Marina Gorrini

Lung injury and degradation of extracellular matrix components by Aspergillus fumigatus serine proteinase

Aspergillus fumigatus produces a variety of extracellular proteinases that are believed to be virulence factors towards Aspergillus-related lung disease. Among Aspergillus proteinases, the serine proteinase is thought to play a major virulent role because of its widespread production. Nevertheless, evidence of direct pulmonary injury caused by the A. fumigatus serine proteinase is still lacking. The purpose of our work was: (1) to provide evidence for a pivotal role of A. fumigatus serine proteinase in producing lung injury in an animal model, and (2) to investigate the broadness of the substrate specificity of the proteinase towards extracellular matrix components. To achieve this aim, the proteinase from an A. fumigatus strain isolated from human airways was purified by a four-step procedure, including cation exchange and hydrophobic interaction. High-performance capillary electrophoresis, SDS-PAGE, determination of K(m) towards synthetic substrates, and inhibitory studies were used to further characterize the A. fumigatus serine proteinase. With reference to extracellular matrix components, the A. fumigatus serine proteinase was shown to degrade human lung elastin at a higher rate than an equimolar amount of human neutrophil elastase. Human lung collagen, type I and type III collagens, as well as fibronectin, were quickly digested by the A. fumigatus serine proteinase. Finally, mice intratracheally injected with the proteinase showed a significant degree of lower respiratory tract destruction. We conclude that the A. fumigatus serine proteinase is capable per se of hydrolyzing the major structural barriers of the lung.

Identification of a novel alpha1-antitrypsin null variant (Q0Cairo).

Alpha1-antitrypsin deficiency (AATD) is a common hereditary disorder associated with high risk of developing pulmonary emphysema early in life and, to a lesser extent, chronic liver disease and cirrhosis. Among Northern Europeans and Northern Americans, more than 95% of individuals with emphysema associated with AATD carry the most frequent AAT deficient gene variants, PI*Z and PI*S. Rare AAT deficient variants account for 2-4% of AATD individuals. We extend the sequence data on AAT by characterizing a novel Null allele detected in 3 subjects: a carrier belonging to an Italian/Egyptian family and 2 members of a family originating from Southern Italy. The mutation raised on a M1 (Ala213) base allele and it is characterized by an A→T transversion at exon III, nt 218, codon 259 (AAA→TAA) (GeneBank accession number AY 256958). The transversion results in a premature stop codon (Lys259AAA→Stop259TAA). The proposed nomenclature of Q0cairo is from the birthplace of the father of first recognized subject. Serum levels and isoelectric focusing of AAT were consistent with the presence of the Null variant.

Screening for Alpha 1 antitrypsin deficiency in Tunisian subjects with obstructive lung disease: a feasibility report.

BackgroundAATD is one of the most common inherited disorders in the World. However, it is generally accepted that AATD in North African populations is not a risk factor for lung and/or liver disease, based on a number of small studies. We therefore planned a screening study for detection of AATD in patients with OLD in a cohort of patients from Kairouan in central Tunisia. Methods: One hundred twenty patients with OLD (asthma, emphysema, COPD) were enrolled in the screening programme. Laboratory diagnosis for AATD was performed according to current diagnostic standards.ResultsWe found that 6/120 OLD patients carried an AAT deficient allele, 1 PI*MZ, 1 PI*MPlowel, 3 PI*MMmalton, 1 PI*MMwurzburg.Conclusionthis pilot study demonstrated that alleles related to deficiency of AAT are not absent in the Tunisian population, and that rare AATD variants prevailed over commonest PI*Z variant. These results would support a larger scale screening for AATD in Tunisia.

