Publications by authors named "Magdalena Dąbrowska"

41 Publications

Paving the way towards precise and safe CRISPR genome editing.

Biotechnol Adv 2021 Mar 27:107737. Epub 2021 Mar 27.

Department of Genome Engineering, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Noskowskiego 12/14, 61-704, Poland. Electronic address:

As the possibilities of CRISPR-Cas9 technology have been revealed, we have entered a new era of research aimed at increasing its specificity and safety. This stage of technology development is necessary not only for its wider application in the clinic but also in basic research to better control the process of genome editing. Research during the past eight years has identified some factors influencing editing outcomes and led to the development of highly specific endonucleases, modified guide RNAs and computational tools supporting experiments. More recently, large-scale experiments revealed a previously overlooked feature: Cas9 can generate reproducible mutation patterns. As a result, it has become apparent that Cas9-induced double-strand break (DSB) repair is nonrandom and can be predicted to some extent. Here, we review the present state of knowledge regarding the specificity and safety of CRISPR-Cas9 technology to define gRNA, protein and target-related problems and solutions. These issues include sequence-specific off-target effects, immune responses, genetic variation and chromatin accessibility. We present new insights into the role of DNA repair in genome editing and define factors influencing editing outcomes. In addition, we propose practical guidelines for increasing the specificity of editing and discuss novel perspectives in improvement of this technology.
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http://dx.doi.org/10.1016/j.biotechadv.2021.107737DOI Listing
March 2021

Surface-Related Kinetic Models for Anaerobic Digestion of Mi-crocrystalline Cellulose: The Role of Particle Size.

Materials (Basel) 2021 Jan 20;14(3). Epub 2021 Jan 20.

Department of Biosystems Engineering, Institute of Mechanical Engineering, Warsaw University of Life Sciences, Nowoursynowska 166, 02-787 Warsaw, Poland.

In this work, for modelling the anaerobic digestion of microcrystalline cellulose, two surface-related models based on cylindrical and spherical particles were developed and compared with the first-order kinetics model. A unique dataset consisting of particles with different sizes, the same crystallinity and polymerisation degree was used to validate the models. Both newly developed models outperformed the first-order kinetics model. Analysis of the kinetic constant data revealed that particle size is a key factor determining the anaerobic digestion kinetics of crystalline cellulose. Hence, crystalline cellulose particle size should be considered in the development and optimization of lignocellulose pre-treatment methods. Further research is necessary for the assessment of impact of the crystalline cellulose particle size and surface properties on the microbial cellulose hydrolysis rate.
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http://dx.doi.org/10.3390/ma14030487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864345PMC
January 2021

The effects of solid lignin on the anaerobic digestion of microcrystalline cellulose and application of smoothing splines for extended data analysis of its inhibitory effects.

Bioresour Technol 2021 Jan 16;320(Pt A):124262. Epub 2020 Oct 16.

Department of Biosystems Engineering, Institute of Mechanical Engineering, Warsaw University of Life Sciences, Nowoursynowska 166, 02-787 Warsaw, Poland.

Lignocellulose is an abundant substrate for biogas production; however, for efficient utilization, proper pre-treatment is required to enhance the biomethane yield and hydrolysis rate significantly. Phenolic compounds from dissolved lignin, produced during alkali pre-treatment, have inhibitory effects on the anaerobic digestion; however, the possible inhibitory effects of solid lignin have not gathered enough interest. Especially, the effect of solid lignin on methanogenesis remains a knowledge gap. In this study, kraft lignin was used as a model solid lignin substrate for its co-digestion with microcrystalline cellulose. A new approach of modelling biomethane production curves using smoothing splines was developed to describe the long-term inhibitory effects of solid lignin on hydrolysis and methanogenesis. The method gives possibility to describe long-term inhibitory effects by using batch instead of continuous test data. Results revealed that kraft lignin showed mild inhibitory effects on methanogens. However lignin impact combined with volatile fatty accumulation can prolong hydrolysis and reactor recovery start-up by 47.3% and 75.3%, respectively. For small dosages of solid lignin adaptation of methanogens is possible.
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http://dx.doi.org/10.1016/j.biortech.2020.124262DOI Listing
January 2021

Influence of Fraction Particle Size of Pure Straw and Blends of Straw with Calcium Carbonate or Cassava Starch on Pelletising Process and Pellet.

Materials (Basel) 2020 Oct 16;13(20). Epub 2020 Oct 16.

Department of Applied Mathematics, Institute of Information Technology, Warsaw University of Life Sciences, Nowoursynowska 166, 02-787 Warsaw, Poland.

