Association between sarcoidosis and HLA polymorphisms in a Czech population from Central Europe: focus on a relationship with clinical outcome and treatment.

Background: Sarcoidosis is an immune-mediated systemic disease with unknown etiology affecting the lung predominantly. The clinical manifestation of sarcoidosis is rather diverse ranging from Löfgren's syndrome to fibrotic disease. Also, it differs among patients with distinct geographical and ethnic origins, consistent with environmental and genetic factors' role in its pathogenesis. Of those, the polymorphic genes of the HLA system have been previously implicated in sarcoidosis. Therefore, we have performed an association study in a well-defined cohort of Czech patients aiming to define how variation in HLA genes, may contribute to disease origin and development. Materials and methods: Total of the 301 Czech unrelated sarcoidosis patients were diagnosed according to international guidelines. In those, HLA typing was performed using next-generation sequencing. The allele frequencies at six HLA loci (HLA-A,-B,-C,-DRB1,-DQA1, and -DQB1) observed in the patients were compared with HLA allele distribution determined in 309 unrelated healthy Czech subjects; sub-analyses of relationships between HLA and distinct sarcoidosis clinical phenotypes were performed. Associations were assessed by two-tailed Fischer's exact test with correction for multiple comparisons. Results: We report two variants, HLA-DQB1*06:02, and HLA-DQB1*06:04, as risk factors for sarcoidosis, and three variants, HLA-DRB1*01:01, HLA-DQA1*03:01, and HLA-DQB1*03:02 as protective factors. HLA-B*08:01, HLA-C*07:01, HLA-DRB1*03:01, HLA-DQA1*05:01, and HLA-DQB1*02:01 variants associated with Löfgren's syndrome, a more benign phenotype. HLA- DRB1*03:01 and HLA-DQA1*05:01 alleles were connected with better prognosis-chest X-ray (CXR) stage 1, disease remission, and non-requirement of corticosteroid treatment. The alleles HLA-DRB1*11:01 and HLA-DQA1*05:05 are associated with more advanced disease represented by the CXR stages 2-4. HLA-DQB1*05:03 associated with sarcoidosis extrapulmonary manifestation. Conclusion: In our Czech cohort, we document some associations between sarcoidosis and HLA previously described in other populations. Further, we suggest novel susceptibility factors for sarcoidosis, such as HLA-DQB1*06:04, and characterize associations between HLA and sarcoidosis clinical phenotypes in Czech patients. Our study also extends the role of the 8.1 ancestral haplotype (HLA-A*01:01∼HLA-B*08:01∼HLA-C*07:01∼HLA-DRB1*03:01∼HLA-DQA1*05:01∼HLA-DQB1*02:01), already implicated in autoimmune diseases, as a possible predictor of better prognosis in sarcoidosis. The general translational application of our newly reported findings for personalized patient care should be validated by an independent study from another, international referral center.

Bronchoalveolar lavage characteristics correlate with HLA tag SNPs in patients with Löfgren's syndrome and other sarcoidosis.

Genetic susceptibility for sarcoidosis and Löfgren's syndrome (LS) has been associated with prognosis. Human leukocyte antigen (HLA)-DRB1*03 is over-represented in LS, and is associated with a good prognosis, whereas HLA-DRB1*15-positive patients have a more chronic course of sarcoidosis. These HLA-DRB1 types can be easily tagged by single nucleotide polymorphisms (SNPs). Our aim was to evaluate the association between these tag SNPs and bronchoalveolar lavage (BAL) characteristics. In 29 patients, both complete HLA-DRB1* locus genotyping and SNP tagging was performed in parallel. HLA-DRB1 type was inferred from the presence of *03 tag rs2040410 allele A and referred to as *03. HLA-DRB1*15 was inferred from the presence of tag SNP rs3135388 allele A and referred to as *15. For BAL analysis, 122 patients with LS and 165 patients with non-LS sarcoidosis were included. BAL lymphocyte subsets were analyzed by flow cytometry. The presence of tag SNPs completely corresponded with HLA-DRB1*03/*15 genotypes in all 29 patients in whom both HLA-DRB1* genotyping and SNP tagging was performed. In all patients together, *03+ /*15- patients showed a higher CD4+ /CD8+ ratio than *03- /*15+ (P = 0·004) and *03- /*15- (P = 0·001). LS patients with *03+ /*15- had a lower BAL lymphocyte count compared to *03- /*15+  patients (P = 0·011). Non-LS sarcoidosis patients with *03+ /*15- patients showed a decreased CD103+ CD4+ /CD4+ ratio compared to *03- /*15+  patients (P = 0·045) and *03- /*15- patients (P = 0·018). We found that HLA-DRB1*03 and HLA-DRB1*15 can be approximated by genotyping of tag SNPs and corresponds with the degree of lymphocytosis and cell phenotypes in BAL in both LS and non-LS sarcoidosis patients.

