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Background Hepatocellular carcinoma (HCC) is a major contributor to the worldwide cancer burden. Recent studies on HCC have demonstrated dramatic alterations in expression of several cytochrome P450 (CYP) family members that play a crucial role in biotransformation of many drugs and other xenobiotics; however, the mechanisms responsible for their deregulation remain unclear. Methods We investigated a potential involvement of miRNAs in downregulation of expression of CYPs observed in HCC tumors. We compared miRNA expression profiles (TaqMan Array Human MicroRNA v3.0 TLDA qPCR) between HCC human patient tumors with strong (CYP-) and weak/no (CYP+) downregulation of drug-metabolizing CYPs. The role of significantly deregulated miRNAs in modulation of expression of the CYPs and associated xenobiotic receptors was then investigated in human liver HepaRG cells transfected with relevant miRNA mimics or inhibitors. Results We identified five differentially expressed miRNAs in CYP- versus CYP+ tumors, namely miR-29c, miR-125b1, miR-505, miR-653 and miR-675. The two most-upregulated miRNAs found in CYP- tumor samples, miR-29c and miR-653, were found to act as efficient suppressors of CYP1A2 or AHR expression. Conclusions Our results revealed a novel role of miR-653 and miR-29c in regulation of expresion of CYPs involved in crucial biotransformation processes in liver, which are often deregulated during liver cancer progression.

The aim of this study was to determine the effect of natural and encapsulated sources of ursolic acid on liver regeneration. Four ursolate sources were tested. Two forms of ursolic acid encapsulates were combined with cyclodextrins, i.e., gamma-CD (gCD) and beta-CD, and two natural sources were adjusted by homogenization (HAP) and micronization of apple peel using Jonagold apples. All ursolate forms were applied intragastrically in daily doses of 20 mg for 7 days. Laboratory rats were fed with standard laboratory diet. Further, gCD and MAP were also tested with a high-fat diet (6 weeks). Partial hepatectomy (PH) was performed 24 hours before the end of the experiment. The concentration of plasma hepatocyte growth factor (HGF) was determined with an immunoassay; simultaneously, the expression of HGF and CYP7A1 in the liver was quantified through qPCR. HGF expression and plasma levels were significantly increased 24 hours after PH in both the HAP (p=0.038) and HFgCD groups (p=0.036), respectively. The correlation between HGF expression and plasma values was significant (p=0.04). The positive effects on liver regeneration were found in both the gCD and HAP forms of ursolic acid, whose effects were confirmed through the upregulation of HGF.

Aim: We studied the influence of coffee consumption on the therapeutic effect of methotrexate (MTX) in patients with rheumatoid arthritis (RA) sorted according to ADORA2A genotypes. Patients & methods: 82 RA patients were dichotomized according to caffeine intake with a threshold of 700 mg/week. Disease activity score 28 (DAS28) was applied (>3.2: high

The newly discovered systematic placement of Bactrodesmium abruptum, the lectotype species of the genus, prompted a re-evaluation of the traditionally broadly conceived genus Bactrodesmium. Fresh material, axenic cultures and new DNA sequence data of five gene regions of six species, i.e. B. abruptum, B. diversum, B. leptopus, B. obovatum, B. pallidum and B. spilomeum, were studied. Bactrodesmium is a strongly resolved lineage in the Savoryellales (Sordariomycetes), supported by Bayesian and Maximum Likelihood methods. The genus Bactrodesmium is emended and delimited to hyphomycetes characterised by sporodochial conidiomata, mononematous often fasciculate conidiophores, holoblastic conidiogenesis and acrogenous, solitary, dry, pigmented, transversely or rarely longitudinally septate conidia. The conidia are seceding rhexolytically, exhibiting multiple secession patterns. An identification key to 35 species accepted in Bactrodesmium is given, providing the most important diagnostic characters. Novel DNA sequence data of B. longisporum and B. stilboideum confirmed their placement in the Sclerococcales (Eurotiomycetes). For other Bactrodesmium, molecular data are available for B. cubense and B. gabretae, which position them in the Dothideomycetes and Leotiomycetes, respectively. All four species are excluded from Bactrodesmium and segregated into new genera, Aphanodesmium, Gamsomyces and Kaseifertia. Classification of 20 other species and varieties not recognised in the genus is discussed. Based on new collections of Dematiosporium aquaticum, the type species of Dematiosporium, the genus is emended to accommodate monodictys-like freshwater lignicolous fungi of the Savoryellales characterised by effuse colonies, holoblastic conidiogenous cells and dictyosporous, pigmented conidia with a pore in each cell. Study of additional new collections, cultures and DNA sequence data revealed several unknown species, which are proposed as taxonomic novelties in the Savoryellales and closely related Pleurotheciales. Ascotaiwania latericolla, Helicoascotaiwania lacustris and Pleurotheciella erumpens are described from terrestrial, lentic and lotic habitats from New Zealand and France, respectively. New combinations are proposed for Helicoascotaiwania farinosa and Neoascotaiwania fusiformis. Relationships and systematics of the Savoryellales are discussed in the light of recent phylogenies and morphological patterns newly linked with the order through cultural studies.

Pregnane X receptor (PXR) is the major regulator of xenobiotic metabolism. PXR itself is controlled by various signaling molecules including glucocorticoids. Moreover, negative feed-back regulation has been proposed at the transcriptional level. We examined the involvement of the 3′-untranslated region (3′-UTR) of NR1I2 mRNA and microRNAs in PXR- and glucocorticoid receptor (GR)-mediated regulation of NR1I2 gene expression. PXR ligands were found to significantly downregulate NR1I2 mRNA expression in a set of 14 human hepatocyte cultures. Similarly, PXR was downregulated by PCN in the C57/BL6 mice liver. In mechanistic studies with the full-length 3′-UTR cloned into luciferase reporter or expression vectors, we showed that the 3′-UTR reduces PXR expression. From the miRNAs tested, miR-18a-5p inhibited both NR1I2 expression and CYP3A4 gene induction. Importantly, we observed significant upregulation of miR-18a-5p expression 6 h after treatment with the PXR ligand rifampicin, which indicates a putative mechanism underlying NR1I2 negative feed-back regulation in hepatic cells. Additionally, glucocorticoids upregulated NR1I2 expression not only through the promoter region but also via 3′-UTR regulation, which likely involves downregulation of miR-18a-5p. We conclude that miR-18a-5p is involved in the down-regulation of NR1I2 expression by its ligands and in the upregulation of NR1I2 mRNA expression by glucocorticoids in hepatic cells., Graphical abstract miR-18a-5p is involved in the down-regulation of pregnane X receptor (PXR) gene (NR1I2) expression by its ligands and in the upregulation NR1I2 mRNA expression by glucocorticoids in hepatic cells. Glucocorticoids and PXR ligands thus regulate NR1I2 expression not only through the promoter region but also via 3ʹ-UTR regulation.Image 1

