Marek Drozdzik

Synthesis, dispersion, and cytocompatibility of graphene oxide and reduced graphene oxide.

The synthesis, characterization, and toxicity of graphene oxide and reduced graphene oxide are reported. Prior to the cytocompatibility tests the stability of the suspensions in a wide range of concentrations (3.125-100 μg/mL) of three different dispersants is studied. Polyethylene glycol (PEG), polyethylene glycol-polypropylene glycol-polyethylene glycol (Pluronic P123), and sodium deoxycholate (DOC) are investigated as the dispersants. The toxicity depends on the type of dispersant and concentration of the nanomaterials in the suspensions. Detailed analysis suggests that graphene oxide functionalized with PEG in the concentration range between 3125 μg/mL and 25 μg/mL exhibits the best biocompatibility with mice fibroblast cells (line L929).

Protein abundance of clinically relevant multidrug transporters along the entire length of the human intestine

Intestinal transporters are crucial determinants in the oral absorption of many drugs. We therefore studied the mRNA expression (N = 33) and absolute protein content (N = 10) of clinically relevant transporters in healthy epithelium of the duodenum, the proximal and distal jejunum and ileum, and the ascending, transversal, descending, and sigmoidal colon of six organ donors (24-54 years). In the small intestine, the abundance of nearly all studied proteins ranged between 0.2 and 1.6 pmol/mg with the exception of those of OCT3 (<0.1 pmol/mg) and PEPT1 (2.6-4.9 pmol/mg) that accounted for ∼50% of all measured transporters. OATP1A2 was not detected in any intestinal segment. ABCB1, ABCG2, PEPT1, and ASBT were significantly more abundant in jejunum and ileum than in colon. In contrast to this, the level of expression of ABCC2, ABCC3, and OCT3 was found to be highest in colon. Site-dependent differences in the levels of gene and protein expression were observed for ABCB1 and ASBT. Significant correlations between mRNA and protein levels have been found for ABCG2, ASBT, OCT3, and PEPT1 in the small intestine. Our data provide further physiological pieces of the puzzle required to predict intestinal drug absorption in humans.

Reduced folate carrier-1 80G>A polymorphism affects methotrexate treatment outcome in rheumatoid arthritis.

The folate antagonist methotrexate (MTX) is a drug currently used in the treatment of rheumatoid arthritis (RA). MTX enters the cells through the reduced folate carrier (RFC-1) and is activated to polyglutamates. Previous studies have shown that RFC-1 expression may influence the efficacy of therapy with MTX. The studies suggest that G80A polymorphism in RFC-1 is associated with altered folate/antifolate levels and the subjects carrying homozygous mutant 80AA genotype tend to have higher plasma folate and MTX concentrations and higher erythrocyte polyglutamate levels compared with those with the wild type or heterozygous genotype. It is possible that this polymorphism might influence MTX treatment outcome in patients with RA. In the present study, we examined the association between RFC-1 G80A polymorphism and treatment outcome in patients with RA administered MTX. The study was carried out on 174 patients diagnosed with RA treated with MTX (7.5–15.0 mg weekly) plus low doses of methylprednisone. The RFC-1 80G>A polymorphism (resulting in a histidine-to-arginine substitution at codon 27 of RFC-1) was detected using a polymerase chain reaction-restriction fragment length polymorphism method. The probability of remission of RA symptoms was 3.32-fold higher in carriers of 80AA genotype as compared with patients with 80GG genotype (P=0.021, OR=3.32, 95% CI: 1.26–8.79). The frequency of A allele among MTX responders was 62.1, compared to 47.8% in a group of poor MTX responders (P=0.013, OR=1.78, 95% CI: 1.13–2.81). Moreover, the increase of aminotransferase activity was noted more frequently in carriers of 80AA genotype. The present data suggest that evaluation of RFC-1 gene 80G>A polymorphism may be a useful tool to optimize MTX therapy in patients with RA.

