Paolo Iadarola

Association between markers of emphysema and more severe chronic obstructive pulmonary disease

Background: The predominant emphysema phenotype is associated with more severe airflow limitation in patients with chronic obstructive pulmonary disease (COPD). A study was undertaken to investigate whether COPD patients, with or without emphysema quantitatively confirmed by high resolution computed tomography (HRCT), have different COPD severity as assessed by the BODE index (body mass index, airflow obstruction, dyspnoea, exercise performance) and inspiratory capacity to total lung capacity ratio (IC/TLC), and by different biological markers of lung parenchymal destruction. Methods: Twenty six outpatients with COPD and eight healthy non-smokers were examined. Each subject underwent HRCT scanning, pulmonary function tests, cell counts, and measurements of neutrophil elastase, matrix metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinase (TIMP)-1 in induced sputum, as well as measurement of desmosine, a marker of elastin degradation in urine, plasma and sputum. Results: Patients with HRCT confirmed emphysema had a higher BODE index and lower IC/TLC ratio than subjects without HRCT confirmed emphysema and controls. Forced expiratory volume in 1 second (FEV1), FEV1/forced vital capacity ratio, and carbon monoxide transfer coefficient were lower, whereas the number of eosinophils, MMP-9, and the MMP-9/TIMP-1 ratio in sputum were higher in patients with emphysema. In COPD patients the number of sputum eosinophils was the biological variable that correlated positively with the HRCT score of emphysema (p = 0.04). Conclusions: These results suggest that COPD associated with HRCT confirmed emphysema is characterised by more severe lung function impairment, more intense airway inflammation and, possibly, more serious systemic dysfunction than COPD not associated with HRCT confirmed emphysema.

Alveolar inflammation in cystic fibrosis

BACKGROUND In infected lungs of the cystic fibrosis (CF) patients, opportunistic pathogens and mutated cystic fibrosis transmembrane conductance regulator protein (CFTR) contribute to chronic airway inflammation that is characterized by neutrophil/macrophage infiltration, cytokine release and ceramide accumulation. We sought to investigate CF lung inflammation in the alveoli. METHODS Lung tissue from 14 CF patients and four healthy individuals was analyzed for numbers of effector cells, elastin and collagen concentrations, inflammatory markers and density of Pseudomonas aeruginosa. Additionally, desmosine and isodesmosine concentrations were determined in 52 urine specimens from CF patients to estimate the burden of elastase activities in respiratory secretions. RESULTS Elastin concentration was significantly decreased and collagen significantly increased in CF alveolar tissues as compared to age-matched, healthy individuals. Elastin split products were significantly increased in urine samples from patients with CF and correlated inversely with age, indicating local tissue remodelling due to elastin degradation by unopposed proteolytic enzymes. Alveolar inflammation was also characterized by a significant cell infiltration of neutrophils, macrophages and T cells, extensive nuclear factor-kappaB and insulin-like growth factor-1 activation in various cell types and increased intercellular adhesion molecule-1 expression, and increased numbers of myofibroblasts. Additionally, ceramide accumulated in type II alveolar epithelial cells, lacking CFTR. P. aeruginosa organisms were rarely present in inflamed alveoli. CONCLUSIONS Chronic inflammation and remodeling is present in alveolar tissues of the CF lung and needs to be addressed by anti-inflammatory therapies.

Adequate energy-protein intake is not enough to improve nutritional and metabolic status in muscle-depleted patients with chronic heart failure

An adequate energy‐protein intake (EPI) when combined with amino acid supplementation may have a positive impact nutritional and metabolic status in patients with chronic heart failure (CHF).