This article is the tenth series of the Fungal Diversity Notes, where 114 taxa distributed in three phyla, ten classes, 30 orders and 53 families are described and illustrated. Taxa described in the present study include one new family (viz. Pseudoberkleasmiaceae in Dothideomycetes), five new genera (Caatingomyces, Cryptoschizotrema, Neoacladium, Paramassaria and Trochilispora) and 71 new species, (viz. Acrogenospora thailandica, Amniculicola aquatica, A. guttulata, Angustimassarina sylvatica, Blackwellomyces lateris, Boubovia gelatinosa, Buellia viridula, Caatingomyces brasiliensis, Calophoma humuli, Camarosporidiella mori, Canalisporium dehongense, Cantharellus brunneopallidus, C. griseotinctus, Castanediella meliponae, Coprinopsis psammophila, Cordyceps succavus, Cortinarius minusculus, C. subscotoides, Diaporthe italiana, D. rumicicola, Diatrypella delonicis, Dictyocheirospora aquadulcis, D. taiwanense, Digitodesmium chiangmaiense, Distoseptispora dehongensis, D. palmarum, Dothiorella styphnolobii, Ellisembia aurea, Falciformispora aquatic, Fomitiporia carpinea, F. lagerstroemiae, Grammothele aurantiaca, G. micropora, Hermatomyces bauhiniae, Jahnula queenslandica, Kamalomyces mangrovei, Lecidella yunnanensis, Micarea squamulosa, Muriphaeosphaeria angustifoliae, Neoacladium indicum, Neodidymelliopsis sambuci, Neosetophoma miscanthi, N. salicis, Nodulosphaeria aquilegiae, N. thalictri, Paramassaria samaneae, Penicillium circulare, P. geumsanense, P. mali-pumilae, P. psychrotrophicum, P. wandoense, Phaeoisaria siamensis, Phaeopoacea asparagicola, Phaeosphaeria penniseti, Plectocarpon galapagoense, Porina sorediata, Pseudoberkleasmium chiangmaiense, Pyrenochaetopsis sinensis, Rhizophydium koreanum, Russula prasina, Sporoschisma chiangraiense, Stigmatomyces chamaemyiae, S. cocksii, S. papei, S. tschirnhausii, S. vikhrevii, Thysanorea uniseptata, Torula breviconidiophora, T. polyseptata, Trochilispora schefflerae and Vaginatispora palmae). Further, twelve new combinations (viz. Cryptoschizotrema cryptotrema, Prolixandromyces australi, P. elongatus, P. falcatus, P. longispinae, P. microveliae, P. neoalardi, P. polhemorum, P. protuberans, P. pseudoveliae, P. tenuistipitis and P. umbonatus), an epitype is chosen for Cantharellus goossensiae, a reference specimen for Acrogenospora sphaerocephala and new synonym Prolixandromyces are designated. Twenty-four new records on new hosts and new geographical distributions are also reported (i.e. Acrostalagmus annulatus, Cantharellus goossensiae, Coprinopsis villosa, Dothiorella plurivora, Dothiorella rhamni, Dothiorella symphoricarposicola, Dictyocheirospora rotunda, Fasciatispora arengae, Grammothele brasiliensis, Lasiodiplodia iraniensis, Lembosia xyliae, Morenoina palmicola, Murispora cicognanii, Neodidymelliopsis farokhinejadii, Neolinocarpon rachidis, Nothophoma quercina, Peroneutypa scoparia, Pestalotiopsis aggestorum, Pilidium concavum, Plagiostoma salicellum, Protofenestella ulmi, Sarocladium kiliense, Tetraploa nagasakiensis and Vaginatispora armatispora).

Hepatocellular carcinoma (HCC) is a major contributor to the worldwide cancer burden. Recent studies on HCC have demonstrated dramatic alterations in expression of several cytochrome P450 (CYP) family members that play a crucial role in biotransformation of many drugs and other xenobiotics; however, the mechanisms responsible for their deregulation remain unclear.We investigated a potential involvement of miRNAs in downregulation of expression of CYPs observed in HCC tumors. We compared miRNA expression profiles (TaqMan Array Human MicroRNA v3.0 TLDA qPCR) between HCC human patient tumors with strong (CYP-) and weak/no (CYP+) downregulation of drug-metabolizing CYPs. The role of significantly deregulated miRNAs in modulation of expression of the CYPs and associated xenobiotic receptors was then investigated in human liver HepaRG cells transfected with relevant miRNA mimics or inhibitors.We identified five differentially expressed miRNAs in CYP- versus CYP+ tumors, namely miR-29c, miR-125b1, miR-505, miR-653 and miR-675. The two most-upregulated miRNAs found in CYP- tumor samples, miR-29c and miR-653, were found to act as efficient suppressors of CYP1A2 or AHR expression.Our results revealed a novel role of miR-653 and miR-29c in regulation of expresion of CYPs involved in crucial biotransformation processes in liver, which are often deregulated during liver cancer progression.

The genera Menisporopsis, Multiguttulispora and Tainosphaeria (Chaetosphaeriaceae) are saprobes inhabiting decaying plant material. This study is based on an integrated morpho-molecular characterisation to assess their generic concepts and explore phylogenetic relationships. Menisporopsis is revealed as polyphyletic, and species with 1-septate conidia and synnemata growing unilaterally along the seta are placed in the new segregate genus Arcuatospora. Codinaea dimorpha and C. triseptata are shown to be congeneric with Multiguttulispora sympodialis, the type species. Two new combinations are proposed: M. sympodialis is found conspecific with M. dimorpha. The Tainosphaeria complex is resolved into three genera. We found that the morphological separation of three groups within the genus is consistent with phylogenetic relationships. Tainosphaeria s. str. is accepted with five species. Tainosphaeria aseptata and T. lunata are transferred to the newly erected Phialoturbella, whereas T. obclavata is revealed as conspecific with Phialogeniculata guadalcanalensis, reducing it to a synonym. A new genus Flectospora is erected for a chloridium-like fungus nested in the Tainosphaeria clade. Based on molecular evidence, we show that asymmetrical, scolecosporous ascospores are a unique teleomorphic characteristic among family members. Therefore, we propose new combinations for Chaetosphaeria hispida in Paragaeumannomyces and Ch. spinosa in the new genus Ericiosphaeria, both exhibiting this rare morphotype.

Dictyochaeta (Chaetosphaeriaceae) is a phialidic dematiaceous hyphomycete with teleomorphs classified in Chaetosphaeria. It is associated with significant variability of asexual morphological traits, which led to its broad delimitation. In the present study, six loci: nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS barcode), nuc 18S rDNA (18S), nuc 28S rDNA (28S), DNA-directed RNA polymerase II second largest subunit gene (RPB2), translation elongation factor 1-α (TEF1-α), and β-tubulin (TUB2), along with comparative morphological and cultivation studies, are used to reevaluate the concept of Dictyochaeta and establish species boundaries. Based on revised species, morphological characteristics of conidia (shape, septation, absence or presence of setulae), collarettes (shape), and setae (presence or absence) and an extension of the conidiogenous cell proved to be important at the generic level. The dual DNA barcoding using ITS and TEF1-α, together with TUB2, facilitated accurate identification of Dictyochaeta species. Thirteen species are accepted, of which seven are characterized in this study; an identification key is provided. It was revealed that D. fuegiana, the type species, is a complex of three distinct species including D. querna and the newly described D. stratosa. Besides, a new species, D. detriticola, and two new combinations, D. callimorpha and D. montana, are proposed. An epitype of D. montana is selected. Dictyochaeta includes saprobes on decaying wood, bark, woody fruits, and fallen leaves. Dictyochaeta is shown to be distantly related to the morphologically similar Codinaea, which is resolved as paraphyletic. Chaetosphaeria talbotii with a Dictyochaeta anamorph represents a novel lineage in the Chaetosphaeriaceae; it is segregated from Dictyochaeta, and a new genus Achrochaeta is proposed. Multigene phylogenetic analysis revealed that D. cylindrospora belongs to the Vermiculariopsiellales, and a new genus Tubulicolla is introduced.

The newly discovered systematic placement of Bactrodesmium abruptum, the lectotype species of the genus, prompted a re-evaluation of the traditionally broadly conceived genus Bactrodesmium. Fresh material, axenic cultures and new DNA sequence data of five gene regions of six species, i.e. B. abruptum, B. diversum, B. leptopus, B. obovatum, B. pallidum and B. spilomeum, were studied. Bactrodesmium is a strongly resolved lineage in the Savoryellales (Sordariomycetes), supported by Bayesian and Maximum Likelihood methods. The genus Bactrodesmium is emended and delimited to hyphomycetes characterised by sporodochial conidiomata, mononematous often fasciculate conidiophores, holoblastic conidiogenesis and acrogenous, solitary, dry, pigmented, transversely or rarely longitudinally septate conidia. The conidia are seceding rhexolytically, exhibiting multiple secession patterns. An identification key to 35 species accepted in Bactrodesmium is given, providing the most important diagnostic characters. Novel DNA sequence data of B. longisporum and B. stilboideum confirmed their placement in the Sclerococcales (Eurotiomycetes). For other Bactrodesmium, molecular data are available for B. cubense and B. gabretae, which position them in the Dothideomycetes and Leotiomycetes, respectively. All four species are excluded from Bactrodesmium and segregated into new genera, Aphanodesmium, Gamsomyces and Kaseifertia. Classification of 20 other species and varieties not recognised in the genus is discussed. Based on new collections of Dematiosporium aquaticum, the type species of Dematiosporium, the genus is emended to accommodate monodictys-like freshwater lignicolous fungi of the Savoryellales characterised by effuse colonies, holoblastic conidiogenous cells and dictyosporous, pigmented conidia with a pore in each cell. Study of additional new collections, cultures and DNA sequence data revealed several unknown species, which are proposed as taxonomic novelties in the Savoryellales and closely related Pleurotheciales. Ascotaiwania latericolla, Helicoascotaiwania lacustris and Pleurotheciella erumpens are described from terrestrial, lentic and lotic habitats from New Zealand and France, respectively. New combinations are proposed for Helicoascotaiwania farinosa and Neoascotaiwania fusiformis. Relationships and systematics of the Savoryellales are discussed in the light of recent phylogenies and morphological patterns newly linked with the order through cultural studies.

