Genetic Programs Between Steroid-Sensitive and Steroid-Insensitive Interstitial Lung Disease.

The effectiveness of corticosteroids (GCs) varies greatly in interstitial lung diseases (ILDs). In this study, we aimed to compare the gene expression profiles of patients with cryptogenic organizing pneumonia (COP), idiopathic pulmonary fibrosis (IPF), and non-specific interstitial pneumonia (NSIP) and identify the molecules and pathways responsible for GCs sensitivity in ILDs. Three datasets (GSE21411, GSE47460, and GSE32537) were selected. Differentially expressed genes (DEGs) among COP, IPF, NSIP, and healthy control (CTRL) groups were identified. Functional enrichment analysis and protein-protein interaction network analysis were performed to examine the potential functions of DEGs. There were 128 DEGs when COP versus CTRL, 257 DEGs when IPF versus CTRL, 205 DEGs when NSIP versus CTRL, and 270 DEGs when COP versus IPF. The DEGs in different ILDs groups were mainly enriched in the inflammatory response. Further pathway analysis showed that "interleukin (IL)-17 signaling pathway" (hsa04657) and "tumor necrosis factor (TNF) signaling pathway" were associated with different types of ILDs. A total of 10 genes associated with inflammatory response were identified as hub genes and their expression levels in the IPF group were higher than those in the COP group. Finally, we identified two GCs' response-related differently expressed genes (FOSL1 and DDIT4). Our bioinformatics analysis demonstrated that the inflammatory response played a pathogenic role in the progression of ILDs. We also illustrated that the inflammatory reaction was more severe in the IPF group compared to the COP group and identified two GCs' response-related differently expressed genes (FOSL1 and DDIT4) in ILDs.

Cell-Specific Response of NSIP- and IPF-Derived Fibroblasts to the Modification of the Elasticity, Biological Properties, and 3D Architecture of the Substrate.

The fibrotic fibroblasts derived from idiopathic pulmonary fibrosis (IPF) and nonspecific interstitial pneumonia (NSIP) are surrounded by specific environments, characterized by increased stiffness, aberrant extracellular matrix (ECM) composition, and altered lung architecture. The presented research was aimed at investigating the effect of biological, physical, and topographical modification of the substrate on the properties of IPF- and NSIP-derived fibroblasts, and searching for the parameters enabling their identification. Soft and stiff polydimethylsiloxane (PDMS) was chosen for the basic substrates, the properties of which were subsequently tuned. To obtain the biological modification of the substrates, they were covered with ECM proteins, laminin, fibronectin, and collagen. The substrates that mimicked the 3D structure of the lungs were prepared using two approaches, resulting in porous structures that resemble natural lung architecture and honeycomb patterns, typical of IPF tissue. The growth of cells on soft and stiff PDMS covered with proteins, traced using fluorescence microscopy, confirmed an altered behavior of healthy and IPF- and NSIP-derived fibroblasts in response to the modified substrate properties, enabling their identification. In turn, differences in the mechanical properties of healthy and fibrotic fibroblasts, determined using atomic force microscopy working in force spectroscopy mode, as well as their growth on 3D-patterned substrates were not sufficient to discriminate between cell lines.

Bimodal fibrosis in a novel mouse model of bleomycin-induced usual interstitial pneumonia.

Idiopathic pulmonary fibrosis is pathologically represented by usual interstitial pneumonia (UIP). Conventional bleomycin models used to study pathogenic mechanisms of pulmonary fibrosis display transient inflammation and fibrosis, so their relevance to UIP is limited. We developed a novel chronic induced-UIP (iUIP) model, inducing fibrosis in D1CC×D1BC transgenic mice by intra-tracheal instillation of bleomycin mixed with microbubbles followed by sonoporation (BMS). A bimodal fibrotic lung disease was observed over 14 wk, with an acute phase similar to nonspecific interstitial pneumonia (NSIP), followed by partial remission and a chronic fibrotic phase with honeycombing similar to UIP. In this secondary phase, we observed poor vascularization despite elevated PDGFRß expression. γ2PF- and MMP7-positive epithelial cells, consistent with an invasive phenotype, were predominantly adjacent to fibrotic areas. Most invasive cells were Scgb1a1 and/or Krt5 positive. This iUIP mouse model displays key features of idiopathic pulmonary fibrosis and has identified potential mechanisms contributing to the onset of NSIP and progression to UIP. The model will provide a useful tool for the assessment of therapeutic interventions to oppose acute and chronic fibrosis.