The aim of this study was to investigate the pressure agglomeration process of wheat straw (WS) and the blends of WS with calcium carbonate (CC) or cassava straw (CS) with a ratio of 6% wt./wt. from seven separate fractions with sizes in the range of 0.21-2.81 mm. The agglomeration was performed at a moisture of 30% wb and a material temperature of 78 °C, with a dose of 0.1 g, in a die of diameter 8 mm and height 80 mm. The effects of the process were evaluated based on the compaction parameters and the pellets' density, tensile strength, and water absorption. The incorporation of additives into the WS improved the pellet process and quality. Refined results were achieved after adding CC, as compared to those achieved after adding CS, and the preferred particle size was in the range of 1.00-1.94 mm. This was because, under the given conditions, the back pressure in the die chamber significantly increased, allowing the achievement of a single pellet density of 800 kg·m. The pellets were resistant to compressive loads and cracked only at tensile strength of 6 MPa and a specific compression work of 6.5 mJ·mm. The addition of CC to the WS improved the strength of the adhesive and the cohesive bonds between the particles. The water absorption for the uncrushed pellets was considerably less than that for crushed pellets, which results in the safer storage of uncrushed pellets and excellent moisture absorption of crushed pellets. The addition of CC to the WS offers benefits in the form of pellet strength with a high water absorption capability. Notably, a study of crushed pellet litter under broiler rearing conditions and an analysis of the operational costs of using WS additives are required for implementing this study.
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http://dx.doi.org/10.3390/ma13204623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7602947PMC
October 2020

Artificial miRNAs targeting CAG repeat expansion in ORFs cause rapid deadenylation and translation inhibition of mutant transcripts.

Cell Mol Life Sci 2021 Feb 21;78(4):1577-1596. Epub 2020 Jul 21.

Department of Molecular Biomedicine, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, Poznan, Poland.

Polyglutamine (polyQ) diseases are incurable neurological disorders caused by CAG repeat expansion in the open reading frames (ORFs) of specific genes. This type of mutation in the HTT gene is responsible for Huntington's disease (HD). CAG repeat-targeting artificial miRNAs (art-miRNAs) were shown as attractive therapeutic approach for polyQ disorders as they caused allele-selective decrease in the level of mutant proteins. Here, using polyQ disease models, we aimed to demonstrate how miRNA-based gene expression regulation is dependent on target sequence features. We show that the silencing efficiency and selectivity of art-miRNAs is influenced by the localization of the CAG repeat tract within transcript and the specific sequence context. Furthermore, we aimed to reveal the events leading to downregulation of mutant polyQ proteins and found very rapid activation of translational repression and HTT transcript deadenylation. Slicer-activity of AGO2 was dispensable in this process, as determined in AGO2 knockout cells generated with CRISPR-Cas9 technology. We also showed highly allele-selective downregulation of huntingtin in human HD neural progenitors (NPs). Taken together, art-miRNA activity may serve as a model of the cooperative activity and targeting of ORF regions by endogenous miRNAs.
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http://dx.doi.org/10.1007/s00018-020-03596-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904544PMC
February 2021

The Correlation between Physical Crosslinking and Water-Soluble Drug Release from Chitosan-Based Microparticles.

Pharmaceutics 2020 May 16;12(5). Epub 2020 May 16.

Department of Pharmaceutical Technology, Medical University of Białystok, Mickiewicza 2c, 15-222 Białystok, Poland.

Microparticles containing water-soluble zidovudine were prepared by spray-drying using chitosan glutamate and beta-glycerophosphate as an ion crosslinker (CF). The Box-Behnken design was applied to optimize the microparticles in terms of their drug loading and release behavior. Physicochemical studies were undertaken to support the results from dissolution tests and to evaluate the impact of the crosslinking ratio on the microparticles' characteristics. The zidovudine dissolution behavior had a complex nature which comprised two phases: an initial burst effect followed with a prolonged release stage. The initial drug release, which can be modulated by the crosslinking degree, was primarily governed by the dissolution of the drug crystals located on the microparticles' surfaces. In turn, the further dissolution stage was related to the drug diffusion from the swollen polymer matrix and was found to correlate with the drug loading. Differential Scanning Calorimetry (DSC) studies revealed the partial incorporation of a non-crystallized drug within the polymer matrix, which correlated with the amount of CF. Although CF influenced the swelling capacity of chitosan glutamate microparticles, surprisingly a higher amount of CF did not impact the time required for 80% of the drug to be released markedly. The formulation with the lowest polymer:CF ratio, 3:1, was selected as optimal, providing satisfactory drug loading and displaying a moderate burst effect within the first 30 min of the study, followed with a prolonged drug release of up to 210 min.
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http://dx.doi.org/10.3390/pharmaceutics12050455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7284795PMC
May 2020

Generation of New Isogenic Models of Huntington's Disease Using CRISPR-Cas9 Technology.

Int J Mol Sci 2020 Mar 8;21(5). Epub 2020 Mar 8.

