Determination of Reference Values of MDR-ABC Transporter Activities in CD3+ Lymphocytes of Healthy Volunteers Using a Flow Cytometry Based Method.

BACKGROUND: MDR transporters are important biomarkers of drug resistance in cancer and in autoimmune conditions. We determined the MDR1, MRP1 and BCRP activity in CD3+ lymphocytes using a flow cytometry based method from 120 healthy volunteers in order to describe normal reference values of the activity of these transporters. The effects of gender and age were also determined. METHODS: The Solvo MDQ Kit™ was used for measurements. In this assay, fluorescent reporter substrates (Calcein-AM for MDR1 and MRP1 and mitoxantrone for BCRP, respectively) are trapped in the cytoplasm and pumped out by MDR proteins depending on the presence or absence of specific inhibitors (verapamil for MDR1 and MRP1, indomethacin for MRP1 and KO134 for BCRP, respectively), allowing for quantitative, standardized assessment. Cell surface staining was applied to select CD3+ cells. RESULTS: MAF values of MRP1 and BCRP are independent from age. MAFC and MAF of MDR1 show negative correlation with the age of the studied subjects (P = 0.003, r = -0.27 and P = 0.0001, r = -0.34, respectively). No difference was detected in any of the four MAF values between men and women. Gender does not affect the presence or lack of correlation between MAF values and age. CONCLUSIONS: The determination of the functional activity of MDR-ABC transporters is achievable using a flow cytometry based standardized method. Having established the normal range of MAF values on CD3+ lymphocytes of a healthy population, our results allow for the development of novel flow cytometry based diagnostic tools. © 2018 International Clinical Cytometry Society.

Differential sensitivity of the species of Candida parapsilosis sensu lato complex against statins.

Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis are human fungal pathogens with clinical importance. The recently reclassified three closely related species have significant variation in virulence, clinical prevalence and susceptibility characteristics to different antifungal compounds. The aim of this study was to investigate the in vitro activity of atorvastatin and fluvastatin against C. metapsilosis, C. orthopsilosis and C. parapsilosis. Susceptibility tests showed that C. parapsilosis was the most sensitive while C. orthopsilosis was the least susceptible species to both drugs. On the basis of the differential sensitivity, we developed a simple, reliable and highly cost-effective plate assay to distinguish these closely related species. Applying this method, 54 isolates belonging to the C. parapsilosis sensu lato complex deposited in Szeged Microbial Collection could be sorted into the three species with 100 % probability.

MDR-ABC transporters: biomarkers in rheumatoid arthritis.

MDR-ABC transporters are widely expressed in cell types relevant to pathogenesis of rheumatoid arthritis. Many reports demonstrate the interaction of small molecule drugs with MDR-ABC transporters. Cell-based assays for disease relevant cell types can be easily gated and could reveal specific drug targets and may increase significance and utilisation of data in clinical practice. Many commonly used DMARDs (e.g. methotrexate, sulfasalazine, leflunomide/teriflunomide, hydroxychloroquine) are ABCG2 substrates. Consequently, the activity of this transporter in patients should be determined to understand the disposition and pharmacokinetics of the therapy. In addition, MDR-ABC transporters transport a variety of endobiotics that play important roles in cell proliferation, cell migration, angiogenesis and inflammation. Therefore, MDR-ABC transporters are important biomarkers in rheumatoid arthritis.

ABCG2 modulates chlorothiazide permeability--in vitro-characterization of its interactions.

We are showing that chlorothiazide, a diuretic, is an ABCG2 substrate. It is a Biopharmaceutics Classification System/Biopharmaceutics Drug Distribution and Classification System (BCS/BDDCS) Class IV drug with low bioavailability. Therefore, we tested if chlorothiazide interacts with major apically located intestinal efflux transporters. Our data show that chlorothiazide is transported by ABCG2 with a Km value of 334.6 µM and does not interact with ABCB1 or ABCC2. The chlorothiazide-ABCG2 interaction results in a vectorial transport in MDCKII-BCRP and Caco-2 cells with efflux ratios of 36 and 8.1 respectively. Inhibition of ABCG2 in Caco-2 cells reduced the efflux ratio to 1.4, suggesting that ABCG2 plays a role in limiting chlorothiazide bioavailability in humans.

European external quality control study on the competence of laboratories to recognize rare sequence variants resulting in unusual genotyping results.

