The Immune Response in the Pathophysiology of Pulmonary Diseases

Chronic obstructive pulmonary disease (COPD) is the world’s third leading cause of death. The number of patients with asthma is increasing in developed countries. We review here the main features of pathophysiology in these obstructive diseases. Tobacco smoke and other air pollution stimulate chronic inflammation in COPD. Asthma is a type 1 hypersensitivity that is a response to various allergens. In both pathologies, chronic inflammatory response leads to airway remodeling, significantly impacting lung function and a patient’s daily activity. Besides imaging techniques, a critical diagnostic tool is a pulmonary function test with characteristic obstructive patterns and respiratory symptoms. Sarcoidosis is discussed as an example of a restrictive disease. Finally, we shortly highlight the direction of current research

PSMB2 and RPL32 are suitable denominators to normalize gene expression profiles in bronchoalveolar cells

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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 (<it>p </it>= 0.004); no change was observed using GAPDH/ACTB (<it>p </it>> 0.05). CCL2 mRNA up-regulation was observed only when PSMB2/RPL32 were used as denominators (<it>p </it>< 0.03).</p> <p>Conclusion</p> <p>PSMB2 and RPL32 are, therefore, suitable reference genes to normalize qRT-PCR in BAL cells in sarcoidosis, and other interstitial lung disease.</p

CCL5/RANTES Gene Polymorphisms in Slavonic Patients with Myocardial Infarction

Coronary artery inflammation is a critical process in the pathogenesis of myocardial infarction (MI). The chemokine CCL5/RANTES (regulated upon activation, normal T cells expressed and secreted) is expressed in advanced atherosclerotic lesions. Functional polymorphisms of the RANTES gene can, therefore, be involved in the pathogenesis of coronary artery disease. We examined the association of polymorphisms in the RANTES gene with myocardial infarction in Slavonic populations of Czech and Russian origin. A total of 467 post-MI patients and 337 control subjects were genotyped for RANTES promoter G-403A (rs2107538) and intron 1.1 T/C (rs2280789) variants by PCR-SSP. Both RANTES genotypes and allele frequencies did not differ between case and control groups. Haplotype-based analysis also failed to reveal an association between MI and investigated markers. Strong linkage disequilibrium was detected between particular RANTES alleles. The data do not support an association between RANTES G-403A polymorphism and MI, as reported previously

The Serum Expression of Selected miRNAs in Pulmonary Sarcoidosis with/without Löfgren’s Syndrome

Purpose. Pulmonary sarcoidosis is associated with dysregulated expression of intracellular miRNAs. There is however only little information on extracellular miRNAs and their association with the disease course in sarcoidosis. We therefore assessed serum miRNAs in sarcoidosis classified according to the presence of Löfgren’s syndrome (LS) as a hallmark of good prognosis in contrast to more advanced disease course. Methods. RT-PCR was used to assess 35 miRNAs in 13 healthy controls and 24 sarcoidosis patients (12 with X-ray (CXR) stage ≤ 1 and LS and 12 with insidious onset and CXR stage ≥ 3). Results. Compared to controls, we consistently observed dysregulated expressions of miR-146, miR-16, miR-425-5p, and miR-93-5p in both sarcoidosis groups irrespective of disease course. Specifically, patients without LS had dysregulated expressions of miR-150-5p, miR-1, and miR-212 compared to controls. Patients with LS had dysregulated expressions of miR-21-5p and miR-340-5p compared to controls. Bioinformatics predicted consistently “Pathways in cancer” to be modulated by both altered profiles in patients with/without LS. Three miRNAs (miR-21-5p, miR-340-5p, and miR-212-3p) differed between our patients with LS and those without LS; their cumulative effect may modulate “TGF-β signalling pathway.” Conclusions. Further study should focus on possible applications of serum miRNAs for diagnostics follow-up and for prognosis

MCP-1 A/G Single Nucleotide Polymorphism in Slovak Patients with Systemic Sclerosis

Recent study in a group of German patients with SSc has implicated the SNP in the MCP-1 gene (−2518 A to G) as a factor of susceptibility to SSc. Reflecting the need for replication of genetic association studies, we investigated if this SNP is associated with SSc in another Caucasian population. MCP-1 −2518 A/G genotypes were determined using PCR-SSP in 46 SSc patients and in 449 healthy subjects, all unrelated and of Slovak (Slavonic) origin. The distribution of MCP-1 −2518 A/G genotypes complied with the Hardy-Weinberg equilibrium both in patient and healthy control groups. There was no difference in MCP-1 −2518∗G allele frequency between SSc patients and healthy subjects (patients: 0.23; controls: 0.24; P>.05). Furthermore, MCP-1 −2518 GG homozygotes were similarly represented among SSc patients and healthy subjects (P>.05). The association of MCP-1 −2518 A/G SNP with SSc observed originally in German population was not replicated in the Slovak population

miR-29a-3p/T-bet Regulatory Circuit Is Altered in T Cells of Patients With Hashimoto’s Thyroiditis