Department of Genome Engineering, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by the expansion of CAG repeats in exon 1 of the huntingtin gene (). Despite its monogenic nature, HD pathogenesis is still not fully understood, and no effective therapy is available to patients. The development of new techniques such as genome engineering has generated new opportunities in the field of disease modeling and enabled the generation of isogenic models with the same genetic background. These models are very valuable for studying the pathogenesis of a disease and for drug screening. Here, we report the generation of a series of homozygous HEK 293T cell lines with different numbers of CAG repeats at the locus and demonstrate their usefulness for testing therapeutic reagents. In addition, using the CRISPR-Cas9 system, we corrected the mutation in HD human induced pluripotent stem cells and generated a knock-out of the gene, thus providing a comprehensive set of isogenic cell lines for HD investigation.
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http://dx.doi.org/10.3390/ijms21051854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084361PMC
March 2020

The influence of scar on the spatio-temporal relationship between electrical and mechanical activation in heart failure patients.

Europace 2020 05;22(5):777-786

Center for Computational Medicine in Cardiology, Università della Svizzera italiana, Via G. Buffi 13, CH-6900 Lugano, Switzerland.

Aims: The aim of this study was to determine the relationship between electrical and mechanical activation in heart failure (HF) patients and whether electromechanical coupling is affected by scar.

Methods And Results: Seventy HF patients referred for cardiac resynchronization therapy or biological therapy underwent endocardial anatomo-electromechanical mapping (AEMM) and delayed-enhancement magnetic resonance (CMR) scans. Area strain and activation times were derived from AEMM data, allowing to correlate mechanical and electrical activation in time and space with unprecedented accuracy. Special attention was paid to the effect of presence of CMR-evidenced scar. Patients were divided into a scar (n = 43) and a non-scar group (n-27). Correlation between time of electrical and mechanical activation was stronger in the non-scar compared to the scar group [R = 0.84 (0.72-0.89) vs. 0.74 (0.52-0.88), respectively; P = 0.01]. The overlap between latest electrical and mechanical activation areas was larger in the absence than in presence of scar [72% (54-81) vs. 56% (36-73), respectively; P = 0.02], with smaller distance between the centroids of the two regions [10.7 (4.9-17.4) vs. 20.3 (6.9-29.4) % of left ventricular radius, P = 0.02].

Conclusion: Scar decreases the association between electrical and mechanical activation, even when scar is remote from late activated regions.
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http://dx.doi.org/10.1093/europace/euz346DOI Listing
May 2020

Gene Therapy for Huntington's Disease Using Targeted Endonucleases.

Methods Mol Biol 2020 ;2056:269-284

Department of Genome Engineering, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.

Huntington's disease (HD) is a hereditary neurological disorder caused by expansion of the CAG repeat tract in the huntingtin gene (HTT). The mutant protein with a long polyglutamine tract is toxic to cells, especially neurons, leading to their progressive degeneration. Similar to many other monogenic diseases, HD is a good target for gene therapy approaches, including the use of programmable endonucleases. Here, we describe a protocol for HTT gene knock out using a modified Cas9 protein (nickase, Cas9n) and a pair of sgRNAs flanking the repeats. Recently, we showed that excision of the CAG repeat tract resulted in a frameshift mutation and premature translation termination. As a model, we used HD patient-derived fibroblasts electroporated with a pair of plasmid vectors expressing CRISPR-Cas9n tools. Efficient HTT inactivation independent of the CAG tract length was confirmed by Western blotting. A modified version of this protocol involving precise excision of the CAG repeats and insertion of a new DNA sequence by homology directed repair may also be used for the generation of new isogenic cellular models of HD.
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http://dx.doi.org/10.1007/978-1-4939-9784-8_17DOI Listing
November 2020

Methotrexate-induced senescence of human colon cancer cells depends on p53 acetylation, but not genomic aberrations.

Anticancer Drugs 2019 04;30(4):374-382

Nencki Institute of Experimental Biology, Warszawa.

Human colon cancer C85 cell response to methotrexate has been documented previously to take on a form of reversible premature senescence. Seeking genomic aberrations encompassing candidate genes whose functional impairment could determine such a response to the drug, an array Comparative Genomic Hybridization method was applied, complemented by expression microarray data set searching. In the C85 cell genome, only short aberrations were identified, classified as focal chromosomal aberrations. 62% of the aberrant regions, selected by referral to normal human colon epithelium, were not carrying any gene. Out of the genes, subject to aberrations, 50% were protein-coding ones. Expression of those that could serve a signaling or a growth-regulatory function was found to be either downregulated or unchanged during C85 cell progression into methotrexate-induced senescence. Lack of extensive chromosomal instability in C85 cells is hypothesized to be attributed to the presence of the wild-type tumor suppressor p53 protein. Although two p53 protein isoforms were detected in C85 cells, stabilization and acetylation of the full-length p53 isoform were shown to underpin progression of the cells into premature senescence upon methotrexate treatment.
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http://dx.doi.org/10.1097/CAD.0000000000000731DOI Listing
April 2019

qEva-CRISPR: a method for quantitative evaluation of CRISPR/Cas-mediated genome editing in target and off-target sites.

Nucleic Acids Res 2018 09;46(17):e101

Department of Molecular Genetics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland.

