Upregulation of citrullination pathway: From Autoimmune to Idiopathic Lung Fibrosis.

BACKGROUND: Increased protein citrullination and peptidylarginine deiminases (PADIs), which catalyze the citrullination process, are central in Rheumatoid arthritis pathogenesis and probably involved in the initial steps towards autoimmunity. Approximately, 10% of RA patients develop clinically significantly ILD. A possible shared role of protein citrullination in rheumatoid arthritis associated interstitial lung disease (RA-ILD), and idiopathic pulmonary fibrosis (IPF) pathogenesis remains unclear. METHODS: We evaluated PADI2 and PADI4 mRNA expression in bronchoalveolar lavage fluid (BALF) cells of 59 patients with IPF, 27 patients RA-ILD and 10 healthy controls. PADI 2 and 4 expression was analyzed by western blot and immunohistochemistry. Citrullinated protein levels were also quantified. RESULTS: PADI4 mRNA and protein levels were higher in RA-ILD and IPF than controls. Furthermore, PADI4 mRNA levels showed an increase among smokers in RA-ILD. PADI4 expression was detected in granulocytes and macrophages in all groups, with the strongest cytoplasmic expression observed in granulocytes in RA-ILD and IPF. PADI2 mRNA and immunostaining of BAL cells, were similar in all groups among smokers. Overall, stronger staining was observed in current smokers. Citrullinated peptides were significantly increased in IPF compared to RA-ILD and controls. In RA-ILD, protein citrullination strongly correlated with PADI4 expression and anti-citrullinated protein antibodies (ACPAs). CONCLUSIONS: These results suggest that the citrullination pathway is upregulated in IPF and in RA-ILD.

Evolution of imaging findings, laboratory and functional parameters in rheumatoid arthritis patients after one year of treatment with anti-TNF-α agents.

OBJECTIVES: To investigate the efficacy and safety of anti-TNF-α agent treatment compared to non-biologic DMARDs in rheumatoid arthritis patients. METHODS: 82 consecutive patients, 29 males, 53 females, aged 42-79, diagnosed with RA and suitable for anti-TNF-α treatment composed two study groups: 42 with pre-existing rheumatoid arthritis-related interstitial lung disease (RA-ILD) and 40 without RA-ILD. Respective control groups consisted of 44 patients with pre-existing RA-ILD and 44 patients without RA-ILD, treated with non-biologic DMARDs. All patients underwent chest high resolution computed tomography (HRCT), pulmonary function tests (PFTs) and peripheral blood biomarkers at baseline and after one year of treatment. RESULTS: There was a significant decrease of air trapping extent and bronchial wall thickening after treatment in RA-ILD and RA-non ILD study groups (p<0.05). This was accompanied by a statistically significant improvement of maximum mid-expiratory flow (MMEF75-25), RV and RV/TLC in both study groups (p<0.05). In the RA-ILD study group ILD extent scores remained unchanged after anti-TNF-α treatment. None of the RA-non ILD group developed new-onset ILD. In both RA-ILD and RA-non ILD control groups, HRCT findings and PFTs did not differ significantly at the one-year follow-up study. Methotrexate (MTX) regression analysis showed in both RA-ILD study and control groups a negative correlation between MTX dose and ILD extent score at one-year and between MTX dose and air trapping extent at baseline and after one year of treatment. CONCLUSIONS: Anti-TNF-α treatment, in contrast to non-biologic DMARDs, there was an improvement of small airways disease. There was no new-onset ILD or exacerbation of preexisting-ILD, especially in patients treated with anti-TNF-α agents, supporting the efficacy and favourable safety profile of this treatment in RA patients.

NLRP3 inflammasome expression in idiopathic pulmonary fibrosis and rheumatoid lung.

