BACKGROUND: Bronchoalveolar lavage (BAL) as complementary method is still used as ancillary tool in diagnosis of interstitial lung diseases. Tobacco smoking has been described to affect the BAL lavage cellular profile. To our knowledge, only few reports have so far investigated CD3+CD4+ and CD3+CD8+ lymphocyte subsets in non-smoking sarcoidosis patients additionally stratified according to CXR stage, and compared them to other non-smoking patients with interstitial lung diseases (ILDs). METHODS: We compared lymphocytes immune phenotypes, subsets, with CD3+, CD3+CD4+ and CD3+CD8+ cell markers, in the non-smoking subjects (n=297) including the patients with pulmonary sarcoidosis (S), idiopathic pulmonary fibrosis (IPF) (n=22), hypersensitivity pneumonitis (HP) (n=15), other interstitial idiopathic pneumonias (OIIPs) (n=39). According to prognosis, the patients with S were divided into four groups: 18 patients with Löfgren's syndrome (LS) in chest X-ray (CXR) ≤1 stage, 64 patients without LS in CXR ≤1 stage, 113 patients in CXR 2 stage and 26 patients with advanced CXR ≥3 stage. RESULTS: After the use of false discovery rate (FDR) correction, relative numbers (%) of CD3+, CD3+CD4+, CD3+CD8+ and CD3+CD4/CD3+CD8 ratio showed the most significant differences between the non-smokers with S (both with/without LS) and the non-smokers with other ILDs (IPF, OIIPs, HP). These lymphocytes subsets were further altered in the non-smokers with CXR stage 2 compared to the non-smokers with other ILDs (IPF, OIIPs, HP). We did not observe any differences in these lymphocyte subsets and CD3+CD4+/CD3+CD8+ ratio between the non-smokers with advanced sarcoidosis stage (CXR ≥3) and the non-smokers with IPF. CONCLUSIONS: Our data on the non-smokers confirmed the presence of the typical BAL cellular profile in sarcoidosis. The BAL cellular profile was helpful namely for differentiation of less advanced sarcoidosis. Its definite diagnostic utility should be the subject of further clinical studies with large numbers of the well characterized patients taking into consideration other clinical factors influencing BAL cellular profile, such as smoking or treatment.

• Klíčová slova
• Sarcoidosis, bronchoalveolar lavage (BAL), lymphocytes, non-smokers,
• Publikační typ
• časopisecké články MeSH

Sarcoidosis is an inflammatory granulomatous disease with unknown etiology driven by cytokines and chemokines. There is limited information regarding the regulation of cytokine/chemokine-receptor network in bronchoalveolar lavage (BAL) cells in pulmonary sarcoidosis, suggesting contribution of miRNAs and transcription factors. We therefore investigated gene expression of 25 inflammation-related miRNAs, 27 cytokines/chemokines/receptors, and a Th1-transcription factor T-bet in unseparated BAL cells obtained from 48 sarcoidosis patients and 14 control subjects using quantitative RT-PCR. We then examined both miRNA-mRNA expressions to enrich relevant relationships. This first study on miRNAs in sarcoid BAL cells detected deregulation of miR-146a, miR-150, miR-202, miR-204, and miR-222 expression comparing to controls. Subanalysis revealed higher number of miR-155, let-7c transcripts in progressing (n = 20) comparing to regressing (n = 28) disease as assessed by 2-year follow-up. Correlation network analysis revealed relationships between microRNAs, transcription factor T-bet, and deregulated cytokine/chemokine-receptor network in sarcoid BAL cells. Furthermore, T-bet showed more pronounced regulatory capability to sarcoidosis-associated cytokines/chemokines/receptors than miRNAs, which may function rather as "fine-tuners" of cytokine/chemokine expression. Our correlation network study implies contribution of both microRNAs and Th1-transcription factor T-bet to the regulation of cytokine/chemokine-receptor network in BAL cells in sarcoidosis. Functional studies are needed to confirm biological relevance of the obtained relationships.

• MeSH
• dospělí MeSH
• genové regulační sítě * MeSH
• lidé středního věku MeSH
• lidé MeSH
• messenger RNA analýza   MeSH
• mikro RNA fyziologie   MeSH
• plicní sarkoidóza imunologie   MeSH
• proteiny T-boxu fyziologie   MeSH
• receptory chemokinů genetika   MeSH
• receptory cytokinové genetika   MeSH
• senioři MeSH
• transkripční faktor T-bet MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• messenger RNA MeSH
• mikro RNA MeSH
• proteiny T-boxu MeSH
• receptory chemokinů MeSH
• receptory cytokinové MeSH
• transkripční faktor T-bet MeSH

BACKGROUND: For accuracy of quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), normalisation with suitable reference genes is required. To date, no reference genes have been validated for expression studies of bronchoalveolar (BAL) cells. The aims of this study were to identify gene(s) with stable mRNA expression in BAL cells irrespective of gender, smoking, BAL cellular composition, lung pathology, treatment; and to assess the influence of reference genes on target gene expression data. RESULTS: The mRNA expression of ten housekeeping genes (ACTB, ARF1, CANX, G6PD, GAPDH, GPS1, GNB2L1, PSMB2, PSMD2, RPL32) was investigated by qRT-PCR in BAL cells from 71 subjects across a spectrum of lung diseases. The analyses were validated in an independent BAL cohort from 63 sarcoidosis patients and 17 control subjects. A second derivative method was used to calculate expression values (CTt); an equivalence test, applets BestKeeper, geNorm and NormFinder were applied to investigate gene expression stability. Of the investigated genes, PSMB2 (CTt +/- SD, 23.66 +/- 0.86) and RPL32 (18.65 +/- 0.92) were the most stable; both were constantly expressed in BAL samples from parallel investigated cohorts irrespective of evaluated variables. Finally, to demonstrate effect of traditional (ACTB/GAPDH) and novel (PSMB2/RPL32) reference genes as denominators, expression of two cytokines known associated with sarcoidosis was investigated in sarcoid BAL cells. While normalization with PSMB2/RPL32 resulted in elevated IFNG mRNA expression (p = 0.004); no change was observed using GAPDH/ACTB (p > 0.05). CCL2 mRNA up-regulation was observed only when PSMB2/RPL32 were used as denominators (p < 0.03). CONCLUSION: PSMB2 and RPL32 are, therefore, suitable reference genes to normalize qRT-PCR in BAL cells in sarcoidosis, and other interstitial lung disease.

• MeSH
• bronchy cytologie   MeSH
• dospělí MeSH
• kouření genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• messenger RNA analýza   MeSH
• mladiství MeSH
• plicní alveoly cytologie   MeSH
• plicní nemoci genetika   MeSH
• polymerázová řetězová reakce s reverzní transkripcí normy   MeSH
• referenční standardy MeSH
• sarkoidóza genetika   MeSH
• stanovení celkové genové exprese normy   MeSH
• studie případů a kontrol MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• messenger RNA MeSH