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31.
Brian J O’Connor Sara Collarini Gianluigi Poli Caterina Brindicci Monica Spinola Daniela Acerbi Peter J Barnes Brian Leaker 《BMC pulmonary medicine》2011,11(1):1-8
Background
Airway Bypass is a catheter-based, bronchoscopic procedure in which new passageways are created that bypass the collapsed airways, enabling trapped air to exit the lungs. The Exhale Airway Stents for Emphysema (EASE) Trial was designed to investigate whether Exhale® Drug-Eluting Stents, placed in new passageways in the lungs, can improve pulmonary function and reduce breathlessness in severely hyperinflated, homogeneous emphysema patients (NCT00391612).Methods/Design
The multi-center, randomized, double-blind, sham-controlled trial design was posted on http://www.clinicaltrials.gov in October 2006. Because Bayesian statistics are used for the analysis, the proposed enrollment ranged from 225 up to 450 subjects at up to 45 institutions. Inclusion criteria are: high resolution CT scan with evidence of homogeneous emphysema, post-bronchodilator pulmonary function tests showing: a ratio of FEV1/FVC < 70%, FEV1≤50% of predicted or FEV1 < 1 liter, RV/TLC≥0.65 at screening, marked dyspnea score ≥2 on the modified Medical Research Council scale of 0-4, a smoking history of at least 20 pack years and stopped smoking for at least 8 weeks prior to enrollment. Following 16 to 20 supervised pulmonary rehabilitation sessions, subjects were randomized 2:1 to receive either a treatment (Exhale® Drug-Eluting Stent) or a sham bronchoscopy. A responder analysis will evaluate the co-primary endpoints of an FVC improvement ≥12% of the patient baseline value and modified Medical Research Council dyspnea scale improvement (reduction) ≥1 point at the 6-month follow-up visit.Discussion
If through the EASE Trial, Airway Bypass is shown to improve pulmonary function and reduce dyspnea while demonstrating an acceptable safety profile, then homogeneous patients will have a minimally invasive treatment option with meaningful clinical benefit.Trial Registration
ClinicalTrials.gov: NCT00391612 相似文献32.
Quaglio E Restelli E Garofoli A Dossena S De Luigi A Tagliavacca L Imperiale D Migheli A Salmona M Sitia R Forloni G Chiesa R 《PloS one》2011,6(4):e19339
The cellular pathways activated by mutant prion protein (PrP) in genetic prion diseases, ultimately leading to neuronal dysfunction and degeneration, are not known. Several mutant PrPs misfold in the early secretory pathway and reside longer in the endoplasmic reticulum (ER) possibly stimulating ER stress-related pathogenic mechanisms. To investigate whether mutant PrP induced maladaptive responses, we checked key elements of the unfolded protein response (UPR) in transgenic mice, primary neurons and transfected cells expressing two different mutant PrPs. Because ER stress favors the formation of untranslocated PrP that might aggregate in the cytosol and impair proteasome function, we also measured the activity of the ubiquitin proteasome system (UPS). Molecular, biochemical and immunohistochemical analyses found no increase in the expression of UPR-regulated genes, such as Grp78/Bip, CHOP/GADD153, or ER stress-dependent splicing of the mRNA encoding the X-box-binding protein 1. No alterations in UPS activity were detected in mutant mouse brains and primary neurons using the Ub(G76V)-GFP reporter and a new fluorogenic peptide for monitoring proteasomal proteolytic activity in vivo. Finally, there was no loss of proteasome function in neurons in which endogenous PrP was forced to accumulate in the cytosol by inhibiting cotranslational translocation. These results indicate that neither ER stress, nor perturbation of proteasome activity plays a major pathogenic role in prion diseases. 相似文献
33.
