17 research outputs found
Quantification of circulating alpha-1-antitrypsin polymers in dried blood spots
No full textBackground: The Z mutation in alpha-antitrypsin (AAT) results in formation of polymeric chains within hepatocytes, with consequent reduction of AAT plasma levels and predisposition to emphysema. A small fraction of Z AAT polymers is also present in the circulation and in tissues, where it further decreases AAT anti-protease activity and may exert pro-inflammatory functions, thus contributing to the pathogenesis of lung disease in AAT deficiency (AATD). As circulating polymers are mainly secreted by the liver, their concentration in the plasma may reflect the extent of polymer accumulation in hepatocytes and the severity of associated liver diseases.
Objectives: Quantification of plasma AAT polymers is a relevant diagnostic and prognostic biomarker in AATD. As most diagnostic flowcharts are performed on dried blood spots (DBS), we have standardized a method for accurate quantification of AAT polymers on DBS.
Methods: We collected DBS from 20 AATD patients (10 PI*MZ and 10 PI*ZZ) and quantified AAT polymers on DBS eluates by a sandwich ELISA based on the polymer-specific 2C1 mAb for capture and an HRP-conjugated anti-AAT polyclonal for detection. Heat-induced polymers of plasma purified AAT were used as standards.
Results: Polymer concentrations determined on DBS from PI*MZ (0,2±0,1 mg/dL) and PI*ZZ (1.1±0,7 mg/dL) subjects were comparable to the levels measured on matched plasma samples.
Conclusions: We developed an assay that determines AAT polymer concentrations in DBS specimens. This assay will give the opportunity to test a high number of cases for plasma polymer content and thereby to evaluate the correlation of this parameter with individual lung and liver clinical manifestations of AATD
Identification and characterisation of twenty-two novel SERPINA1 pathological mutations
No full textBackground: Alpha-1 antitrypsin (AAT) is a serine protease inhibitor, encoded by the highly polymorphic SERPINA1 gene. Mutations in the SERPINA1 gene can lead to AAT deficiency (AATD), which is associated with a substantially increased risk of lung and liver disease.
Objectives: Here we reported 22 novel AAT pathological mutations that we discovered during the Italian and Irish targeted detection programs of AATD during the last years.
Methods: We performed the determination of AAT serum levels by a rate immune nephelometric method. The phenotype was determined by isoelectric focusing analysis. DNA was isolated from whole peripheral blood, dried blood spot (DBS) samples or buccal swabs using a commercial extraction kit. The new mutations were identified by sequencing exonic and part of the intronic portions of the SERPINA1 gene by Sanger methods or Next Generation Sequencing. Predictive algorithm have been applied to them.
Results: We found 22 previously unidentified SERPINA1 pathological mutations. Three of them are Null mutations, the others are non-synonymous mutations causing protein deficiency. According to clinical data, the protein structural context and residue conservation, the variants have been classified as “pathogenic”, “likely pathogenic” and “likely benign”.
Conclusions: We added twenty-two more mutations to the list of SERPINA1 alleles. Moreover, we underlined that the laboratory diagnosis of AATD is not just a matter of degree, because the precise determination of the deficiency and Null alleles carried by an AATD individual may help to evaluate the risk for the lung disease
Analysis of long term CD4+CD25highCD127- T-reg cells kinetics in peripheral blood of lung transplant recipients
No full textAbstract Background The role of CD4+CD25highCD127− T-reg cells in solid-organ Transplant (Tx) acceptance has been extensively studied. In previous studies on kidney and liver recipients, peripheral T-reg cell counts were associated to graft survival, while in lung Tx, there is limited evidence for similar findings. This study aims to analyze long term peripheral kinetics of T-reg-cells in a cohort of lung recipients and tests its association to several clinical variables. Methods From jan 2009 to dec 2014, 137 lung Tx recipients were submitted to an immunological follow up (median: 105.9 months (6.7–310.5)). Immunological follow up consisted of a complete blood peripheral immuno-phenotype, inclusive of CD4+CD25highCD127− T and FOXP3+ cells. We tested the association between T-reg and relevant variables by linear OR regression models for repeated measures, adjusting for time from Tx. Also, by ordered logistic models for panel data, the association between Chronic Lung Allograft Dysfuncton (CLAD) onset/progression and T-reg counts in the previous 3 months was tested. Results Among all variables analyzed at multivariate analysis: Bronchiolitis Obliterans Syndrome (OR −6.51, p < 0.001), Restrictive Allograft Syndrome (OR −5.19, p = 0.04) and Extracorporeal photopheresis (OR −5.65, p < 0.001) were significantly associated to T-reg cell. T-reg cell counts progressively decreased according to the severity of CLAD. Furthermore, patients with higher mean T-reg counts in a trimester had a significantly lower risk (OR 0.97, p = 0.012) of presenting CLAD or progressing in the graft dysfunction in the following trimester. Conclusions Our present data confirm animal observations on the possible role of T-reg in the evolution of CLAD
Comparison among populations with severe and intermediate alpha1-antitrypsin deficiency and chronic obstructive pulmonary disease
No full textBACKGROUND: Severe alpha1-antitrypsin (AAT) deficiency (AATD) is associated with a high risk of airflow obstruction and emphysema. The risk of lung disease in those with intermediate AAT deficiency is unclear. Our aims were to compare pulmonary function, time of onset of symptoms, and indicators of quality of life among patients with severe AATD (PI*ZZ), patients with intermediate AATD (PI*MZ) from the Italian Registry of AATD with a chronic obstructive pulmonary disease (COPD) cohort of patients without AATD (PI*MM). METHODS: We considered a total of 613 patients: 330 with the PI*ZZ genotype, 183 with the PI*MZ genotype and 100 with the PI*MM genotype. Radiological exams, pulmonary function test, and measurement of quality of life have been performed on all cohorts of patients. RESULTS: The three populations differ significantly in terms of age at COPD/AATD diagnosis (P=0.00001), respiratory function (FEV1, FVC, DLCO P<0.001), quality of life (P=0.0001) and smoking history (P<0.0001). PI*ZZ genotype had 24.9 times a higher likelihood of developing airflow obstruction. The MZ genotype is not associated with a significant early risk of airflow obstruction. CONCLUSIONS: The comparison of populations with PI*ZZ, MZ and MM genotypes allows to delineate the role of alpha1-antitrypsin deficiency on respiratory function and on the impact on quality of life, in relation to other risk factors. These results highlight the crucial role of primary and secondary prevention on smoking habits in PI*MZ subjects and the importance of an early diagnosis
Different clinical suspect that brings to the diagnosis of alpha1-antitrypsin deficiency
No full text
Adipose Mesenchymal Extracellular Vesicles as Alpha-1-Antitrypsin Physiological Delivery Systems for Lung Regeneration
No full textAccumulating 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