Oral Amino Acid Supplements Improve Exercise Capacities in Elderly Patients with Chronic Heart Failure

We investigated whether 30 days of oral supplementation with a special mixture of amino acids (AAs), together with conventional therapy, could improve exercise capacity in elderly outpatients with chronic heart failure (CHF). A group of 95 outpatients (12 women and 83 men; New York Heart Association class II-III) aged 65-74 years were studied. This was a randomized, double-blind, placebo-controlled study. The patients performed a basal exercise test and were then randomly assigned to a special oral nutritional mixture of AAs 4 g twice daily (n = 43) or placebo (n = 42). After 30 days we repeated the exercise test. In both tests we measured the following: oxygen consumption (VO2), CO2 production (VCO2), minute ventilation (VE), oxygen cost of ventilation (VO2/VE), CO2 elimination per liter of ventilation (VCO2/VE), respiratory exchange ratio (RER; calculated as VCO2/VO2), oxygen pulse (VO2/heart rate [HR]) and anaerobic metabolism during exercise (ANA-VO2). At day 30, exercise capacity in the AA group had improved (+11 +/- 8 W, p <0.01; +67.5 +/- 44 seconds, p <0.02). This improvement was associated with both reduced circulatory dysfunction and increased peripheral oxygen availability. Indeed, peak VO2 increased by 1.2 +/- 1.1 mL/kg per min (+12.7% +/- 13%; p<0.02) and VO2/HR improved by 1.5 +/- 1.4 mL O2 per heartbeat (p <0.05). ANA-VO2 was reduced by >50% in patients on AAs (from 20.2 +/- 10 mL/kg at day 0 to 10.9 +/- 5 mL/kg at day 30; p <0.02). These variables did not significantly change for patients who received placebo. In conclusion, the study showed that oral AA supplementation, in conjunction with standard pharmacologic therapy, appears to increase exercise capacity by improving circulatory function, muscle oxygen consumption, and aerobic production of energy in elderly outpatients with CHF.

High levels of desmosines in urine and plasma of patients with pseudoxanthoma elasticum

Background  Pseudoxanthoma elasticum (PXE), a rare heritable disorder caused by mutations of the ABCC6 gene, is characterized by fragmentation and mineralization of elastic fibres. We determined the extent of degradation of elastin by measuring and comparing the amount of desmosines in plasma and urine of PXE patients, healthy carriers and normal subjects.

Branched-Chain Amino Acids May Improve Recovery From a Vegetative or Minimally Conscious State in Patients With Traumatic Brain Injury: A Pilot Study

OBJECTIVE To investigate whether supplementation with branched-chain amino acids (BCAAs) may improve recovery of patients with a posttraumatic vegetative or minimally conscious state. DESIGN Patients were randomly assigned to 15 days of intravenous BCAA supplementation (n=22; 19.6g/d) or an isonitrogenous placebo (n=19). SETTING Tertiary care rehabilitation setting. PARTICIPANTS Patients (N=41; 29 men, 12 women; mean age, 49.5+/-21 y) with a posttraumatic vegetative or minimally conscious state, 47+/-24 days after the index traumatic event. INTERVENTION Supplementation with BCAAs. MAIN OUTCOME MEASURE Disability Rating Scale (DRS) as log(10)DRS. RESULTS Fifteen days after admission to the rehabilitation department, the log(10)DRS score improved significantly only in patients who had received BCAAs (log(10)DRS score, 1.365+/-0.08 to 1.294+/-0.05; P<.001), while the log(10)DRS score in the placebo recipients remained virtually unchanged (log(10)DRS score, 1.373+/-0.03 to 1.37+/-0.03; P not significant). The difference in improvement of log(10)DRS score between the 2 groups was highly significant (P<.000). Moreover, 68.2% (n=15) of treated patients achieved a log(10)DRS point score of .477 or higher (3 as geometric mean) that allowed them to exit the vegetative or minimally conscious state. CONCLUSIONS Supplemented BCAAs may improve the recovery from a vegetative or minimally conscious state in patients with posttraumatic vegetative or minimally conscious state.

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (NADP): amino acid sequence of the subunits from isoenzyme I and structural relationship with isoenzyme II

The structural relationship between isoenzymes I and II of chloroplast glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NADP+ oxidoreductase (phosphorylating) EC 1.2.1.13) has been established at the protein level. The complete primary structure of subunits A and B of glyceraldehyde-3-phosphate dehydrogenase I from Spinacia oleracea has been determined by sequence analysis of the corresponding tryptic peptides, aligned by fragments derived from cyanogen bromide and Staphylococcus proteinase V8 digestions and by partially sequencing each intact subunit. Subunit A has an Mr of 36,225 and consists of 337 amino acid residues, whilst subunit B (Mr 39,355) consists of 368 residues. The amino acid sequence of subunit B, as determined through direct analysis of the protein, is identical to that recently deduced at cDNA level (Brinkmann et al. (1989) Plant Mol. Biol. 13, 81-94). The two subunits share a common portion of amino acid sequence which differs by 66 amino acid residues. Subunit B has an extra C-terminal sequence of 31 amino acid residues. Chloroplast glyceraldehyde-3-phosphate dehydrogenase II was partially characterized by sequencing the N-terminal portion of the intact protein and some of its tryptic peptides. The sequences of all the examined fragments fit precisely that of the corresponding regions of subunit A from glyceraldehyde-3-phosphate dehydrogenase I.

