Molecular diagnostics of alpha-1-antitrypsine deficiency in clinical practice

Niedobór alfa-1-antytrypsyny (AATD, alpha-1-antitrypsin deficiency), inhibitora proteaz serynowych, jest genetycznie uwarunkowanym defektem zwiększającym ryzyko rozwoju chorób płuc i wątroby. Na podstawie wyników badań epidemiologicznych z ostatnich lat wykazano, że większość osób z tym deficytem pozostaje wciąż niezdiagnozowana. Pełna diagnostyka kliniczna AATD oparta jest na kombinacji analiz ilościowych i jakościowych. Badaniem wstępnym wykonywanym u osób z podejrzeniem deficytu jest pomiar stężenia inhibitora w surowicy/osoczu krwi. Pełną weryfikację rozpoznania klinicznego AATD zapewnia jednak dopiero identyfikacja fenotypu lub genotypu alfa-1-antytrypsyny. Spośród wielu technik fenotypowania i genotypowania wariantów AAT, uznawanych przez referencyjne ośrodki diagnostyczne na świecie, ogniskowanie izoelektryczne, real-time PCR i analiza restrykcyjna produktu PCR (RFLP-PCR, restriction fragment-length polymorphism) stosowane są najpowszechniej. W Polsce wciąż nie wykonuje się rutynowo badań w kierunku niedoboru alfa-1-antytrypsyny. Celem niniejszej pracy poglądowej jest przedstawienie najważniejszych metod diagnozowania AATD, ze szczegółowym omówieniem zalet i problemów związanych z wyborem danej techniki oznaczania wariantów AAT. Omówiono również zalecany algorytm postępowania diagnostycznego.The deficiency of serine protease inhibitor, alpha-1-antitrypsin (AATD), is genetically determined defect that increases the risk of lung and liver disease development. The results of recent epidemiological studies indicate the overwhelming majority of individuals with alpha-1-antitrypsin deficiency still remain undiagnosed. The complete laboratory diagnosis of AATD is based on combination of quantitative and qualitative methods. The measurment of plasma/serum AAT concentration is always the initial test performed in the clinically suspected individuals. Nevertheless, only the AAT phenotype or genotype identification allows the full medical verification of the diagnosis. Among the various techniques of either AAT variant phenotyping or genotyping accepted by reference medical centers worldwide, the isoelectric focusing, real-time-PCR and restriction fragment-length polymorphism PCR (RFLP-PCR) are “considered the most effective” performed the most commonly. The AAT diagnostics in Poland still awaits for introduction into clinical routine. The aim of present review is to outline the major methods of AATD diagnosis and discuss with the special issuing of their potential benefits and disadvantages

Current methods to detect EGFR gene mutations as predictive factor for targeted therapies in non-small cell lung cancer — is there a „golden standard” in diagnostics?

Zgodnie z aktualnymi zaleceniami polskimi i międzynarodowymi, wykrywanie mutacji somatycznych genu EGFR powinno być nieodzownym etapem rutynowej diagnostyki chorych na zaawansowaną postać gruczołowego raka płuca, u których rozważana jest terapia inhibitorami kinazy tyrozynowej. Klasyczne metody analizy genetycznej, takie jak sekwencjonowanie metodą Sangera, prezentują jednak ograniczoną efektywność diagnostyczną ze względu na molekularną heterogenność materiałów tkankowych/ /cytologicznych od chorych na raka płuca. Coraz większe znaczenie w praktyce laboratoryjnej zdobywają natomiast specjalistyczne techniki analizy mutacji o wysokiej czułości detekcji, przede wszystkim oparte na PCR w czasie rzeczywistym. Stały, dynamiczny rozwój technologii stosowanych w biologii molekularnej, zwłaszcza opracowanie metod sekwencjonowania nowej generacji, pozwala oczekiwać stosunkowo szybkiego wdrożenia równoległej analizy wielu biomarkerów predykcyjnych dla nowych terapii ukierunkowanych molekularnie, a także nieinwazyjnej diagnostyki mutacji genu EGFR w pozakomórkowej frakcji DNA izolowanego z krwi obwodowej.According to current Polish and international recommendations, detection of EGFR gene somatic mutations is the essential part of routine diagnostic algorithm in advanced NSCLC patients considered for tyrosine kinase inhibitor therapy. Molecular heterogeneity of tumor tissue and cytology materials used for molecular diagnostics is challenging for classic methods of genetic analysis, such as Sanger sequencing, driving the development and implementation of specialized, highly sensitive techniques for mutations detection. Constant, dynamic progress in molecular biology techniques, particularly development of next-generation sequencing, should enable clinical implementation of simultaneous multiple therapeutic biomarkers analysis as well as non-invasive EGFR mutations diagnostic based on free-circulating DNA isolated from blood of non-small cell lung cancer patients

Expression of insulin-like growth factor-I (IGF-I) in alveolar macrophages and lymphocytes obtained by bronchoalveolar lavage (BAL) in interstitial lung diseases (ILD). Assessment of IGF-I as a potential local mitogen and antiapoptotic cytokine.

