Interactions between SERPINA1 PiMZ genotype, occupational exposure and lung function decline.

OBJECTIVES: We evaluated interactions between SERPINA1 PiMZ genotype, associated with intermediate α1-antitrysin deficiency, with outdoor particulate matter ≤10 µm (PM10), and occupational exposure to vapours, dusts, gases and fumes (VGDF), and their effects on annual change in lung function. METHODS: Pre-bronchodilator spirometry was performed in 3739 adults of the Swiss Cohort Study on Air Pollution and Lung Disease in Adults (SAPALDIA) for whom SERPINA1 genotypes were available. At baseline in 1991, participants were aged 18-62 years; follow-up measurements were conducted from 2001 to 2003. In linear mixed regression models of annual change in lung function, multiplicative interactions were evaluated between PiMZ genotype (PiMM as reference) and change in PM10 (µg/m(3)), and VGDF exposure (high-level, low-level or no exposure as reference) during follow-up. RESULTS: Annual declines in forced expiratory flow at 25-75% of forced vital capacity (FEF25-75%) (-82 mL/s, 95% CI -125 to -39) and forced expiratory volume in 1 s over forced vital capacity (FEV1/FVC) (-0.3%, 95% CI -0.6% to 0.0%) in association with VGDF exposure were observed only in PiMZ carriers (Pinteraction<0.0001 and Pinteraction=0.03, respectively). A three-way interaction between PiMZ genotype, smoking and VGDF exposure was identified such that VGDF-associated FEF25-75% decline was observed only in ever smoking PiMZ carriers (Pinteraction=0.01). No interactions were identified between PiMZ genotype and outdoor PM10. CONCLUSIONS: SERPINA1 PiMZ genotype, in combination with smoking, modified the association between occupational VGDF exposure and longitudinal change in lung function, suggesting that interactions between these factors are relevant for lung function decline. These novel findings warrant replication in larger studies.

Laboratory testing of individuals with severe alpha1-antitrypsin deficiency in three European centres.

alpha(1)-Antitrypsin (AT) deficiency is a hereditary disorder that may lead to early-onset emphysema, and chronic liver disease later in life. Although there are validated methods for testing, the vast majority of alpha(1)-AT-deficient individuals remain undiagnosed. Recommendations have been published for the testing and diagnosis of alpha( 1)-AT deficiency; however, guidelines on best practice are not well established. In our article, we review the developments in diagnostic techniques that have taken place in recent years, and describe the practices used in our three European centres. The determination of the level of alpha(1)-AT and genotyping are reported as the main diagnostic steps, whereas isoelectric focusing (also referred to as phenotyping) is reserved for confirmatory analysis. The following recommendations for best practice are put forward: detection of all PiZZ and other severe deficiency individuals; automated genotyping; preparation of reference standards; quality control programmes; development of standard operating procedure documents; and standardised methods for the collection of dried blood samples. Closer cooperation between laboratories and the sharing of knowledge are recommended, with the objectives of improving the efficiency of the diagnosis of severe alpha(1)-AT deficiency, increasing the numbers of individuals who are detected with the disorder, and assisting the establishment of new patient identification programmes.

Plasmapheresis for treatment of pulmonary alveolar proteinosis.

Whole lung lavage (WLL) is currently the standard therapy for pulmonary alveolar proteinosis (PAP). Nevertheless, some PAP patients respond poorly to WLL or require it frequently. The present paper reports a patient with autoimmune PAP with persistent disease despite three WLL treatments over 10 months. Plasmapheresis with ten 1.5-L plasma exchanges was performed, which lowered the serum granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibody level from 250 microg mL(-1) to 156 microg mL(-1) but did not improve respiratory impairment. Further WLL therapy was required and transiently effective. Serum GM-CSF autoantibody levels declined progressively, reaching a value of 56 microg mL(-1) 80 weeks after completion of plasmapheresis. However, this decrease was not accompanied by clinical improvement and the patient required additional WLL therapy. The results confirm that minor reductions in serum granulocyte-macrophage colony-stimulating factor autoantibody levels from plasmapheresis are not reflected in clinical improvement in the severity of lung disease in pulmonary alveolar proteinosis.