Summary Background FTO, a gene recently discovered in genomewide associated studies for type 2 diabetes mellitus (T2D), play an important role in the management of energy homeostasis, nucleic acid demethylation and regulation of body fat mass by lipolysis. The aim of this study was to analyze the association of FTO rs8050136 A>C genetic variant with clinical and biochemical parameters of T2D in the population of West Balkan region (Bosnians and Herzegovinians and Kosovars). Methods The study included 638 patients with T2D and prediabetes and 360 healthy controls of both genders, aged from 40 to 65 years. Patients were recruited at the Clinical Centre University of Sarajevo, University Hospital of Clinical Centre in Banja Luka, General Hospital in Tešanj and Health Centre in Prizren. Genotyping of analyzed FTO polymorphism rs8050136 A>C was performed by qPCR allelic discrimination. Results Genotype frequencies of the analyzed polymorphism were comparable between patients with T2D, prediabetic patients, and healthy population. Logistic regression analyses didn’t show significant association of FTO rs8050136 A allele with increased risk of T2D. However, risk A allele was significantly associated with higher levels of HbA1c, insulin, HOMA-IR index, diastolic blood pressure, and inflammatory markers (fibrinogen and leukocytes) as well as showed tendency of association with increased values of obesity markers (BMI, waist and hip circumference). Conclusions Results of our study showed a significant association of FTO genetic variant rs8050136 A>C with the major markers of insulin resistance, obesity and inflammation, opening new avenues for solving many unclear questions in the pathogenesis of T2D.

Colorectal cancer, one of the most frequent types of cancer worldwide, has a high mortality rate. Irinotecan (CPT-11) has been approved for the treatment of advanced or metastatic disease either as a single agent or, more commonly, as part of combined chemotherapeutic regimens. Treatment with irinotecan is often accompanied by severe toxicity (e.g. neutropenia and diarrhea) that can result in treatment interruption or cessation, thus jeopardizing the patient’s prognosis and quality of life. Irinotecan is bioactivated into its metabolite SN-38, which is subsequently detoxified by uridine diphosphate-glucuronosyl transferases (mainly UGT1A1). Further, ABC transporters (i.e. ABCB1, ABCC1–ABCC6, and ABCG2) are responsible for drug efflux into bile and urine whereas OATP transporters (SLCO1B1) enable its influx from blood into hepatocytes. Genetic polymorphisms in these enzymes/pumps may result in increased systemic SN-38 level, directly correlating with toxicity. Contemporary research is focused on the clinical implementation of genetic screenings for validated gene variations prior to treatment onset, allowing tailored individual doses or treatment regimens.

Cytochrome P450 family 1 (CYP1) enzymes contribute both to metabolism of xenobiotics and to the control of endogenous levels of ligands of the aryl hydrocarbon receptor (AhR). Their activities, similar to other CYPs, can be altered in tumor tissues. Here, we examined a possible role of proliferative/survival pathways signaling, which is often deregulated in tumor cells, and possible links with p300 histone acetyltransferase (a transcriptional co-activator) in the control of CYP1 expression, focusing particularly on CYP1A1. Using cell models derived from human liver, we observed that the induction of CYP1A1 expression, as well as other CYP1 enzymes, was reduced in exponentially growing cells, as compared with their non-dividing counterparts. The siRNA-mediated inhibition of proliferation/pro-survival signaling pathway effectors (such as β-catenin and/or Hippo pathway effectors YAP/TAZ) increased the AhR ligand-induced CYP1A1 mRNA levels in liver HepaRG cells, and/or in colon carcinoma HCT-116 cells. The activation of proliferative Wnt/β-catenin signaling in HCT-116 cells reduced both the induction of CYP1 enzymes and the binding of p300 to the promoter of CYP1A1 or CYP1B1 genes. These results seem to indicate that aberrant proliferative signaling in tumor cells could suppress induction of CYP1A1 (or other CYP1 enzymes) via competition for p300 binding. This mechanism could be involved in modulation of the metabolism of both endogenous and exogenous substrates of CYP1A1 (and other CYP1 enzymes), with possible further consequences for alterations of the AhR signaling in tumor cells, or additional functional roles of CYP1 enzymes.

Hepatocellular carcinoma (HCC) remains a highly prevalent and deadly disease, being among the top causes of cancer-related deaths worldwide. Despite the fact that the liver is the major site of biotransformation, studies on drug metabolizing enzymes in HCC are scarce. It is known that malignant transformation of hepatocytes leads to a significant alteration of their metabolic functions and overall deregulation of gene expression. Advanced stages of the disease are thus frequently associated with liver failure, and severe alteration of drug metabolism. However, the impact of dysregulation of metabolic enzymes on therapeutic efficacy and toxicity in HCC patients is largely unknown. Here we demonstrate a significant down-regulation in European Caucasian patients of cytochromes P450 (CYPs), the major xenobiotic-metabolizing enzymes, in HCC tumour samples as compared to their surrounding non-cancerous (reference) tissue. Moreover, we report for the first time the association of the unique CYP profiles with specific transcriptome changes, and interesting correlations with expression levels of nuclear receptors and with the histological grade of the tumours. Integrated analysis has suggested certain co-expression profiles of CYPs with lncRNAs that need to be further characterized. Patients with large tumours with down-regulated CYPs could be more vulnerable to drug toxicity; on the other hand, such tumours would eliminate drugs more slowly and should be more sensitive to pharmacotherapy (except in the case of pro-drugs where activation is necessary).

OBJECTIVE The treatment of rheumatoid arthritis (RA) patients with methotrexate (MTX) is linked to the development or progression of rheumatoid nodules. The aim of this study was to determine whether folate and adenosine pathways-related single nucleotide polymorphisms might be predictive of increased nodule formation in RA patients treated with oral MTX. METHODS A total of 185 Caucasian RA patients were enrolled in this cross-sectional study, all of whom fulfilled the 1987 RA criteria of the American College of Rheumatology; each patient had a history of MTX treatment. RESULTS A higher frequency of the MTHFR 1298AA genotype was found in 17 (70.8%) of 24 patients with general nodules [odds ratio (OR)=3.08, 95% confidence interval (CI): 1.20-7.69] and in 14 (73.7%) of 19 patients who developed nodules during MTX treatment (OR=3.55, 95% CI: 1.22-10.32). In contrast, a negative association with nodules during MTX treatment (OR=0.29, 95% CI: 0.08-1.10) was found for 19 (79.2%) patients with the TT genotype (rs2298383) in the adenosine A2a receptor gene (ADORA2A). However, the significance did not remain upon correction for multiple testing. The combination of MTHFR 1298AA along with ADORA2A rs2298383 CC or CT genotypes occurring in one-third of RA patients showed a higher frequency of general nodules 15/59 (25.4%) as well as developing nodules during MTX treatment 13/59 (22.0%) in comparison with the overall studied group: 24/185 (13.0%) and 19/185 (10.3%), respectively. CONCLUSION This exploratory study indicates for the first time a plausible association of adenosine and folate pathways single nucleotide polymorphisms in nodules' etiopathogenesis.