C reactive protein and alpha1-antitrypsin: relationship between levels and gene variants

The first step in laboratory diagnosis of alpha1-antitrypsin deficiency (AATD) is the determination of alpha1-antitrypsin (AAT) serum levels; these levels in turn are influenced by the inflammatory status. C reactive protein (CRP) has been proposed as a marker of systemic inflammation. Single nucleotide polymorphisms (SNPs) in the CRP gene have been associated with differences in baseline CRP levels. The purpose of this study was to investigate the relationship between CRP and AAT in the AATD diagnostic setting and to verify whether variations in the CRP gene could influence CRP. We determined AAT and CRP levels in 362 consecutive dried blood spot (DBS) samples submitted for AATD diagnosis and genotyped 3 CRP gene SNPs (rs1205, rs3093077, and rs3091244) associated with variations in serum CRP concentrations. To this aim, we developed a method to measure CRP in a DBS with a good correlation with CRP measurement in serum (r2=0.9927). We showed then that systemic inflammatory status parallels increased levels of AAT (80% of subjects with intermediate AATD and a CRP>0.8 mg/dL had an AAT level above the cut-off of 113 mg/dL) and that this increase might mask the presence of AATD variants. No association was detected between CRP levels and the 3 CRP gene polymorphisms. Simultaneous determination of CRP and AAT is useful in the correct diagnosis of heterozygotes carrying intermediate AATD genotypes; their genetic influence on the CRP level is negligible.

Plasma sRAGE and N-(carboxymethyl) lysine in patients with CHF and/or COPD.

Knowledge of the role of the receptor for advanced glycation end products (RAGE), particularly its soluble form (sRAGE), and of its advanced glycation end product (AGE) ligand, N‐(carboxymethyl)lysine adducts (CML), is limited in chronic heart failure (CHF) and in chronic obstructive pulmonary disease (COPD). We evaluated whether the AGE/RAGE system is activated in stable CHF and COPD, and whether plasma sRAGE and CML levels are affected by clinical and functional parameters.

α1-Antitrypsin in serum determined by capillary isoelectric focusing

A capillary isoelectric focusing (CIEF) method using bare fused‐silica capillaries filled with polyethylene oxide (PEO) and carrier ampholyte solutions in the pH 3.5—5.0 range has been developed for the identification of alpha1‐antitrypsin (α1AT) phenotypes in human serum. This novel procedure was routinely applied to the study of serum samples of five controls whose α1AT phenotype was previously identified and of twelve subjects whose α1AT phenotype was unknown. The results obtained allowed us to confirm or identify the α1AT phenotype in all sera tested. This procedure seems particularly suitable for identification of α1AT variants associated with diseases of clinical relevance.

SP-A binds alpha1-antitrypsin in vitro and reduces the association rate constant for neutrophil elastase.

Backgroundα1-antitrypsin and surfactant protein-A (SP-A) are major lung defense proteins. With the hypothesis that SP-A could bind α1-antitrypsin, we designed a series of in vitro experiments aimed at investigating the nature and consequences of such an interaction.Methods and resultsAt an α1-antitrypsin:SP-A molar ratio of 1:1, the interaction resulted in a calcium-dependent decrease of 84.6% in the association rate constant of α1-antitrypsin for neutrophil elastase. The findings were similar when SP-A was coupled with the Z variant of α1-antitrypsin. The carbohydrate recognition domain of SP-A appeared to be a major determinant of the interaction, by recognizing α1-antitrypsin carbohydrate chains. However, binding of SP-A carbohydrate chains to the α1-antitrypsin amino acid backbone and interaction between carbohydrates of both proteins are also possible. Gel filtration chromatography and turnover per inactivation experiments indicated that one part of SP-A binds several molar parts of α1-antitrypsin.ConclusionWe conclude that the binding of SP-A to α1-antitrypsin results in a decrease of the inhibition of neutrophil elastase. This interaction could have potential implications in the physiologic regulation of α1-antitrypsin activity, in the pathogenesis of pulmonary emphysema, and in the defense against infectious agents.

How can we improve the detection of Alpha1-antitrypsin deficiency?

