Comparative proteomic analysis of bronchoalveolar lavage of familial and sporadic cases of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by progressive deterioration of the alveolar integrity. Among IPF identified phenotypes, that of familial (f-)IPF is usually associated with several gene mutations which are seldom observed in sporadic (s-)IPF. This study aimed at investigating the molecular patterns and variability in f-IPF and s-IPF patients through a differential proteomic analysis. Protein patterns of bronchoalveolar lavage fluid (BALF) samples from 10 familial and 17 sporadic IPF patients were compared using 2D electrophoresis and mass spectrometry. Principal component analysis (PCA) was applied to proteomic data and an enrichment analysis was also performed to characterize specific pathogenic mechanisms and to identify potential biomarkers. BALF samples from f-IPF showed 87 protein spots differentially expressed than those from s-IPF samples; once identified, these spots revealed 22 unique proteins. The functional analysis showed that the endothelial reticulum stress probably plays a central pathogenetic role in f-IPF with an up-regulation of proteins involved in wounding and immune responses, coagulation system, and ion homeostasis. Up-regulated proteins in the s-IPF group were those involved in the oxidative stress response. PCA analysis of differentially expressed proteins clearly distinguished f-IPF from s-IPF patients, and in agreement with radiological and histological patterns, pointed out a higher heterogeneity in f-IPF than s-IPF samples. The 'Slit/Robo signaling', 'clathrin-coated vesicle' and 'cytoskeleton remodelling', were extrapolated by 'pathways analysis' and the results of 'diseases (by biomarkers)' highlighted a 'connective tissue and autoimmune disease', two aspects of increasing interest in IPF.

A functional proteomics approach to the comprehension of sarcoidosis.

Pulmonary sarcoidosis (Sar) is an idiopathic disease histologically typified by non-caseating epitheliod cell sarcoid granulomas. A cohort of 37 Sar patients with chronic persistent pulmonary disease was described in this study. BAL protein profiles from 9 of these Sar patients were compared with those from 8 smoker (SC) and 10 no-smoker controls (NSC) by proteomic approach. Principal Component Analysis was performed to clusterize the samples in the corresponding conditions highlighting a differential pattern profiles primarily in Sar than SC. Spot identification reveals thirty-four unique proteins involved in lipid, mineral, and vitamin Dmetabolism, and immuneregulation of macrophage function. Enrichment analysis has been elaborated by MetaCore, revealing 14-3-3ε, α1-antitrypsin, GSTP1, and ApoA1 as "central hubs". Process Network as well as Pathway Maps underline proteins involved in immune response and inflammation induced by complement system, innate inflammatory response and IL-6signalling. Disease Biomarker Network highlights Tuberculosis and COPD as pathologies that share biomarkers with sarcoidosis. In conclusion, Sar protein expression profile seems more similar to that of NSC than SC, conversely to other ILDs. Moreover, Disease Biomarker Network revealed several common features between Sar and TB, exhorting to orientate the future proteomics investigations also in comparative BALF analysis of Sar and TB.

Towards a functional proteomics approach to the comprehension of idiopathic pulmonary fibrosis, sarcoidosis, systemic sclerosis and pulmonary Langerhans cell histiocytosis.

Bronchoalveolar lavage fluid of patients with four interstitial lung diseases (sarcoidosis, idiopathic pulmonary fibrosis, pulmonary Langerhans cell histiocytosis, fibrosis associated to systemic sclerosis) and smoker and non smoker control subjects were compared in a proteomic study. Principal component analysis was used to statistically verify the association between differentially expressed proteins and the conditions analyzed. Pathway and functional analysis by MetaCore and DAVID software revealed possible regulatory factors involved in specific "process networks" like regulation of stress and inflammatory responses. Immune response by alternative complement pathways, protein folding, Slit-Robo signaling and blood coagulation were "pathway maps" possibly associated with interstitial lung diseases pathogenesis. Four interesting proteins plastin 2, annexin A3, 14-3-3ε and S10A6 (calcyclin) were validated by Western blot analysis. In conclusion, we identified proteins that could be directly or indirectly linked to the pathophysiology of the different interstitial lung diseases. Multivariate analysis allowed us to classify samples in groups corresponding to the different conditions analyzed and based on their differential protein expression profiles. Finally, functional and pathway analysis defined the potential function and relations among identified proteins, including low abundance molecules present in the MetaCore database. BIOLOGICAL SIGNIFICANCE: This is the first study where different interstitial lung diseases such as sarcoidosis, idiopathic pulmonary fibrosis, pulmonary Langerhans cell histiocytosis, fibrosis associated to systemic sclerosis and smoker and non smoker control subjects were compared in a proteomic study to highlight their common pathways. We decided to report not only principal component analysis, used to statistically verify the association between differentially expressed proteins and the conditions analyzed, but also functional analysis general results, considering all differential proteins potentially involved in these conditions, to speculate about possible common pathogenetic pathways involved in fibrotic lung damage.

Utility of spiral CAT scan in the follow-up of patients with pulmonary Langerhans cell histiocytosis.