Salmona M Capobianco R Colombo L De Luigi A Rossi G Mangieri M Giaccone G Quaglio E Chiesa R Donati MB Tagliavini F Forloni G 《Journal of virology》2005,79(17):11225-11230
To investigate whether plasminogen may feature in scrapie infection, we inoculated plasminogen-deficient (Plg(-/-)), heterozygous plasminogen-deficient (Plg(+/-)), and wild-type (Plg(+/+)) mice by the intracerebral or intraperitoneal (i.p.) route with the RML scrapie strain and monitored the onset of neurological signs of disease, survival time, brain, and accumulation of scrapie disease-associated forms of the prion protein (PrP(Sc)). Only after i.p. inoculation, a slight, although significant, difference in survival (P < 0.05) between Plg(-/-) and Plg(+/+) mice was observed. Neuropathological examination and Western blot analysis were carried out when the first signs of disease appeared in Plg(+/+) animals (175 days after i.p. inoculation) and when mice reached the terminal stage of illness. At the onset of symptoms, PrP(Sc) accumulation was higher in the brain and spleen of Plg(+/+) and Plg(+/-) mice than in those of Plg(-/-) mice, and these differences were paralleled by differences in the severity of spongiform changes and astrogliosis in the cerebral cortex and subcortical gray structures. Immunohistochemical analysis of the spleens before inoculation did not show any impairment of the immune system affecting follicular dendritic or lymphoid cells in Plg(-/-) mice. Once the disease progressed and mice began to die of infection, differences were no longer apparent in either brains or spleens. In conclusion, our data indicate that plasminogen has no major effect on the survival of scrapie agent-infected mice. 相似文献
34.
Cytosolic prion protein (PrP) is not toxic in N2a cells and primary neurons expressing pathogenic PrP mutations 总被引:4,自引:0,他引:4
Fioriti L Dossena S Stewart LR Stewart RS Harris DA Forloni G Chiesa R 《The Journal of biological chemistry》2005,280(12):11320-11328
Inherited prion diseases are linked to mutations in the prion protein (PrP) gene, which favor conversion of PrP into a conformationally altered, pathogenic isoform. The cellular mechanism by which this process causes neurological dysfunction is unknown. It has been proposed that neuronal death can be triggered by accumulation of PrP in the cytosol because of impairment of proteasomal degradation of misfolded PrP molecules retrotranslocated from the endoplasmic reticulum (Ma, J., Wollmann, R., and Lindquist, S. (2002) Science 298, 1781-1785). To test whether this neurotoxic mechanism is operative in inherited prion diseases, we evaluated the effect of proteasome inhibitors on the viability of transfected N2a cells and primary neurons expressing mouse PrP homologues of the D178N and nine octapeptide mutations. We found that the inhibitors caused accumulation of an unglycosylated, aggregated form of PrP exclusively in transfected N2a expressing PrP from the cytomegalovirus promoter. This form contained an uncleaved signal peptide, indicating that it represented polypeptide chains that had failed to translocate into the ER lumen during synthesis, rather than retrogradely translocated PrP. Quantification of N2a viability in the presence of proteasome inhibitors demonstrated that accumulation of this form was not toxic. No evidence of cytosolic PrP was found in cerebellar granule neurons from transgenic mice expressing wild-type or mutant PrPs from the endogenous promoter, nor were these neurons more susceptible to proteasome inhibitor toxicity than neurons from PrP knock-out mice. Our analysis fails to confirm the previous observation that mislocation of PrP in the cytosol is neurotoxic, and argues against the hypothesis that perturbation of PrP metabolism through the proteasomal pathway plays a pathogenic role in prion diseases. 相似文献
35.
La Piana G Marzulli D Gorgoglione V Lofrumento NE 《Archives of biochemistry and biophysics》2005,436(1):91-100
Cytochrome c (cyto-c) added to isolated mitochondria promotes the oxidation of extra-mitochondrial NADH and the reduction of molecular oxygen associated to the generation of an electrochemical membrane potential available for ATP synthesis. The electron transport pathway activated by exogenous cyto-c molecules is completely distinct from the one catalyzed by the respiratory chain. Dextran sulfate (500 kDa), known to interact with porin (the voltage-dependent anion channel), other than to inhibit the release of ATP synthesized inside the mitochondria, greatly decreases the activity of exogenous NADH/cyto-c system of intact mitochondria but has no effect on the reconstituted system made of mitoplasts and external membrane preparations. The results obtained are consistent with the existence of specific contact sites containing cytochrome oxidase and porin, as components of the inner and the outer membrane respectively, involved in the oxidation of cytosolic NADH. The proposal is put forward that the bi-trans-membrane electron transport chain activated by cytosolic cyto-c becomes, in physio-pathological conditions: (i) functional in removing the excess of cytosolic NADH; (ii) essential for cell survival in the presence of an impairment of the first three respiratory complexes; and (iii) an additional source of energy at the beginning of apoptosis. 相似文献
36.