Association of Nrf2-encoding NFE2L2 haplotypes with Parkinson's disease

BackgroundOxidative stress is heavily implicated in the pathogenic process of Parkinsons disease. Varying capacity to detoxify radical oxygen species through induction of phase II antioxidant enzymes in substantia nigra may influence disease risk. Here, we hypothesize that variation in NFE2L2 and KEAP1, the genes encoding the two major regulators of the phase II response, may affect the risk of Parkinsons disease.MethodsThe study included a Swedish discovery case-control material (165 cases and 190 controls) and a Polish replication case-control material (192 cases and 192 controls). Eight tag single nucleotide polymorphisms representing the variation in NFE2L2 and three representing the variation in KEAP1 were chosen using HapMap data and were genotyped using TaqMan Allelic Discrimination.ResultsWe identified a protective NFE2L2 haplotype in both of our European case-control materials. Each haplotype allele was associated with five years later age at onset of the disease (p = 0.001) in the Swedish material, and decreased risk of PD (p = 2 × 10-6), with an odds ratio of 0.4 (95% CI 0.3-0.6) for heterozygous and 0.2 (95% CI 0.1-0.4) for homozygous carriers, in the Polish material. The identified haplotype includes a functional promoter haplotype previously associated with high transcriptional activity. Genetic variation in KEAP1 did not show any associations.ConclusionThese data suggest that variation in NFE2L2 modifies the Parkinsons disease process and provide another link between oxidative stress and neurodegeneration.

677C>T and 1298A>C MTHFR polymorphisms affect methotrexate treatment outcome in rheumatoid arthritis

INTRODUCTION Methotrexate (MTX), widely used in the treatment of rheumatoid arthritis (RA), inhibits dihydrofolate reductase and folate-dependent enzymes. Methylenetetrahydrofolate reductase (MTHFR) is involved in folate metabolism and has been shown to be polymorphic, affecting the enzyme activity. METHODS To examine the association between 677C>T and 1298A>C MTHFR polymorphisms and MTX efficacy in the treatment of RA, a total of 174 RA patients, treated with MTX plus methylprednisone 4 mg and folic acid 5 mg were analyzed. RESULTS In univariate regression analysis model, the MTHFR 677T allele was associated with significantly higher frequency of remission, whereas in the case of the 1298C allele, a tendency for higher remission rate was observed. In multivariate regression analysis, the presence of both 677T and 1298C alleles was associated with an increased frequency of remission. CONCLUSION The results of our study suggest that the MTHFR 677T and 1298C alleles may be associated with an increased rate of RA remission in patients treated with MTX receiving high doses of folic acid supplementation.

The association of functional catechol-O-methyltransferase haplotypes with risk of Parkinson's disease, levodopa treatment response, and complications.

Introduction Differences in catechol-O-methyltransferase (COMT) activity and genotype may determine individual variations in the therapeutic response to levodopa or Parkinsons disease (PD) susceptibility. The role of functional COMT haplotypes in PD susceptibility and treatment response has not been examined. Objectives In this case–control study, we investigated the association of the most common COMT gene haplotypes (formed by single nucleotide polymorphisms (SNPs): rs6269:A>G; rs4633C>T; rs4818:C>G; and rs4680:A>G) with PD risk and the association of the COMT haplotypes with the dose and complications of levodopa therapy in PD patients. Methods A total of 679 study participants (322 PD and 357 controls) were included. Each participant was genotyped for four SNPs in the COMT gene, located in a common haploblock, that has been shown to influence COMT enzymatic activity. The influence of COMT haplotypes on the dose of levodopa administered during fifth year of treatment, and occurrence of motor complications were examined in PD patients. The EH program (Jurg Ott, Rockefeller University, New York, USA) was used to estimate haplotype frequencies. Results The estimated frequencies of low (A_C_C_G) and medium (A_T_C_A) activity haplotypes tended to be slightly lower among PD patients when compared with controls (P=0.09, G_C_G_G-high activity haplotype as reference). The frequency of G_C_G_G (high activity) haplotype carriers was higher in late onset PD patients (P=0.04) compared with controls. The mean levodopa dose increased with the activity of the functional haplotypes (low<medium<high). Doses prescribed for G_C_G_G (high activity) haplotype carriers (mean 604.2±261.9 mg) were significantly higher than those for the noncarriers (mean 512.2±133.5 mg, P<0.05). The COMT haplotype seemed to have little influence on the development of levodopa-induced dyskinesias. Conclusion Our study showed a possible association of functional COMT haplotypes with the risk of PD. Both nonsynonymous and synonymous SNPs within functional COMT haplotype blocks may be more relevant than individual SNPs in conferring PD susceptibility. The doses of levodopa treatment can be influenced by specific COMT haplotypes and this may be useful in instituting individualized therapy for PD patients.