Letter to the editor with respect to: Lima A, Bernardes M, Azevedo R, Seabra V and Medeiros R. Moving toward personalized medicine in rheumatoid arthritis: SNPs in methotrexate intracellular pathways are associated with methotrexate therapeutic outcome. Pharmacogenomics 17(15), 1649–1674 (2016).

Some single-nucleotide polymorphisms (SNPs) might be predictive of methotrexate (MTX) therapeutic outcome in rheumatoid arthritis (RA). The aim of this study was to determine whether SNPs in the methylenetetrahydrofolate reductase (MTHFR) gene are predictive of MTX response. Comparison was made using EULAR response criteria and according to the change of DAS28 (∆DAS28) after a 6-month MTX treatment in RA patient cohort. The two SNPs C677T (rs1801133) and A1298C (rs1801131) have been genotyped. A total of 120 patients were enrolled in the study, and all of them fulfilled the American College of Rheumatology 1987 RA criteria and are currently or previously taking MTX oral treatment, either as a monotherapy (n = 65) or in a combination with other disease-modifying antirheumatic drugs (n = 55). Genotyping was performed using qPCR allelic discrimination. We did not found any association of C677T and A1298C genotypes with MTX treatment inefficacy in dominant model (OR 1.23, 95 % CI 0.57–2.65, P = 0.697; and OR 0.98, 95 % CI 0.47–2.14, P = 1.0, respectively), or in recessive and codominant models. However, when ∆DAS28 after a 6-month therapy was used as a measure of treatment efficacy, the 677CT and 1298AC genotypes were found to be significantly associated with less favorable response to MTX (P = 0.025 and P = 0.043, respectively). In addition, even lower ∆DAS28 was determined for double-mutated 677CT–1298AC heterozygotes. It means that a synergistic effect of 677CT and 1298AC genotypes was observed. Nevertheless, the DAS28 baseline was lower here comparing to other genotypes. Unexpectedly, quite the opposite trend—i.e., better response to MTX—was found in genotypes 677CC–1298CC and 677TT–1298AA. It is an intriguing finding, because these double-mutated homozygotes are known for their low MTHFR-specific activity. Global significance was P = 0.013, η 2 = 0.160—i.e., large-size effect. Thus, our data show greater ability of 677CC–1298CC and 677TT–1298AA genotypes to respond to MTX treatment.

Elevated levels of circulating angiogenic cytokines and increased expression of genes encoding angiogenic factors have been reported in recent years in patients with chronic lymphocytic leukemia (CLL) but data regarding prognostic and predictive significance are still limited. Therefore, in the present study based upon our prior pilot results, we measured mRNA expressions of angiopoietin-2 (Ang-2), fibroblast growth factor-2 (FGF-2) and endoglin (CD105) by reverse transcription quantitative PCR in purified CD19+ cells from 70 untreated CLL patients (median age, 63 years; males, 64%; Rai III/IV stages, 29 %; unmutated IgVH genes, 60 %) and evaluated their possible association with established prognostic factors and clinical course of the disease. Higher expression of Ang-2 was significantly associated with unmutated IgVH genes (n = 55, p = 0.003). Higher CD105 expression was significantly associated with unmutated IgVH genes (n = 55, p < 0.001), high CD38 expression (n = 66, p = 0.022), high ZAP-70 expression (n = 66, p = 0.010), Rai stage I-IV (n = 70, p < 0.001), progressive clinical course of CLL (n = 70, p = 0.001) and shorter time to treatment (n = 70; p < 0.001). Expression of FGF-2 was not significantly associated with any of the prognostic markers. These results indicate that elevated expression of Ang-2 and in particular CD105 by CLL cells is associated with unfavorable prognostic features and clinical outcome; thus, both cytokines appear to play an important role in biology and progression of CLL and warrant further investigation.

Multiple myeloma still remains incurable in the majority of cases prompting a further search for new and better prognostic markers. Emerging evidence has suggested that circulating microRNAs can serve as minimally invasive biomarkers for multiple myeloma and monoclonal gammopathy of undetermined significance. In this study, a global analysis of serum microRNAs by TaqMan Low Density Arrays was performed, followed by quantitative real-time PCR. The analyses revealed five deregulated microRNAs: miR-744, miR-130a, miR-34a, let-7d and let-7e in monoclonal gammopathy of undetermined significance, newly diagnosed and relapsed multiple myeloma when compared to healthy donors. Multivariate logistical regression analysis showed that a combination of miR-34a and let-7e can distinguish multiple myeloma from healthy donors with a sensitivity of 80.6% and a specificity of 86.7%, and monoclonal gammopathy of undetermined significance from healthy donors with a sensitivity of 91.1% and a specificity of 96.7%. Furthermore, lower levels of miR-744 and let-7e were associated with shorter overall survival and remission of myeloma patients. One-year mortality rates for miR-744 and let-7e were 41.9% and 34.6% for the ‘low’ expression and 3.3% and 3.9% for the ‘high’ expression groups, respectively. Median time of remission for both miR-744 and let-7e was approximately 11 months for the ‘low’ expression and approximately 47 months for the ‘high’ expression groups of myeloma patients These data demonstrate that expression patterns of circulating microRNAs are altered in multiple myeloma and monoclonal gammopathy of undetermined significance and miR-744 with let-7e are associated with survival of myeloma patients.

Renal cell carcinoma (RCC) is the most common neoplasm of adult kidney accounting for about 3 % of adult malignancies. MicroRNAs (miRNAs) are a class of naturally occurring, short non-coding RNAs that regulate gene expression at the post-transcriptional level. We determined global miRNA expression profiles of RCC and parallel renal parenchyma tissues by using quantitative reverse transcriptase-polymerase chain reaction-based TaqMan low-density arrays. Afterward, we validated the difference in miR-210 expression levels on the larger group of RCC patients (35 RCC versus 10 non-tumorous parenchyma samples). Functional in vitro experiments were performed on ACHN and CAKI-2 RCC cell lines transfected with miRNA-210 inhibitor. Cell viability, apoptosis, cell cycle, scratch wound migration assay, and invasion assay (xCELLigence) were performed. We have identified original ccRCC-specific miRNA signature in clinical samples (73 miRNAs were significantly downregulated and five miRNAs upregulated (P

MicroRNAs (miRNAs) constitute a robust regulatory network with post-transcriptional regulatory efficiency for almost one half of human coding genes, including oncogenes and tumour suppressors. We determined the expression profile of 667 miRNAs in colorectal cancer (CRC) tissues and paired non-tumoural tissues and identified 42 differentially expressed miRNAs. We chose miR-215, miR-375, miR-378, miR-422a and miR-135b for further validation on an independent cohort of 125 clinically characterized CRC patients and for in vitro analyses. MiR-215, miR-375, miR-378 and miR-422a were significantly decreased, whereas miR-135b was increased in CRC tumour tissues. Levels of miR-215 and miR-422a correlated with clinical stage. MiR-135b was associated with higher pre-operative serum levels of CEA and CA19-9. In vitro analyses showed that ectopic expression of miR-215 decreases viability and migration, increases apoptosis and promotes cell cycle arrest in DLD-1 and HCT-116 colon cancer cell lines. Similarly, overexpression of miR-375 and inhibition of miR-135b led to decreased viability. Finally, restoration of miR-378, miR-422a and miR-375 inhibited G1/S transition. These findings indicate that miR-378, miR-375, miR-422a and miR-215 play an important role in CRC as tumour suppressors, whereas miR-135b functions as an oncogene; both groups of miRNA contribute to CRC pathogenesis.