Pulmonary Langerhans cell histiocytosis (PLCH) is a rare disease that occurs almost exclusively in smokers, generally young adults between 20 and 40 years old. Prognostic biomarkers of the disease are lacking. This study describes the clinical-radiological features of a group of PLCH patients and applies a semi-quantitative CT score of the chest to verify the prognostic value of radiological findings in this disease. Clinical-radiological and immunological data from 12 Caucasian patients (6M, 7 smokers and 5 ex-smokers, mean age 36±8 years) were recorded at onset and after a follow-up period of 4 years. Application of the semi-quantitative CT score revealed a prevalently cystic pattern at onset and follow-up in the majority of the patients. Patients with a prevalently nodular pattern developed cystic lesions during follow-up. Interestingly, significant correlations were found between the extent of cystic lesions and DLCO values at onset (time 0: p<0.05) and at the end of follow-up (time 1, p<0.05) and with FEV1 values at time 0 (p<0.05) and time 1 (p<0.05). Patients with progressive functional decline were those with CT evidence of severe cystic alterations. The results suggest that high resolution CT scan of the chest is mandatory for characterizing PLCH patients at diagnosis and during follow-up. The proposed CT score of the chest showed potential prognostic value.

Analysis of macrophage migration inhibitory factor (MIF) in patients with idiopathic pulmonary fibrosis.

By proteomic approach we previously characterised bronchoalveolar lavage (BAL) protein profiles of patients with idiopathic pulmonary fibrosis (IPF), sarcoidosis and systemic sclerosis. Among differently expressed proteins we identified macrophage migration inhibitory factor (MIF), a multi-function pleiotropic cytokine. This study was performed to validate our findings by a further proteomic approach and ELISA in a larger population of patients and controls. MIF expression in lung tissue was also evaluated by immunohistochemistry. MIF was identified in all 2-DE gels of IPF patients and it was significantly increased compared to controls (p<0.05). This result was confirmed by ELISA: MIF concentrations were significantly higher in IPF patients than controls (p<0.001) and were directly correlated with neutrophil percentages (p=0.0095). Immunohistochemical analysis revealed enhanced expression in bronchiolar epithelium, alveolar epithelium, and fibroblastic foci. In conclusion, MIF is a pleiotropic cytokine that could be involved in the pathogenesis of IPF, being particularly abundant in BAL of these patients and mainly expressed in the areas of active fibrosis.

CD4+CD25+ lymphocyte subsets in chronic graft versus host disease patients undergoing extracorporeal photochemotherapy.

Extracorporeal photochemotherapy (ECP) has been used successfully for the treatment of chronic Graft versus Host Disease (cGvHD). However, the mechanism by which ECP exerts its protective effects remains elusive. Some recent observations have suggested a possible role of certain subsets of T lymphocytes with immunosuppressive properties (T-regulatory cells) that coexpress CD4 and high levels of the interleukin-2 receptor chain: CD4+CD25+ T lymphocytes. We studied whether ECP affects the percentage of these cells in the peripheral blood of patients with cGvHD. The study population consisted of 14 patients with cGvHD refractory to systemic steroids. On enrollment in each cycle of ECP, patients underwent clinical examination, blood chemistry analysis and other instrumental procedures to document and assess involvement of the various organs and systems. For cytofluorimetric identification and phenotyping of CD4+CD25+ T lymphocytes, peripheral blood samples were collected in EDTA anticoagulant before ECP, after 48 hours, and after 6 and 12 months from the start of treatment. The 14 patients in this study received a total of more than 300 cycles of ECP, with only minor side effects. The clinical outcome was negative in 2 patients and positive in 12 patients. Within subject analysis indicated that the percentage of CD4+CD25+ T lymphocytes before ECP and after 12 months of treatment was significantly increased. Our study confirms that changes in the percentage of CD4+CD25+ T cells induced by ECP could be a central aspect in the cascade of immune events leading to the immunological and clinical effects of this treatment in patients with cGvHD.

Clinical, laboratory and radiological findings in pulmonary fibrosis with and without connective tissue disease.

Uncertainty exists over whether to consider "lone" idiopathic pulmonary fibrosis (LIPF) and pulmonary fibrosis associated with connective tissue disorders (PFCTD) as significantly different entities. We retrospectively analysed data collected at the time of first diagnosis in 17 patients with LIPF and in 14 patients with PFCTD and compared survival in the two groups. At first evaluation, the time from onset of respiratory symptoms, spirometric volumes and the diffusing capacity for carbon monoxide were not significantly different between the two groups. However, arterial oxygen tension was significantly lower in LIPF than in PFCTD (63 +/- 3 vs 88 +/- 3 mmHg, p < 0.001). The radiological profusion scores relative to the upper and middle lung fields were significantly higher in LIPF than in PFCTD (upper regions: 6.9 +/- 0.6 vs 3.4 +/- 0.6, p < 0.005 - middle regions: 7.1 +/- 0.5 vs 4.8 +/- 0.7, p < 0.025), whereas the scores relative to the lower fields were similar (7.4 +/- 0.4 in LIPF and 8.4 +/- 0.6 in PFCTD). Survival since onset of respiratory symptoms was significantly better in the PFCTD than in LIPF patients, with a hazard ratio of 4.16 (95% CI 1.12-15.58, p=0.034). Thus, in our series of patients, those with LIPF had a more severe disease than those with PFCTD as shown by the higher frequency of hypoxaemia, the more diffuse pulmonary involvement demonstrated by the chest X-ray and the decreased survival.