Desmosine as a biomarker of elastin degradation in COPD: current status and future directions.

Desmosine (DES) and isodesmosine (IDES) are two unusual, tetrafunctional, pyridinium ring-containing amino acids involved in elastin cross-linking. Being amino acids unique to mature, cross-linked elastin, they are useful for discriminating peptides derived from elastin breakdown from precursor elastin peptides. According to these features, DES and IDES have been extensively discussed as potentially attractive indicators of elevated lung elastic fibre turnover and markers of the effectiveness of agents with the potential to reduce elastin breakdown. In the present manuscript, immunology-based and separation methods for the evaluation of DES and IDES are discussed, along with studies reporting increased levels of urine excretion in chronic obstructive pulmonary disease (COPD) patients with and without alpha(1)-antitrypsin deficiency. The results of the application of DES and IDES as surrogate end-points in early clinical trials in COPD are also reported. Finally, recent advances in detection techniques, including liquid chromatography tandem mass spectrometry and high-performance capillary electrophoresis with laser-induced fluorescence, are discussed. These techniques allow detection of DES and IDES at very low concentration in body fluids other than urine, such as plasma or sputum, and will help the understanding of whether DES and IDES are potentially useful in monitoring therapeutic intervention in COPD.

Secondary outputs of alpha1-antitrypsin deficiency targeted detection programme.

Targeted detection programmes are recommended to identify subjects affected by severe alpha1-antitrypsin deficiency (AATD). Guidelines are available to address physicians towards subjects at high risk for AATD. We wanted to investigate the clinical characteristics of subjects enrolled in the programme, who result as not being affected by severe AATD; this information is not available in the present literature. We elaborated data contained in the questionnaires accompanying the samples of 2127 Italian subjects submitted for AATD detection in a period spanning 11 years (1996-2006). A total of 588 subjects were eligible to enter this study: PI*MM subjects and subjects with intermediate AATD, referred for lung disease, were characterised by a relatively young mean age, and a high proportion (31.2% and 28.6%, respectively) were never smokers. Fifty percent or more had symptoms of chronic bronchitis, but without obstruction. Only a minority belonged to most severe GOLD stages. The mean levels of AAT varied as a function of the presence or absence of airflow obstruction in intermediate AATD subjects, but not in PI*MM. Individuals enrolled in AATD detection programmes represent an interesting cohort both for public health and research purposes.

Alpha-1 antitrypsin deficiency in Italy: regional differences of the PIS and PIZ deficiency alleles.

BACKGROUND: Critical to the effective diagnosis and management of disease is information on its prevalence in a particular geographic area such as Italy. Alpha-1 antitrypsin deficiency (AAT Deficiency) is one of the most common serious hereditary diseases in the world, but its prevalence varies markedly from one country to another. AAT Deficiency affects at least 120.5 million carriers and deficient subjects worldwide for the two most prevalent deficiency alleles PIS and PIZ. This genetic disease is known to exist in Italy and is related to a high risk for development of jaundice in infants, liver disease in children and adults, and pulmonary emphysema in adults. METHODS: Studies on the genetic epidemiology of AAT Deficiency has resulted in the development of a unique database that permits a unique analysis of the geographic distribution in 14 different regions located at random from Piemonte to Sicilia. RESULTS: The use of Hardy-Weinberg statistical analysis to evaluate the distribution of these two deficiency alleles has demonstrated striking differences in the frequencies of these two deficiency alleles in these 14 different regions with 23/84 pair wise combinations significantly different (P=0.05) for PIS, and 5/84 combinations for PIZ. CONCLUSIONS: These findings demonstrate differences that impact the standards of care and diagnosis of AAT Deficiency in Italy since the prevalence of these deficiency alleles is not uniform throughout the country.