Genome editing technology based on engineered nucleases has been increasingly applied for targeted modification of genes in a variety of cell types and organisms. However, the methods currently used for evaluating the editing efficiency still suffer from many limitations, including preferential detection of some mutation types, sensitivity to polymorphisms that hamper mismatch detection, lack of multiplex capability, or sensitivity to assay conditions. Here, we describe qEva-CRISPR, a new quantitative method that overcomes these limitations and allows simultaneous (multiplex) analysis of CRISPR/Cas9-induced modifications in a target and the corresponding off-targets or in several different targets. We demonstrate all of the advantages of the qEva-CRISPR method using a number of sgRNAs targeting the TP53, VEGFA, CCR5, EMX1 and HTT genes in different cell lines and under different experimental conditions. Unlike other methods, qEva-CRISPR detects all types of mutations, including point mutations and large deletions, and its sensitivity does not depend on the mutation type. Moreover, this approach allows for successful analysis of targets located in 'difficult' genomic regions. In conclusion, qEva-CRISPR may become a method of choice for unbiased sgRNA screening to evaluate experimental conditions that affect genome editing or to distinguish homology-directed repair from non-homologous end joining.
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http://dx.doi.org/10.1093/nar/gky505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158505PMC
September 2018

Precise Excision of the CAG Tract from the Huntingtin Gene by Cas9 Nickases.

Front Neurosci 2018 26;12:75. Epub 2018 Feb 26.

Department of Genome Engineering, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.

Huntington's disease (HD) is a progressive autosomal dominant neurodegenerative disorder caused by the expansion of CAG repeats in the first exon of the huntingtin gene (). The accumulation of polyglutamine-rich huntingtin proteins affects various cellular functions and causes selective degeneration of neurons in the striatum. Therapeutic strategies used to date to silence the expression of mutant include antisense oligonucleotides, RNA interference-based approaches and, recently, genome editing with the CRISPR/Cas9 system. Here, we demonstrate that the CAG repeat tract can be precisely excised from the gene with the use of the paired Cas9 nickase strategy. As a model, we used HD patient-derived fibroblasts with varied numbers of CAG repeats. The repeat excision inactivated the gene and abrogated huntingtin synthesis in a CAG repeat length-independent manner. Because Cas9 nickases are known to be safe and specific, our approach provides an attractive treatment tool for HD that can be extended to other polyQ disorders.
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http://dx.doi.org/10.3389/fnins.2018.00075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834764PMC
February 2018

Crystal structures of nematode (parasitic T. spiralis and free living C. elegans), compared to mammalian, thymidylate synthases (TS). Molecular docking and molecular dynamics simulations in search for nematode-specific inhibitors of TS.

J Mol Graph Model 2017 10 12;77:33-50. Epub 2017 Aug 12.

Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warszawa, Poland.

Three crystal structures are presented of nematode thymidylate synthases (TS), including Caenorhabditis elegans (Ce) enzyme without ligands and its ternary complex with dUMP and Raltitrexed, and binary complex of Trichinella spiralis (Ts) enzyme with dUMP. In search of differences potentially relevant for the development of species-specific inhibitors of the nematode enzyme, a comparison was made of the present Ce and Ts enzyme structures, as well as binary complex of Ce enzyme with dUMP, with the corresponding mammalian (human, mouse and rat) enzyme crystal structures. To complement the comparison, tCONCOORD computations were performed to evaluate dynamic behaviors of mammalian and nematode TS structures. Finally, comparative molecular docking combined with molecular dynamics and free energy of binding calculations were carried out to search for ligands showing selective affinity to T. spiralis TS. Despite an overall strong similarity in structure and dynamics of nematode vs mammalian TSs, a pool of ligands demonstrating predictively a strong and selective binding to TsTS has been delimited. These compounds, the E63 family, locate in the dimerization interface of TsTS where they exert species-specific interactions with certain non-conserved residues, including hydrogen bonds with Thr174 and hydrophobic contacts with Phe192, Cys191 and Tyr152. The E63 family of ligands opens the possibility of future development of selective inhibitors of TsTS and effective agents against trichinellosis.
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http://dx.doi.org/10.1016/j.jmgm.2017.08.008DOI Listing
October 2017

Oxidative stress and inhibition of nitric oxide generation underlie methotrexate-induced senescence in human colon cancer cells.

Mech Ageing Dev 2018 03 21;170:22-29. Epub 2017 Jul 21.

Laboratory of Molecular Basis of Ageing, Department of Biochemistry, Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur St., 02-093 Warszawa, Poland.