The Z deficiency in α1-antitrypsin (A1ATD) is an under-recognized condition. Alpha1-antitrypsin (A1AT) is the main protein in the α1-globulin fraction of serum protein electrophoresis (SPE); however, evaluation of the α1-globulin protein fraction has received very little attention. Serum Z-type A1AT manifests in polymeric forms, but their interference with quantitative immunoassays has not been reported. Here, 214 894 samples were evaluated by SPE at the G. Fracastoro Hospital of Verona, Italy. Patients with an A1AT level ≤ 0.92 g/L were recalled to complete A1ATD diagnosis. In parallel, to qualitatively and quantitatively characterize A1AT, sera samples from 10 PiZZ and 10 PiMM subjects obtained at the National Institute of Tuberculosis and Lung Diseases in Warsaw, Poland, were subjected to non-denaturing 7.5% PAGE and 7.5% SDS-PAGE followed by Western blot. Moreover, purified A1AT was heated at 60°C and analyzed by a non-denaturing PAGE and 4–15% gradient SDS-PAGE followed by Western blot as well as by isolelectrofocusing and nephelometry. A total of 966 samples manifested percentages ≤ 2.8 or a double band in the alpha1-zone. According to the nephelometry data, 23 samples were classified as severe (A1AT ≤ 0.49 g/L) and 462 as intermediate (A1AT >0.49≤ 1.0 g/L) A1ATD. Twenty subjects agreed to complete the diagnosis and an additional 21 subjects agreed to family screening. We detected 9 cases with severe and 26 with intermediate A1ATD. Parallel experiments revealed that polymerization of M-type A1AT, when measured by nephelometry or isolelectrofocusing, yields inaccurate results, leading to the erroneous impression that it was Z type and not M-type A1AT. We illustrate the need for confirmation of Z A1AT values by “state of the art” method. Clinicians should consider a more in-depth investigation of A1ATD in patients when they exhibit serum polymers and low α1-globulin protein levels by SPE.

Case-finding for alpha1-antitrypsin deficiency in Kazakh patients with COPD

BackgroundAlpha-1-antitrypsin deficiency (AATD) is an under-diagnosed condition in patients with chronic obstructive pulmonary disease (COPD). The aim of this study was to screen for AATD in Kazakh patients with COPD using dried blood spot specimens.MethodsThe alpha1-antitrypsin (AAT) concentration was determined by nephelometry, PCR was used to detect PiS and PiZ alleles; and isoelectric focusing was used to confirm questionable genotype results and detect rare AAT variants.ResultsTo this aim, 187 Kazakh subjects with COPD were recruited. Blood samples were collected as dried blood spot. Genotyping of 187 samples revealed 3 (1.6%) PI*MZ and 1 (0.53%) PI*MS, Phenotyping identified also two sample (1.1%) with phenotype PiMI. Allelic frequencies of pathological mutations Z, S and I resulted 0.8%, 0.3%, 0.5%, respectively, in COPD Kazakh population.ConclusionThis study proved that AATD is present in the Kazakh population. These results support the general concept of targeted screening for AAT deficiency in countries like Kazakhstan, with a large population of COPD patients and low awareness among care-givers about this genetic condition.

Inherited alpha1-antitrypsin deficiency: is the level the key message?

Fifty-one years ago, two scientists from the Malmö University, Carl Bertil Laurell and Sten Eriksson, first reported five individuals affected by inherited alpha1-antitrypsin ( 1-AT) deficiency [1]. Of particular interest, this original nucleus of described patients, already included the spectrum of clinical phenotypic variability associated with this genetic disorder: absence of any respiratory disease, juvenile or late pulmonary emphysema, disseminated bronchiectasis. Since then, knowledge on the related epidemiology, pathophysiology, pathogenesis, clinical course, and treatment has dramatically improved, as evidenced by the authoritative published review articles [2,3]. Still, there are a number of open questions awaiting on answer. In particular, we currently have relatively solid information on the worldwide epidemiology of 1-AT; this information has led to the consensus that 1-AT is likely the most common rare respiratory disorder [4]. Nevertheless, the diagnostic rate for 1-AT is still dramatically low, with no more than 5—10% of expected individuals actually being recognized. This is true in all developed countries, irrespective of the local prevalence of the disorder and the quality of the diagnostic programs in place. Therefore, it has never been more evident that ‘‘. . . inherited 1-AT is not a rare condition, but a condition rarely diagnosed’’ [5]. We are thus heartened by the fact that the opening article in a thematic series of the Revue de Maladies Respiratoires was devoted to the laboratory diagnosis for inherited 1-AT deficiency. In this paper, Balduyck et al. provide an exhaustive review of the methodologies used to perform a ( s p p