BACKGROUND: Depending on the method used, rare sequence variants adjacent to the single nucleotide polymorphism (SNP) of interest may cause unusual or erroneous genotyping results. Because such rare variants are known for many genes commonly tested in diagnostic laboratories, we organized a proficiency study to assess their influence on the accuracy of reported laboratory results. METHODS: Four external quality control materials were processed and sent to 283 laboratories through 3 EQA organizers for analysis of the prothrombin 20210G>A mutation. Two of these quality control materials contained sequence variants introduced by site-directed mutagenesis. RESULTS: One hundred eighty-nine laboratories participated in the study. When samples gave a usual result with the method applied, the error rate was 5.1%. Detailed analysis showed that more than 70% of the failures were reported from only 9 laboratories. Allele-specific amplification-based PCR had a much higher error rate than other methods (18.3% vs 2.9%). The variants 20209C>T and [20175T>G; 20179_20180delAC] resulted in unusual genotyping results in 67 and 85 laboratories, respectively. Eighty-three (54.6%) of these unusual results were not recognized, 32 (21.1%) were attributed to technical issues, and only 37 (24.3%) were recognized as another sequence variant. CONCLUSIONS: Our findings revealed that some of the participating laboratories were not able to recognize and correctly interpret unusual genotyping results caused by rare SNPs. Our study indicates that the majority of the failures could be avoided by improved training and careful selection and validation of the methods applied.

Certification of reference materials for detection of the human prothrombin gene G20210A sequence variant.

BACKGROUND: There is a need for reference materials (RMs) in the field of genetic testing for verification of test results obtained in patients and probands. For the frequent genetic variation G20210A in the prothrombin gene, it has been shown that purified plasmids containing the gene fragment harbouring the mutation constitute good candidate RMs. METHODS: Plasmid-type RMs were characterised for homogeneity, stability, sequence identity and fitness for purpose. Their certification required the use of different real-time PCR methods for genotyping and quantification of the plasmid copy number. RESULTS: Homogeneity, stability and fitness for the purpose of the plasmids could be demonstrated. The long-term stability (up to 24 months) of the materials was confirmed by highly sensitive and specific quantitative real-time PCR methods. CONCLUSIONS: New types of certified RMs (CRMs) for genetic testing of the human prothrombin gene G20210A sequence variant are available. Their fitness for purpose was demonstrated and no evidence was found that they would not work with other methods as long as these are targeting the whole or parts of the prothrombin gene fragment inserted into the plasmids. The described CRMs support the efforts of the international community in development, validation and harmonisation of tests for molecular genetic testing.

Cytochrome P450 2C9 polymorphism and acenocoumarol therapy.

BACKGROUND: Oral anticoagulants, in Hungary acenocoumarol being the one exclusively used, have a low therapeutic index and a high bleeding complication rate. The cytochrome P450 2C9 enzyme plays an important role in their metabolism. AIM: To investigate the influence of CYP2C9 polymorphism on the occurrence of bleeding complications related to acenocoumarol therapy. METHODS: Genotyping of 421 patients (183 men and 238 women, mean age 66.2+/-11.8 years), who had been taking acenocoumarol for at least 6 months, was performed. Based on patient history and laboratory data, the correlations between genotype and acenocoumarol dose and bleeding complications were retrospectively analysed. RESULTS: In 145 patients bearing alleles with reduced activity (CYP2C9*2 and/or *3), the optimal dose of acenocoumarol was significantly (p<0.001) lower than in patients with the wild type allele (2.12+/-0.96 mg/day and 2.90+/-1.46 mg/day, respectively). In comparison with wild type allele patients, the mean daily acenocoumarol dose was lower in the CYP2C9*2 group, and the lowest in *3 bearers. Although the occurrence of minor bleeding complications in patients with the variant allele was significantly (p <0.005) higher (OR=1.99 [CI: 1.20-3.33]) than in other patients, there was no difference in major bleeding complications. CONCLUSIONS: Patients bearing CYP2C9 alleles with reduced enzymatic activity have a lower acenocoumarol requirement. In patients with CYP2C9*2 and *3 alleles the frequency of minor bleeding complications and the occurrence of high INR values were significantly higher, but there was no difference in the rate of major bleedings.

Reference materials (RMs) for analysis of the human factor II (prothrombin) gene G20210A mutation.