Delayed contrast dynamics as marker of regional impairment in pulmonary fibrosis using 5D MRI - a pilot study.

OBJECTIVE: To analyse delayed contrast dynamics of fibrotic lesions in interstitial lung disease (ILD) using five dimensional (5D) MRI and to correlate contrast dynamics with disease severity. METHODS: 20 patients (mean age: 71 years; M:F, 13:7), with chronic fibrosing ILD: n = 12 idiopathic pulmonary fibrosis (IPF) and n = 8 non-IPF, underwent thin-section multislice CT as part of the standard diagnostic workup and additionally MRI of the lung. 2 min after contrast injection, a radial gradient echo sequence with golden-angle spacing was acquired during 5 min of free-breathing, followed by 5D image reconstruction. Disease was categorized as severe or non-severe according to CT morphological regional severity. For each patient, 10 lesions were analysed. RESULTS: IPF lesions showed later peak enhancement compared to non-IPF (severe: p = 0.01, non-severe: p = 0.003). Severe lesions showed later peak enhancement compared to non-severe lesions, in non-IPF (p = 0.04), but not in IPF (p = 0.35). There was a tendency towards higher accumulation and washout rates in IPF compared to non-IPF in non-severe disease. Severe lesions had lower washout rate than non-severe ones in both IPF (p = 0.003) and non-IPF (p = 0.005). Continuous contrast agent accumulation, without washout, was found only in IPF lesions. CONCLUSIONS: Contrast agent dynamics are influenced by type and severity of pulmonary fibrosis, which might enable a more thorough characterisation of disease burden. The regional impairment is of particular interest in the context of antifibrotic treatments and was characterised using a non-invasive, non-irradiating, free-breathing method. ADVANCES IN KNOWLEDGE: Delayed contrast enhancement patterns allow the assessment of regional lung impairment which could represent different disease stages or phenotypes in ILD.

A subpopulation of airway epithelial cells that express hepatocyte nuclear factor 4α - its implication in the development of non-terminal respiratory unit-type lung adenocarcinoma.

A normal counterpart and precancerous lesion that non-terminal respiratory unit (TRU) lung adenocarcinomas (LADCs) develop from have not been clarified. Non-TRU LADCs specifically express hepatocyte nuclear factor 4α (HNF4α). Thus, we have been interested in airway epithelial cells that express HNF4α as the potential precursor of non-TRU LADC. The purposes of the present study are to report the frequency and distribution of HNF4α-expressing cells at the different airway levels, and to investigate the potential significance of the expression of HNF4α in the histogenesis of non-TRU LADC with a special reference to the relationship to bronchiolar metaplasia in idiopathic interstitial pneumonia. We herein identified a minor subpopulation of epithelial cells that express HNF4α in a physiological state. This subpopulation was mainly located in the terminal bronchioles and had the appearance of ciliated cells, which were mutually exclusive from Clara cells and others that strongly expressed thyroid transcription factor 1. Furthermore, the expression of HNF4α was similar in bronchiolar metaplastic lesions and the terminal bronchioles, and some of the metaplastic lesions showed an unequivocally higher frequency and expression level of HNF4α, which was comparable to non-TRU LADC. In summary, this is the first study to describe a subpopulation of ciliated cells that express HNF4α as a potential normal counterpart for non-TRU LADCs and suggests that bronchiolar metaplastic lesions that strongly express HNF4α are a precancerous lesion for non-TRU LADCs.

Calgranulin B and KL-6 in Bronchoalveolar Lavage of Patients with IPF and NSIP.

Idiopathic pulmonary fibrosis (IPF) and non-specific interstitial pneumonia (NSIP) are the most frequent idiopathic interstitial pneumonias. The aim of this study was to evaluate concentrations of calgranulin B and Krebs von den Lungen 6 (KL-6) in bronchoalveolar lavage (BAL) fluid of patients with IPF and idiopathic NSIP (i-NSIP) with fibrotic pattern. Thirty patients with IPF (68.73 ± 8.63 years), 30 with i-NSIP (68.33 ± 7.45 years), and healthy controls were included in the study. Calgranulin B and KL-6 both proved to be significantly higher in BAL of IPF and i-NSIP patients than in healthy controls (p < 0.05). Calgranulin B showed several significant correlations with functional parameters (oxygen demand at rest, 6-min walking test (6MWT), and PFTs); KL-6 was correlated with oxygen demand at rest and during 6MWT. Patients with higher concentrations of both biomarkers (> 75th percentile) had more advanced disease with lower values of FEV1%, FVC%, RV%, TLC%, DLCO% of predicted, distance walked in 6MWT, and BAL neutrophil percentage. Calgranulin B and KL-6 in BAL proved to be reliable biomarkers of IPF and i-NSIP and to have prognostic meaning, discriminating severe and advanced patients. The combination of the two biomarkers can facilitate the stratification of severity.