Increased frequency of CFTR gene mutations in sarcoidosis: a case/control association study.

A complete screening of the CFTR gene by DGGE and DNA sequencing was performed in patients with sarcoidosis. In 8/26 cases, missense and splicing CFTR gene mutations were found, a significant difference over controls (9/89) from the same population (P = 0.014). The odds ratio for a person with a CFTR gene mutation to develop the disease is 3.95 (1.18 < OR < 13.26). Seven different CFTR gene mutations were observed: R75Q, R347P, 621 + 3 A/G, 1898 + 3 A/G, L997F, G1069R, and a novel mutation which was detected in this study, I991V. R75Q mutation was present in 3/26 patients, a significant increase (P = 0. 01) in cases over controls, indicating its preferential association with sarcoidosis. A trend towards disease progression was observed in patients with CFTR gene mutations compared to patients without mutations. These data suggest that CFTR gene mutations predispose to the development of sarcoidosis.

Bioengineering: alpha 1-proteinase inhibitor site-specific mutagenesis. The prospect for improving the inhibitor.

alpha 1-Proteinase inhibitor (alpha 1-PI) augmentation therapy has been licensed for treatment of alpha 1-PI-deficient individuals with pulmonary emphysema. The currently available product is purified from pooled human plasma. To obtain larger amounts of protein free from possible unknown plasma contaminants, human alpha 1-PI has been produced by recombinant DNA. Since wild-type alpha 1-PI is susceptible to oxidative impairment, several alpha 1-PI variants in which the active site oxidation-sensitive residue is replaced by inert residues have been constructed. This article is aimed at reviewing the history, biological efficacy, advantages, disadvantages, and concerns linked to alpha 1-PI recombinant DNA and site-specific mutagenesis technology.

Elevated serum procollagen III aminopeptide levels in sarcoidosis.

Procollagen III aminopeptide (P-III-P), a peptide released during the conversion of type III procollagen to type III collagen, is considered a potential marker of fibroblast activity in a variety of pulmonary and extrapulmonary diseases. The aim of the present article was to investigate the levels of P-III-P in serum samples (sP-III-P) from a large number of sarcoid patients, in particular looking at its relationship with other markers of disease activity and its presumed role as a marker of pulmonary fibrosis. sP-III-P has been radioimmunoassayed in an overall series of 57 patients and the levels were higher (19.18 +/- 9.17 ng/ml) than in 25 age- and sex-matched controls (11.32 +/- 2.15 ng/ml; p less than 0.001). The elevation was neither sex-related nor related to obvious liver sarcoid localization. Although sP-III-P levels were slightly higher in patients with stage II, there was no significant difference in patients with stage I or III. We found a positive relationship with serum angiotensin-converting enzyme (S-ACE) levels (p less than 0.04), but not with other markers of disease activity (67Ga uptake, bronchoalveolar lavage [BAL] lymphocyte percent, vital capacity, and lung diffusing capacity). The relationship with S-ACE was confirmed in a longitudinal follow-up study, where sP-III-P strictly paralleled the S-ACE behavior. Finally, the initial sP-III-P levels did not predict cases either with disease relapse or resistance to corticosteroid treatment. We conclude that, in our study, sP-III-P levels failed to characterize sarcoid patients with radiologic fibrotic pattern (stage III), and, in addition, were unable to predict which patients would have a poor prognosis. Rather, they reflect a metabolic activity of sarcoid granuloma cells. Thus, the usefulness of sP-III-P in the treatment of patients with sarcoid may be considered similar to that of S-ACE.

Tumor necrosis factor gene complex in COPD and disseminated bronchiectasis.