The response of human colon cancer C85 cells to methotrexate takes the form of reversible growth arrest of the type of stress-induced senescence. In the present study it is shown that during C85 cell progression into methotrexate-induced senescence, dihydrofolate reductase, the primary intracellular target for the drug, is stabilized at the protein level and its enzymatic activity, assayed in crude cellular extracts, decreases by 2-fold. Dihydrofolate reductase inhibition results in an increase in dihydrobiopterin level and an ultimate decrease in the tetrahydrobiopterin: dihydrobiopterin ratio in senescent cells. Endothelial nitric oxide synthase expression declines. Despite concomitant upregulation of inducible nitric oxide synthase expression, no nitric oxide generation in senescent cells is detected. Progressing oxidative stress accompanies establishment of the state of senescence. DNA damage, in the form of double strand-breaks, occurs at the highest level at the senescence initiation phase and decreases as cells progress into the senescence maintenance phase.
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http://dx.doi.org/10.1016/j.mad.2017.07.006DOI Listing
March 2018

E2F site in the essential promoter region does not confer S phase-specific transcription of the ABCC10 gene in human prostate cancer cells.

Acta Biochim Pol 2017 13;64(2):371-374. Epub 2017 Jun 13.

Memorial Sloan-Kettering Cancer Center, New York, NY10021, USA.

ABCC10 (MRP7) plays a role in cellular detoxification and resistance to anticancer drugs. Since ABCC10 gene transcription in human prostate cancer CWR22Rv1 cells was found dependent on E2F binding sequence motif, ABCC10 expression in G and S phases of the cell cycle of CWR22Rv1 cells, was analyzed. The cells were synchronized in G phase by double thymidine block and in S phase by thymidine/mimosine double block. ABCC10 mRNA level was found to be similar in S phase-synchronized and asynchronous cell populations. In G phase it decreased by 2.4- to 3-fold. It is thus inferred, that ABCC10 expression in CWR22Rv1 cells is not S phase-specific but is primarily associated with cell proliferation.
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http://dx.doi.org/10.18388/abp.2017_1521DOI Listing
August 2017

Wnt signaling in regulation of biological functions of the nurse cell harboring Trichinella spp.

Parasit Vectors 2016 09 2;9(1):483. Epub 2016 Sep 2.

Laboratory of Comparative Enzymology, Department of Biochemistry, Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur St., Warsaw, 02-093, Poland.

Background: The nurse cell (NC) constitutes in mammalian skeletal muscles a confined intracellular niche to support the metabolic needs of muscle larvae of Trichinella spp. encapsulating species. The main biological functions of NC were identified as hypermitogenic growth arrest and pro-inflammatory phenotype, both inferred to depend on AP-1 (activator protein 1) transcription factor. Since those functions, as well as AP-1 activity, are known to be regulated among other pathways, also by Wnt (Wingless-Type of Mouse Mammary Tumor Virus Integration Site) signaling, transcription profiling of molecules participating in Wnt signaling cascades in NC, was performed.

Methods: Wnt signaling-involved gene expression level was measured by quantitative RT-PCR approach with the use of Qiagen RT(2) Profiler PCR Arrays and complemented by that obtained by searching microarray data sets characterizing NC transcriptome.

Results: The genes involved in inhibition of canonical Wnt/β-catenin signaling cascade as well as leading to β-catenin degradation were found expressed in NC at high level, indicating inhibition of this cascade activity. High expression in NC of genes transmitting the signal of Wnt non-canonical signaling cascades leading to activation of AP-1 transcription factor, points to predominant role of non-canonical Wnt signaling in a long term maintenance of NC biological functions.

Conclusions: Canonical Wnt/β-catenin signaling cascade is postulated to play a role at the early stages of NC formation when muscle regeneration process is triggered. Following mis-differentiation of infected myofiber and setting of NC functional specificity, are inferred to be controlled among other pathways, by Wnt non-canonical signaling cascades.
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http://dx.doi.org/10.1186/s13071-016-1770-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5010673PMC
September 2016

Synthesis, reactivity and biological activity of N(4)-boronated derivatives of 2'-deoxycytidine.

Bioorg Med Chem 2014 Aug 16;22(15):3906-12. Epub 2014 Jun 16.

Rzeszów University of Technology, Faculty of Chemistry, Bioorganic Chemistry Laboratory, 6 Powstańców Warszawy Ave., 35-959 Rzeszów, Poland. Electronic address:

By seeking new stable boron-containing nucleoside derivatives, potential BNCT boron delivery agents, a novel synthetic approach was tested, aimed at a boron attachment via a single bond to an aliphatic carbon of sp(3) hybridization. The latter allowed successful modification of deoxycytidine in the reaction with 2-(iodomethyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane of the deoxynucleoside amino group. For new compounds, detailed NMR, LDI HRMS (Laser Desorption/Ionization High-Resolution Mass Spectrometry) analyses along with in vivo phosphorylation studies, toxicity assays and DFT modelling are presented.
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http://dx.doi.org/10.1016/j.bmc.2014.06.014DOI Listing
August 2014

Trichinella pseudospiralis vs. T. spiralis thymidylate synthase gene structure and T. pseudospiralis thymidylate synthase retrogene sequence.

Parasit Vectors 2014 Apr 9;7:175. Epub 2014 Apr 9.

Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur Street, 02-093 Warszawa, Poland.