The Scientific Committee of Molecular Biology Techniques (C-MBT) in Clinical Chemistry of the IFCC has initiated a joint project in co-operation with the European Commission, Joint Research Centre, Institute of Reference Materials and Measurements to develop and produce plasmid-type reference materials (RMs) for the analysis of the human prothrombin gene G20210A mutation. Although DNA tests have a high impact on clinical decision-making and the number of tests performed in diagnostic laboratories is high, issues of quality and quality assurance exist, and currently only a few RMs for clinical genetic testing are available. A gene fragment chosen was produced that spans all primer annealing sites published to date. Both the wild-type and mutant alleles of this gene fragment were cloned into a pUC18 plasmid and two plasmid RMs were produced. In addition, a mixture of both plasmids was produced to mimic the heterozygous genotype. The present study describes the performance of these reference materials in a commutability study, in which they were tested by nine different methods in 13 expert laboratories. This series of plasmid RMs are, to the best of our knowledge, the first plasmid-type clinical genetic RMs introduced worldwide.

[Significance of cytochrome P450 2C9 genotype for the bleeding complications in patients treated with acenocoumarol]. / A citokróm P450 2C9 genotípus jelentosége acenocoumarollal kezelt betegek vérzéses szövodményeire.

INTRODUCTION: For the primary and secondary prevention of thromboembolic events are used the oral anticoagulants, the drugs having a low therapeutic index and frequent bleeding complication rate. Establishing the proper therapeutic dose of these drugs for different patients is complicated by a variety of conditions, such as the comorbidity, age, other drugs used, diet, and pharmacogenetic factors. One of the latters is the polymorphism of the cytochrome P450 CYP2C9 enzyme. AIM: The influence of CYP2C9 polymorphism on the effectiveness of the--in Hungary for oral anticoagulation exclusively used--acenocoumarol therapy and on the occurrence of bleeding complications was investigated. METHODS: Genotyping of 421 patients including 183 men and 238 women, (mean age 66.2 +/- 11.8 years) who took acenocoumarol (Syncumar) for at least 6 months was performed. Based on anamnestic and laboratory data, the correlation between the genotype and the acenocoumarol dose and bleeding complications were retrospectively analysed. RESULTS: The frequency-distribution for the CYP2C9*1, *2, and *3 alleles were found to be: 0.814, 0.110, and 0.076, respectively. In the 145 patients bearing the alleles with reduced activity (CYP2C9*2 and/or *3), the optimised dose of the acenocoumarol was significantly (p < 0.001) lower than in patients with the wild type allele (2.12 +/- 0.96 mg/day and 2.90 +/- 1.45 mg/day, respectively). Although the occurrence of minor bleeding complications in the former group was significantly (p < 0.005) higher [OR = 1.99 (CI: 1.20-3.33)], there was no difference in major bleeding complications. In patients taking an acenocoumarol dose lower than 2 mg/day, the occurrence of an INR value higher than 6 in the anamnesis was significantly (p < 0.05) more frequent. Evaluating separately the variant alleles we have concluded, that in the presence of allele *2 a lower acenocoumarol dose was required than in wild-type subjects, and even lower in the presence of allele *3. CONCLUSIONS: The frequency-distribution of the CYP2C9 alleles was as reported by others. In patients bearing alleles with reduced enzymatic activity, the occurrence of minor bleeding complications and the INR values higher than 6 were significantly more frequent. In patients with a lower acenocoumarol demand at the introduction of this therapy, a caution is required. In order to test the hypothesis that before the initiation of acenocoumarol therapy the determination of CYP2C9 polymorphism is cost-effective and could improve the optimization of anticoagulation and reduce the risk of bleeding complications a large prospective randomised trial is required.

The nitric oxide synthase-3 codon 298 polymorphism is not associated with late-onset sporadic Alzheimer's dementia and Lewy body disease in a sample from Hungary.

An association study was performed between apolipoprotein E (apoE) polymorphism and the common structural polymorphism Glu/Asp at codon 298 of the nitric oxide synthase (NOS3) gene in late-onset sporadic Alzheimer's dementia probands (LOAD), diffuse Lewy body dementia cases (DLBD) and controls in a Hungarian sample. The frequency of individuals who carried the apoE epsilon4 allele was significantly more common in both dementia groups (LOAD, 20%; DLBD, 27%; control, 8%; control versus DLBD, chi2=13.264, degrees of freedom=2, P<0.001; control versus LOAD, chi2=6.628, degrees of freedom=2, P<0.036). However, there were no significant differences in the NOS3 genotype and allele distributions between the LOAD, DLBD and control groups. The apoE status has been found to be independent from the NOS3 codon 298 polymorphism in the examined cohort. Despite the facts that NOS3 is associated with neuritic sprouting, and aberrant neuronal and glial expression of the same molecule has been found in neurodegenerative diseases, it is unlikely that the polymorphism Glu/Asp of the NOS3 gene is involved in the development of LOAD and DLBD.