Systematic review of drug effects in humans and models with surfactant-processing disease.

Fibrotic interstitial pneumonias are a group of rare diseases characterised by distortion of lung interstitium. Patients with mutations in surfactant-processing genes, such as surfactant protein C (SFTPC), surfactant protein A1 and A2 (SFTPA1 and A2), ATP binding cassette A3 (ABCA3) and Hermansky-Pudlak syndrome (HPS1, 2 and 4), develop progressive pulmonary fibrosis, often culminating in fatal respiratory insufficiency. Although many mutations have been described, little is known about the optimal treatment strategy for fibrotic interstitial pneumonia patients with surfactant-processing mutations.We performed a systematic literature review of studies that described a drug effect in patients, cell or mouse models with a surfactant-processing mutation. In total, 73 articles were selected, consisting of 55 interstitial lung disease case reports/series, two clinical trials and 16 cell or mouse studies. Clinical effect parameters included lung function, radiological characteristics and clinical symptoms, while experimental outcome parameters included chemokine/cytokine expression, surfactant trafficking, necrosis and apoptosis. SP600125, a c-jun N-terminal kinase (JNK) inhibitor, hydroxychloroquine and 4-phenylbutyric acid were most frequently studied in disease models and lead to variable outcomes, suggesting that outcome is mutation dependent.This systematic review summarises effect parameters for future studies on surfactant-processing disorders in disease models and provides directions for future trials in affected patients.

Expression Profile of Six RNA-Binding Proteins in Pulmonary Sarcoidosis.

BACKGROUND: Sarcoidosis is characterised by up-regulation of cytokines and chemokine ligands/receptors and proteolytic enzymes. This pro-inflammatory profile is regulated post-transcriptionally by RNA-binding proteins (RBPs). We investigated in vivo expression of six RBPs (AUF1, HuR, NCL, TIA, TIAR, PCBP2) and two inhibitors of proteolytic enzymes (RECK, PTEN) in pulmonary sarcoidosis and compared it to the expression in four control groups of healthy individuals and patients with other respiratory diseases: chronic obstructive pulmonary disease (COPD), asthma and idiopathic interstitial pneumonias (IIPs). METHODS: RT-PCR was used to quantify the mRNAs in bronchoalveolar (BA) cells obtained from 50 sarcoidosis patients, 23 healthy controls, 30 COPD, 19 asthmatic and 19 IIPs patients. Flow cytometry was used to assess intracellular protein expression of AUF1 and HuR in peripheral blood T lymphocytes (PBTLs) obtained from 9 sarcoidosis patients and 6 healthy controls. RESULTS: Taking the stringent conditions for multiple comparisons into consideration, we consistently observed in the primary analysis including all patients regardless of smoking status as well as in the subsequent sub-analysis limited for never smokers that the BA mRNA expression of AUF1 (p<0.001), TIA (p<0.001), NCL (p<0.01) and RECK (p<0.05) was decreased in sarcoidosis compared to healthy controls. TIA mRNA was also decreased in sarcoidosis compared to both obstructive pulmonary diseases (COPD and asthma; p<0.001) but not compared to IIPs. There were several positive correlations between RECK mRNA and RBP mRNAs in BA cells. Also sarcoidosis CD3+, CD4+ and CD8+ PBTLs displayed lower mean fluorescence intensity of AUF1 (p≤0.02) and HuR (p≤0.03) proteins than control healthy PBTLs. CONCLUSION: mRNA expressions of three RBPs (AUF1, TIA and NCL) and their potential target mRNA encoding RECK in BA cells and additionally protein expression of AUF1 and HuR in PBTLs were down-regulated in our sarcoidosis patients compared to healthy individuals. Its significance, e.g. for stability of mRNAs encoding pro-inflammatory factors, should be further explored in sarcoidosis.

Lung diffusing capacity for nitric oxide as a marker of fibrotic changes in idiopathic interstitial pneumonias.