The aim of this study is to evaluate the biologic importance and prognostic significance of selected clinicopathological parameters in patients with oral (OSCC) and oropharyngeal (OPSCC) squamous cell carcinoma, with emphasis on smoking, protein p16(INK4a) (p16) expression, and human papillomavirus (HPV) status.The study sample consisted of 48 patients with OSCC and 44 patients with OPSCC. Half of the patients were nonsmokers and the other half were gender-, age- and tumor localization-matched smokers. p16 expression was detected in 17/48 (35 %) OSCCs and in 36/44 (82 %) OPSCCs and HPV DNA was present in 7/48 (15 %) OSCCs and in 35/44 (80 %) OPSCCs. The sensitivity and specificity of p16 expression for HPV DNA presence were 0.74 and 0.88, respectively. The OPSCCs were more frequently basaloid (p0.001) while the OSCCs were more frequently conventional (p0.000001). The OSCCs were more likely to recur locally and to be the cause of death (p = 0.009 in both parameters).The HPV-positive tumors were more frequently localized in oropharynx, were basaloid SCCs and were p16- and HPV-positive (p0.000001 in all 4 parameters). The HPV-negative tumors were more frequently localized in oral cavity (p0.000001), more frequently asociated with local, regional and locoregional recurence (p = 0.011, p = 0.019 and p = 0.030, respectively) and with tumor-related death (p = 0.003). There was no significant difference with regard to smoking history (p0.05). The survival of patients with HPV-positive tumors was significantly longer (median 112 months; 95% CI 54 - 112 months) than that of patients with HPV-negative tumors (median 17 months; 95% CI 12 - 39 months) (p0.001). The HPV status of OSCC/OPSCC is an important biological and prognostic parameter and should be examined in all cases, using PCR or immunohistochemical detection of surrogate marker p16. Smoking itself does not seem to be an important prognostic factor.

Background: Aberrant hypermethylation of tumour suppressor genes (TSGs) occurring in hepatocellular carcinoma (HCC) could provide a mean of molecular characterisation of this cancer. The aim of this study was to investigate promoter methylation and gene expression of selected TSGs in HCC to identify candidate genes for further validation as potential biomarkers. Methods: Methylation-specific multiplex ligation-dependent probe amplification method was used to measure the methylation status of 25 TSGs in 49 HCC samples and 36 corresponding non-cancerous liver tissue samples. Relative expression of the differentially methylated genes was assessed at the mRNA level using quantitative PCR. Results: We observed a significantly higher methylation in genes WT1, PAX5, PAX6, PYCARD and GATA5 in HCC compared with control samples. The expression of PAX5 was significantly decreased by methylation; conversely methylation of WT1 was associated with higher mRNA levels. Methylation of GATA5 was significantly associated with overall survival and methylation of WT1 and PAX5 significantly varied between patients with ALBI score 1 vs. 2+3. Moreover, PAX5 was significantly more methylated in patients with tumour grade 2+3 vs. grade 1, and methylation of the PAX5 correlated with the patient’s age at the time of diagnosis. Conclusions: HCC evince aberrant promoter methylation of WT1, PAX5, PAX6, PYCARD and GATA5 genes. Correlation between GATA5, WT1 and PAX5 methylation and clinical/histological parameters is suggestive of applicability of these markers in non-invasive (epi)genetic testing in HCC.

Adefovir (PMEA) and tenofovir (PMPA) and their prodrugs, adefovir dipivoxil (bisPOM-PMEA) and tenofovir disoproxil (bisPOC-PMPA), were subjected to a detailed study of their potential to inhibit the activities of human liver microsomal cytochromes P450 (CYP). The inhibition of marker enzyme activities of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 was examined with high-performance liquid chromatography (HPLC) or spectroscopic (fluorescence, luminescence) detection. Adefovir and adefovir dipivoxil did not significantly influence activities of most CYP enzymes. The activity of CYP3A4 was inhibited by adefovir dipivoxil at concentrations over 100 microM. Adefovir and its prodrug inhibited CYP2C9 at concentrations below 100 microM; inhibition by adefovir was of the uncompetitive (at the lower inhibitor concentrations) or of the competitive nature with a Ki = 420 microM. Tenofovir and tenofovir disoproxil influenced the activity of CYP2C9, and competitive inhibition was found with Ki = 580 and 395 microM, respectively. Tenofovir disoproxil was shown to inhibit microsomal CYP2E1 activities by a mixed-type inhibition with Ki values at about 140 microM. The results indicate the possibility of an influence of the compounds tested on the respective CYP activities when used at high doses.

MicroRNAs are short non-coding ribonucleic acid molecules that regulate gene expression at the post-transcriptional level thus affecting important physiological as well as pathophysiological processes in the organism, for example cell differentiation, proliferation, apoptosis, and metabolism. They are involved in pathogenesis of many diseases including cancer. Many microRNAs are tissue or organ-specific which implies their possible potential as biomarkers or maybe even therapeutical agents as documented by microRNA research interest rising exponentially during last years. Among all, microRNAs are important also for physiological function of the kidney and they are involved in various renal disorders. Today research is focused mainly on renal and urinary tract carcinogenesis, acute kidney injury, chronic renal diseases (polycystic kidney disease) or renal complications of systemic diseases such as diabetic or hypertension nephropathy and autoimmune kidney injury including acute allograft rejection after kidney transplantation. The review summarizes current information about microRNA effect on kidney development and function and also on the most common kidney diseases.

1. Nucleotide analogues comprise an important class of drugs used in treatment of viral infections but also cancer. These drugs affect the structural integrity of DNA and activate different pathways and processes in the cell and may directly or indirectly influence the drug metabolizing system. Adefovir dipivoxil (AD) and tenofovir disoproxil (TD) are nucleotide analogues approved for the treatment of chronic hepatitis B and/or HIV/AIDS infection. 2. To evaluate the risk of their drug-drug interactions on the level of drug metabolism, an effect of both compounds on cytochromes P450 expression was studied using cDNA microarrays, real-time RT-PCR and immunoblotting. Mice were given intraperitoneally 25 mg/kg of AD or TD, respectively. As a positive control, a combination of prototypic cytochromes P450 (CYP) inducers, phenobarbital and β-naphthoflavone was chosen. 3. The data obtained showed a significant CYP induction in the positive control group, but no clinically significant induction of CYP genes by AD or TD was observed. Our results support the evidence of safety of AD and TD with respect to drug-drug interactions based on enzyme induction. These findings are important as a plethora of new antivirals of different types are being tested and introduced to clinical practice, mostly to be used in combinations.

Clinical behavior of neuroblastoma (NBL) is remarkably heterogeneous, as it ranges from spontaneous regression to aggressive clinical phenotype and death. There is increasing body of evidence demonstrating that microRNAs could be considered the potential biomarkers for clinical applications in NBL. In this report, we focus on molecular characterization of high-risk as well as low-risk and intermediate-risk NBL cases in the context of the microRNA expression profile that is specific for the given risk category of the disease. We investigated a total of 30 NBL patients, out of whom there were 19 patients with low- to intermediate-risk and 11 with high-risk NBLs as defined by the Clinical Oncology Group. We determined the expression profiles of 754 microRNAs (miRNAs), whereas the miRNA expression levels were normalized to RNU44, mean expression levels were calculated, and data were analyzed by use of the microarray biostatistical approaches. We identified the signature of 38 miRNAs differentially expressed between these groups of NBL patients (P < 0.05): 17 miRNAs were upregulated and 21 miRNAs were downregulated in the tumors of high-risk NBL patients. We confirm some of the previous observations and we report several new microRNAs associated with aggressive NBL, both being relevant subjects for further translational validation and functional studies.

Background Rectal cancer accounts for approximately one third of all colorectal cancers (CRC), which belong among leading causes of cancer deaths worldwide. Standard treatment for locally advanced rectal cancer (cT3/4 and/or cN+) includes neoadjuvant chemoradiotherapy with fluoropyrimidines (capecitabine or 5-fluorouracil) followed by radical surgical resection. Unfortunately, a significant proportion of tumors do not respond enough to the neoadjuvant treatment and these patients are at risk of relapse. MicroRNAs (miRNAs) are small non-coding RNAs playing significant roles in the pathogenesis of many cancers including rectal cancer. MiRNAs could present the new predictive biomarkers for rectal cancer patients. Methods We selected 20 patients who underwent neoadjuvant chemoradiotherapy for advanced rectal cancer and whose tumors were classified as most sensitive or resistant to the treatment. These two groups were compared using large-scale miRNA expression profiling. Results Expression levels of 8 miRNAs significantly differed between two groups. MiR-215, miR-190b and miR-29b-2* have been overexpressed in non-responders, and let-7e, miR-196b, miR-450a, miR-450b-5p and miR-99a* have shown higher expression levels in responders. Using these miRNAs 9 of 10 responders and 9 of 10 non-responders (p < 0.05) have been correctly classified. Conclusions Our pilot study suggests that miRNAs are part of the mechanisms that are involved in response of rectal cancer to the chemoradiotherapy and that miRNAs may be promising predictive biomarkers for such patients. In most miRNAs we identified (miR-215, miR-99a*, miR-196b, miR-450b-5p and let-7e), the connection between their expression and radioresistance or chemoresistance to inhibitors of thymidylate synthetase was already established.