TP53 rs1042522 and rs8064946 variants in myocardial infarction.

OBJECTIVE: This study investigated the hypothesis that the single nucleotide polymorphisms (SNPs) of TP53 gene are related to a risk of myocardial infarction. METHODS: The coding SNP at codon 72 (rs1042522) and non-coding rs8064946 SNP were genotyped by polymerase chain reaction with sequence specific primers in 205 Czech patients with myocardial infarction and 148 Czech control subjects. RESULTS: The distribution of both SNPs was in agreement with the Hardy-Weinberg equilibrium and was similar to other European populations. Our power analysis showed 96 % of probability to detect an odd ratio equal to 2. Neither rs1042522 nor rs8064946 were associated with the risk of myocardial infarction. The haplotypes combined of rs1042522 and rs8064946 were not associated with myocardial infarction in the present study. CONCLUSION: The TP53 SNPs are not strongly associated with genetic predisposition to myocardial infarction (Tab. 3, Fig. 3, Ref. 23).

ANTIPHOSPHOLIPID SYNDROME AND MONOCYTES: NEW ASPECTS.

After discovery of antiphospholipid syndrome (APS) our understanding of molecular mechanisms of living matter has become more sophisticated and on this way monocytes has become crucial player, particularly in pathogenesis of APS. Thrombotic and non-thrombotic complications of APS could be explained by monocytes' activation too. But mechanisms underlying their activation are poorly investigated. So we aimed to determine transcriptional activity of monocytes after exposing them to low concentrations of lipopolysaccharide (LPS) and LPS+ATP using comparative of RT-PCR. Our study included eleven women suffering from recurrent miscarriages and APS (mean age 30±5,6 years). Nine healthy women (mean age of 29±8,5 years) without a positive family history of APS, autoimmune diseases and thrombosis were chosen as a control group. The results showed increasing levels of TLR2, IL-23, CCL2, CXCL10, IL-1ß and IL-6 in APS cells, while in healthy cells LPS resulted in IL-6 and STAT3 elevated mRNAs. Double stimulation of APS cells resulted in decreased mRNA levels of CCL-2, IL-1ß, and mRNA NLRP3 in healthy cells. At the same time TLR2 mRNAs were elevated in both groups after double stimulation. Thus increased sensitivity of APS cells to LPS may contribute to thrombus formation. Low concentration of ATP diminishes LPS-induced inflammatory state of APS monocytes, which might be one of potential regulatory mechanisms.

A novel HLA-B allele, HLA-B*35:279, identified by sequencing-based typing in a Czech patient.

The identification of a novel HLA-B*35:279 allele in a Czech patient is described. This allele is identical to the B*35:03:01 variant except the G/A nucleotide exchange at position 652 of the HLA-B gene that corresponds to the amino acid substitution from valine to isoleucine in alpha 3 domain of the HLA-B antigen.

Transcriptional activity of neutrophils exposed to high doses of colchicine: short communication.

Colchicine is an antimitotic drug which binds to tubulin and at high doses results in cytoskeleton disruption. Colchicine is believed to be an anti-inflammatory agent, though its modulatory effects on the level and transcriptional activity of genes is still a matter of debate. There is growing evidence that alterations in the cytoskeleton exert specific effects on the expression of various genes. This study was undertaken to analyze whether disrupting the microtubule cytoskeleton by colchicine modulates transcriptional levels of MEFV, NF-κB p65, NLRP3, HMGB1, and caspase-3 in neutrophils from patients with familial Mediterranean fever (FMF) and healthy subjects. In the present study, colchicine caused increased expression of NLRP3 (p=0.007) and MEFV (p=0.03), but had no effect on caspase-3, NF-κB p65 and HMGB1 genes in healthy neutrophils. FMF neutrophils were less responsive to the drug treatment. This study supports the hypothesis that, being an anti-inflammatory agent, colchicine at relatively high concentrations might lead to the activation of pro-inflammatory signalling pathways in neutrophils.