Lung diffusing capacity for carbon monoxide (DLCO) is decreased in both usual interstitial pneumonia-idiopathic pulmonary fibrosis (UIP-IPF) and nonspecific interstitial pneumonia (NSIP), but is moderately related to computed tomography (CT)-determined fibrotic changes. This may be due to the relative insensitivity of DLCO to changes in alveolar membrane diffusive conductance (DMCO). The purpose of this study was to determine whether measurement of lung diffusing capacity for nitric oxide (DLNO) better reflects fibrotic changes than DLCO DLNO-DLCO were measured simultaneously in 30 patients with UIP-IPF and 30 with NSIP. Eighty-one matched healthy subjects served as a control group. The amount of pulmonary fibrosis was estimated by CT volumetric analysis of visually bounded areas showing reticular opacities and honeycombing. DMCO and pulmonary capillary volume (VC) were calculated. DLNO was below the lower limit of normal in all patients irrespective of extent and nature of disease, whereas DLCO was within the normal range in a nonnegligible number of patients. Both DLNO and DLCO were significantly correlated with visual assessment of fibrosis but DLNO more closely than DLCO DMCO was also below the lower limit of normal in all UIP-IPF and NSIP patients and significantly correlated with fibrosis extent in both diseases, whereas VC was weakly correlated with fibrosis in UIP-IPF and uncorrelated in NSIP, with normal values in half of patients. In conclusion, measurement of DLNO may provide a more sensitive evaluation of fibrotic changes than DLCO in either UIP-IPF or NSIP, because it better reflects DMCO.

Increased ectodomain shedding of cell adhesion molecule 1 as a cause of type II alveolar epithelial cell apoptosis in patients with idiopathic interstitial pneumonia.

BACKGROUND: Lung alveolar epithelial cell (AEC) apoptosis has attracted attention as an early pathogenic event in the development of idiopathic interstitial pneumonia (IIP); however, the causative mechanism remains unclear. Cell adhesion molecule 1 (CADM1) is an AEC adhesion molecule in the immunoglobulin superfamily. It generates a membrane-associated C-terminal fragment, αCTF, through protease-mediated ectodomain shedding, termed α-shedding. Increased CADM1 α-shedding contributes to AEC apoptosis in emphysematous lungs. METHODS: Formalin-fixed, paraffin-embedded lung lobes (n = 39) from 36 autopsied patients with IIP were classified as acute IIP (n = 10), fibrosing-type nonspecific IIP (f-NSIP, n = 10), cryptogenic organizing IIP (n = 9), and usual IIP (n = 10). CADM1 expression in the lung sections was examined by western blotting and compared with control lungs (n = 10). The rate of CADM1 α-shedding was calculated as the relative amount of αCTF to full-length CADM1, and the full-length CADM1 level was estimated per epithelial cell by normalization to cytokeratin 7, a lung epithelial marker. Apoptotic AECs were detected by immunohistochemistry for single-stranded DNA (ssDNA). NCI-H441 and A549 human lung epithelial cells were transfected with small interfering RNA (siRNA) to silence CADM1 expression and analyzed by terminal nucleotide nick end labeling assays. RESULTS: The rate of CADM1 α-shedding was higher in all IIP subtypes than in the control (P ≤ 0.019), and the full-length CADM1 level was lower in f-NSIP (P = 0.007). The α-shedding rate and full-length CADM1 level were correlated with each other (P = 0.015) and with the proportion of ssDNA-positive AECs (P ≤ 0.024). NCI-H441 cells transfected with siRNA exhibited a 61 % lower rate of expression of full-length CADM1 and a 17-fold increased proportion of apoptotic cells. Similar results were obtained with A549 cells. CONCLUSIONS: CADM1 α-shedding appeared to be increased in all four IIP subtypes and consequently contributed to AEC apoptosis by decreasing the full-length CADM1 level. This mechanism particularly impacted f-NSIP. The molecular mechanism causing AEC apoptosis may be similar between IIP and emphysema.

Innate immunity alterations in idiopathic interstitial pneumonias and rheumatoid arthritis-associated interstitial lung diseases.