Dipolar waves are distinct hallmarks of both the secondary and tertiary structures of alpha-helical proteins that are immobilized in membrane bilayers or embedded in anisotropic media. We present a simple, semi-empirical approach that exploits the modulation of the amplitude and average of dipolar waves to determine the topology of alpha-helical proteins. Moreover, we describe the application of this method for the structural determination of a detergent solubilized membrane protein, phospholamban (PLB) that is involved in calcium regulation of cardiac muscle. When combined with high-resolution solid-state NMR data, this method can serve as a fast route for determining the topology of helical membrane proteins solubilized in detergent micelles. 相似文献
37.
Tringali C Papini N Fusi P Croci G Borsani G Preti A Tortora P Tettamanti G Venerando B Monti E 《The Journal of biological chemistry》2004,279(5):3169-3179
Recombinant human cytosolic sialidase (HsNEU2), expressed in Escherichia coli, was purified to homogeneity, and its substrate specificity was studied. HsNEU2 hydrolyzed 4-methylumbelliferyl alpha-NeuAc, alpha 2-->3 sialyllactose, glycoproteins (fetuin, alpha-acid glycoprotein, transferrin, and bovine submaxillary gland mucin), micellar gangliosides GD1a, GD1b, GT1b, and alpha 2-->3 paragloboside, and vesicular GM3. alpha 2-->6 sialyllactose, colominic acid, GM1 oligosaccharide, whereas micellar GM2 and GM1 were resistant. The optimal pH was 5.6, kinetics Michaelis-Menten type, V(max) varying from 250 IU/mg protein (GD1a) to 0.7 IU/mg protein (alpha(1)-acid glycoprotein), and K(m) in the millimolar range. HsNEU2 was activated by detergents (Triton X-100) only with gangliosidic substrates; the change of GM3 from vesicular to mixed micellar aggregation led to a 8.5-fold V(max) increase. HsNEU2 acted on gangliosides (GD1a, GM1, and GM2) at nanomolar concentrations. With these dispersions (studied in detailed on GM1), where monomers are bound to the tube wall or dilutedly associated (1:2000, mol/mol) to Triton X-100 micelles, the V(max) values were 25 and 72 microIU/mg protein, and K(m) was 10 and 15 x 10(-9) m, respectively. Remarkably, GM1 and GM2 were recognized only as monomers. HsNEU2 worked at pH 7.0 with an efficiency (compared with that at pH 5.6) ranging from 4% (on GD1a) to 64% (on alpha(1)-acid glycoprotein), from 7% (on GD1a) to 45% (on GM3) in the presence of Triton X-100, and from 30 to 40% on GM1 monomeric dispersion. These results show that HsNEU2 differentially recognizes the type of sialosyl linkage, the aglycone part of the substrate, and the supramolecular organization (monomer/micelle/vesicle) of gangliosides. The last ability might be relevant in sialidase interactions with gangliosides under physiological conditions. 相似文献
38.