[Study of gene expression of transcription factors T cells during aging].

The level of expression of transcription factor genes (GATA-3, TBX21, IL23A), and changing of oxidative modification of proteins in young and elderly healthy persons was studied. The results of evaluation of gene expression, GATA-3 in lymphocytes showed the increased expression of GATA-3 in elderly people in comparison with the young. Women demonstrated higher expression of GATA-3 in compare with men. Study of IL-2p showed reduced levels of expression in aged humans compared to young. TVH21 expression level showed a reduction expression in both men and women. Comparative analysis of protein oxidation in blood plasma of young and elderly people showed an increase in the intensity of oxidative modification of proteins in the elderly.

A novel HLA-DRB1 allele, HLA-DRB1*13:116, identified by sequencing-based typing in a member of the Czech National Marrow Donor Registry.

We describe the identification of a novel HLA-DRB1 allele, DRB1*13:116, in a member of the Czech National Marrow Donor Registry. The novel allele differs from the known DRB1*13:17 variant by a nucleotide exchange at position 227 (T/A) of the coding HLA-DRB1 sequence, which causes an amino acid substitution (Phe47Tyr) in the HLA-DR beta 1 chain.

Colchicine modulates expression of pro-inflammatory genes in neutrophils from patients with familial Mediterranean fever and healthy subjects.

Colchicine (Col) is a microtubule depolymerizing drug, widely used for treatment of familial Mediterranean fever (FMF). Mechanisms by which Col exerts its beneficial effects are not yet completely understood, especially with respect to gene expression in polymorphonuclear neutrophils (PMNs), the main effector cells in acute inflammatory attacks of FMF. This study was, therefore, designed to elucidate possible modulatory effect of Col on expression of inflammation-related genes in circulating PMNs from 16 FMF patients in the remission period and 11 healthy subjects. In vitro effect of Col exposure (1 microg/ml) on expression of 8 selected genes was examined using quantitative real-time RT-PCR. Col up-regulated expression of IL-8 and IL-1beta genes in FMF (13-fold and 2.7-fold, p less than 0.05, respectively) and healthy (3-fold and 6.5-fold, p less than 0.05, respectively) PMNs, and down-regulated caspase-1 in FMF neutrophils (3-fold, p less than 0.05). In FMF PMNs treated with Col mRNAs of IL-8 (51-fold, p less than 0.01) and c-FOS (7-fold, p less than 0.05) transcripts were elevated compared to those from healthy subjects. By contrast, caspase-1 mRNA was decreased in FMF neutrophils compared to healthy cells (1.6-fold, p less than 0.05). Hereby, we provide evidence that, at least in vitro, Col displays pro-inflammatory potential in respect to IL-1beta and IL-8 genes. At the same time, our findings implicate suppression of caspase-1 expression by Col as a potential mechanism for its effects in FMF treatment.

MBL2 gene variation affecting serum MBL is associated with prosthetic joint infection in Czech patients after total joint arthroplasty.

Prosthetic joint infection (PJI) is a serious complication of the total joint arthroplasty (TJA). Serum mannose-binding lectin (MBL), a pattern recognition receptor, is involved in antibacterial immune response. This study investigated whether functional variants of the MBL2 gene may be associated with the risk of PJI. MBL2 -550 (H/L, rs11003125), MBL2 -221 (Y/X, rs7096206) and MBL2 +54 (G/A, rs1800450) single nucleotide polymorphisms (SNP) were genotyped in 112 PJI patients and two control groups: 245 patients with aseptic TJA and 196 Czech population controls without TJA. Serum MBL concentration was assessed in PJI patients (n = 92) and aseptic TJA controls (n = 56). The distribution of MBL2 genotypes complied with the Hardy-Weinberg equilibrium in all investigated groups. Importantly, MBL2 -550 L allele (allelic frequency, 0.72) and LL genotype (genotype frequency, 0.51) were more frequent among PJI patients compared to aseptic TJA controls (L allele: 0.63, P = 0.016, P(c) = 0.048; LL genotype: 0.39, P = 0.037, P(c) > 0.05) and to Czech population controls (L allele: 0.61, P = 0.010, P(c) = 0.030; LL genotype: 0.35, P = 0.006, P(c) = 0.018), respectively. Regarding MBL protein, the MBL2 -550 L carriers presented with lower serum MBL concentrations than non-carriers (median; 593 vs 1876 ng/ml; P < 0.01). Similarly, the carriage of MBL2 -221 X and 54 A alleles was associated with lower serum MBL concentrations (P < 0.01). In conclusion, MBL2 -550 genetic variant(s) associated with low serum concentration of MBL protein can increase the risk of PJI.