In this study we investigated the implication of NLRP3 inflammasomes in the pathogenesis of idiopathic pulmonary fibrosis (IPF) and rheumatoid arthritis-usual interstitial pneumonia (RA-UIP).NLRP3 inflammasome activation at baseline and following stimulation with lipopolysaccharide/ATP was evaluated by measuring interleukin (IL)-1ß and IL-18 levels released in the bronchoalveolar lavage fluid (BALF) fluid and by cultures of BALF cells. IL-1ß and IL-18 levels were significantly elevated in the BALF and BALF macrophage cultures from RA-UIP patients, consistent with pre-existing inflammasome activation in these patients. In contrast, in IPF, BALF levels of IL-1ß were significantly less elevated relative to RA-UIP and IL-18 was lower than controls. Furthermore, upon inflammasome stimulation, IPF BALF macrophage cultures failed to upregulate IL-1ß and partly IL-18 secretion, in contrast to controls, which showed robust IL-1ß and IL-18 upregulation. Interestingly, RA-UIP BALF cell cultures treated with lipopolysaccharide/ATP showed a potent stimulation of IL-18 secretion but not IL-1ß, the latter being already elevated in the unstimulated cultures, while examination of the intracellular IL-1ß levels in RA-UIP BALF cells upon NLRP3 inflammasome stimulation showed a significant upregulation of IL-1ß suggesting the NLRP3 pathway could be further activated.Taken together, our results suggest distinct inflammasome activation profiles between autoimmune and idiopathic lung fibrosis.

Detection and genotype analysis of human papillomavirus in non-small cell lung cancer patients.

Although the role of human papillomavirus (HPV) in the development of uterine cervical cancer is well established, the role of HPV in lung carcinogenesis remains controversial. The detection rates of HPV DNA are subject to a wide variation from 0 to 100%. This is partly influenced by the detection techniques employed. To elucidate the impact of HPV infection on lung parenchyma, we analyzed 100 non-small cell lung cancer (NSCLC) specimens (39 squamous cell carcinomas, 50 adenocarcinomas, 5 samples with characteristics of both squamous cell and adenocarcinoma, 5 undifferentiated and 1 large cell carcinoma) from the region of Crete, Greece. Sixteen non-cancerous samples served as the negative controls. DNA was extracted from 100 paraffin-embedded tissue sections obtained from NSCLC patients. The specimens were examined for the detection of HPV DNA by Real-Time PCR using GP5+/GP6+ primers. Furthermore, the HPV-positive samples were subjected to genotyping. In contrast to the absence of viral genomes in the control samples, HPV DNA was detected in 19 NSCLC specimens (19%). In particular, 4 squamous cell carcinomas, 12 adenocarcinomas, 1 sample with characteristics of both squamous cell and adenocarcinoma, and 2 undifferentiated samples were HPV-positive. The distribution of HPV genotypes was as follows: HPV 16: eight cases (42.1%); HPV 11: three cases (15.8%); HPV 6: one case (5.2%); HPV 59: one case (5.2%); HPV 33: two cases (10.5%); HPV 31: two cases (10.5%) and HPV 18: two cases (10.5%). The presence of HPV in the tumor samples provides evidence of the potential role of HPV in NSCLC and strongly argues for additional research on this issue.

Association of human herpes, papilloma and polyoma virus families with bladder cancer.

The aim of the present study was to assess the possible etiologic role of human papillomavirus (HPV), human herpes virus (HHV) and the human polyoma virus families (BKV and JCV) in the tumourigenesis of bladder cancer. Thirty biopsy specimens from patients with different grades and stages of bladder cancer, who underwent transurethral bladder cancer resection, and 30 normal bladder mucosa specimens were analysed using polymerase chain reaction (PCR) for the detection of the above three virus family members. The presence of HPV was determined in all specimens with nested PCR and real-time quantitative PCR. All cancerous specimens, including the control group, were found to be negative both by PCR and real-time qPCR for the presence of HPV DNA, whilst all samples examined by PCR tested negative for the presence of HSV-1,2 Varicella zoster virus and HSV-7 DNA. Cytomegalovirus, HHV-6 and HHV-8 exhibited similar incidence in sample positivity in both cancerous and healthy tissues. EBV showed a higher prevalence in bladder cancer specimens compared to healthy tissue (p = 0.048), whilst BKV and JCV were detected only in tumour samples. The presence of EBV in a significant proportion of bladder tumours indicates the etiological role of this virus in cancer tumourigenesis.

Expression of SDF-1/CXCR4 axis in bone marrow mesenchymal stem cells derived from rheumatoid arthritis-usual interstitial pneumonia.

