Analysis of bronchoalveolar lavage fluid proteome from systemic sclerosis patients with or without functional, clinical and radiological signs of lung fibrosis

Lung fibrosis is a major cause of mortality and morbidity in systemic sclerosis (SSc). However, its pathogenesis still needs to be elucidated. We examined whether the alteration of certain proteins in bronchoalveolar lavage fluid (BALF) might have a protective or a causative role in the lung fibrogenesis process. For this purpose we compared the BALF protein profile obtained from nine SSc patients with lung fibrosis (SSc(Fib+)) with that obtained from six SSc patients without pulmonary fibrosis (SSc(Fib-)) by two-dimensional gel electrophoresis (2-DE). Only spots and spot-trains that were consistently expressed in a different way in the two study groups were taken into consideration. In total, 47 spots and spot-trains, corresponding to 30 previously identified proteins in human BALF, showed no significant variation between SSc(Fib+ )patients and SSc(Fib- )patients, whereas 24 spots showed a reproducible significant variation in the two study groups. These latter spots corresponded to 11 proteins or protein fragments, including serum albumin fragments (13 spots), 5 previously recognized proteins (7 spots), and 4 proteins (3 spots) that had not been previously described in human BALF maps, namely calumenin, cytohesin-2, cystatin SN, and mitochondrial DNA topoisomerase 1 (mtDNA TOP1). Mass analysis did not determine one protein-spot. The two study groups revealed a significant difference in BALF protein composition. Whereas levels of glutathione S-transferase P (GSTP), Cu–Zn superoxide dismutase (SOD) and cystatin SN were downregulated in SSc(Fib+ )patients compared with SSc(Fib- )patients, we observed a significant upregulation of α1-acid glycoprotein, haptoglobin-α chain, calgranulin (Cal) B, cytohesin-2, calumenin, and mtDNA TOP1 in SSc(Fib+ )patients. Some of these proteins (GSTP, Cu–Zn SOD, and cystatin SN) seem to be involved in mechanisms that protect lungs against injury or inflammation, whereas others (Cal B, cytohesin-2, and calumenin) seem to be involved in mechanisms that drive lung fibrogenesis. Even if the 2-DE analysis of BALF did not provide an exhaustive identification of all BALF proteins, especially those of low molecular mass, it allows the identification of proteins that might have a role in lung fibrogenesis. Further longitudinal studies on larger cohorts of patients will be necessary to assess their usefulness as predictive markers of disease

Using Heat as a Tracer to Characterize Streambed Water Fluxes of the Brenta River (Italy)

This work deals with the characterization of the spatial and temporal variability of water exchange fluxes from/to the Brenta river streambed (Veneto, Italy), critically important to regional water resources management. The aquifer system is structurally quite complex. It evolves from a large undifferentiated aquifer near adjacent mountain ranges to a wellstructured multi-layer system made up of six well-defined confined aquifers which constitute the noteworthy subsurface reservoirs of the area. Both the undifferentiated and the multiaquifer systems are heavily exploited for urban, industrial and agricultural uses. A three-dimensional groundwater flow model of the multi-aquifer system of the Central Veneto has been set up within a 3,300-km(2) area. The model integrates a large amount of available geological, geophysical, climatic and hydrologic data, has been specifically developed to analyze the behavior of such large, complex and well-monitored sedimentary system and to aid sustainable large-scale water resources management. A hydrothermal model of the river and of the underlying aquifer was implemented to improve the estimation, obtained for the 3D flow model, of the water exchange between the Brenta river and its underlying aquifer systems. The contribution of streambed exchanges is quantified by comparing simulations of water flow and heat transport to observed temperature and levels in river and in groundwater

Systemic inflammatory response and downmodulation of peripheral CD25(+)Foxp3(+) T-regulatory cells in patients undergoing radiofrequency thermal ablation for lung cancer

Radiofrequency thermal ablation (RFTA) is a local tumor-destructing technique that can potentially modulate the host immune response through mechanisms that are not clearly defined. We assessed whether RFTA could affect multiple systemic inflammatory and immunological parameters, including CD25+Foxp+ cells, in patients with primary or metastatic lung tumors. Three days after RFTA, a moderate and temporary systemic inflammatory response developed, as demonstrated by the increase in peripheral neutrophils and monocytes and in plasma levels of proinflammatory chemokines (MIP-1alpha, MIP-1beta, eotaxin, and interleukin[IL]-8) and acute phase reactants (complement C3 and C4, serum amyloid, alpha1 antichymotrypsin, and C-reactive protein). Moreover, we found a concomitant release of the anti-inflammatory factor IL-10. Thirty days after RFTA, a significant reduction in CD25+Foxp3+ counts with an increase in CD4+ T-cell proliferation and number of interferon-gamma-secreting cells was observed. The reduction in CD25+Foxp3+ cells lasted up to 90 days after treatment. The use of RFTA in lung cancer patients has an immunomodulatory activity: it induces a self-limiting systemic inflammation early and later a reduction of circulating CD25+Foxp3+ Tregs. In addition to tumor ablation, downmodulation of this regulatory subset might be an important mechanism involved in the long-term clinical efficacy of RFTA