Background: Thymidylate synthase is a housekeeping gene, designated ancient due to its role in DNA synthesis and ubiquitous phyletic distribution. The genomic sequences were characterized coding for thymidylate synthase in two species of the genus Trichinella, an encapsulating T. spiralis and a non-encapsulating T. pseudospiralis.

Methods: Based on the sequence of parasitic nematode Trichinella spiralis thymidylate synthase cDNA, PCR techniques were employed.

Results: Each of the respective gene structures encompassed 6 exons and 5 introns located in conserved sites. Comparison with the corresponding gene structures of other eukaryotic species revealed lack of common introns that would be shared among selected fungi, nematodes, mammals and plants. The two deduced amino acid sequences were 96% identical. In addition to the thymidylate synthase gene, the intron-less retrocopy, i.e. a processed pseudogene, with sequence identical to the T. spiralis gene coding region, was found to be present within the T. pseudospiralis genome. This pseudogene, instead of the gene, was confirmed by RT-PCR to be expressed in the parasite muscle larvae.

Conclusions: Intron load, as well as distribution of exon and intron phases in thymidylate synthase genes from various sources, point against the theory of gene assembly by the primordial exon shuffling and support the theory of evolutionary late intron insertion into spliceosomal genes. Thymidylate synthase pseudogene expressed in T. pseudospiralis muscle larvae is designated a retrogene.
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http://dx.doi.org/10.1186/1756-3305-7-175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4022200PMC
April 2014

Balancing between bleeding and thromboembolism after percutaneous coronary intervention in patients with atrial fibrillation. Could triple anticoagulant therapy be a solution?

Postepy Kardiol Interwencyjnej 2013 16;9(3):234-40. Epub 2013 Sep 16.

3 Division of Cardiology, 3 Department of Cardiology, Medical University of Silesia, Katowice, Poland.

Introduction: Atrial fibrillation (AF) has nowadays become a common disease as it comes along with medical procedures propagation in the ageing population with coexistent diseases. Hence a need for use of combined anticoagulant and antithrombotic therapy has arisen. According to the 2010 ESC guidelines on myocardial revascularization, short-term triple antithrombotic therapy after percutaneous coronary intervention (PCI) should be given if compelling indications exist.

Aim: To assess bleeding and thromboembolic events depending on the antithrombotic regimen in short- and long-term follow-up in patients with AF after PCI with stent implantation.

Material And Methods: A 12-month prospective, non-randomized registry was conducted in the 3(rd) Department of Cardiology in the Upper Silesian Medical Center in Katowice from October 2008 to April 2011. One hundred and four patients in two groups - on triple therapy (TT; aspirin + clopidogrel + vitamin K antagonists (VKA; warfarin or acenocoumarol) n = 44) and on dual therapy (DT; aspirin + clopidogrel; n = 60) - were assessed 30 days and 12 months after angioplasty.

Results: All bleeding events occurred more often in the triple anticoagulated group in 30 days (TT 20.5% vs. DT 6.7%; p = 0.03) and after 12 months (TT 38.9% vs. DT 17.2%, p = 0.09). The difference in major bleeding events was not significant after 30 days (TT 9.1% vs. DT 3.3%; p = NS) or 12 months (TT 11.1% vs. DT 6.9%; p = NS). Thromboembolic events after 30 days (DT 5.0% vs. TT 2.3%) and 12 months (TT 11.1% vs. DT 3.4%) were comparable. The percentage of deaths after 30 days (DT 1.7% vs. TT 0.0%, p = NS) increased after 12 months (DT 13.8% vs. TT 0.0%, p = 0.09).

Conclusions: Significantly higher risk of bleeding on TT becomes blurred by a tendency to increased mortality in patients on DT.
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http://dx.doi.org/10.5114/pwki.2013.37501DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3915991PMC
February 2014

Gene expression profiling of murine T-cell lymphoblastic lymphoma identifies deregulation of S-phase initiating genes.

Leuk Res 2013 Oct 26;37(10):1383-90. Epub 2013 Jul 26.

Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.

In a search for genes and pathways implicated in T-cell lymphoblastic lymphoma (T-LBL) development, we used a murine lymphoma model, where mice of the NMRI-inbred strain were inoculated with murine leukemia virus mutants. The resulting tumors were analyzed by integration analysis and global gene expression profiling to determine the effect of the retroviral integrations on the nearby genes, and the deregulated pathways in the tumors. Gene expression profiling identified increased expression of genes involved in the minichromosome maintenance and origin of recognition pathway as well as downregulation in negative regulators of G1/S transition, indicating increased S-phase initiation in murine T-LBLs.
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http://dx.doi.org/10.1016/j.leukres.2013.04.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4057920PMC
October 2013

MicroRNA expression profiling identifies molecular signatures associated with anaplastic large cell lymphoma.

Blood 2013 Sep 25;122(12):2083-92. Epub 2013 Jun 25.

Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE;

Anaplastic large-cell lymphomas (ALCLs) encompass at least 2 systemic diseases distinguished by the presence or absence of anaplastic lymphoma kinase (ALK) expression. We performed genome-wide microRNA (miRNA) profiling on 33 ALK-positive (ALK[+]) ALCLs, 25 ALK-negative (ALK[-]) ALCLs, 9 angioimmunoblastic T-cell lymphomas, 11 peripheral T-cell lymphomas not otherwise specified (PTCLNOS), and normal T cells, and demonstrated that ALCLs express many of the miRNAs that are highly expressed in normal T cells with the prominent exception of miR-146a. Unsupervised hierarchical clustering demonstrated distinct clustering of ALCL, PTCL-NOS, and the AITL subtype of PTCL. Cases of ALK(+) ALCL and ALK(-) ALCL were interspersed in unsupervised analysis, suggesting a close relationship at the molecular level. We identified an miRNA signature of 7 miRNAs (5 upregulated: miR-512-3p, miR-886-5p, miR-886-3p, miR-708, miR-135b; 2 downregulated: miR-146a, miR-155) significantly associated with ALK(+) ALCL cases. In addition, we derived an 11-miRNA signature (4 upregulated: miR-210, miR-197, miR-191, miR-512-3p; 7 downregulated: miR-451, miR-146a, miR-22, miR-455-3p, miR-455-5p, miR-143, miR-494) that differentiates ALK(-) ALCL from other PTCLs. Our in vitro studies identified a set of 32 miRNAs associated with ALK expression. Of these, the miR-17∼92 cluster and its paralogues were also highly expressed in ALK(+) ALCL and may represent important downstream effectors of the ALK oncogenic pathway.
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http://dx.doi.org/10.1182/blood-2012-08-447375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3778551PMC
September 2013

Inflammatory phenotype of the nurse cell harboring Trichinella spp.

Vet Parasitol 2013 May 5;194(2-4):150-4. Epub 2013 Feb 5.

Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur Street, 02-093 Warsaw, Poland.

The nurse cell (NC), formed from muscle cells upon infection with the parasitic nematode Trichinella spp. constitutes a confined habitat for muscle larvae of encapsulating species. Signaling pathway-directed analysis of microarray data allowed identification of the stage of NC cell cycle arrest as being of G1-like type, accompanied by cellular senescence. In accord with the specificity of senescent cellular systems, up-regulation of pro-inflammatory molecules was also found within the NC preparations. Potential immune-related activities associated with NCs as inferred from the aforementioned analysis, are reviewed herein. Transcriptional data suggest that the NC which harbors the larvae may exhibit the following immune-related functions: (i) production of complement components, (ii) antigen presentation and phagocytosis, (iii) pro-inflammatory cytokine secretion, (iv) oxidative stress generation and (v) eicosanoid synthesis.
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http://dx.doi.org/10.1016/j.vetpar.2013.01.043DOI Listing
May 2013

[Hyponatremic hypervolemia in heart failure--the hot spot for vasopressin receptor antagonists?].

Wiad Lek 2012 ;65(1):19-30

Klinice Chorób Wewnetrznych i Farmakologii Klinicznej Katedry Farmakologii, Slaski Uniwersytet Medyczny w Katowicach.

Dilutional hyponatremia is the most common electrolyte disorder occurring in heart failure (HF). The relation between hyponatremia and increased morbidity and mortality is widely evidenced. Treatment of this condition is circumscribed by strict correlation of electrolyte and water balance, though. Neurohormonal activation in HF is the effect of compensatory mechanisms due to insufficient effective blood volume. One of the factors is increased serum argininovasopressin (AVP) concentration. The principal effects of this action are free water retention via V2 receptor and vasoconstriction by V(1A) receptor. Stimulation of V(1A) receptor also contributes to heart muscle remodeling. Diuretics are still the basic treatment of hypervolemia. At the same time they have a very unfavorable side effect in the form of exacerbating hyponatremia. The new direction of investigations occurred after noticing the spectacular aquaretic effect of antagonizing AVP receptors. The effect is new group of drugs--vasopressin receptor antagonists called the vaptans. Unlike the diuretics, they cause free water excretion without electrolyte loss. The registration includes in-hospital treatment of states of hyponatremia (conivaptan, tolvaptan, mozavaptan), the two other (lixivaptan, satavaptan) are undergoing research. Apart from improving clinical status, there is still no evidence of improving the patients' survival. Further research in this field is necessary to demonstrate whether there is prognosis amelioration in short-term and long-term patients' observation.
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August 2012

HCV coinfection possibly promotes left ventricular dysfunction development: analysis of brain natriuretic peptide serum levels in HCV/HIV-coinfected and HIV-monoinfected patients.

Eur J Gastroenterol Hepatol 2012 Nov;24(11):1308-12

Department of Hepatology and Acquired Immunodeficiencies, Warsaw Medical University, Warsaw, Poland.