BACKGROUND: This is a prospective cohort study elucidating innate immunity in idiopathic pulmonary fibrosis (IPF), cryptogenic organizing pneumonia (COP), rheumatoid arthritis-associated usual interstitial pneumonia (RA-UIP) and RA-associated non specific interstitial pneumonia (RA-NSIP). METHODS: 23 IPF subjects, 9 COP subjects, 5 RA-UIP subjects, 8 RA-NSIP subjects were enrolled. 10 subjects were excluded. 19 healthy subjects served as controls. Blood and bronchoalveolar lavage (BAL) were obtained. Natural killer (NK) and NKT cells, NK cells apoptosis and the expression of triggering receptor expressed on myeloid cells type 1 (TREM-1) were assessed. Tumor necrosis factor-α (TNF-α) production was measured in cell cultures after stimulation with lipopolysaccharide endotoxin (LPS) and Pam3CysSK3, and in BAL. Surface expression of Toll-like receptors (TLR) 2 and 4 on peripheral blood monocytes (PBMC's) and circulating NK cells was also assessed. RESULTS: RA-NSIP had low blood NKs, marginally insignificant (p=0.07). These NKs poorly produced TNF-α after LPS stimulation. TLR's expression on NK cells was similar throughout disease groups and controls. PBMC's mainly from IPF patients exhibited low TNF-α production after LPS stimulation but not after Pam3CysSK3 stimulation, while TLR4 expression on PBMC's was found normal in all study groups. TLR2 expression on PBMC's was increased in IPF, but mainly in COP, RA-UIP and RA-NSIP (p=0.015). TREM-1 expression was significant on COP monocytes and on COP neutrophils versus controls. RA-NSIP monocytes also exhibited TREM-1 expression (p=0.07). Decreased TNF-α concentration in BAL was finally observed in IPF and RA-UIP. CONCLUSIONS: Innate immunity in the lungs and the peripheral circulation in IPF and RA-UIP are similar and more fibrotic than in RA-NSIP which is characterized by NK cell depletion and dysfunction. TREM-1 and TLR's likely affect patterns of inflammation in various interstitial lung diseases.

[The expressions and meanings of BMP-7 and TGF-ß in idiopathic pulmonary fibrosis and idiopathic nonspecific interstitial pneumonia].

OBJECTIVE: To investigate the expressions of cytokines in idiopathic pulmonary fibrosis (IPF) and in idiopathic nonspecific interstitial pneumonia (INSIP); To discuss expressions and meanings of bone morphogenetic protein 7 (BMP-7) and transforming growth factor beta (TGF-ß) in IPF and IPF. METHODS: Selected 47 cases of idiopathic interstitial pneumonia (IIP), which were diagnosed by clinical-radiologic-pathologic (CRP), and classified into two groups which were group IPF (25 IPF) and group INSIP (22 INSIP, including 6 cellular pattern and 16 fibrosing pattern). The normal lung tissues were collected as the control group: The fresh tissues were made to detect more than 114 kinds of cytokines' expressions via Oligo GEArray gene microarray technology. Made a tissue microarray which applied EnVision immunohistochemistry technology to detect the expressions of BMP-7 and TGF-ß in both kinds of IIPs. The two groups of patients were followed-up visited around 5 to 8 years and the survival curves were evaluated by Kaplan-Meier method. RESULTS: According to gene microarray results, these two groups were up-expression in TGF family,IL family and TNF family. Most of BMP members were down-expression, in comparison with the control group, except BMP-5,BMP-8B and BMP-15. As the tissue microarray results demonstrated, compared with normal lung tissues,BMP-7 expressed decreasingly in IPF and INSIP groups (t1 = 27.618, P < 0.001; t2 = -12.404, P < 0.001). The expression of IPF were lower than INSIP (t = 5.387, P < 0.05); In INSIP group, patients of cellular pattern expressed BMP-7 more than fibrosing pattern's (t = -5.341, P < 0.001). There were dramatically increasing expressions of TGF-ß in IPF and INSIP, when compared with the control group (t1 = 23.393, P < 0.001; t2 = -13.445, P < 0.001) and it presented negative correlation with BMP-7(group IPF: r = -0.771, P < 0.001; group INSIP: r = -0.729, P < 0.001). (3) Clinical follow-up data showed, the stability(improvement), deterioration and death rates of the group IPF and the group INSIP were, respectively, 0(0%), 2 (8%), 23 (92%) and 15 (68.1%), 3 (13.6%), 4 (18.2%). The results were statistically significant (all P < 0.05). The median survival time of the part with higher BMP-7 expression and the part with relatively lower BMP-7 expression, in the group IPF, were 110.8 and 66.4 months (t = -2.686, P < 0.05); In the group INSIP, were 146.4 and 74.9 months (t = -3.037, P < 0.05). CONCLUSIONS: Cellular cytokines presented different expression profiles in IPF and INSIP patients. Differently with highly activated TGF-ß, BMP-7 was inhibited in IIP patients, which would remind the degree of fibrosis and prognosis of IIP. BMP-7 would be expected to be a novel target for IIP pathogenesis and prognostic research.