Interleukin-6 promoter polymorphism and plasma levels in patients with schizophrenia.

Schizophrenia is a severe psychiatric disease with inflammatory component. Several studies indicated the increased blood levels of proinflammatory interleukin-6 cytokine in schizophrenia. However, only limited studies explored the relationship between excess production and genetic variations of this cytokine in schizophrenia, and the results were controversial. Here, we investigated possible association of the interleukin-6 gene (IL6) rs1800795 (-174G/C) polymorphism with schizophrenia and relationship between this polymorphism and interleukin-6 protein (IL-6) blood levels. This polymorphism was found by other researchers to associate with different transcription rates and different plasma levels of IL-6. A total of 208 unrelated Armenians were genotyped by polymerase chain reaction with sequence-specific primers, and IL-6 levels were assessed by enzyme-linked immunosorbent assay. The IL6 rs1800795 alleles and genotypes in both groups were in Hardy-Weinberg (H-W) equilibrium. We found that rs1800795*C allele [38% vs 24%, P = 0.002, odds ratio (OR) = 1.95, 95% confidence interval (CI): 1.18-2.14] and its carriers (62% vs 42%, P = 0.003, OR = 2.28, 95% CI: 1.13-1.94) were more frequent in patients than in controls. IL-6 in patients was 1.5-fold higher than in controls (mean ± SD: 6.41 ± 2.47 pg/ml vs 4.15 ± 1.42 pg/ml, P = 1.9E-19). In both groups, higher IL-6 in rs1800795 GG compared to rs1800795*C allele carriers was observed (GG vs GC + CC, patients: 7.02 ± 2.83 pg/ml vs 5.39 ± 1.2 pg/ml, P = 0.0006; controls: 5.21 ± 1.17 pg/ml vs 3.38 ± 1.03 pg/ml, P = 1.6E-15). In conclusion, we report an association of IL6 rs1800795 and higher IL-6 with schizophrenia. We also conclude that IL6 rs1800795*C allele is linked to increased IL-6 blood levels and may be a risk factor for schizophrenia development at least in Armenian population.

A pilot randomized trial comparing long-term effects of red and white wines on biomarkers of atherosclerosis (in vino veritas: IVV trial).

BACKGROUND: Since early 90', growing body of evidence indicates that the Mediterranean diet with mild to moderate consumption of wine, mostly red wine, has a protective effect on cardiovascular diseases. Several mechanisms have been discussed to participate in the beneficial effect of red wine, such as antioxidant or vasodilating activity. However, later it has been shown that also other alcoholic beverages have a protective effect on atherosclerosis. Up to now, data from the prospective, long-term, head-to-head comparisons of the effects of different drinks on markers of atherosclerosis are insufficient. METHODS: The IVV (in vino veritas) study is a long-term, prospective, multicenter, randomized trial comparing the effect of red and white wines on the markers of atherosclerosis. One hundred and twenty healthy subjects with mild to moderate risk of atherosclerosis will be randomized to regular consumption of red wine (Pinot Noir) or white wine (Chardonnay-Pinot) for one year. The primary endpoint is the level of HDL-cholesterol at one year, while secondary endpoints are levels of other markers of atherosclerosis (LDL-cholesterol, C-reactive protein, myeloperoxidase, advanced oxidation protein product, interleukins 6 and 18, matrix metalloproteinases, glutathione s-transferase, monocyte chemoattractant protein 1, soluble CD40L). CONCLUSION: The IVV trial is the first study focusing on the long-term prospective comparison of the effects of red and white wines consumption on HDL-cholesterol and other markers of atherosclerosis. Results of the IVV trial may extend our understanding of the widely discussed "French paradox" (Tab. 1, Ref. 21)

MicroRNAs: Emerging Regulators of Immune-Mediated Diseases.