BACKGROUND: Tumor necrosis factor (TNF) is a potent proinflammatory cytokine with increased levels in the sputum of COPD subjects. Two biallelic TNF gene complex polymorphisms have been described: LtalphaNcoI, in the first intron of the lymphotoxin alpha (previously referred to as TNF-beta) gene, and TNF-308, in the promoter region of the TNF-alpha gene. Higher levels of TNF production are associated with allele 1 of LtalphaNcoI (LtalphaNcoI*1) and with allele 2 of TNF-308 (TNF-308*2). STUDY OBJECTIVES: To study the frequencies of the two TNF gene complex polymorphisms in patients with COPD and bronchiectasis. DESIGN: Association study. SUBJECTS AND METHODS: We studied the frequencies of these polymorphisms in 66 subjects with COPD and in 23 subjects with disseminated bronchiectasis and compared them to the frequencies in 98 healthy control subjects and 45 subjects with nonobstructive pulmonary disease. Genomic DNA samples were extracted, and TNF-alpha and LtalphaNcoI polymorphisms were detected after polymerase chain reaction by restriction digestion. RESULTS: We found the following frequencies: the TNF-308*2 allele was detected in 11% of COPD individuals, 15% of bronchiectasis patients, 10% of healthy control subjects, and 18% of subjects with nonobstructive pulmonary disease. The LtalphaNcoI*1 allele was detected in 28% of COPD individuals, 30% of bronchiectasis patients, 29% of healthy control subjects, and 29% of subjects with nonobstructive pulmonary disease. We found evidence of linkage disequilibrium between the two loci (Delta = 0.068). CONCLUSIONS: We conclude that the TNF gene complex, at least in Caucasoid individuals and for the considered polymorphisms, does not seem to play a major role as genetic risk factor in COPD and bronchiectasis.

HLA class I, II, and III polymorphism in Italian patients with sarcoidosis. The Pavia-Padova Sarcoidosis Study Group.

We studied the HLA polymorphisms (class I, II, and III) in 107 Italian patients with biopsy specimen-proven sarcoidosis in order to investigate the immunogenetic background of this disease. The mean age of onset of the disease was 36.08 +/- 12.4 years. Four patients (3.73 percent) were in radiologic stage 0, 38 patients (35.51 percent) were in radiologic stage I, 40 patients (37.38 percent) were in stage II, and 25 (23.36 percent) were in stage III. Thirty-eight patients (35.51 percent) had one or more extrapulmonary localization(s) of the disease. Positive association between sarcoidosis and HLA-B8 (chi 2 = 6.07, p = 0.0127, RR = 1.91) was confirmed. Regarding the age of onset of the disease, HLA-B35 was more frequent (chi 2 = 7.34, p = 0.0056, pc < 0.05, RR = 4.62) in patients with early onset of symptoms and/or signs, before the mean age of 36 years. With reference to the radiologic stage of the disease, HLA class II marker DR3 was more frequent in patients with stage I (chi 2 = 7.22, p = 0.0061, pc < 0.05, RR = 7.08). No significant relationship was found between sarcoidosis and HLA class III markers. These results seem to confirm an association of sarcoidosis with HLA classic genes and can sustain the hypothesis of a genetic heterogeneity of this disease.

Proteinase/proteinase inhibitor imbalance in sputum sol phases from patients with chronic obstructive pulmonary disease. Suggestions for a key role played by antileukoprotease.