Zanusso G Farinazzo A Prelli F Fiorini M Gelati M Ferrari S Righetti PG Rizzuto N Frangione B Monaco S 《The Journal of biological chemistry》2004,279(37):38936-38942
In prion diseases, the cellular prion protein (PrP(C)) is converted to an insoluble and protease-resistant abnormal isoform termed PrP(Sc). In different prion strains, PrP(Sc) shows distinct sites of endogenous or exogenous proteolysis generating a core fragment named PrP27-30. Sporadic Creutzfeldt-Jakob disease (sCJD), the most frequent human prion disease, clinically presents with a variety of neurological signs. As yet, the clinical variability observed in sCJD has not been fully explained by molecular studies relating two major types of PrP27-30 with unglycosylated peptides of 21 (type 1) and 19 kDa (type 2) and the amino acid methionine or valine at position 129. Recently, smaller C-terminal fragments migrating at 12 and 13 kDa have been detected in different sCJD phenotypes, but their significance remains unclear. By using two-dimensional immunoblot with anti-PrP antibodies, we identified two novel groups of protease-resistant PrP fragments in sCJD brain tissues. All sCJD cases with type 1 PrP27-30, in addition to MM subjects with type 2 PrP27-30, were characterized by the presence of unglycosylated PrP fragments of 16-17 kDa. Conversely, brain homogenates from patients VV and MV with type 2 PrP27-30 contained fully glycosylated PrP fragments, which after deglycosylation migrated at 17.5-18 kDa. Interestingly, PrP species of 17.5-18 kDa matched deglycosylated forms of the C1 PrP(C) fragment and were associated with tissue PrP deposition as plaque-like aggregates or amyloid plaques. These data show the presence of multiple PrP(Sc) conformations in sCJD and, in addition, shed new light on the correlation between sCJD phenotypes and disease-associated PrP molecules. 相似文献
39.
Searching for markers of Creutzfeldt-Jakob disease in cerebrospinal fluid by two-dimensional mapping
Piubelli C Fiorini M Zanusso G Milli A Fasoli E Monaco S Righetti PG 《Proteomics》2006,6(Z1):S256-S261
Differential proteomic analysis has been performed on the cerebrospinal fluid (CSF) of six healthy and six patients suffering form sporadic Creutzfeldt-Jakob disease (sCJD), age- and sex-matched, after immuno-subtraction of albumin and immunoglobulins. These maps have revealed 28 polypeptide chains differentially modulated in the sCJD samples, of which 10 appeared to be up-regulated, the remaining 18 being down-regulated. Among those, 13 could be identified upon digestion and MALDI-TOF, MS analysis. In addition, the strong modulation of cystatin C was also confirmed by immunoblot analysis and the highly altered level of the 14-3-3 proteins that escaped detection by 2-D mapping, could be assessed by Western blots and immuno-detection of monomeric and homo- and hetero-dimeric 14-3-3 isotypes. In search for a panel of potential markers for sCJD, we highlight cystatin C, 14-3-3 proteins, transferrin, ubiquitin, Apoliprotein J and perhaps some of the still unidentified, but strongly modulated polypeptide chains detected in the differential map. 相似文献
40.
Biasini E Massignan T Fioriti L Rossi V Dossena S Salmona M Forloni G Bonetto V Chiesa R 《Proteomics》2006,6(9):2823-2834
Inherited prion diseases are linked to insertional and point mutations in the prion protein (PrP) gene, which favor conversion of PrP into a conformationally altered, pathogenic isoform. The cellular mechanism by which this process causes neurological dysfunction is unknown. Transgenic (Tg) (PG14) mice express a mouse PrP homolog of a nine-octapeptide insertion associated with an inherited prion disorder. These mice develop a progressive neurological syndrome characterized by ataxia and cerebellar atrophy due to synaptic degeneration in the molecular layer and massive apoptosis of granule neurons. To investigate the molecular events that may contribute to neurological dysfunction, we carried out a differential proteomic analysis of cerebella from Tg(PG14) mice at the preclinical, onset, and symptomatic phases of their neurological illness. 2-D maps of cerebellar proteins from Tg(PG14) mice were compared to those obtained from age-matched Tg(WT) mice that express wild-type PrP and remain healthy. Proteins whose levels were significantly modified in at least one stage of the Tg(PG14) disease were identified by PMF. Analysis detected a preclinical decrease of the calcium/calmodulin-dependent phosphatase calcineurin (CaN) in granule neurons, suggesting that dysregulation of CaN activity induced by mutant PrP may be responsible for the cerebellar dysfunction in Tg(PG14) mice. 相似文献