MicroRNAs (miRNAs) represent the most abundant class of regulators of gene expression in humans: they regulate one-third of human protein-coding genes. These small noncoding ∼22-nucleotides (nt)-long RNAs originate by multistep process from miRNA genes localized in the genomic DNA. To date, more than 1420 miRNAs have been identified in humans (miRBase v17). The main mechanism of miRNA action is the posttranscriptional regulation via RNA interference with their target mRNAs. The majority of target mRNAs (more than 80%) undergo degradation after recognition by complementary miRNA; the translational inhibition with little or no influence on mRNA levels has been also reported. Each miRNA may suppress multiple mRNA targets (average ∼200), and at the same time, one mRNA can be targeted by many miRNAs enabling to control a spectrum wide range of cellular processes. Recently, the role of miRNAs in the development of immune cells and the maintenance of immune system homeostasis gained attention, and the involvement of miRNAs in the pathogenesis of several immune system diseases has emerged. This review focuses on the role of miRNAs in autoimmune disorders (systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease and psoriasis), inflammatory pathologies of distinct organ (atherosclerosis, osteoarthritis and atopic eczema) and/or systemic locations such as allergy. The role of miRNAs, their predicted and known mRNA targets and description of their actions in physiological immune reactions and in the pathological processes ongoing in immune-mediated human disorders will be discussed. Finally, miRNA-based diagnostics and therapeutic potentials will be highlighted.

Functional variant ANXA11 R230C: true marker of protection and candidate disease modifier in sarcoidosis.

In the recent genome-wide association study the polymorphisms of annexin A11 (ANXA11) gene were associated with susceptibility to sarcoidosis. Beside the replication of this finding and analysis of local ANXA11 expression in bronchoalveolar lavage cells, we wondered whether 'leading' ANXA11 rs1049550 (R230C) variant might also be related to the clinical manifestation of sarcoidosis. The study included 245 Czech patients with sarcoidosis and 254 healthy control subjects. The frequency of ANXA11(*)T allele was significantly lower in patients with sarcoidosis (35%) compared with controls (42%, P=0.04, odds ratio=0.77). Furthermore, ANXA11(*)T allele was less frequent in patients with the infiltration of lung parenchyma by comparison with those with isolated hilar lymphadenopathy (P=0.01). In line with the previous observation, ANXA11 mRNA expression was not deregulated in sarcoidosis and was independent from rs1049550 variant. In conclusion, ANXA11 rs1049550 single nucleotide polymorphism is the susceptibility marker in sarcoidosis, at least in Caucasians. Its role as a disease modifier should be independently replicated.

T-helper cell type-1 transcription factor T-bet is upregulated in pulmonary sarcoidosis.

Upregulation of genes for interferon (IFN)-γ and CXC chemokine receptor (CXCR)3 expression, two crucial molecules in sarcoid inflammation and granuloma formation, is directly controlled by the T-helper (Th)1 transcription factor T-bet (T-box, expressed in T-cells). However, there is no information on T-bet expression in sarcoidosis or its relationship with "sarcoidosis-associated" genes. Therefore, we investigated expression of T-bet mRNA and, in parallel, a spectrum of genes known to be involved in sarcoidosis pathogenesis. Transcripts were determined in bronchoalveolar lavage (BAL) cells from 62 sarcoidosis patients and 25 controls by quantitative RT-PCR; T-bet protein was localised by immunohistochemistry. Patient's BAL cells expressed higher mRNA T-bet levels than those of controls (mean ± sd fold change 3.64 ± 1.72; p = 0.00006). T-bet mRNA expression did not vary between clinical phenotypes as assessed by chest radiography stage, presence/absence of Löfgren's syndrome, extrapulmonary/pulmonary involvement or progressing/remitting disease (p > 0.05). T-bet mRNA expression correlated with expression of IFN-γ, CC chemokine ligand 5, CXC chemokine ligand (CXC)10, interleukin (IL)-2 receptor/IL-15 receptor ß, CXCR3 and CXCR6 (p < 0.01). T-bet protein was localised to alveolar macrophages and lymphocytes, tissue multinucleated giant cells, macrophages and lymphocytes. In pulmonary sarcoidosis, T-bet upregulation is associated with changes in expression of IFN-γ, CXCR3 and chemokines/receptors involved in the pathogenesis of sarcoidosis, which suggests a role for T-bet in this Th1 disease, including modulation of some sarcoidosis-associated genes.