Background There is no standard serum biomarker used for diagnosis or early detection of recurrence for renal cell carcinoma (RCC) patients. MicroRNAs (miRNAs) are abundant and highly stable in blood serum, and have been recently described as powerful circulating biomarkers in a wide range of solid cancers. Our aim was to identify miRNA signature that can distinguish the blood serum of RCC patients and matched healthy controls and validate identified miRNAs as potential biomarkers for RCC. Methods In the screening phase of the study, blood serum of 15 RCC patients and 12 matched healthy controls were analyzed by use of the TaqMan Low-Density Arrays enabling parallel identification of expression levels of 667 miRNAs through qRT-PCR-based approach. In the validation phase, identified miRNAs were further evaluated on the independent group of 90 RCC patients and 35 matched healthy controls by use of individual qRT-PCR assays and statistically evaluated. Results We identified 30 miRNAs differentially expressed between serum of RCC patients and healthy controls: 19 miRNAs were up-regulated and 11 miRNAs were down-regulated in RCC patients. MiR-378, miR-451 and miR-150 were further evaluated in the independent group of patients, and two of them were successfully validated: levels of miR-378 were increased (p = 0.0003, AUC = 0.71), miR-451 levels were decreased (p < 0.0001, AUC = 0.77) in serum of RCC patients. Combination of miR-378 and miR-451 enable identification of RCC serum with the sensitivity of 81%, specificity 83% and AUC = 0.86. Conclusions Circulating miRNAs in serum are promising biomarkers in RCC.

Renal cell carcinoma (RCC) is the most common neoplasm of adult kidney. One of the important unmet medical needs in RCC is prognostic biomarker enabling identification of patients at high risk of relapse after nephrectomy. MicroRNAs (miRNAs) constitute a robust regulatory network with posttranscriptional regulatory efficiency for almost one-half of human coding genes, including oncogenes and tumor suppressors. To identify potential prognostic miRNAs, we analyzed expression profiles in tumors of different prognostic groups of RCC patients. Seventy-seven patients with clear cell RCC and detailed clinicopathological data were enrolled in a single-center study. Global miRNA expression profiles were obtained by use of TaqMan Low Density Arrays (754 parallel quantitative reverse-transcriptase polymerase chain reactions (qRT-PCR) reactions). For validation of identified miRNAs individual miRNA TaqMan assays were performed in an independent group of patients. We identified tumor relapse-signature based on the expression of 64 miRNAs differentially expressed between relapse-free RCC patients and RCC patients who developed relapse (20 miRNAs were increased, 44 miRNAs were decreased). In the validation phase of the study, we successfully confirmed that expression levels of miR-143, miR-26a, miR-145, miR-10b, miR-195, and miR-126 are lower in the tumors of RCC patients who developed tumor relapse, moreover, the lowest levels of these miRNAs we observed in primary metastatic tumors. By using Kaplan-Meier analysis, we identified that miR-127-3p, miR-145, and miR-126 are significantly correlated with relapse-free survival of nonmetastatic RCC patients. If further validated, we suggest that identified miRNAs might be used for identification of RCC patients at high risk of early relapse after nephrectomy in clinical practice.

IgM monoclonal gammopathies are a group of diseases characterized by increased level of IgM immunoglobulin produced by one clone of B cells. These diseases range trom benign (monoclonal gammopathy ot undetermined significance, MGUS) to malignant, such as Waldenstrom macroglobulinemia (WM) or to a lesser extent multiple myeloma (MM) [1,2]. The criteria that differentiate WM from IgM-MGUS are based on the extent of bone marrow (BM) involvement, amount of serum concentration of the M-proicin, presence or absence of symptomatic disease or more recently, MYD88 (L265P) or CXCR4 mutations [3-6]. Despite that, new criteria for the differential diagnosis between these conditions are still needed, circulating microRNAs (miRNAs) being one of them. Circulating miRNAs are present in different body fluids; they reflect physiological or pathologi cal conditions and can be used tor patient classification [7,8]. Thus, we aimed to investigate the ability of serum miRNAs to distinguish WM from IgM-MGUS as well as IgM-MM patients and healthy donors (HD).

The aim of the study was to investigate the role of high-risk human papillomavirus (HR-HPV) infection in the etiopathogenesis of oral (OSCC) and oropharyngeal (OPSCC) squamous cell carcinoma in non-smoking and non-drinking patients (NSNDP). Twenty-four OSCCs and 22 OPSCCs were analyzed by immunohistochemistry for p16INK4a protein (p16) expression and by chromogene in situ hybridization (CISH) and polymerase chain reaction (PCR) for HR-HPV DNA presence. The series included 23 males and 23 females aged 35–93 years. p16 expression was seen in 7 out of 24 (29%) OSCCs and in 22 out of 22 (100%) OPSCCs. Using CISH, HR-HPV DNA was observed in 6 out of 24 (25%) OSCCs and in 21 out of 22 (95%) OPSCCs. HPV DNA was found in 3 out of 24 (13%) OSCCs and in 18 out of 22 (82%) OPSCCs using PCR. HPV 16 and 33 were detected in 16 and in two cases, respectively. Compared with OSCCs, OPSCCs more frequently showed basaloid morphology (p

Cytochrome P450 2E1 (CYP2E1), the alcohol-inducible member of the cytochrome P450 super family, plays an important role in both physiological and pathophysiological processes. The present study focused on the induction of human CYP2E1 transcription by the anti-inflammatory cytokine interleukin-4 (IL-4) in human hepatoma B16A2 cells and revealed that this regulation is mediated by two independent pathways. RNA interference and overexpression of STAT6, indicated that the JAK-STAT signaling pathway is involved in IL-4-dependent induction and mutagenesis revealed the presence of a STAT6 binding site in CYP2E1 proximal promoter region (-583/-574-bp). However, inhibition of the JAK-STAT6 pathway using JAK1 siRNA constructs could only partially inhibit the induction of CYP2E1 promoter constructs indicating the presence of a second IL-4 responsive element. Indeed by using a series of truncated CYP2E1 promoter constructs a second more distal IL-4 responsive element (-1604/-1428-bp) was identified, which was further shown to involve the activation of IRS1/2. This induction was dependent on the transcription factor NFATc1 as IL-4-induced CYP2E1 expression was altered by silencing or overexpressing NFATc1. A NFATc1 binding site was identified in the second distal IL-4 responsive element (-1551/-1545-bp) by chromatin immunoprecipitation (ChIP) analysis. Finally simultaneous siRNA-mediated down-regulation of both STAT6 and NFATc1 or mutation of both STAT6 and NFATc1 binding sites abolished the IL-4-dependent transcriptional induction of CYP2E1, demonstrating that both pathways are required for maximal activation. In conclusion, the present study indicates that the induction of CYP2E1 transcription by IL-4 is mediated through two independent parallel pathways, involving JAK-STAT6 and IRS1/2 and NFATc1.