Frequency distribution of thiopurine S-methyltransferase alleles in a Polish population

Thiopurine S-methyltransferase (TPMT) is an enzyme that catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathioprine. TPMT activity exhibits an interindividual variability mainly a result of genetic polymorphism. Patients with intermediate or deficient TPMT activity are at risk for toxicity after receiving standard doses of thiopurine drugs. It has previously been reported that 3 variant alleles:TPMT*2, *3A, and *3C are responsible for over 95% cases of lower enzyme activity. The purpose of this study was to determine the frequency of TPMT variant alleles in a Polish population. DNA samples were obtained from 358 unrelated healthy Polish subjects of white origin, and TPMT genetic polymorphism was determined using PCR-RFLP and allele-specific PCR methods. The results showed that allelic frequencies were 0.4% for TPMT*2, 2.7% for TPMT*3A, and 0.1% for TPMT*3C, respectively. A TPMT*3B allele was not found in the studied population. The general pattern of TPMT allele disposition in the Polish population is similar to those determined for other white populations, but the frequency of total variant alleles is lower than in other European populations studied to date.

TPMT but not ITPA gene polymorphism influences the risk of azathioprine intolerance in renal transplant recipients

PurposeThiopurine drugs have to be withdrawn in 10–30% of cases due to side effects, and it has been presented that genetic factors may be responsible for some of reported toxicity cases. Among polymorphic enzymes of thiopurines’ metabolic pathway, thiopurine S-methyltransferase (TPMT) has been studied most extensively, and some recent studies point to inosine triphosphate pyrophosphohydrolase (ITPA) polymorphism as an additional toxicity risk factor.MethodsThe aim of the current study was to evaluate an association between TPMT and ITPA gene polymorphisms and drug intolerance in a cohort of 157 renal transplant recipients treated with azathioprine (AZA). Each subject was genotyped for the presence of variant TPMT (*2, *3A, *3B, and *3C) and ITPA (94C>A and IVS2+21A>C) alleles.ResultsMean AZA dose, mean white-blood-cell count, and platelet count in the course of treatment were lower in carriers of variant TPMT alleles compared to patients with TPMT wild-type genotype. Leukocyte numbers fell below 4.0 × 109/L in 41.2% of TPMT heterozygous renal transplant recipients, compared to only 18.0% of wild-type patients (P < 0.01). In contrast, ITPA genotype did not influence AZA dose, hematological parameters, or leucopenia risk.ConclusionsOur results suggest that routine genotyping of renal transplant recipients for TPMT variants may be useful in reducing the risk of AZA-related myelotoxicity, but there is not enough evidence to introduce ITPA testing into clinical practice.

CARD15 variants in patients with sporadic Parkinson's disease

Recent reports have proven the importance of genetic factors and inflammation in the pathogenesis of sporadic Parkinsons disease (PD). In the current study, the frequency of CARD15/NOD2 gene variants (R702W, G908R, L1007fs), previously associated with Crohns disease--a common inflammatory bowel disease, have been examined in a group of 308 sporadic PD patients and 220 healthy controls. Significantly higher frequency of total CARD15 variant alleles in PD patients (13.0%) compared to the controls (8.0%, p<0.02) was observed. 24.0% of PD patients carried at least one CARD15 variant allele compared to 15.5% of healthy controls (p<0.02, OR=1.73). The results of the study suggest, that the polymorphism in CARD15/NOD2 gene may be a risk factor for sporadic PD development, and support the concept of inflammatory pathogenesis of PD.

Analysis of LRRK2 G2019S and I2020T mutations in Parkinson's disease

Mutations in the leucine-rich repeat kinase 2 (LRRK2), encoding dardarin protein, have been demonstrated to be linked to autosomal dominant Parkinsons disease (PD). In the present study the entire exon 41 of LRRK2 gene was evaluated in a series of 174 PD patients recruited from Polish population, aged at the time of diagnosis 54.0 ± 39.1 years, 21 of them had positive family history of PD with mean onset of the disease of 51.9 ± 11.7 years as well as in 190 healthy controls aged 73.7 ± 6.0 years. The mutations were evaluated by direct sequencing for mutations in exon 41 of LRRK2 gene. In the studied patients no known mutations in exon 41 of LRRK2 gene, including G2019S and I2020T were found, both in PD patients as well as in the controls. It can be concluded that the G2019S and I2020T mutations in exon 41 of LRRK2 gene are rare causes of Parkinson disease in a Polish population.