Little is known about IGF-I expression in the alveolar lymphocytes (AL), and about local role of IGF-I in physiological conditions and in interstitial lung diseases. Bronchoalveolar lavage was carried out in patients with silicosis, asbestosis, idiopathic pulmonary fibrosis (IPF) and sarcoidosis, as well as in control subjects (n = 13, 9, 12, 56, 15, resp). Alveolar macrophages (AM) and lymphocytes (AL) were studied for (1) IGF-I, BCL-2, Fas and Fas Ligand expression and (2) cell cycle (incl. sub-G1 peak of late apoptosis) with propidium iodide (PI). Flow cytometry (FC) and immunocytochemistry were used. AL early apoptosis was detected by Annexin V FITC/PI staining. IGF-I was present in AL of all tested groups. The number of IGF-I positive AL was significantly higher in IPF (52 +/- 6.7%) and in later (II and III) stages of sarcoidosis (39 +/- 7.8 vs 16 +/- 4.0% in controls, p < 0.05). Increased BCL-2 expression in AL was detected in IPF and sarcoidosis. In all tested groups, AL were almost exclusively Fas+ T cells. Generally, a low number of AL entered apoptosis; no significant differences were found between patient groups, except decreased apoptosis rate in sarcoidosis (0.60 +/- 0.17 vs 1.15 +/- 0.33% in controls, p < 0.05). Proportion of AL positive for IGF-I was significantly correlated with parameters reflecting AL and AM cell proliferation and BCL-2 expression (e.g. AL IGF-I+ vs AM in S phase of cell cycle: r(S) = +0.50, p = 0.001), but not with apoptosis. The results show that human alveolar lymphocytes express IGF-I in normal conditions, as well as in ILD. The proportion of IGF-I+ lymphocytes was significantly increased in IPF and at later stages of sarcoidosis. In our material there was no evidence for profibrogenic or antiapoptotic activity of IGF-I. We suggest that IGF-I originating from AL may be locally active as a mitogen for alveolar macrophages and lymphocytes in ILD

Expression of insulin-like growth factor-I (IGF-I) in alveolar macrophages and lymphocytes obtained by bronchoalveolar lavage (BAL) in interstitial lung diseases (ILD) : assessment of IGF-I as a potential local mitogen and antiapoptotic cytokine

Little is known about IGF-I expression in the alveolar lymphocytes (AL), and about local role of IGF-I in physiological conditions and in interstitial lung diseases. Bronchoalveolar lavage was carried out in patients with silicosis, asbestosis, idiopathic pulmonary fibrosis (IPF) and sarcoidosis, as well as in control subjects (n = 13, 9, 12, 56, 15, resp). Alveolar macrophages (AM) and lymphocytes (AL) were studied for (1) IGF-I, BCL-2, Fas and Fas Ligand expression and (2) cell cycle (incl. sub-G1 peak of late apoptosis) with propidium iodide (PI). Flow cytometry (FC) and immunocytochemistry were used. AL early apoptosis was detected by Annexin V FITC/PI staining. IGF-I was present in AL of all tested groups. The number of IGF-I positive AL was significantly higher in IPF (52 +/- 6.7%) and in later (II and III) stages of sarcoidosis (39 +/- 7.8 vs 16 +/- 4.0% in controls, p &lt; 0.05). Increased BCL-2 expression in AL was detected in IPF and sarcoidosis. In all tested groups, AL were almost exclusively Fas+ T cells. Generally, a low number of AL entered apoptosis; no significant differences were found between patient groups, except decreased apoptosis rate in sarcoidosis (0.60 +/- 0.17 vs 1.15 +/- 0.33% in controls, p &lt; 0.05). Proportion of AL positive for IGF-I was significantly correlated with parameters reflecting AL and AM cell proliferation and BCL-2 expression (e.g. AL IGF-I+ vs AM in S phase of cell cycle: r(S) = +0.50, p = 0.001), but not with apoptosis. The results show that human alveolar lymphocytes express IGF-I in normal conditions, as well as in ILD. The proportion of IGF-I+ lymphocytes was significantly increased in IPF and at later stages of sarcoidosis. In our material there was no evidence for profibrogenic or antiapoptotic activity of IGF-I. We suggest that IGF-I originating from AL may be locally active as a mitogen for alveolar macrophages and lymphocytes in ILD