In order to characterize the imbalance between proteinases and proteinase inhibitors in sputum sol phases, we studied 25 patients (mean age, 59 +/- 11 yr) with exacerbated chronic obstructive pulmonary disease (COPD). An aliquot of sputum was used for bacteriologic determinations, and the remainder was centrifuged in order to obtain gel and sol phases. On the basis of the bacteriologic data, patients were divided into colonized patients (14) and noncolonized patients (11). All of the major inhibitors were immunologically detectable in sol phases without a significant difference between colonized and noncolonized patients (alpha 1-proteinase inhibitor [alpha 1-PI], 2.56 microM +/- 0.53 microM and 2.39 microM +/- 0.72 microM; alpha 2-macroglobulin [alpha 2-MG], 0.21 microM +/- 0.07 microM and 0.16 microM +/- 0.05 microM; antileukoprotease (ALP), 1.78 microM +/- 0.57 microM and 1.53 microM +/- 0.6 microM, respectively [mean +/- SE]). With regard to proteinase activities, both free elastase-like and free chymotrypsin-like activities were detectable in the majority of patients (15/25) (0.59 microM +/- 0.15 microM and 0.74 microM +/- 0.15 microM for elastase-like activity [ELA], and 0.010 microM +/- 0.003 microM and 0.017 microM +/- 0.007 microM for chymotrypsin-like activity [CLA], respectively [mean +/- SE]). The inhibitory profile of proteinase activities, performed by means of a panel of inhibitors, allowed us to assign specific activities mainly to neutrophil elastase and cathepsin G (Cat G). Next we looked at the relationships between inhibitors and proteinase activities. We found a significant negative correlation between neutrophil elastase activity and ALP (r = -0.58; p < 0.01). In confirmation of this suggestion, sol phases were divided into samples (15) with detectable ELA (> 0.50 microM) and samples (10) with no detectable ELA (< 0.18 microM). Levels of alpha 1-PI and alpha 2-MG did not differ significantly between the two groups, whereas ALP values were higher in the group with no detectable ELA (3.12 microM +/- 0.69 microM) than in the other group (0.58 microM +/- 0.21 microM; p < 0.001). We conclude that most sputum sol phases from patients with exacerbated COPD have a high burden of free neutrophil elastase and Cat G. Antileukoprotease seems to be the major naturally occurring inhibitor effective in the modulation of proteinase activities in bronchial secretions under these conditions.

Lung cancer in young adults.

OBJECTIVE: To define the basis for the conflicting reports on the prognosis of lung cancer in young adults. DESIGN: Retrospective review of lung cancer patients between 1977 and 1988. SETTING: Medical centers in Chicago (Northwestern Memorial Hospital), northern Israel (Rambam Medical Center), and northern Italy (S. Anna and U. of Pavia Hospitals). PATIENTS: Patients were < or = 45 years of age with a diagnosis of primary lung cancer identified from tumor registry records, pathology reports, and hospital charts, plus a sample of patients > 45 years of age. MEASUREMENTS AND MAIN RESULTS: In Chicago, younger patients had a higher incidence of chest pain, fever, and neurologic symptoms at presentation than the older patients, and fewer were asymptomatic. They also had more lower lobe lesions on chest roentgenogram, a higher incidence of adenocarcinoma, more advanced disease, an increased likelihood of receiving chemotherapy, and reduced survival (p < 0.03). The poorer prognosis was due to more advanced disease at presentation. In Israel, younger patients more frequently presented with stage I disease than the older patients and they had a higher incidence of adenocarcinoma, an increased likelihood of receiving treatment especially surgery, and better survival (p < 0.02). There were no differences between the two age groups for symptoms, symptom duration, and chest roentgenogram findings. Compared with the younger patients in Chicago and Israel, those from northern Italy had more squamous cell cancers and fewer adenocarcinomas, more commonly presented with stage I or II disease, received radiation therapy less frequently, and were given supportive care more often. Survival was low and comparable to that reported from Chicago. CONCLUSION: Differences exist in the clinical characteristics, pathologic findings, and prognosis of younger and older patients with lung cancer from the same region and of younger patients from different regions. The difference in prognosis is related in part to the stage of disease at presentation and the ability to undergo resectional surgery.

Enzymatic modification of natural compounds with pharmacological properties.