ObjectiveHashimoto’s thyroiditis (HT) is a common autoimmune thyroid disorder that frequently evolves from asymptomatic, T-cell mediated chronic inflammation toward overt hypothyroidism. Previously, we have demonstrated a role for T-bet, a T helper 1/CD8+ T cell transcription factor (TF), and FoxP3, a regulatory T cell TF, in disease progression and severity, but the basis behind their altered mRNA expression remains unknown. In this study, we aimed to leverage the role for microRNAs, representing negative transcriptional regulators, across the spectrum of HT clinical presentations using the same, well-characterized RNA sample cohort.MethodTen hypothyroid, untreated patients (hypoHT), 10 hypothyroid cases rendered euthyroid by l-thyroxine therapy (substHT), 11 spontaneously euthyroid HT subjects (euHT), and 10 healthy controls (ctrl) were probed for three candidate immunoregulatory miRNA (miR-9-5p, miR-29a-3p, and miR-210-3p) using quantitative real-time PCR measurements. Data were normalized to U6snRNA and fold difference in expression calculated by the efficiency corrected 2−ΔΔCt model.ResultsCompared to healthy controls, peripheral blood (PB) T cells of HT patients exhibited significantly diminished miR-29a-3p expression levels [median expression levels (IQR), HT vs CTRL, 0.62 (0.44–1.01) vs 1.373 (0.63–2.7), P = 0.046], and a similar, but not significant decline in miR-210-3p abundance [HT vs CTRL, 0.64 (0.39–1.31) vs 1.2 (0.5–2.56), P = 0.24, Wilcoxon test]. A significant inverse correlation was observed between the two differentially expressed transcripts, T-bet mRNA and miR-29a-3p. Moreover, altered miR-29a-3p/T-bet expression in T cells of untreated HT patients was related to low serum FT4, high serum thyrotropin, and decreased thyroid volumes. Of note, miR-210-3p expression was positively correlated to HIF1α, and inversely to FoxP3 mRNA levels, but no evidence of differential expression for any of these miRNA–mRNA pairs was observed. Finally, miR-9-5p expression levels were no different in HT vs control comparisons, or related to clinicopathological features.ConclusionT cell miR-29a-3p is downregulated in HT patients and associated with clinical and biochemical parameters of progressive thyroid injury, plausibly subsequent to altered control of T-bet expression in PB T cells. As such miR-29a-3p/T-bet axis should be further explored as a biomarker or as a plausible target for therapeutic interventions in HT

The Expression of T Cell FOXP3 and T-Bet Is Upregulated in Severe but Not Euthyroid Hashimoto’s Thyroiditis

Hashimoto’s thyroiditis (HT) is an organ-specific autoimmune disorder characterized by progressive thyroid failure. Th1 and Treg subset of CD4+ cells have been implicated in the pathogenesis; however, less is known about their respective roles across the spectrum of HT clinical presentations. To shed more light on CD4+ subsets role in HT, we investigated the mRNA expression levels of several Th1/Treg-associated transcription factors (T-bet/ETS1, HIF1α/BLIMP1/FOXP3) in peripheral blood T cells of 10 hypothyroid, untreated HT patients, 10 hypothyroid patients undergoing hormone replacement therapy, 12 euthyroid HT subjects, and 11 healthy controls by the qRT-PCR. Compared to euthyroid HT patients and controls, both hypothyroid (2.34-fold difference versus controls, P<0.01) and thyroxine-supplemented patients (2.5-fold, P<0.001) showed an increased FOXP3 mRNA expression in T cells. Similarly, mRNA expression levels of T-bet were upregulated in severely affected but not in euthyroid HT subjects (2.37-fold and 3.2-fold, hypothyroid and thyroxine-supplemented HT patients versus controls, resp., P<0.01). By contrast, no differences in mRNA expression levels of ETS1, BLIMP1, and HIF1α were observed across the study groups. In summary, severe but not euthyroid HT was associated with robust upregulation of T-bet and FOXP3 mRNA in peripheral T cells, independent of the thyroid hormone status but proportional to disease activity