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Alpha1-antitrypsin (AAT) is a serine protease inhibitor that is encoded by the highly polymorphic SERPINA1 gene. Mutations in this gene can lead to AAT deficiency (AATD), which is associated with an increased risk of lung and/or liver disease. On the basis of electrophoretic migration, AAT variants are named with capital letters; M (medium) signifies the normal protein. Among pathological variants, the M-like ones represent a heterogeneous group of rare allelic variants that exhibit the same electrophoretic pattern as the M wild-type protein, which makes them difficult to detect with routine methods. In order to avoid their misdiagnosis, the present study defines and validates effective methods for the detection of two pathogenic M-like variants, Mwurzburg and Mwhitstable. Comparison of protein phenotypes using isoelectric focusing of samples that presented the Mwurzburg variant, as revealed by exons 5 sequencing, identified a particular electrophoretic pattern amenable to the Mwurzburg protein. The specific phenotyping pattern was retrospectively validated, thus enabling the detection of 16 patients with Mwurzburg variant among the subjects already tested but not sequenced according to our diagnostic algorithm. The Mwhitstable allele was detected by intron 4 sequencing of SERPINA1 gene. Mwurzburg and Mwhitstable are often misdiagnosed and the introduction of diagnostic improvements can help the clinical management, especially in patients with established lung disease without any other reported risk factors. [ABSTRACT FROM AUTHOR]

Alpha1-antitrypsin deficiency (AATD) is an inherited condition that predisposes individuals to an increased risk of developing lung and liver disease. Even though AATD is one of the most widespread inherited diseases in Caucasian populations, only a minority of affected individuals has been detected. Whereas methods have been validated for AATD testing, there is no universally-established algorithm for the detection and diagnosis of the disorder. In order to compare different methods for diagnosing AATD, we carried out a systematic review of the literature on AATD diagnostic algorithms. Complete biochemical and molecular analyses of 5,352 samples processed in our laboratory were retrospectively studied using each of the selected algorithms. When applying the diagnostic algorithms to the same samples, the frequency of False Negatives varied from 1.94 to 12.9%, the frequency of True Negatives was 62.91% for each algorithm and the frequency of True Positives ranged from 24.19 to 35.15%. We, therefore, highlighted some differences among Negative Predictive Values, ranging from 0.83 to 0.97. Accordingly, the sensitivity of each algorithm ranged between 0.61 and 0.95. We also postulated 1.108 g/L as optimal AAT cut-off value, in absence of inflammatory status, which points to the possible presence of genetic AATD. The choice of the diagnostic algorithm has a significant impact on the correct diagnosis of AATD, which is essential for appropriate treatment and medical care. The fairly large number of possible false negative diagnoses revealed by the present paper should also warn clinicians of negative results in patients with clinically-suspected AATD. [ABSTRACT FROM AUTHOR]

Alpha-1-antitrypsin deficiency is a genetic disorder caused by mutations in the SERPINA1 gene encoding alpha-1-antitrypsin (AAT), the major serine protease inhibitor in plasma. Reduced AAT levels are associated with elevated risk of developing emphysema mainly due to uncontrolled activity of neutrophil elastase in the lungs. The prevalent Z-AAT mutant and many rare pathogenic AAT variants also predispose to liver disease due to their accumulation as polymeric chains in hepatocytes. Part of these polymers are secreted into the bloodstream and could represent biomarkers of intra-hepatic accumulation. Moreover, being inactive, they further lower lung protection against proteases. Aim of our study is to accurately quantify the percentage of circulating polymers (CP) in a cohort of subjects with different SERPINA1 genotypes. CP concentration was measured in plasma or Dried Blood Spot (DBS) by a sensitive sandwich ELISA based on capture by the polymer-specific 2C1 monoclonal antibody. CP were significantly elevated in patients with the prevalent PI*SZ and PI*ZZ genotypes, with considerable intra-genotype variability. Notably, higher percentage of polymers was observed in association with elevated C-reactive protein. CP levels were also increased in carriers of the Mmalton variant, and of Mprocida, I, Plowell and Mherleen in heterozygosity with Z-AAT. These findings highlight the importance of implementing CP quantification in a clinical laboratory. Indeed, the variable amount of CP in patients with the same genotype may correlate with the variable severity of the associated lung and liver diseases. Moreover, CP can reveal the polymerogenic potential of newly discovered ultrarare AAT variants. [ABSTRACT FROM AUTHOR]

Background : Alpha-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. Methods: The 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. Results: To 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. Conclusion: This 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. [ABSTRACT FROM AUTHOR]

Accumulating evidence shows that Mesenchymal Stem/Stromal Cells (MSCs) exert their therapeutic effects by the release of secretome, made of both soluble proteins and nano/microstructured extracellular vesicles (EVs). In this work, for the first time, we proved by a proteomic investigation that adipose-derived (AD)-MSC-secretome contains alpha-1-antitrypsin (AAT), the main elastase inhibitor in the lung, 72 other proteins involved in protease/antiprotease balance, and 46 proteins involved in the response to bacteria. By secretome fractionation, we proved that AAT is present both in the soluble fraction of secretome and aggregated and/or adsorbed on the surface of EVs, that can act as natural carriers promoting AAT in vivo stability and activity. To modulate secretome composition, AD-MSCs were cultured in different stimulating conditions, such as serum starvation or chemicals (IL-1β and/or dexamethasone) and the expression of the gene encoding for AAT was increased. By testing in vitro the anti-elastase activity of MSC-secretome, a dose-dependent effect was observed; chemical stimulation of AD-MSCs did not increase their secretome anti-elastase activity. Finally, MSC-secretome showed anti-bacterial activity on Gram-negative bacteria, especially for Klebsiella pneumoniae. These preliminary results, in addition to the already demonstrated immunomodulation, pave the way for the use of MSC-secretome in the treatment of AAT-deficiency lung diseases. [ABSTRACT FROM AUTHOR]