Joanna Lapczuk

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.

Association of transcription factor 7-like 2 (TCF7L2) gene polymorphism with posttransplant diabetes mellitus in kidney transplant patients medicated with tacrolimus

New onset posttransplant diabetes mellitus (PTDM) has a high incidence after kidney transplantation in patients medicated with tacrolimus. PTDM can adversely affect patient and graft survival. The pathophysiology of PTDM closely mimics type 2 diabetes mellitus (T2DM). One of the possible genetic factors predisposing individuals to PTDM might be a polymorphism in the transcription factor 7-like 2 gene (TCF7L2). This polymorphism has previously been associated with increased risk of T2DM in the general population. Therefore, the present study aimed to evaluate TCF7L2 polymorphisms in PTDM in kidney transplant patients medicated with tacrolimus. Non-diabetic kidney transplant patients medicated with tacrolimus (n = 234) were genotyped for the presence of TCF7L2 gene variants (rs12255372 and rs7903146) using TaqMan probes. Of the 234 patients, 66 patients had developed PTDM and 168 had not. Frequencies of the studied single nucleotide polymorphisms (SNPs) did not differ significantly between the study groups. Moreover, haplotype analyses failed to detect any associations between TCF7L2 haplotypes and PTDM. However, in late-onset PTDM (developed later that 2 weeks from transplantation), frequencies of the rs7903146 TT genotype and T minor allele were significantly increased compared to non-PTDM controls (17.9% vs. 5.9%, p = 0.017, OR: 4.13, 95% CI: 1.19-14.33 for TT genotype, 39.3% vs. 25.9%, p = 0.038 for T allele). If the application of TCF7L2 rs7903146 SNPs as a marker for PTDM is confirmed by further independent studies, replacing tacrolimus with other immunosuppressants could be warranted in patients at high risk of PTDM, as diagnosed by TCF7L2 genotyping.

Mesoporous Silica Nanospheres Functionalized by Tio2 as a Photoactive Antibacterial Agent

In this contribution we present comparative study on synthesis, bio-characterization and antibacterial properties of mesoporous silica nanospheres modified by titanium dioxide. Mesoporous silica nanospheres functionalized by titania were studied as light activated antibacterial agents. The analysis of the antibacterial effects on E. coli ATCC 25922 shows strong enhancement during the visible and ultraviolet light irradiation in respect to the commercial catalyst and sample free from the nanomaterials. In darkness the mesoporous silica/titania nanostructures revealed low antibacterial activity dependent on the stirring intensity of the suspension containing nanomaterials and bacteria. The nanomaterials toxicity was determined on the amount of lactate dehydrogenase released from mouse fibroblast cells L929 with LDH assay. Sample was characterized in details by means of high resolution transmission electron microscopy (HR-TEM), Raman spectroscopy, XRD and BET Isotherm.

Antibacterial performance of nanocrystallined titania confined in mesoporous silica nanotubes

In this paper, we study synthesis and characteristics of mesoporous silica nanotubes modified by titanium dioxide, as well as their antimicrobial properties and influence on mitochondrial activity of mouse fibroblast L929. Nanocrystalized titania is confined in mesopores of silica nanotubes and its light activated antibacterial response is revealed. The analysis of the antibacterial effect on Escherichia coli. (ATCC 25922) shows strong enhancement during irradiation with the artificial visible and ultraviolet light in respect to the commercial catalyst and control sample free from the nanomaterials. In darkness, the mesoporous silica/titania nanostructures exhibited antibacterial activity dependent on the stirring speed of the suspension containing nanomaterials. Obtained micrograph proved internalization of the sample into the microorganism trough the cell membrane. The analysis of the mitochondrial activity and amount of lactate dehydrogenase released from mouse fibroblast cells L929 in the presence of the sample were determined with LDH and WST1 assays, respectively. The synthesized silica/titania antibacterial agent also exhibits pronounced photoinduced inactivation of the bacterial growth under the artificial visible and UV light irritation in respect to the commercial catalyst. Additionally, mesoporous silica/titania nanotubes were characterized in details by means of high resolution transmission electron microscopy (HR-TEM), XRD and BET Isotherm.

Protein Abundance of Clinically Relevant Drug‐Metabolizing Enzymes in the Human Liver and Intestine: A Comparative Analysis in Paired Tissue Specimens

This work revises and complements existing findings on the distribution of drug‐metabolizing enzymes in the first‐pass effect organs. We explored gene expression (quantitative polymerase chain reaction) and protein abundance (liquid chromatography/ tandem mass spectrometry) of CYP1A2, CYP2B6, CYP2C8/9/19, CYP2D6, CYP2E1, CYP3A4/5, UGT1A1/3, UGT2B7/15 in the liver, duodenum, jejunum, ileum, and colon in paired tissues from nine organ donors. All proteins were detected in the liver, but in the intestine CYP2C9/19, CYP2D6, CYP3A4/5, UGT1A1/3, and UGT2B7 were found. CYP3A4 showed comparable abundance in the liver and jejunum, whereas other enzymes were markedly higher in the hepatic tissue. Nearly all detected enzymes showed their highest abundance in the jejunum. Significant correlations between mRNA and protein levels in liver or intestine were found for most enzymes. CYP3A4 and CYP3A5 protein abundance, but not other enzymes, were significantly correlated in the liver and the small intestine. Our data may contribute to an improved understanding of hepatic and intestinal drug metabolism.