Detection of specific antibodies in immune complexes of farmer's lung patients.

The authors examined 23 precipitin-positive symptomatic patients with Farmer's Lung(FL) and compared them to different groups of exposed asymptomatic precipitin-positive(EAPP) and precipitin- negative(EAPN) farmers. The sera were tested using several techniques (i.e., immunodiffusion and ELISA for specific antibodies; polyethylene glycol [PEG] for circulating immune complexes [CIC]) in an attempt to find an in vitro test correlated with the disease which could also provide an insight into the pathogenic mechanisms of Farmer's Lung. Circulating immune complexes formed by IgG were significantly higher in Farmer's Lung patients than in EAPP subjects. In polyethlyene glycol precipitates from Farmer's Lung patients, specific antibodies found by ELISA correlated well with serum positivity, but they were not found in EAPP subjects. The possibility that the circulating immune complexes found were Ig aggregates was ruled out, as was the possibility that the antibodies found in the polyethylene glycol precipitate were also due to an unspecific link. The authors suggest that the circulating immune complexes of Farmer's Lung patients contain specific specific antibodies and that since their composition is different in EAPP subjects, these circulating immune complexes may play a role in the pathogenesis of the disease.

Imparied interferon-gamma production by peripheral blood mononuclear cells and effects of calcitriol in pulmonary sarcoidosis.

Pulmonary sarcoidosis (S) is a granulomatous disease of unknown etiology characterized by spontaneous release of cytokines and 1,25-dihydroxyvitamin D3 (calcitriol) at the sites of granulomatous reaction. Stimulated by our previous findings that high levels of interferon-gamma (IFN-gamma) occur in this disease and that calcitriol reduces IFN-gamma production by peripheral blood mononuclear cells (PBMC) from normal subjects, we designed the present study to evaluate IFN-gamma production and the effect of calcitriol on the release of this cytokine by PBMC in S patients. The cells were stimulated with staphylococcal enterotoxin A (SEA) and A23187 calcium ionophore. Our results show that SEA- and A23187-stimulated PBMC from patients with S released significantly less IFN-gamma than those from control subjects. Calcitriol at 10(-6) M and 10(-9) M concentrations reduced IFN-gamma production by SEA-stimulated PBMC but this inhibitory effect was lower in S patients than controls. With A23187 we observed different behaviour at the various doses: at low doses calcitriol was as effective as in controls, but at 10(-6) M it was significantly less inhibitory in S than in healthy subjects.

Mast cells in bronchoalveolar lavage in sarcoidosis: correlation with alveolar lymphocytes.

Today it is believed that mast cells (MC) are important not only in IgE-mediated reactions, but also in delayed hypersensitivity reactions, and that their functions are mediated by factors released by T lymphocytes. Recent studies have shown their presence in bronchoalveolar lavage (BAL) of patients with asthma and interstitial lung disease. MC have been identified by us in the BAL of patients with sarcoidosis and lung cancer, and in controls. A statistically significant correlation has been found between MC and lymphocytes, CD3+ and CD4+ cells present in BAL, thus supporting the hypothesis of interactions between T lymphocytes and MC in immune reactions at the alveolar level.

Alveolar lymphocytes in the follow-up of sarcoidosis.

Bronchoalveolar lavage (BAL) was performed in a group of 25 patients with pulmonary sarcoidosis in order to monitor variations in alveolar lymphocytes (AL) in comparison to the clinical evolution of the disease. Two BAL were performed in each patient, the second 6-12 months after the first (8.9 +/- 3, M +/- SD). Twelve patients were classified as improved and 13 as unimproved on the basis of a score of changes in the clinical picture, x-ray, respiratory function, immunological and biochemical tests. There was a significant decrease (p less than 0.001) of the percentage of AL in 66% of cases in the improved group, but not in the unimproved group, where, on the contrary, it was possible to observe an increase in the percentage of AL in 46% of these cases (n.s.). In 9 of 11 patients followed for 1-5 years with at least 3 BAL, AL behaviour correlated with clinical evolution. On the basis of the parameters studied, it was possible to show a correlation between lymphocytes, alveolitis and clinical evolution, especially in patients with an improved course. While a single BAL does not seem to be sufficient to show the course of the disease, repeated long-term washings allow a more accurate staging of sarcoidosis and can give early warning of relapses even when the conventional parameters (clinical, radiological and functional) are unchanged. Our aim was to look for a simpler parameter, which could be used routinely by even unspecialized laboratories, who are not able to type lymphocytes.