Sputum trace metals are biomarkers of inflammatory and suppurative lung disease.

BACKGROUND: Induced sputum cytology and protein biomarkers can be used to assess airways inflammation. Increases in sputum iron have been described in inflammatory lung disease. We hypothesized that other sputum metals may be affected by airways inflammation and investigated their potential value as biomarkers. METHODS: Sputum was obtained from 20 healthy control subjects and from patients with inflammatory pulmonary diseases (23 with cystic fibrosis [CF], 16 with bronchiectasis, 17 with asthma, and 23 with COPD), and iron, zinc, manganese, and copper were measured. Fourteen patients with CF were also studied through an exacerbation cycle. RESULTS: Sputum zinc and iron were elevated in CF and non-CF bronchiectasis vs controls (P < .001, zinc; P < .01 iron). Manganese was elevated in asthma (P < .01) and bronchiectasis (P < .05) vs controls. Copper was elevated in CF vs controls (P < .05). Zinc decreased (P < .01) following treatment of CF exacerbation. In subjects with CF zinc levels correlated with other biomarkers. CONCLUSIONS: These results suggest a relationship of high concentrations of total zinc and iron with airways inflammation in CF and non-CF bronchiectasis, with longitudinal changes being observed in CF. Further work is required to elucidate potential inflammatory mechanisms related to these observations.

SELDI-TOF biomarker signatures for cystic fibrosis, asthma and chronic obstructive pulmonary disease.

OBJECTIVES: The aim of this work was to establish protein profiles in serum and nasal epithelial cells of cystic fibrosis individuals in comparison with controls, asthma and chronic obstructive pulmonary disease patients for specific biomarker signatures identification. DESIGN AND METHODS: Protein extracts were analyzed by Surface Enhanced Laser Desorption/Ionization Time-Of-Flight Mass-Spectrometry (SELDI-TOF-MS). RESULTS: The mass spectra revealed a set of peaks with differential expression in serum and nasal cells among the different groups studied, resulting into peak signatures representative/specific of each pathology. Logistic regressions were applied to those peaks; sensitivity, specificity, Youden's indexes and area under the curve (AUC) of the respective receiver operating characteristic (ROC) curves were compared. DISCUSSION: Multivariate analysis demonstrated that combination of peaks has a better predictive value than the individual ones. These protein signatures may serve as diagnostic/prognostic markers for the studied diseases with common clinical features, or as follow-up assessment markers of therapeutic interventions.

Sputum proteomics in inflammatory and suppurative respiratory diseases.

RATIONALE: Markers of inflammatory activity are important for assessment and management of many respiratory diseases. Markers that are currently unrecognized may be more valuable than those presently believed to be useful. OBJECTIVES: To identify potential biomarkers of suppurative and inflammatory lung disease in induced sputum samples. METHODS: Induced sputum was collected from 20 healthy control subjects, 24 patients with asthma, 24 with chronic obstructive pulmonary disease, 28 with cystic fibrosis (CF), and 19 with bronchiectasis. Twelve patients with CF had sputum sampled before and after antibiotic therapy for an infective exacerbation. The fluid phase of induced sputum was analyzed by surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectroscopy on three protein array surfaces. Some protein markers were selected for identification, and relevant ELISA assays sought. For 12 patients with CF, both SELDI-TOF and ELISA monitored changes in inflammatory responses during infective exacerbations. MEASUREMENTS AND MAIN RESULTS: SELDI-TOF identified potential biomarkers that differentiated each of the disease groups from healthy control subjects: at a significance of P < 0.01, there were 105 for asthma, 113 for chronic obstructive pulmonary disease, 381 for CF, and 377 for bronchiectasis. Peaks selected for protein identification yielded calgranulin A, calgranulin B, calgranulin C, Clara cell secretory protein, lysosyme c, proline rich salivary peptide, cystatin s, and hemoglobin alpha. On treatment of an infective CF exacerbation, SELDI-TOF determined falls in levels of calgranulin A and calgranulin B that were mirrored by ELISA-measured falls in calprotectin (heterodimer of calgranulins A and B). CONCLUSIONS: Proteomic screening of sputum yields potential biomarkers of inflammation. The early development of a clinically relevant assay from such data is demonstrated.

Biomarkers for cystic fibrosis lung disease: application of SELDI-TOF mass spectrometry to BAL fluid.

BACKGROUND: For cystic fibrosis (CF) patients there is a lack of good assays of disease activity and response to new therapeutic interventions, including gene therapy. Current measures of airways inflammation severity are insensitive or non-specific. METHODS: Bronchoalveolar lavage fluid from 39 CF children and 38 respiratory disease controls was obtained at bronchoscopy and analysed by surface enhanced laser desorption ionisation time of flight (SELDI-TOF) mass spectrometry. Recognized proteins were assessed for CF disease specificity. Individual protein identification of specific peaks was performed. RESULTS: 1277 proteins/peptides, >4 kDa, were detected using 12 different surfaces and binding conditions. 202 proteins/peptides were differentially expressed in the CF samples (p<0.001), 167 up-regulated and 35 down-regulated. The most discriminatory biomarker had a mass of 5.163 kDa. The most abundant, with a mass of 10.6 kDa, was identified as s100 A8 (calgranulin A). CONCLUSIONS: The application of SELDI-TOF mass spectrometry allows evaluation of proteins in BAL fluid avoiding the limitations of only analysing predetermined proteins and potentially identifying proteins not previously appreciated as biomarkers. Its application to cystic fibrosis should enable appropriate evaluation of evolving illness, of gene therapy and other new therapies.

Characterization of the ovine ortholog of secretory leukoprotease inhibitor.

There is great interest in the use of the sheep as a model for the investigation of inflammation in the lung. The serine antiproteases secretory leukoprotease inhibitor (SLPI) and elafin are important "alarm antiproteases" in the lung and have potentially important roles in the innate immune response. SLPI was first characterized in man and subsequently in murine, porcine, and rat tissues. Here we present the first data concerning the gene and cDNA sequence encoding for the ovine ortholog of SLPI, a protein of 132 amino acids with 66% sequence identity at the amino acid level with human SLPI. A 24-amino-acid signal sequence signifies that, like the other mammalian orthologs, ovine SLPI is a secreted protein. Tissue distribution of expression is demonstrated by reverse transcription polymerase chain reaction (RT-PCR) and shows features similar to SLPI expression in other mammals, specifically at mucosal surfaces such as the upper respiratory and intestinal tracts, and also the skin, liver, and kidney. This distribution lends credence to SLPI having important roles in innate immunity. We have also cloned the ovine SLPI cDNA into an expression vector and expressed the ovine SLPI protein in vitro. This has enabled us to demonstrate that ovine SLPI is correctly processed (Western blot analysis and SELDI-TOF mass spectrometry analysis) and has biological antihuman neutrophil elastase activity. In summary, the ovine ortholog of SLPI shows similarities to other members of the SLPI family and has all the features of a modulator of innate immunity.