Gene expression profile analysis for different idiopathic interstitial pneumonias subtypes.

OBJECTIVE: Idiopathic interstitial pneumonias (IIPs) are a group of diffuse parenchymal lung diseases of unknown etiology characterized by the presence of various degrees of inflammation and fibrosis. We aimed to screen the differences among IIPs subtypes in the gene level by using the microarray expression profiles of normal lung tissue and IIPs tissue for the key genes associated with early diagnosis and treatment of IIPs. METHODS: The gene expression profile of six kinds of IIPs (GSE 32537) subtypes tissue and normal lung tissues were downloaded. The differentially expressed genes (DEGs) in different IIPs subtypes were selected by using the expression profiling. In addition, the screened DEGs were further analyzed by function annotation, pathway analysis, and interaction network analysis to reveal the differences among these subtypes. RESULTS: The gene expression analysis showed that nine genes including SERPINA3, IL1R2, CBS, MGAM, SLCO4A1, S100A12, FPR1, SDR16C5, and MT1X in six subtypes of IIPs were significantly increased. There were significant differences in DEGs among six subtypes of IIPs, and the DEGs of some IIPs subtypes involved in immune, inflammatory response and cell adhesion processes. Moreover, the PPI network analysis indicated that SERPINA3 played an important role in the molecular mechanisms of IIPs. CONCLUSION: This comprehensive description of altered gene expression in different subtypes of IIPs underscores the complex biological processes characteristic of different subtypes of IIPs and may provide a foundation for future research into this devastating disease.

Stromal cells can be cultured and characterized from diagnostic bronchoalveolar fluid samples obtained from patients with various types of interstitial lung diseases.

Increased proliferation of stromal cells is a typical feature encountered in several lung diseases. The objective of this study was to evaluate the success of standardized process for culturing stromal cells from small volumes of diagnostic bronchoalveolar lavage (BAL) fluid samples collected from various patients and to characterize the cultured cells. Small volumes (average 15 mL) of BAL fluid samples were collected from 98 patients who underwent bronchoscopy and BAL for diagnostic purposes. The cells were cultured in vitro and characterized by immunohistochemistry, electron microscopy, flow cytometry and differentiation tests. Cells could be cultured from 62% of samples with the success rate varying with the disease (p = 0.003). Cultures from samples of the patients with idiopathic pulmonary fibrosis, non-specific interstitial pneumonia, connective tissue disorder associated interstitial lung disease and allergic alveolitis had a higher success rate than samples derived from control lung (p < 0.001, 0.03, 0.03 and 0.044, respectively). Smokers had a higher success rate compared with non-smokers (p = 0.035). The cultured cells were fibroblasts or myofibroblasts, but shared also similarities with progenitor-type cells. The study shows that mesenchymal cells can be cultured and studied from small volumes of diagnostic BAL fluid samples from patients with several different types of lung diseases.

Expression of toll-like receptor 2 and 4 is increased in the respiratory epithelial cells of chronic idiopathic interstitial pneumonia patients.

BACKGROUND: Idiopathic interstitial pneumonia (IIP) is characterized by chronic interstitial inflammation and fibrosis. Although mounting evidence has suggested that toll-like receptor (TLR) 2 and TLR4 are involved in the pathogenesis of non-infectious lung injury in vitro and in mouse models, their roles in human IIP remain unknown. METHODS: To address this issue, we investigated the expression patterns of TLR2 and TLR4 by immunohistochemistry in resected lung tissues from patients with usual interstitial pneumonia (UIP) or nonspecific interstitial pneumonia (NSIP). RESULTS: Type II pneumocytes, bronchial epithelial cells (BECs), and alveolar macrophages accounted for the majority of TLR2- and TLR4-expressing cells in both UIP and NSIP. The numbers of TLR2 and TLR4-positive respiratory epithelial (RE) cells, including type II pneumocytes and BECs, were significantly greater in UIP and NSIP than in the control. In particular, the numbers of TLR2-positive RE cells were much greater in UIP than in NSIP. The intensities of TLR2 and TLR4 expression in type II pneumocytes were also significantly stronger in UIP and NSIP than in the control. A comparison of the TLR expression patterns between the fibroblastic and fibrotic areas in UIP indicated that the numbers TLR2 and TLR4-positive RE cells were similar in fibroblastic areas, whereas the TLR2-positive RE cells outnumbered the TLR4-positive RE cells in the fibrotic areas. CONCLUSIONS: This study demonstrates that RE cells over-express TLR2 and TLR4 in the lungs of IIP patients. These findings suggest that high expression of TLRs may contribute to the pathogenesis of human IIP.