OBJECTIVES: To explore the SDF-1/ CXCR4 axis as driving mechanism of bone marrow mesenchymal stem-cells to the injured lung in patients with rheumatoid arthritis associated usual interstitial pneumonia (RA-UIP). METHODS: We evaluated the m-RNA expression of SDF-1 and CXCR4 with real-time PCR in bone marrow mesenchymal stem cells of 7 RA-UIP and 10 RA patients without lung involvement. RESULTS: The axis was not expressed in RA whereas both SDF-1 and CXCR4 were expressed in RA-UIP [1.93 (1.32, 2.00) and 0.008 (0, 0.01)] respectively. CONCLUSIONS: The development of pulmonary fibrosis in RA may be considered as the key event for the migration of stem cells to the injured lung through the SDF-1/CXCR4 axis.

HRCT findings in secondhand smokers with respiratory symptoms.

PURPOSE: The present study aims to investigate the occurrence and severity of HRCT abnormalities in symptomatic never-smokers, passively exposed to cigarette smoke. MATERIALS AND METHODS: A total of 135 never-smokers with respiratory symptoms, without underlying lung disease, underwent paired inspiratory-expiratory HRCT and completed the secondhand smoking (SHS) exposure scale questionnaire. Individuals passively exposed to tobacco consisted the secondhand smoking group (SHS group) (n = 68); the remaining 67 controls were never exposed to SHS. Statistical analysis was performed using the Kolmogorov-Smirnov, x2 and Pearson Point-Biserial correlation tests. P < 0.05 was statistically significant. RESULTS: HRCT findings in SHS group included bronchial wall thickening (98.5%), mild cylindrical bronchiectasis (44%), ground-glass opacities (17.6%), and mosaic attenuation pattern (53%). Air trapping occurred in 65% of expiratory scans. Differences in occurrence of findings between SHS group participants and controls were statistically significant (p < 0.0001). There was significant correlation between exposure to tobacco smoke in hours/day and in number of cigarettes/day and the presence of mosaic attenuation pattern, ground-glass opacities, cylindrical bronchiectasis and air trapping, respectively (p < 0.05). Cumulative SHS exposure in years was also strongly correlated with the presence of ground-glass opacities, mosaic attenuation pattern, cylindrical bronchiectasis, and air trapping, as well as with the frequency of respiratory symptoms (p < 0.05). CONCLUSION: HRCT findings attributed to SHS are mostly related to airway involvement and are correlated with the duration and grade of exposure to environmental tobacco smoke. SHS followed by lifestyle adjustments should be considered in symptomatic non-smokers with such HRCT findings.

Physiologic effects of stress dose corticosteroids in in-hospital cardiac arrest (CORTICA): A randomized clinical trial.

Aim: Postresuscitation hemodynamics are associated with hospital mortality/functional outcome. We sought to determine whether low-dose steroids started during and continued after cardiopulmonary resuscitation (CPR) affect postresuscitation hemodynamics and other physiological variables in vasopressor-requiring, in-hospital cardiac arrest. Methods: We conducted a two-center, randomized, double-blind trial of patients with adrenaline (epinephrine)-requiring cardiac arrest. Patients were randomized to receive either methylprednisolone 40 mg (steroids group) or normal saline-placebo (control group) during the first CPR cycle post-enrollment. Postresuscitation shock was treated with hydrocortisone 240 mg daily for 7 days maximum and gradual taper (steroids group), or saline-placebo (control group). Primary outcomes were arterial pressure and central-venous oxygen saturation (ScvO2) within 72 hours post-ROSC. Results: Eighty nine of 98 controls and 80 of 86 steroids group patients with ROSC were treated as randomized. Primary outcome data were collected from 100 patients with ROSC (control, n = 54; steroids, n = 46). In intention-to-treat mixed-model analyses, there was no significant effect of group on arterial pressure, marginal mean (95% confidence interval) for mean arterial pressure, steroids vs. control: 74 (68-80) vs. 72 (66-79) mmHg] and ScvO2 [71 (68-75)% vs. 69 (65-73)%], cardiac index [2.8 (2.5-3.1) vs. 2.9 (2.5-3.2) L/min/m2], and serum cytokine concentrations [e.g. interleukin-6, 89.1 (42.8-133.9) vs. 75.7 (52.1-152.3) pg/mL] determined within 72 hours post-ROSC (P = 0.12-0.86). There was no between-group difference in body temperature, echocardiographic variables, prefrontal blood flow index/cerebral autoregulation, organ failure-free days, and hazard for poor in-hospital/functional outcome, and adverse events (P = 0.08->0.99). Conclusions: Our results do not support the use of low-dose corticosteroids in in-hospital cardiac arrest.Trial Registration:ClinicalTrials.gov number: NCT02790788 ( https://www.clinicaltrials.gov ).