Expression of clinically relevant drug-metabolizing enzymes along the human intestine and their correlation to drug transporters and nuclear receptors: An intra-subject analysis

The oral bioavailability of many drugs is highly influenced not only by hepatic but also by intestinal biotransformation. To estimate the impact of intestinal phase I and II metabolism on oral drug absorption, knowledge on the expression levels of the respective enzymes is an essential prerequisite. In addition, the potential interplay of metabolism and transport contributes to drug disposition. Both mechanisms may be subjected to coordinative regulation by nuclear receptors, leading to unwanted drug‐drug interactions due to induction of intestinal metabolism and transport. Thus, it was the aim of this study to comprehensively analyse the regional expression of clinically relevant phase I and II enzymes along the entire human intestine and to correlate these data to expression data of drug transporters and nuclear receptors of pharmacokinetic relevance. Gene expression of 11 drug‐metabolizing enzymes (CYP2B6, 2C8, 2C9, 2C19, 2D6, 3A4, 3A5, SULT1A, UGT1A, UGT2B7, UGT2B15) was studied in duodenum, jejunum, ileum and colon from six organ donors by real‐time RT‐PCR. Enzyme expression was correlated with expression data of the nuclear receptors PXR, CAR and FXR as well as drug transporters observed in the same cohort. Intestinal expression of all studied metabolizing enzymes was significantly higher in the small intestine compared to colonic tissue. CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4/5, SULT1A, UGT1A and UGT2B7 expression increased from the duodenum to jejunum but was markedly lower in the ileum. In the small intestine, that is, the predominant site of drug absorption, the highest expression has been observed for CYP3A4, CYP2C9, SULT1A and UGT1A. In addition, significant correlations were found between several enzymes and PXR as well as ABC transporters in the small intestine. In conclusion, the observed substantial site‐dependent intestinal expression of several enzymes may explain regional differences in intestinal drug absorption. The detected correlations between intestinal enzymes, transporters and nuclear receptors provide indirect evidence for their coordinative expression, regulation and function in the human small intestine.

Single Nucletide Polymorphisms in Gene of IL-1Beta in Bronchial Asthma

ABSTRACT Bronchial asthma is a common chronic lung disease that is driven by abnormal inflammatory reactions in the airways in response to the complex interaction between genetics and environmental factors. The underlying inflammation in asthma is manifested by prominent eosinophil infiltration and Th2 inflammatory mediators; however the pro-inflammatory cytokines, particularly IL-1β, have also been found in increased amount in the sputum and BAL in individuals with bronchial asthma, especially in more severe state. Therefore, IL-1β is considered to be of importance in pathogenesis of this condition as the protein level of the cytokine is finely regulated by variety of factors, including genetics. The IL1B gene displays many SNPs both in promoter and coding regions, which have been associated with IL-1β production. In the current case-control study we investigated -511C>T (rs16944) promoter polymorphism and +3953C>T (rs1143634) silent polymorphism in exon 5 of IL1B and their haplotypes as candidate risk factors of Bronchial asthma in Bulgarian population. We genotyped 47 patients with bronchial asthma and 174 control individuals using Taqman genotyping assay for IL1B -511C>T SNP and PCR-RFLP-based method for +3953C>TSNP. We did not observe statistically significant differences in genotype frequencies of IL1B -511C>T and IL1B +3953C>T between controls and patients with asthma (p=0.065 and p=0.987). However, the minor T allele of IL1B -511C>T was less frequently found in the controls (0.305) compared to the patients with asthma (0.415, p=0.0002). Carriers of IL1B -511T allele (TT or TC genotypes) appeared to have 2.25-fold higher risk for Bronchial asthma (95% CI: 1.127–4.498, p=0.019). The performed estimations of IL1B haplotypes did not reveal any difference in the haplotype frequencies between controls and patients with asthma (p=0.270). However, the T_C haplotype, constructed by alleles found to determine enhanced expression of IL-1β, appeared to be associated with higher risk of asthma (OR 1.78, 1.04–3.03, p=0.035) compared to the most common C_C haplotype. Based on the results of the current study we suggest that the -511C>T promoter polymorphism and +3953C>T silent polymorphism in exon 5 of IL1B may influence the genetic predisposition of Bronchial asthma in Bulgarian population, as the carriers of alleles and haplotypes supposed to define higher IL-1β protein levels are more susceptible for this lung diseases.