Comparative proteome analysis of lung tissue from patients with idiopathic pulmonary fibrosis (IPF), non-specific interstitial pneumonia (NSIP) and organ donors.

Among the idiopathic interstitial pneumonias (IIP), the two entities IPF and NSIP seem to be clinically related, but NSIP has a better outcome. The proteomic signatures which distinguish NSIP from IPF remain still elusive. We therefore performed comparative proteomic analysis of peripheral lung tissue from patients with sporadic IPF (n=14) and fibrotic NSIP (fNSIP, n=8) and organ donors (Controls, n=10), by using the 2-dimensional DIGE technique and MALDI-TOF-MS. The study revealed that the proteomic profiles of IPF and fNSIP were quite similar. Among the upregulated proteins in IPF and fNSIP were stress-induced genes involved in the ER stress-pathway, whereas downregulated proteins in IPF and fNSIP included antiapoptotic factors and antifibrotic molecules. The comparison fNSIP versus IPF indicated upregulation of subunits of the proteasome activator complex and antioxidant enzymes of the peroxiredoxin family. We conclude, that only few protein expression changes exist between IPF and fNSIP, and that epithelial ER- and oxidative stress play a major role in the pathogenesis of both diseases. In contrast to IPF, intracellular clearance of ROS and misfolded protein carbonyls seem to be enhanced in fNSIP due to enhanced expression of antioxidant acting proteins, and may explain the better outcome and survival in patients with fNSIP. BIOLOGICAL SIGNIFICANCE: IPF and fibrotic NSIP (fNSIP) belong to the idiopathic interstitial pneumonias and are usually fatal, but fNSIP has a better outcome. In order to identify molecular mechanisms and differences between IPF and fNSIP, we herein present results of a comparative proteome analysis of IPF, fNSIP and control lung tissue. Our data including validation experiments suggest that ER stress and a general stress-response as well as the decline of antioxidant capacity in alveolar epithelium is key in the pathogenesis of IPF and fNSIP. In addition, we could observe a signature of an increased alveolar epithelial protection against oxidative and ER-stress in fNSIP as compared to IPF, which could help to explain the better outcome of fNSIP patients.

A possible role of galectin-9 in the pulmonary fibrosis of patients with interstitial pneumonia.

BACKGROUND: Galectin-9 (Gal-9) is a ß-galactoside-binding protein that induces biological reactions, such as cell activation, chemoattraction, and apoptosis. We evaluated the role of Gal-9 in the pathogenesis of interstitial pneumonia (IP). METHODS: Gal-9 levels in the bronchoalveolar lavage fluid (BALF) of patients with IP associated with collagen vascular disease (CVD-IP) and with idiopathic interstitial pneumonias (IIPs), including idiopathic pulmonary fibrosis (IPF) and idiopathic nonspecific interstitial pneumonia (idiopathic NSIP), were estimated by enzyme-linked immunosorbent assay. Gal-9 expression in the lungs of these patients was evaluated using immunohistochemistry. The effect of Gal-9 on the growth and apoptosis of human lung fibroblast cells was assessed in vitro. RESULTS: Gal-9 levels in the BALF were significantly higher in patients with CVD-IP than in patients with IIPs. Gal-9 levels significantly correlated with both the total cell count and the absolute number of lymphocytes in the BALF of patients with IIPs and CVD-IP. Strong reactivity with anti-Gal-9 antibody was observed in the cytoplasm of alveolar macrophages, lymphocytes, and type II pneumocytes in the lungs of patients with IP. Gal-9 expression in those cells was more remarkable in patients with CVD-IP than in patients with IPF and idiopathic NSIP. Gal-9 suppressed the growth of human lung fibroblast cells in a dose- dependent manner. Gal-9 induced apoptosis of human lung fibroblast cells in a dose-dependent manner. CONCLUSIONS: Our findings suggest that the expression level of Gal-9 in the lung is increased in patients with CVD-IP and that Gal-9 may have a protective role against pulmonary fibrosis of these patients.

Diffuse alveolar damage of the lungs in forensic autopsies: assessment of histopathological stages and causes of death.