2-DE and MALDI-TOF-MS for a comparative analysis of proteins expressed in different cellular models of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal, neurodegenerative disorder characterized by the selective loss of motor neurons from the spinal cord and brain. About 10% of ALS cases are familial (FALS), and in 20% of these cases the disease has been linked to mutations in the Cu,Zn-SOD1 gene. Although the molecular mechanisms causing these forms of ALS are still unclear, evidence has been provided that motor neurons injuries associated with mutant superoxide dismutase (SOD1)-related FALS result from a toxic gain-in-fuction of the mutated enzyme. To understand better the role of these mutations in the pathophysiology of FALS we have compared the pattern of proteins expressed in human neuroblastoma SH-SY5Y cell line with those of cell lines transfected with plasmids expressing the wild-type human SOD1 and the H46R and G93A mutants. 2-DE coupled to MALDI-TOF-MS were the proteomic tools used for identification of differentially expressed proteins. These included cytoskeletal proteins, proteins that regulate energetic metabolism and intracellular redox conditions, and the ubiquitin proteasome system. The proteomic approach allowed to expand the knowledge on the pattern of proteins, with altered expression, which we should focus on, for a better understanding of the possible mechanism involved in mutated-SOD1 toxicity. The cellular models considered in this work have also evidenced biochemical characteristics common to other SOD1-mutated cellular lines connected to the pathogenesis of ALS

2-DE AND MALDI-TOF-MS FOR A COMPARATIVE ANALYSIS OF PROTEINS EXPRESSED IN DIFFERENT CELLULAR MODELS OF AMYOTROPHIC LATERAL SCLEROSIS

Amyotrophic lateral sclerosis (ALS) is a fatal, neurodegenerative disorder characterized by the selective loss of motor neurons from the spinal cord and brain. About 10% of ALS cases are familial (FALS), and in 20% of these cases the disease has been linked to mutations in the Cu,Zn-SOD1 gene. Although the molecular mechanisms causing these forms of ALS are still unclear, evidence has been provided that motor neurons injuries associated with mutant superoxide dismutase (SOD1)-related FALS result from a toxic gain-in-fuction of the mutated enzyme. To understand better the role of these mutations in the pathophysiology of FALS we have compared the pattern of proteins expressed in human neuroblastoma SH-SY5Ycell line with those of cell lines transfected with plasmids expressing the wild-type human SOD1 and the H46R and G93A mutants. 2-DE coupled to MALDI-TOFMS were the proteomic tools used for identification of differentially expressed proteins. These included cytoskeletal proteins, proteins that regulate energetic metabolism and intracellular redox conditions, and the ubiquitin proteasome system. The proteomic approach allowed to expand the knowledge on the pattern of proteins, with altered expression, which we should focus on, for a better understanding of the possible mechanism involved in mutated-SOD1 toxicity. The cellular models considered in this work have also evidenced biochemical characteristics common to other SOD1-mutated cellular lines connected to the pathogenesis of ALS

Bronchoalveolar Lavage Fluid Proteome in Bronchiolitis Obliterans Syndrome: Possible Role for Surfactant Protein A in Disease Onset

Bronchiolitis obliterans syndrome (BOS) affects long-term survival of lung transplant (Tx) recipients (LTRs), with no consistently effective treatment strategy. Identifying early markers of BOS is of paramount importance for improving graft survival. Methods: We used 2-dimensional gel electrophoresis and protein identification by mass spectrometry to compare the protein profile of bronchoalveolar lavage fluid (BALf) in two groups of LTRs: one composed of patients with BOS and the other composed of patients with good graft function at 5 years post-surgery (stable LTRs). Based on the hypothesis that only proteins of lung origin could represent reliable BOS markers, we also evaluated paired plasma samples. Proteins of interest were also assessed in the BALf of control subjects and results confirmed by dot- blot analysis. Results: Among 11 differentially expressed proteins, we identified 2 locally produced factors: peroxiredoxin II (PRXII), exclusively expressed in BOS; and surfactant protein A (SP-A), expressed consistently less in BOS patients than in stable LTRs. PRXII expression was never observed in BALf from control subjects, whereas SP-A was present in higher amounts compared with stable LTRs and BOS patients. Finally, the time course of SP-A was studied in 5 LTRs who subsequently developed BOS. A reduction in BALf SP-A content was detectable early after Tx, preceding BOS onset in 4 of 5 patients. Conclusions: Our results suggest that testing SP-A levels in BALf could predict LTR patients who are at higher risk of BOS development