Role of VEGF-stromal cell-derived factor-1alpha/CXCL12 axis in pleural effusion of lung cancer.

CONTEXT AND OBJECTIVE: It has been suggested that stromal cell-derived factor-1alpha ((SDF-1alpha) or CXCL12, both transcripts, TR1 and TR2) and its cognate receptor CXCR4 may regulate cancer metastasis. We have investigated the role of vascular endothelial growth factor (VEGF), angiopoietins (Ang-1 and Ang-2) and the biological axis of CXCL12-CXCR4, in patients with malignant pleural effusions (PEs). MATERIAL AND METHODS: Twenty five patients, seven with transudative PEs due to heart failure and 18 with exudative malignant PEs (7 with small cell lung cancer (SCLC) and 11 with nonsmall cell lung cancer (NSCLC)) were included in the study. Expression analysis of the mediators was performed in pleural fluid pellet using real-time reverse transcription-PCR. Protein expression has been evaluated by western blot analysis. RESULTS: SDF-TR1 (P = 0.02) but not SDF-TR2 (P = 0.23) or CXCR4 levels (P = 0.23) were higher in malignant PEs than in transudates. SDF-TR1 (P = 0.04) and SDF- TR2 levels (P = 0.04) but not CXCR4 levels (P = 0.123) were higher in SCLC PEs than in heart failure PEs. SDF-TR1 (P = 0.03) but not SDF-TR2 levels (P = 0.6) and CXCR4 levels (P = 0.4) were higher in NSCLC PEs than in transudates. Ang-1 has not been expressed in PEs, whereas no significant difference has been detected in VEGF and Ang-2 expression between malignant PEs and transudates. However, protein expression showed increased VEGF and SDF expression in malignant PEs. CONCLUSIONS: These results suggest that elevated SDF-1alpha/CXCL12 levels would be suggestive of a link to metastasis and may participate in pleural trafficking in lung cancer.

Expression analysis of angiogenic growth factors and biological axis CXCL12/CXCR4 axis in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is associated with aberrant repair, persistence of collagen deposition, and the development of vascular remodeling. However, the role of angiogenesis in the pathogenesis of IPF is still undetermined. The aim of this study was to evaluate the combined mRNA expression of vascular endothelial growth factor A (VEGFA), fibroblast growth factor 2 (FGF2), insulin-like growth factor 1 (IGF1) epidermal growth factor (EGF), and its receptor (EGFR) in lung tissue obtained from IPF patients. We have also investigated the expression of chemokine CXCL12/stromal cell-derived factor-1 (SDF-1) and its receptor, CXCR4, to identify alterations that maybe implicated in the pathogenesis of IPF. The subjects studied consisted of two distinct groups: patients with IPF (n = 25) and subjects (control) undergoing thoracic surgery for reasons other than interstitial lung disease (n = 10). Expression analysis of the aforementioned growth factors and biological axis CXCL12/CXR4 analysis were performed using real-time RT-PCR. IGF-1, EGF, and FGF2 mRNA levels are significantly decreased in the patients compared to the controls (p = 0.028, p = 0.023 and p = 0.009, respectively). SDF1-TR1 and SDF1-TR2 transcript levels were significantly lower in patients compared to controls (p = 0.017 and p = 0.001). Significant coexpression of VEGF mRNA with IGF mRNA was observed in the group of the patients (p = 0.017). An additional coexpression of VEGF mRNA with SDF1-TR1 mRNA was demonstrated(p = 0.030). Our results show a downregulation in angiogenetic mechanisms in IPF. However, our results should be further verified by measuring other angiogenetic pathways in more samples.

