (LPS)-stimulated monocytes/alveolar macrophages (AMs). Sarcoidosis is a granulomatous disease that is driven by the action of tumor necrosis factor (TNF)-α and other proinflammatory cytokines. In this study, we aimed to investigate the effects of POF on the production of TNF-α, interleukin (IL)-1β, IL-6, IL-8, IL-10, and the soluble TNF receptors (sTNFRs) 1 and 2 from AMs in sarcoidosis, and we also compared them with those of dexamethasone (DEX). Methods AMs from 14 patients with sarcoidosis were cultured for 24 h with RPMI medium alone or with LPS (100 ng/mL), and with POF at concentrations of 0.01, 0.1, and 1 mmol/L, or with 0.1 mmol/L DEX. Cytokines in the culture supernatants were analyzed by enzyme-linked immunosorbent assay. Results The results showed that POF induced a dose-dependent suppression of the spontaneous TNF-α release from AMs in sarcoidosis (p Conclusions Compared with DEX, POF may improve therapeutic regimens in patients with sarcoidosis either by sparing or by replacing corticosteroids. However, the precise clinical value of POF in the treatment of sarcoidosis and other lung diseases will have to be determined in further clinical trials.

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.

A single nucleotide polymorphism (SNP) rs35705950 in the promoter of Mucin 5B (MUC5B) has been reported to be associated with idiopathic pulmonary fibrosis (IPF) mainly in Caucasian populations. This study was conducted to confirm the association between rs35705950 and IPF in a Japanese population.Genomic DNA was extracted from blood samples in 384 Japanese and 137 German subjects, and rs35705950 was detected by commercially available genotyping assay.The genotype distributions of rs35705950 in Japanese patients with IPF, nonspecific interstitial pneumonia (NSIP) and healthy subjects (HS) were significantly different from those in the German counterparts (P0.001, P0.001 and P = 0.010, respectively). The rs35705950 T allele frequencies in patients with IPF, NSIP and HS were 3.4%, 1.7% and 0.8%, respectively in the Japanese, while they were 33.1%, 27.4% and 4.3%, respectively in the German cohort. The T allele frequencies in patients with IPF were significantly higher than those in HS both in the Japanese (P = 0.031) and German (P0.001) cohorts.The association between rs35705950 and IPF was also present in this Japanese cohort, but was not as strong as the German counterpart. To our knowledge, this is the first study to successfully validate the association between rs35705950 and IPF in a Japanese ethnicity.

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.

Bronchoalveolar lavage (BAL) has become a standard diagnostic procedure for the majority of patients with interstitial disease. The technique is safe, minimally invasive, and reveals specific information in some disorders such as pulmonary alveolar proteinosis, Langerhans cell histiocytosis, alveolar hemorrhage, malignant infiltrates, or dust exposure. Here BAL can often replace lung biopsy. The results of BAL cell differentials with a lymphocytic, a neutrophilic, an eosinophilic, or a mixed cellular pattern can be used as an adjunct to diagnosis. If a BAL finding is compatible with the suspected diagnosis in the context of an appropriate disease history and clinical and radiologic findings, this can then be sufficient for disease confirmation. Recent research focused on the pathogenesis of various types of interstitial lung disorders. In this regard, BAL findings contributed to the characterization of idiopathic pulmonary fibrosis (IPF) as a condition with a predominant T-helper-2 (Th2) cytokine profile, whereas BAL findings in sarcoidosis and hypersensitivity pneumonitis are characterized by a Th1-dominant profile. The clinical value of BAL to assess the activity of disease processes and to provide prognostic information is still under debate. The routine performance of serial BAL is not recommended at present.

Background Soluble TNF receptors (sTNFR1 and sTNFR2) are inhibitors of TNF and can block TNF bioactivity. TNF plays an important role in the development of extrinsic allergic alveolitis (EAA). Objective To evaluate whether sTNFR1 and sTNFR2 are locally increased in EAA. Methods We measured sTNFR1 and sTNFR2 in bronchoalveolar lavage fluid (BALF) and serum from nine EAA patients and 11 control subjects using an ELISA method. Results BALF sTNFR1 and sTNFR2 levels were 0.24 ± 0.04 ng/mL and 0.59 ± 0.16 ng/mL in EAA patients, and thus significantly elevated in comparison with the controls (0.13 ± 0.02 ng/mL and 0.08 ± 0.04 ng/mL, both P

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.

The aim of this study was to investigate the relationship between release of tumor necrosis factor-alpha (TNF-alpha) by alveolar macrophages (AM) and the phenotypic characteristics of bronchoalveolar lavage (BAL) cells in sarcoidosis. We studied the spontaneous release of TNF-alpha by AM in vitro and the phenotypic characteristics of freshly recovered BAL T-cells and AM in 31 individuals (13 with active sarcoidosis, nine with inactive sarcoidosis, and nine normal controls). TNF-alpha was measured by enzyme-linked immunosorbent assay (ELISA) in supernatants from unstimulated AM after 24 h culture. Phenotypic markers of BAL cells were determined by an immunocytochemical assay. AM of patients with active sarcoidosis released more TNF-alpha (1,355 +/- 133 pg/ml/ 10(6) AM/24 h) than those of the inactive group (651 +/- 142 pg/ml/10(6) AM/24 h) or the normal controls (425 +/- 121 pg/ml/10(6) AM/24 h), with p0.001 for both comparisons. The amount of TNF-alpha released correlated positively with the percentage expression of CD4 (r = 0.72) and CD25 (r = 0.70) by lymphocytes, and of CD14 (r = 0.63), VLA-4 (r = 0.59), FRD1 (r = 0.67) and 27E10 (r = 0.67) by AM, with p0.001 for all correlations. In conclusion, this relationship suggests that these antigens may be considered as cellular activation markers, and that some of these AM antigens may indirectly characterize the AM phenotype that is capable of producing TNF-alpha.

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.