Profiling the Proteome of Exhaled Breath Condensate in Healthy Smokers and COPD Patients by LC-MS/MS

Three pools of exhaled breath condensate (EBC) from non-smokers plus healthy smokers (NS + HS, n = 45); chronic obstructive pulmonary disease (COPD) without emphysema (COPD, n = 15) and subjects with pulmonary emphysema associated with α1-antitrypsin deficiency (AATD, n = 23) were used for an exploratory proteomic study aimed at generating fingerprints of these groups that can be used in future pathophysiological and perhaps even clinical research. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was the platform applied for this hypothesis-free investigation. Analysis of pooled specimens resulted in the production of a “fingerprint” made of 44 proteins for NS/HS; 17 for COPD and 15 for the group of AATD subjects. Several inflammatory cytokines (IL-1α, IL-1β, IL-2; IL-12, α and β subunits, IL-15, interferon α and γ, tumor necrosis factor α); Type I and II cytokeratins; two SP-A isoforms; Calgranulin A and B and α1-antitrypsin were detected and validated through the use of surface enhanced laser-desorption ionization mass spectrometry (SELDI-MS) and/or by Western blot (WB) analysis. These results are the prelude of quantitative studies aimed at identifying which of these proteins hold promise as identifiers of differences that could distinguish healthy subjects from patients.

Proteomic analysis of exhaled breath condensate from single patients with pulmonary emphysema associated to α1-antitrypsin deficiency

The non-invasive character of exhaled breath (EBC) collection makes this fluid attractive for monitoring the respiratory tract by the measurement of various compounds. Because EBC is likely to reflect the composition of the airway-lining fluid, it can provide valuable information on possible disease states. Aim of our study was to apply proteomic technology to the study of EBC samples collected from single patients with pulmonary emphysema associated to alpha(1)-antitrypsin deficiency. The protein profiles from EBC of twenty patients and of twenty-five healthy individuals, used as controls, have been analyzed in parallel by a combination of 1-DE, 2-DE, micro-HPLC and MS. These sensitive techniques allowed to identify a number of cytokines and cytokeratins. Their level was found to be higher in patients than in controls.

Ex vivo evaluation of prolidase loaded chitosan nanoparticles for the enzyme replacement therapy

Prolidase loaded chitosan nanoparticles were set up in order to suggest an innovative therapeutic approach for Prolidase Deficiency (PD), a rare autosomal inherited disorder of the connective tissue. The satisfactory drug loading efficiency (42.6+/-2.1%) as well as the suitable physical characteristics (mean diameter of 365.5+/-35.1 nm and a positive zeta-potential of 17.94+/-0.12 mV) was achieved. In order to verify the compatibility of the chitosan nanoparticles with cells, the influence of the nanoparticles on the growth and the viability (MTT assay) of cultured skin fibroblasts were determined: the nanoparticles showed a good biocompatibility up to 5 microg of chitosan/10,000 fibroblasts. Uptake of chitosan nanoparticles by fibroblasts was verified by confocal microscopy using FITC-labelled chitosan nanoparticles. The ex vivo experiments were performed by incubating different amounts of prolidase loaded chitosan nanoparticles with skin human fibroblasts from PD patients for scheduled times. The restored prolidase activity was quantitatively monitored by a capillary electrophoretic method and confirmed by cells morphological observations. Standing from the nanoparticles internalization, the enzymatic activity was progressively restored reaching the best value (about 66%) after 5 days of co-incubation. Moreover, prolidase loaded chitosan nanoparticles permitted to restore prolidase activity in PD fibroblasts for a prolonged period of time (8 days).