Upregulation of stromal cell derived factor-1alpha in collagen vascular diseases-associated interstitial pneumonias (CVDs-IPs).

OBJECTIVE: We speculated that distinct angiogenic profiles are involved in idiopathic interstitial pneumonias (IIPs) in comparison with interstitial pneumonias associated with collagen vascular disease (CVD-IPs). This hypothesis was investigated by measuring the expression of a cardinal biologic axis, the vascular endothelial growth factor (VEGF)-stromal derived growth factor [SDF-1alpha, transcripts 1 and 2 (TR1 and TR2)] and receptor, CXCR4 and the angiogenetic receptors CXCR2 and CXCR3 in bronchoalveolar lavage fluid (BALF) in both conditions. METHODS: We studied prospectively 25 patients with fibrotic IIPs (f-IIPs) [20 with idiopathic pulmonary fibrosis (IPF) and 5 with idiopathic non-specific interstitial pneumonia (NSIP)] and 16 patients with CVD-IPs. mRNA expression was measured by Real-Time RT-PCR and protein was evaluated by Western Blotting. RESULTS: A significantly greater value has been detected in SDF-1alpha-TR1 mRNA expression levels of CVD-IPs (p=0.05) in comparison with IPF group. A similar trend has been also detected in protein expression in favor of CVD-IP group. In addition, VEGF mRNA levels have been found significantly increased in CVD-IPs in comparison with the NSIP group (p=0.05). No significant difference has been found in SDF-1alpha-TR2-CXCR4 mRNA and CXCR2-CXCR3 between the two groups. CONCLUSION: These results showed increased expression of SDF-1alpha in CVD-IPs, suggesting different angiogenic procedures. Further studies are needed in order to better explore the angiogenetic pathway in these disorders.

Role of Smoking in the Evolution of Cardiovascular Magnetic Resonance and Laboratory Findings of Acute Myocarditis.

PURPOSE: The purpose is to investigate cardiac magnetic resonance and laboratory findings in patients with clinically suspected acute myocarditis and re-assess the evolution of findings in relation to clinical parameters and smoking habits. METHODS: We prospectively analyzed 68 consecutive patients (4 females, 64 males, median age 25 years) at baseline and 51 patients 12 months later with regard to age, symptoms, and signs, smoking history, cardiac troponin I, erythrocyte sedimentation rate, c-reactive protein blood levels, electrocardiography changes, and cardiac magnetic resonance findings. Statistical analysis included group comparisons and linear regression between clinical parameters and the obtained data. RESULTS: A statistically significant correlation was recorded between smoking and late gadolinium enhancement extent, both at baseline and follow-up study. Late gadolinium enhancement extent was positively associated with cardiac troponin I serum levels and c-reactive protein and negatively with left ventricular ejection fraction at baseline study. Myocardial segments 4 and 5 were most frequently involved. Late gadolinium enhancement persisted in 96% of patients with no significant extent change at 12-month follow-up, while improved. CONCLUSIONS: A strong correlation was recorded between smoking patients with acute myocarditis and extent both at baseline and follow-up cardiac magnetic resonance. Myocardial segments 4 and 5 involvement was most prevalent. Late gadolinium enhancement persisted at follow-up, its incidence was higher than that reported in other studies and did not have an impact on the patient's clinical status or cardiac function. However, longer-term follow-up is highly recommended in these patients.

NLRP3/Caspase-1 inflammasome activation is decreased in alveolar macrophages in patients with lung cancer.

Lung cancer (LC) remains the leading cause of cancer-related mortality. The interaction of cancer cells with their microenvironment, results in tumor escape or elimination. Alveolar macrophages (AMs) play a significant role in lung immunoregulation, however their role in LC has been outshined by the study of tumor associated macrophages. Inflammasomes are key components of innate immune responses and can exert either tumor-suppressive or oncogenic functions, while their role in lung cancer is largely unknown. We thus investigated the NLRP3 pathway in Bronchoalveolar Lavage derived alveolar macrophages and peripheral blood leukocytes from patients with primary lung cancer and healthy individuals. IL-1ß and IL-18 secretion was significantly higher in unstimulated peripheral blood leukocytes from LC patients, while IL-1ß secretion could be further increased upon NLRP3 stimulation. In contrast, in LC AMs, we observed a different profile of IL-1ß secretion, characterized mainly by the impairment of IL-1ß production in NLRP3 stimulated cells. AMs also exhibited an impaired TLR4/LPS pathway as shown by the reduced induction of IL-6 and TNF-α. Our results support the hypothesis of tumour induced immunosuppression in the lung microenvironment and may provide novel targets for cancer immunotherapy.