INTRODUCTION: Diffuse alveolar damage (DAD) is a morphological prototype of acute interstitial pneumonia. Hospital autopsies or open-lung biopsies are used to monitor common alveolar damage and hyaline membrane (HM) development histopathologically. The aim of this study was to detect histopathological profiles and frequency of DAD and HM in adult forensic autopsies. MATERIALS AND METHODS: In total, 6813 reports with histopathological samples in 12,504 cases on which an autopsy was performed between 2006 and 2008 were investigated. Sixty-six individuals >18 years of age who were diagnosed with DAD were included. Hematoxylin- and eosin-stained lung preparations were reexamined in line with the 2002 American Thoracic Society/European Respiratory Society idiopathic interstitial pneumonia consensus criteria. RESULTS: Histopathological examination revealed that 50 cases (75.7%) were in the exudative phase and 16 (24.2%) were in the proliferative phase. Only the rate of alveolar exudate/oedema in exudative phase cases (P = 0.003); those of alveolar histiocytic desquamation (P = 0.037), alveolar fibrosis (P = 0.017), chronic inflammation (P = 0.02), and alveolar fibrin (P = 0.001) in proliferative cases were significantly higher. The presence of alveolar fibrin was the only independent variable in favour of proliferative cases (P = 0.016). CONCLUSION: The detection of all DAD morphological criteria with the same intensity is not always possible in each case. Forensic autopsies may provide a favourable means for expanding our knowledge about acute lung damage, DAD, and interstitial lung disease.

Rescue therapy with a pumpless extracorporeal lung assist device in a patient with acute interstitial lung disease and severe refractory hypercapnia.

Idiopathic interstitial pneumonia frequently causes severe pulmonary restriction that in turn makes mechanical ventilation difficult. We report the case of a 44-year-old woman who developed a refractory severe hypercapnic respiratory failure (P(aCO(2)) 281 mm Hg, pH 6.77) despite mechanical ventilation with high inspiratory pressure and PEEP. A pumpless extracorporeal lung assist device, Novalung, was used as rescue therapy for carbon dioxide removal, enabling lung-protective ventilation and normalization of life-threatening acidosis. Open lung biopsy revealed an idiopathic interstitial pneumonia with histological features of a nonspecific interstitial pneumonia. Corticosteroid therapy led to progressive improvement of pulmonary function, soon permitting cessation of mechanical ventilation and extracorporeal therapy. The patient was discharged from the intensive care unit after 20 days. This case demonstrates the successful use of pumpless extracorporeal lung assist as an alternative device to pump-driven extracorporeal membrane oxygenation in severe hypercapnic respiratory failure secondary to nonspecific interstitial pneumonia.

Prognostic value of immunohistochemical surfactant protein A expression in regenerative/hyperplastic alveolar epithelial cells in idiopathic interstitial pneumonias.

BACKGROUND: It is difficult to predict survival in patients with idiopathic pulmonary fibrosis. Recently, several proteins, such as surfactant protein (SP) and KL-6, have been reported to be useful biologic markers for prediction of prognosis for interstitial pneumonias. It is not clear whether there is any relationship between expression of these proteins in regenerative/hyperplastic alveolar epithelial cells and prognosis of idiopathic interstitial pneumonias (IIPs). OBJECTIVES: This study aimed to elucidate the clinical significance of the expression of such lung secretory proteins as SP-A and KL-6 in lung tissues of patients with IIPs. METHODS: We retrospectively investigated the immunohistochemical expression of SP-A, KL-6, cytokeratin (CK), and epithelial membrane antigen (EMA) in alveolar epithelial cells in lung tissues obtained from surgical lung biopsy in 43 patients with IIPs, and analyzed the correlation between expression of these markers and the prognosis of each IIP patient. CK and EMA were used as general markers for epithelial cells. RESULTS: In patients with usual interstitial pneumonia (UIP), the ratio of SP-A positive epithelial cells to all alveolar epithelial cells (SP-A positive ratio) in the collapsed and mural fibrosis areas varied, ranging from cases where almost all alveolar epithelial cells expressed SP-A to cases where only a few did. On the other hand, in many patients with nonspecific interstitial pneumonia (NSIP), many of the alveolar epithelial cells in the diseased areas expressed SP-A. The SP-A positive ratio was significantly lower in patients who died from progression of UIP than in patients with UIP who remained stable or deteriorated but did not die. In NSIP patients, a similar tendency was noted between the SP-A positive ratio and prognosis. CONCLUSIONS: The results suggest that the paucity of immunohistochemical SP-A expression in alveolar epithelial cells in diseased areas (i.e. regenerative/hyperplastic alveolar epithelial cells) may predict a worse prognosis for patients with IIPs, especially patients with UIP. A prospective study is needed to confirm these results.