Glycosides of various classes of natural products are widely distributed in nature, where they are often present esterified with aliphatic and aromatic acids at specific OH's of their sugar moieties. Many of these compounds are pharmacologically important molecules or possess other interesting properties. For instance, ginsenosides (e.g., 3) are therapeutic dammarane-type oligoglycosides isolated from the water-soluble portion of the dried roots and leaves of Panax ginseng C.A. Meyer (Aralianceae), a plant widely used in traditional Chinese medicine. In recent years, we have exploited the regioselectivity of lipases and proteases in organic solvents for the synthesis of specific esters of ginsenosides as well as the selectivity of the beta-1,4-galactosyltransferase from bovine colostrum to obtain new glycosyl derivatives of these compounds. The application of these two enzymatic methodologies has also been exemplified with other natural compounds with pharmacological properties: digitonin (5), colchicoside (6), and flavonoid glycosides.

Eosinophilic pleural effusion due to mesalamine. Report of a rare occurrence.

Mesalamine-induced lung toxicity has often been described. We report on a case of a patient who underwent mesalamine treatment, though in the absence of established criteria required for diagnosing Crohn's disease (CD) or ulcerative colitis (UC). He developed an adverse respiratory reaction to the drug, thus definitely proving its lung damaging capacity. The clinical presentation included eosinophilic pleural effusion, a feature never previously described in association with mesalamine intake.

Effects of MR889, a cyclic thiolic neutrophil elastase inhibitor, on rheologic properties of mucus.

MR889 is a synthetic inhibitor of human neutrophil elastase with potential in clinical conditions characterized by a high load of this proteolytic enzyme, mainly chronic obstructive pulmonary disease. On the basis of its mechanism of inhibition, it has been suggested that MR889, upon reaction with elastase, would generate new free thiol groups. The aim of this study was to investigate whether MR889, upon reaction with elastase, may modify rheologic properties, i.e. apparent viscosity and elasticity, of both human sputum and porcine stomach mucus, in ex-vivo and in vitro experiments. MR889 10(-4) M alone had no effect on rheologic properties of samples, whereas an agent with free thiol group, n-acetylcysteine 10(-4) M reduced both viscosity and elasticity. MR889 10(-4) M upon reaction with elastase 0.03 nMol (giving 83% of elastase inhibition) had no effect on rheologic properties of samples, whereas upon reaction with elastase 0.15 nMol (49% inhibition) yielded a marked decrease in viscosity. This result would suggest that MR889, in the presence of a high load of elastase, may acquire direct mucus modifying capacity, likely because of its greater utilization upon reaction with the enzyme.

Simultaneous determination of Pseudomonas aeruginosa elastase, human leukocyte elastase and cathepsin G activities by micellar electrokinetic chromatography.

Micellar electrokinetic chromatography (MEKC) is a new method for analysing proteolytic activities simultaneously present in incubation mixtures. Here we demonstrate that MEKC differentiates between the enzymatic activities of Pseudomonas aeruginosa elastase (PsE) and human leukocyte elastase (HLE) or cathepsin G (Cat G) in assays using the chromogenic peptide substrates Suc-Ala-Ala-Ala-NA or Suc-Ala-Ala-Pro-Phe-NA, respectively (where Suc = succinyl and NA = 4-nitroaniline/u-nitroanilide). When PsE and Cat G were incubated at equimolar ratio with Suc-Ala-Ala-Pro-Phe-NA, the PsE-specific cleavage products PheNA and Suc-Ala-Ala-Pro were detected whereas inhibition of the metalloproteinase PsE with EDTA resulted in detection of NA and Suc-Ala-Ala-Pro-Phe only. Similarly, when PsE and HLE were incubated at equimolar ratio with Suc-Ala-Ala-Ala-NA, the PsE-specific cleavage products Suc-Ala and Ala-Ala-NA were detected whereas at an PsE-HLE ratio 1:50, both the PsE-specific and the HLE-specific cleavage products NA and Suc-Ala-Ala-Ala were separated. MEKC also allowed determination of the kinetic constants for the interactions of PsE, Cat G and HLE with the substrates considered.