Clinical applications of mesenchymal stem cells in chronic lung diseases.

Mesenchymal stem (stromal) cells (MSCs) are multipotent stromal cells that have the ability to modulate immune response to tissue injury and promote repair in vivo. The therapeutic potential of ex vivo expanded MSCs are currently under investigation for a variety of chronic and acute lung diseases. This review summarizes the encouraging results regarding the safety of MSCs administration from recent and current clinical trials for idiopathic pulmonary fibrosis, acute respiratory distress syndrome, and chronic obstructive pulmonary disease. It also reviews the early preliminary data extracted by the same trials regarding the efficacy of MSCs in the aforementioned lung diseases.

Toll-like receptors and autophagy in interstitial lung diseases.

Interstitial lung diseases (ILDs) include a number of diseases whose pathogenesis still is not fully understood. Idiopathic pulmonary fibrosis (IPF), the most frequent and severe form of ILDs is an epithelial-driven disease and the treatment consists of the use of antifibrotic agents. In the rest of ILDs an inflammation-driven pathway is believed to be the main pathogenetic mechanism and treatment consists of the use of immunomodulatory agents. In both groups it is believed that infection can play an important role in the development and progression of the diseases. The immune system can recognize exogenous threats or endogenous stress through specialized receptors namely pattern recognition receptors (PRRs) which in turn, initiate downstream signaling pathways to control immune responses. Recently, a link between PRRs and autophagy, a specialized biological process involved in maintaining cellular homeostasis but also involved in various immunologic processes, has been described. In this review, we focus on the reciprocal influences of PRRs with particular emphasis on Toll-like receptors and autophagy in modulating innate immune responses.

Aberrant expression of miR-21, miR-376c and miR-145 and their target host genes in Merkel cell polyomavirus-positive non-small cell lung cancer.

Merkel Cell Polyoma Virus (MCPyV) infection has been associated with non-small cell lung cancer (NSCLC). Viruses can manipulate cellular miRNAs or have a profound impact on cellular miRNA expression to control host regulatory pathways. In this study, we evaluated the expression profiles of cancer-associated and virally affected host microRNAs miR-21, miR-145, miR-146a, miR-155, miR-302c, miR-367 and miR-376c in a series of NSCLC tissue samples as well as in samples from "healthy" sites, distant from the tumour region that were either positive or negative for MCPyV DNA. miR-21 and miR-376c were significantly upregulated whereas miR-145 was significantly downregulated in the MCPyV+ve samples compared to the MCPyV-ve tumour samples. Overall, miR-21 and miR-376c expression was higher in tumour compared to healthy tissue samples. No association was observed between the miR-155, miR-146a, miR-302c and miR-367 levels and the presence of MCPyV. The expression of miR-21 target genes (Pten, Bcl-2, Daxx, Pkr, Timp3), miR-376c (Grb2, Alk7, Mmp9) and miR-145 (Oct-4, Sox2, Fascin1) and their associated pathways (Braf, Akt-1, Akt-2, Bax, Hif1a, p53) was altered between MCPyV+ve tumor samples and their corresponding controls. These results show a novel association between miR-21, miR-376c and miR-145 and their host target genes with the presence of MCPyV, suggesting a mechanism of virus-specific microRNA signature in NSCLC.

MiR-185/AKT and miR-29a/collagen 1a pathways are activated in IPF BAL cells.