Enhanced expression of Fas Ligand (FasL) in the lower airways of patients with fibrotic interstitial lung diseases (ILDs)

The exact role of FasL, and particularly its soluble and membrane-bound forms, in the development of chronic ILDs and lung fibrosis has not been extensively explored. We aimed at analyzing membrane-bound FasL expression on alveolar macrophages (AM) and lymphocytes (AL) as well as soluble FasL (sFasL) levels in bronchoalveolar lavage (BAL) from ILDs patients, incl. pulmonary sarcoidosis (PS), hypersensitivity pneumonitis (HP), silicosis, asbestosis, idiopathic pulmonary fibrosis (IPF), nonspecific interstitial pneumonia (NSIP), and healthy subjects (n = 89, 12, 7, 8, 23, 6, 17, respectively). In IPF, significantly increased percentage of AM FasL+ and CD8+FasL+ cells as well as sFasL levels in BAL were found. Increased sFasL levels were also observed in HP. NSIP and asbestosis were characterized by higher AM FasL+ relative number; CD8+FasL+ population was expanded in asbestosis only. There was a significant decline in AL FasL+ percentage in PS and HP. Vital capacity was negatively correlated with sFasL levels, AM FasL+ and CD8+FasL+ cell relative count. CD4+FasL+ and CD8+FasL+ percentage strongly correlated with BAL neutrophilia, an unfavorable prognostic factor in lung fibrosis. The concurrent comparative BAL analysis of FasL expression indicates that FasL+ AM and AL (mainly Tc cells) comprise an important element of the fibrotic process, mostly in IPF. FasL might play a crucial role in other fibrosis-complicated ILDs, like NSIP and asbestosis. (Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 4, pp. 636&#8211;645

Molecular Diagnostics of Alpha-1-Antitrypsine Deficiency in Clinical Practice

The deficiency of serine protease inhibitor, alpha-1-antitrypsin (AATD), is genetically determined defect that increases the risk of lung and liver disease development. The results of recent epidemiological studies indicate the overwhelming majority of individuals with alpha-1-antitrypsin deficiency still remain undiagnosed. The complete laboratory diagnosis of AATD is based on combination of quantitative and qualitative methods. The measurment of plasma/serum AAT concentration is always the initial test performed in the clinically suspected individuals. Nevertheless, only the AAT phenotype or genotype identification allows the full medical verification of the diagnosis. Among the various techniques of either AAT variant phenotyping or genotyping accepted by reference medical centers worldwide, the isoelectric focusing, real-time-PCR and restriction fragment-length polymorphism PCR (RFLP-PCR) are “considered the most effective” performed the most commonly. The AAT diagnostics in Poland still awaits for introduction into clinical routine. The aim of present review is to outline the major methods of AATD diagnosis and discuss with the special issuing of their potential benefits and disadvantages

Highlights from the ERS Congress in Amsterdam, 24&#8211;28 September, 2011

W 2011 roku nie zmienił się skład zarządu grupy European Respiratory Society do spraw intensywnej terapii w pneumonologii—jej przewodniczącym jest Paolo Pelosi (Genua, Włochy) [...

Coexistence of Borrelia burgdorferi s. l. genospecies within Ixodes ricinus ticks from central and eastern Poland

The purpose of the study was to assess the prevalence and coinfection rates of Borrelia burgdorferisensu lato genotypes inIxodes ricinus(L.) ticks sampled from diverse localities in central and eastern regions of Poland. In years 2009-2011, questingnymphs and adults ofI. ricinuswere collected using a flagging method at 18 localities representing distinct ecosystem types:urban green areas, suburban forests and rural woodlands. Molecular detection of B. burgdorferis.l. genospecies was based onamplification of a flagene using nested PCR technique, subsequent PCR-RFLP analysis and bidirectional sequencing. It wasrevealed that 45 samples (2.1%) harboured two different B. burgdorferis.l. genospecies, whereas triple infections with variousspirochetes was found in 11 (0.5%) individuals. Generally, the highest average coinfection rates were evidenced in arachnidsgathered at rural woodlands, intermediate at suburban forests, while the lowest were recorded at urban green areas. Overall, sin-gle spirochete infections were noted in 16.3% (n = 352/2,153) ticks. Importantly, it is the first report evidencing the occurrenceof Borrelia miyamotoi(0.3%, n = 7/2153) in I. ricinuspopulations within central Poland. Circumstantial variability of B. burgdorferis.l. genospecies in the common tick individuals sampled at various habitat types in central and eastern Polandwas displayed. The coexistence of two or three different spirochete genospecies in single adult ticks, as well as the presence ofB. miyamotoiwere demonstrated. Therefore, further studies uncovering the co-circulation of the tested bacteria and other humanpathogens in I. ricinusticks are require