CYP2C9 is an important drug-metabolizing enzyme that metabolizes, e. g., warfarin, antidiabetics, and antiphlogistics. However, the endogenous regulation of this enzyme is largely unknown. In this study, we examined the role of GATA transcription factors in the gene expression of CYP2C9. We investigated four putative GATA binding sites within the first 200 base pairs of CYP2C9 promoter at the positions I: -173/-170, II: -167/-164, III: -118/ -115, and IV: -106/-103. Luciferase activity driven by a wildtype CYP2C9 promoter construct was strongly up-regulated in Huh-7 cells upon cotransfection with expression plasmids for GATA-2 and GATA-4, whereas mutations introduced into GATA binding site III or I and II reduced this induction to a significant extent. Electrophoretic mobility shift assays revealed specific binding of GATA-4 and GATA-6 to the oligonucleotides containing GATA binding sites I and II. Furthermore, the association of GATA-4 with CYP2C9 promoter was confirmed by chromatin immunoprecipitation assays in HepG2 cells. Taken together, these data strongly suggest an involvement of liver-specific transcription factor GATA-4 in the transcriptional regulation of CYP2C9. Swedish Research Council; Stockholm County Council; Danish Agency of Science, Technology and Innovation; Lundbeck Foundation; Portuguese Foundation for Science and Technology [SFRH/BPD/34152/2006 IBB/CBME]

Aims Cytochrome P4502C19 (CYP2C19) is an important enzyme involved in the metabolism of antiulcer drugs and antidepressants. However, despite the well documented drug-dependent variability of CYP2C19 expression, the mechanisms underlying the regulation of the enzyme remain unknown. In this study we investigated whether the transcription factor family GATA is involved in the regulation of CYP2C19 gene expression. Main methods We identified a novel putative GATA binding site at position –165/–156 within the CYP2C19 gene promoter. 5′-Deletion fragments of the CYP2C19 promoter containing wild type or mutant variants of this GATA binding site were co-transfected with expression vectors encoding the transcription factors GATA-4 or GATA-2 and analyzed using dual luciferase gene reporter assays in HepG2 and Huh-7 hepatoma cells. Electrophoretic Mobility Shift Assay (EMSA) and Chromatin Immunoprecipitations (ChIP) were performed to proof a sequence-specific interaction of GATA proteins with the putative GATA binding site. Key findings The wild type fragments of CYP2C19 promoter were highly upregulated by GATA-4 and GATA-2 in luciferase gene reporter assay, whereas mutations introduced into the GATA binding sites caused a significant activity loss. Similar attenuation was observed upon co-transfection of GATA-4 with a known co-regulator of GATA activity, FOG-2. EMSA analysis revealed a sequence-specific binding of GATA-4 and GATA-6 to the wild type GATA binding site. In addition, the association of GATA-4 with the CYP2C19 promoter was confirmed by ChIP analysis. Significance These data indicate that GATA-4 plays an important role in the transcriptional regulation of CYP2C19 expression.

Poor outcome of extramedullary disease in multiple myeloma patients and lack of outcome predictors prompt continued search for new markers of the disease. In this report, we show circulating microRNA distinguishing multiple myeloma patients with extramedullary disease from myeloma patients without such manifestation and from healthy donors. MicroRNA-130a was identified by TaqMan Low Density Arrays and verified by quantitative PCR on 144 serum samples (59 multiple myeloma, 55 myeloma with extramedullary disease, 30 healthy donors) in test and validation cohorts as being down-regulated in myeloma patients with extramedullary disease. Circulating microRNA-130a distinguished myeloma patients with extramedullary disease from healthy donors with specificity of 90.0% and sensitivity of 77.1%, patients with extramedullary disease from newly diagnosed multiple myeloma patients with specificity of 77.1% and sensitivity of 34.3% in the test cohort and with specificity of 91.7% and sensitivity of 30.0% in the validation cohort of patients. Circulating microRNA-130a in patients with extramedullary myeloma was associated with bone marrow plasma cells infiltration. Further, microRNA-130a was decreased in bone marrow plasma cells obtained from patients with extramedullary myeloma in comparison to bone marrow plasma cells of myeloma patients without such manifestation, but it was increased in tumor site plasma cells of patients with extramedullary disease compared to bone marrow plasma cells of such patients (p

Background Some single nucleotide polymorphisms (SNPs) might be predictive of methotrexate (MTX) therapeutic outcome in rheumatoid arthritis (RA). Objectives The aim of this study was to determine whether SNPs in key MTX pathway genes might be predictive of an adverse drug reactions (ADR) and MTX discontinuation in RA patients treated with peroral MTX. Methods There were 185 patients enrolled in the retrospective study, all of whom fulfilled the 1987 RA criteria of the American College of Rheumatology. The patients were currently or previously treated with peroral MTX. Genotyping was performed by quantitative PCR with allelic discrimination using commercial TaqMan (allele-specific) assays (Life Technologies, USA): ( ADORA2A , rs2298383), ( ADORA2A , rs3761422), ( ADORA2A , rs2267076), ( ADORA2A , rs2236624), ( AMPD1 , rs17602729), ( ATIC , rs2372536), ( ITPA , rs1127354), ( MTHFD1 , rs2236225), ( MTHFR , rs1801131), ( MTHFR , rs1801133), ( SLC01B1 , rs4149056). Results Clinical data were available in 185 patients: mean age 58.7 years, SD ±12.17, age of 29-85 years. 30.3% of patients were male. MTX treatment was discontinued in 33 (17.8%) patients owing to ADR (hepatopathy, n=12; infection, n=10; dyspepsia, n=9; leucopenia, n=1; alopecia, n=1). 83 patients were treated by MTX monotherapy or MTX together with glucocorticoids. Other patients were treated by MTX in a combination with conventional synthetic or biologic DMARDs. Presence and absence of ADR were evaluated in relation to the identified genotypes. We revealed no evidence to support association of the presence of ADR and the following SNPs: ATIC rs2372536, AMPD1 rs17602729, ITPA rs1127354, SNPs of ADORA2A (rs2298383, rs3761422, rs2267076, and rs2236624), MTHFD1 rs2236225, MTHFR rs1801131, MTHFR rs1801133 and SLC01B1 rs4149056. Stratification of different ADRs in ITPA 94CC and ITPA 94CA genotypes showed association with dyspepsia P=0.01 using Kendall9s test, in case of MTX DMARDs monotherapy P=0.046 respectively. ITPA 94AA genotype have not been found. Conclusions In conclusion, we found for the first time that ITPA 94CC genotype is associated with a higher risk of dyspepsia in Caucasian RA patients treated with peroral MTX. Acknowledgements This work was supported by a research project MH CZ - DRO (UHHK, 00179906) to T.S. and supported by research project PRVOUK P37-08 (from Charles University). Disclosure of Interest None declared

To determine a possible influence of acyclic nucleoside phosphonates on FMO3 and CYP activity at molecular level in vitro.Inhibition of individual CYP and FMO3 activities in the reconstituted system and in artificial membranes (bactosomes) was studied.Activity of FMO3 was inhibited by PMPA and bisPOC-PMPA even at low levels of these drugs (below 100 microM). In reconstituted system with CYP2C9, no inhibition of CYP2C9 activity was observed. On the other hand, experiments based on membrane coexpressed system showed a modest extent of inhibition (for PMEA, PMPA, bisPOM-PMEA and bisPOC-PMPA the level of inhibition was 77.8%; 74.1%; 64.2% and 68.6%, respectively at 400 microM).PMPA and bisPOC-PMPA are able to inhibit FMO3 activity at relatively low levels (10-100 microM) indicating a relatively specific interaction. Activity of CYP2C9 was affected when using the membranes coexpressed with NADPHcytochrome P450 reductase. This is probably due to more natural conditions for maintaining the CYP activity in bacterial membranes. As the inhibitor concentration in the systemic circulation does not exceed 2 microM, the probability of a significant in vivo effect of adefovir, tenofovir and the respective prodrugs on the microsomal system of cytochromes P450 and FMO3 is relatively low.