MicroRNA signatures of BAL cells and alveolar macrophages are currently lacking in IPF. Here we sought to investigate the expression of fibrosis-related microRNAs in the cellular component of the BAL in IPF. We thus focused on microRNAs previously associated with fibrosis (miR-29a, miR-29b, miR-29c, let-7d, and miR-21) and rapid IPF progression (miR-185, miR-210, miR-302c-3p miR-376c and miR-423-5p). Among the tested microRNAs miR-29a and miR-185 were found significantly downregulated in IPF while miR-302c-3p and miR-376c were not expressed by BAL cells. Importantly, the downregulation of miR-29a inversely correlated with the significantly increased levels of COL1A1 mRNA in IPF BAL cells. Collagen 1 a was found mainly overexpressed in alveolar macrophages and not other cell types of the BAL by immunofluorescence. In view of the downregulation of miR-185, we tested the response of THP-1 macrophages to profibrotic cytokine TGFb and observed the downregulation of miR-185. Conversely, proinflammatory stimulation lead to miR-185 upregulation. Upon examination of the mRNA levels of known miR-185 targets AKT1, DNMT1 and HMGA2, no significant correlations were observed in the BAL cells. However, increased levels of total AKT and AKTser473 phosphorylation were observed in the IPF BAL cells. Furthermore, miR-185 inhibition in THP-1 macrophages resulted in significant increase of AKTser473 phosphorylation. Our study highlights the importance of BAL microRNA signatures in IPF and identifies significant differences in miR-185/AKT and miR-29a/collagen axes in the BAL cells of IPF patients.

Expression profiles of Toll-like receptors in non-small cell lung cancer and idiopathic pulmonary fibrosis.

Patients with idiopathic pulmonary fibrosis (IPF) have a higher incidence of lung cancer. The role of Toll-like receptors (TLRs), a key component of the innate immunity, in interstitial lung diseases (ILDs) and lung cancer pathogenesis is not clarified. TLR2, TLR3, TLR4, TLR7, TLR8 and TLR9 mRNA expression was quantitatively measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR) in bronchoalveolar lavage fluid (BALF) of 16 IPF patients, 16 non-small cell lung cancer (NSCLC) patients and 9 control subjects. TLR2, TLR3, TLR4 and TLR9 protein expression was assessed on BALF T-lymphocytes using flow cytometry. TLR3 mRNA expression was significantly higher in NSCLC compared to IPF (p=0.023) and controls (p=0.001). TLR7 mRNA expression levels were significantly higher in both NSCLC and IPF groups compared to controls (p=0.029, p=0.009). TLR9 expression at the mRNA level was significantly higher in both NSCLC and IPF groups compared to controls (p=0.01, p=0.001). Finally, TLR2 mRNA expression was significantly higher in IPF patients compared to controls (p=0.042). Flow cytometry revealed decreased TLR3 and TLR9 expression in IPF patients compared to the NSCLC group (p=0.02, p=0.014) and decreased TLR9 expression in IPF compared with the controls (p=0.04). TLR2 protein expression was significantly higher in IPF patients compared to NSCLC (p=0.04). Increased expression of endosomal TLRs in NSCLC patients and elevated expression of TLR2 in pulmonary fibrosis are the main results of this study. These results do not provide support for a common TLR pathway hypothesis between NSCLC and IPF.

Investigation of Telomerase/Telomeres system in Bone Marrow Mesenchymal Stem Cells derived from IPF and RA-UIP.

OBJECTIVE: Idiopathic Pulmonary Fibrosis and Rheumatoid Arthritis associated usual interstitial pneumonia seem to have the same poor outcome as there is not an effective treatment. The aim of the study is to explore the reparative ability of bone marrow mesenchymal stem cells by evaluating the system telomerase/telomeres and propose a novel therapeutic approach. METHODS: BM-MSCs were studied in 6 IPF patients, 7 patients with RA-UIP and 6 healthy controls. We evaluated the telomere length as well as the mRNA expression of both components of telomerase (human telomerase reverse transcriptase, h-TERT and RNA template complementary to the telomeric loss DNA, h-TERC). RESULTS: We found that BM-MSCs from IPF, RA-UIP cases do not present smaller telomere length than the controls (p = 0.170). There was no significant difference regarding the expression of both h-TERT and h-TERC genes between patients and healthy controls (p = 0.107 and p = 0.634 respectively). CONCLUSIONS: We demonstrated same telomere length and telomerase expression in BM-MSCs of both IPF and RA-UIP which could explain similarities in pathogenesis and prognosis. Maintenance of telomere length in these cells could have future implication in cell replacement treatment with stem cells of these devastating lung disorders.