HLA-C locus and genetic susceptibility to psoriatic arthritis in Romanian population.

We determined the distribution of human leukocyte antigen-C (HLA-C) allelic groups in a cohort of psoriatic arthritis (PsA) patients and a control population of Romanian ethnicity. A nominal association of HLA-C*06 with susceptibility to PsA was observed [P = 0.014, p(corr) > 0.05, odds ratio (OR) 2.1, 95% confidence interval (CI) 1.08-4.46]. When subanalyzing data according to PsA clinical phenotypes, association was noticed between HLA-C*06 and PsA with psoriasis onset before 40 years (p(corr) = 0.013, OR 3.7, 95% CI 1.58-9). This first report from Romania confirmed the association of HLA-C*06 with type I psoriasis in PsA patients. Other study findings, such as the relationship between HLA-C*06 and spondylitis or the protective effect of HLA-C*07 for the polyarthritis clinical phenotype of PsA, are of preliminary character and require verification.

Genetic variants of the inflammatory C-reactive protein and schizophrenia in Armenian population: a pilot study.

C-reactive protein (CRP) is an inflammation marker implicated in the pathogenesis of schizophrenia. To investigate association of the CRP rs1417938, rs1800947, rs1205 variants with susceptibility to schizophrenia 208 unrelated Armenians (103 patients and 105 healthy controls) were genotyped. In this pilot study, none of studied variants was associated with schizophrenia.

MCP-1 -2518 A/G gene polymorphism is associated with blood pressure in ischemic heart disease asymptomatic subjects.

Monocyte chemoattractant protein-1 (MCP-1), one of the key inflammatory chemokines, plays an important role in the initiation of atherosclerosis, and represents a risk for coronary artery disease and myocardial infarction. A recent animal study showed that MCP-1 gene might be a candidate gene for salt-sensitive hypertension in Dahl salt sensitive rats. This effect has not been yet studied in asymptomatic humans. We tested the MCP-1 -2518 A/G single nucleotide polymorphism (SNP) in 66 hypertensive ischemic heart disease asymptomatic subjects. Inflammatory markers, classic risk factors and absolute cardiovascular risk (SCORE system) were also investigated in these subjects. Our results showed that both, systolic and diastolic values of blood pressure were associated with MCP-1 -2518 A/G SNP at the level of both, genotype and allele frequencies. Subjects with mutant G allele had higher levels of both values of blood pressure, systolic (p = 0.035) and diastolic (p = 0.040) than subjects with allele A. Statistically significantly higher levels of both values of blood pressure, systolic (p = 0.037) and diastolic (p = 0.021) were found also in IHD asymptomatic subjects with AG and GG genotypes. Subjects with AG and GG genotypes had also an increased absolute cardiovascular risk (1.62% vs 3.17%; p = 0.004) and an increasing trend for elevated plasma level of high-sensitive CRP (2.858 vs 2.062 mg/l; p = 0.076). We did not find any significant correlation between the serum level of MCP-1 and blood pressure. To our best knowledge, this is the first study concerning the association between MCP-1 polymorphism and arterial blood pressure in IHD asymptomatic subjects. These results indicate that the expression of MCP-1 may be increased before the onset of hypertension but further observations from larger cohorts are needed to confirm this finding (Tab. 6, Ref. 41).

Distribution of HLA-B27 in Romanian spondyloarthritides patients.

The analysis of 310 Romanian spondyloarthritides patients confirmed the association of the HLA-B27 marker with the susceptibility to different diseases of this group. For ankylosing spondylitis, the HLA-B27 frequency in Romanian patients (72.1%) was similar to that found in several regions in the Mediterranean area.