[Pleiotropic effect of the micronized fenofibrate: the reduction of plasma chlamydia pneumoniae antibody levels in patients with coronary artery disease]. / A mikronizált fenofibrát pleiotrop hatása: chlamydia pneumoniae ellenanyagszintek csökkenése ischaemiás szívbetegségben szenvedókben.

INTRODUCTION: A growing amount of data suggest that atherosclerosis is an inflammatory disease and in it's development Chlamydia pneumoniae infection may contribute. Recent studies have shown that administration of micronized fenofibrate reduces the plasma levels of several markers of the inflammatory response. AIM: The aim of the authors was to evaluate the effect of micronized fenofibrate on the lipids and Chlamydia pneumoniae antibody levels of 20 patients with coronary artery disease. METHODS: The plasma total cholesterol, HDL-cholesterol, triglyceride, lipoprotein (a), ApoA1, ApoB, fibrinogen and Chlamydia Ig A, IgG and IgM antibody concentrations were examined. The patients were on strict lipid lowering diet and were treated by daily 200 mg micronized fenofibrate for 3 weeks. RESULTS: A significant reduction of the total cholesterol (18.3%), LDL cholesterol (17.7%), triglyceride (37.8%), ApoB (18.4%), fibrinogen (16.1%) levels was observed. The concentration of HDL cholesterol (17.0%) and ApoA1 (12.2%) showed a significant elevation. The Chlamydia pneumoniae IgM antibody (characterizing the acute infections) was not detectable. The IgA antibody level decreased from 13.0 EIU to 12.3 EIU (p < 0.05) and IgG from 85.1 EIU to 78.7 EIU (p < 0.05). CONCLUSIONS: Beside the expected lipid lowering effect of the micronized fenofibrate a significant reduction in the plasma Chlamydia pneumoniae IgA and IgG antibody levels was observed supporting the anti-inflammatory, pleiotropic effect of this drug.

Lack of influence of the apolipoprotein E genotype on the outcome of selegiline treatment in Alzheimer's disease.

The objective of our study was to investigate whether an interaction exists between apolipoprotein E (APOE) genotype and the response of patients with Alzheimer's disease (AD) to selegiline treatment, and whether APOE genotype independently affects the rate of AD progression. A 48-week multicenter double-blind trial was undertaken on 43 patients with mild to moderate AD. Primary efficacy measures were the AD Assessment Scale (ADAS), an 11-item cognitive subscale of ADAS (ADAS-Cog/11) and the Mini Mental State Examination. Secondary outcome measures were Clinical Global Impression of severity and CGI of change scales. The therapeutic response to selegiline was not affected by APOE genotype. Our results revealed that the APOE4 allele carrier AD probands did not respond better to selegiline treatment than the APOE2-3 patients, i.e. APOE status did not influence the therapeutic outcome of selegiline treatment.

Alpha2-macroglobulin exon 24 (Val-1000-Ile) polymorphism is not associated with late-onset sporadic Alzheimer's dementia in the Hungarian population.

Several lines of biochemical evidence support a role of alpha2-macroglobulin (A2M) in the pathogenesis of Alzheimer's dementia (AD). A2M participates in the general defence mechanism against proteinases and it is supposed to be involved in the degradation of beta-amyloid peptide (betaAP). Furthermore, A2M has been shown to reduce betaAP fibril formation, and it is upregulated in the acute-phase inflammatory response like the process occurring in the AD brain. The exon 18 splice acceptor deletion polymorphism and the exon 24 (Val-1000-Ile) GG genotype were reported to be associated with AD, but the results are contradictory. Since the Hungarian population is genetically distinct from the other European ethnic groups, we examined whether the risk for developing AD is increased in the A2M GG carriers. The interaction of apolipoprotein E (apoE) and A2M polymorphisms was also examined. The distribution of A2M genotypes and alleles in the entire data set was consistent with the previous negative observations in which A and G allelic frequencies were comparable in both groups (72% and 28% in the AD population, and 72% and 28% in the control population, respectively). The GG genotype was over-represented (14%) only in the apoE epsilon4 non-carrier subgroup of AD probands (7% in the control group), but the difference was not significant. Our data suggest that, although A2M has an important role in the AD-specific neurodegenerative process, its exon 24 Val-1000-Ile polymorphism is not likely to be associated with late-onset sporadic AD in the Hungarian population.