Background MicroRNAs (miRNAs) are a class of short, non-coding, single stranded RNAs regulating a broad spectrum of processes. Circulating miRNAs are an important emerging biomarker in cancer as well as a possible non-invasive diagnostic solution for a wide range of clinical disorders due to their high stability and association with disease state, although their source still remains uncertain. In multiple myeloma (MM), a plasma cell malignancy, circulating miRNAs have been reported to have a diagnostic and prognostic potential. It is therefore plausible to assume that they are involved in pathogenesis of this disease and thus could be used as diagnostic tool not only for MM, its extramedullary (EM) form but also for monitoring the clinical course of the disease. Therefore, in this study, we aimed to identify such miRNAs. Methods Screening analysis of 667 miRNAs was performed on 5 EM serum samples, 5 newly diagnosed MM samples and 6 healthy donors (HD) serum samples using TaqMan Low Density Arrays (TLDA) from Life Technologies. QPCR was performed for miR-130a on 118 serum samples obtained in Brno from newly diagnosed MM patients (pts) (35 pts), primary and secondary EM (35 pts), relapsed MM (18 pts) and HD (30). Further, 45 serum samples (12 diagnostic and 33 follow-up) of pts reaching VGPR/better response, enrolled in Italian CRD/MEL-200 and EMN-02 studies were used for circulating miRNA estimation. Receiver Operating Characteristic (ROC) analysis was used to calculate specificity and sensitivity of the miRNA as a biomarker. Biochemical characteristics were also available for EM and MM pts from Brno. P values Results TLDA profiling revealed 14 deregulated miRNAs (all p Further, in the analysis of miR-130a dynamics in follow-up samples from Italy, we observed increase of miR-130a levels in 8/12 MM pts during the follow-up sampling (p Conclusions In this study, miR-130a was decreased in serum samples of pts developing EM disease and distinguished EM pts from newly diagnosed MM pts and relapsed/progressed MM pts with specificity over 90%. Further, we observed increased level of miR-130a in the follow-up samples of MM pts. It suggests that miR-130a could be associated with EM disease; however, underlying biology and origin of miR-130a still remains to be explored. Work was supported by grants IGA NT 12130, NT 14575 and NT 13190. Disclosures Palumbo: Amgen: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Array BioPharma: Honoraria; Genmab A/S: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Onyx Pharmaceuticals: Consultancy, Honoraria; Sanofi Aventis: Honoraria.

Background Identifying genetic predictors of methotrexate (MTX) therapy response in patients with rheumatoid arthritis (RA) may have importance for clinical benefit optimalization. MTX therapeutic effect is achieved by inhibiting enzymes of the folate and adenosine pathways . MTX response is mainly influenced by 5,10-methylenetetrahydrofolate reductase (MTHFR) enzyme activity. Objectives The aim of the study was to determine whether single nucleotide polymorphisms (SNPs) in the MTHFR gene are predictive of MTX response according to the change of DAS28 after a 6-month MTX treatment in RA patient cohort of the East Bohemian population. The two SNPs 677C>T (rs1801133) and 1298A>C (rs1801131) of the MTHFR gene have been genotyped. Methods Monocentric, regional, retrospective and prospective, cross-sectional study. The 118 patients (mean age 57 years, SD ±12.7, age of 27- 83 years, and 31 male – 26%) were enrolled in study, all of whom fulfilled the American College of Rheumatology (ACR) 1987 criteria, and currently or previously taking MTX oral treatment, either as monotherapy (n=16) or in combination with DMRDs or corticoids (n=102). The concomitant treatment were sulfasalazin (n=18), leflunomide (n=10), hydrochloroquin (n=18), cyclosporine (n=8), biologics (n=7) and glucocorticoids (n=86). Treatment outcome was evaluated using DAS28 score and based on EULAR criteria at the beginning of MTX treatment, prospectively at entry into study or retrospectively from patients9 file (in case of patients with history of MTX treatment) and after a 6-month therapy. Genotyping assays: Leukocyte genomic DNA will be extracted from whole blood using a QIAamp DNA Blood Mini Kit (Quiagen). Genotyping was performed by qPCR allelic discrimination using commercial TaqMan (allele-specific) assays (LifeTechnologies) or sequencing. Statistical analysis was carried out in PASW® 18 software. Statistical dependences were tested by GLM (Generalized Linear Models), with bootstrap procedure. Results Results of the study thus show that higher dose of MTX lead to better response of DAS28, i.e. 1.19 DAS28/10mg MTX (p=0.02). In case of polymorphism 677C>T, mean response on MTX treatment (expressed by decrease of DAS 28 after a 6-months treatment) in CC homozygotes was found 1.59 DAS/10mg MTX (median of MTX dose); CI 95% = (0.12, 3.06); p=0.034, in CT heterozygotes 0.70 DAS28/10mg; CI 95% = (-0.82, 2.22); p=0.36, and in homozygotes TT 1.83 DAS 28/10mg; CI 95% = (-1.70, 5.37); p=0.31. Regarding 1298A>C polymorphism, in AA homozygotes DAS 28 changed by 1.92 DAS28/10mg; CI 95% =(0.43, 3.41); p=0.012, in AC heterozygotes 0.43 DAS28/10mg; CI 95% = (-1.08, 1.94); p=0.57, and in CC homozygotes 1.33 DAS 28/10mg; CI 95% = (-1.59, 4.24); p=0.37. Conclusions Our data shows that significant response to MTX treatment was found only in case of 677CC and 1298AA homozygous patients. Significant decrease of DAS 28 was associated with MTX dose in 677CC and 1298AA homozygotes. These results indicate the association of MTHFR polymorphisms with MTX response. Acknowledgements Supported by MH CZ - DRO (UHHK, 00179906) Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.4365

Glioblastoma multiforme (GBM) is the most malignant primary brain tumor. The prognosis of GBM patients varies considerably and the histopathological examination is not sufficient for individual risk estimation. MicroRNAs (miRNAs) are small, non-coding RNAs that function as post-transcriptional regulators of gene expression and were repeatedly proved to play important roles in pathogenesis of GBM. In our study, we performed global miRNA expression profiling of 58 glioblastoma tissue samples obtained during surgical resections and 10 non-tumor brain tissues. The subsequent analysis revealed 28 significantly deregulated miRNAs in GBM tissue, which were able to precisely classify all examined samples. Correlation with clinical data led to identification of six-miRNA signature significantly associated with progression free survival [hazard ratio (HR) 1.98, 95% confidence interval (CI) 1.33-2.94, P < 0.001] and overa+ll survival (HR 2.86, 95% CI 1.91-4.29, P < 0.001). O(6)-methylguanine-DNA methyltransferase methylation status was evaluated as reference method and Risk Score based on six-miRNA signature indicated significant superiority in prediction of clinical outcome in GBM patients. Multivariate Cox analysis indicated that the Risk Score based on six-miRNA signature is an independent prognostic classifier of GBM patients. We suggest that the Risk Score presents promising prognostic algorithm with potential for individualized treatment decisions in clinical management of GBM patients.

We describe Caucasian monozygotic twin brothers with rheumatoid arthritis (RA) and discuss influence of predictors to methotrexate (MTX) outcome treatment. Single nucleotide polymorphisms (SNPs) of the MTX metabolic pathways were genotyped. Twins have multiple mutations: a CC mutation of SNP 1298A>C in methylenetetrahydrofolate reductase (MTHFR) gene, CC mutations of three SNPs in the adenosine receptor gene ADORA2A (rs3761422_4217241T>C, rs2267076_4221164T>C, rs2236624_4226593T>C), and a heterozygous genotype in SNPs ATIC_rs2372536_347C>G, MTHFD1_rs2236225_1958G>A. These mutations are known to predict a worse outcome of MTX treatment. The twins had different lifestyles (alcohol drinking and smoking in Twin 1, regular coffee consumption in Twin 2), but a very similar clinical presentation of the outset of RA, radiographic scoring according to the Sharp/van der Heijde method with an almost identical antibodies presentation. The period of the patients before anti-TNFα treatment was characterized by unsuccessful per oral MTX pharmacotherapy in both cases (a low effect of MTX in Twin 1; an early discontinuation of MTX due to an adverse event in Twin 2). In both twins, the outcome of well-controlled anti-TNFα treatment (co-medication with MTX in Twin 1) for 10 years was expressed as low disease activity measured using composite index DAS28. It is interesting that Twin 2 had an unfavorable radiographic scoring after a 10-year follow-up than Twin 1 in spite of the comparable DAS28 in Twin 2 and smoking in Twin 1. In conclusion, co-medication of MTX with biologics may impact on RA radiographic progression despite predicted bad MTX outcome based on pharmacogenetic analysis.