Objective: Hepatitis C virus (HCV) has been already linked to possible myocarditis and cardiomyopathy development. The brain natriuretic peptide (BNP) is a sensitive biomarker of left ventricular dysfunction. The present study aimed to evaluate the potential risk of cardiac injury in HIV-infected and HCV/HIV-coinfected patients with or without antiretroviral (ARV) therapy by comparing BNP serum levels in the groups studied.

Methods: Eighty HIV-infected patients (65 men, 15 women, mean age 40 years; 29 with HCV coinfection, 48 on combined ARV therapy) were included in the cross-sectional study. BNP serum levels were evaluated by enzyme-linked immunosorbent assay. The BNP cut-off level for possible heart failure was 42 fmol/l as in an immunocompetent population.

Results: Seventy-eight (97.5%) patients studied had a BNP concentration above 42 fmol/l; seven patients (8.7%) had a concentration above 168 fmol/l associated with a worse outcome. There was no difference in the mean BNP serum levels in ARV-treated and untreated patients. However, the mean BNP serum level was significantly higher in HCV/HIV-coinfected patients in comparison with HIV-monoinfected patients (160.0 ± 130.9 vs. 81.9 ± 37.2 fmol/l; P<0.0001). There was no relationship between BNP serum levels and HIV viral load, CD4 cell count, sex, age, and abacavir or protease inhibitors use.

Conclusion: A significant association was found between HCV coinfection and BNP serum level in HIV-infected patients. HCV coinfection possibly enhances the risk of left ventricular dysfunction development in this vulnerable population.
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http://dx.doi.org/10.1097/MEG.0b013e32835702c6DOI Listing
November 2012

The anemia prevalence and the association between complete blood count analysis and renal function parameters in HIV-1-infected patients.

Curr HIV Res 2012 Apr;10(3):247-51

Department of Hepatology and Acquired Immunodeficiences, Warsaw Medical University, 37 Wolska Street, 01-201 Warsaw, Poland.

Objectives: To determine the anemia prevalence and the correlation between complete blood count (CBC) analysis and renal function parameters in HIV-1-infected population.

Methods: It was a single-center study set in Warsaw (Poland) over a 3-year period. The study was performed in 214 adult HIV-1- infected patients (180 males and 34 females, aged from 20 to 69 years old, mean age 39.55 years, 130 on combined antiretroviral therapy, cART). Glomerular filtration rate (GFR) was estimated using the re-expressed Modification of Diet in Renal Disease (MDRD) and the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formulas. In statistical analyses U Mann-Whitney and Spearman correlation test as logistic regression analysis was used.

Results: 25.2% of studied patients were anemic. In all of them, estimated GFR (eGFR) was positively correlated with red blood cells (RBC) and platelet (PLT) count, and negatively correlated with mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC). All these correlations were statistically significant (p < 0.001) and independent of gender and used type of eGFR formula. In logistic regression analysis, lower eGFR strongly predicted lower RBC and PLT levels (p < 0.0001; OR 0.08, 95%CI: 0.03, 0.22 and OR 0.99, 95%CI: 0.987, 0.993, respectively).

Conclusions: Our findings suggest a strong association between CBC and renal function in ARV-treated HIV-infected patients who fulfilled the criteria of anemia. Consequently, eGFR in all HIV-infected subjects with anemia, especially on treatment with nephrotoxic drugs and concomitant thrombocytopenia, should be monitored more frequently then standardly recommended every 3-6 months.
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http://dx.doi.org/10.2174/157016212800618147DOI Listing
April 2012

Immunofluorescent localization of thymidylate synthase in the development of Trichinella spiralis and Caenorhabditis elegans.

Mol Biochem Parasitol 2012 May 13;183(1):63-9. Epub 2012 Feb 13.

Nencki Institute of Experimental Biology, Polish Academy of Sciences, 02-093 Warszawa, Poland.

Localization of thymidylate synthase protein in Trichinella spiralis and Caenorhabditis elegans development was followed with the use of confocal microscopy, revealing similar expression patterns in both nematode species. In T. spiralis premature muscle larvae and C. elegans dauer, L3 and L4 larvae, thymidylate synthase was detected in the nerve ring and gonad primordia, as well as T. spiralis stichosome and C. elegans pharyngeal glandular cells. In developmentally arrested T. spiralis muscle larvae, the enzyme was found localized to the gonad primordia and stichosome. High enzyme level was also observed in the embryos developing in uteri of T. spiralis female adult and C. elegans hermaphrodite forms. In the case of T. spiralis adult forms, thymidylate synthase was detected in stichosome, along esophagus wall, as well as in egg and sperm cells. While the enzyme protein present in the embryos remains in accord with its known association with proliferating systems, thymidylate synthase presence in the nerve ring, and reproductive and secretory (T. spiralis stichosomal and C. elegans pharyngeal glandular cells) systems, points to a state of cell cycle-arrest, also known to preserve the enzyme protein.
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http://dx.doi.org/10.1016/j.molbiopara.2012.02.002DOI Listing
May 2012

Plasma HIV-RNA is the key determinant of long-term antibody persistence after Yellow fever immunization in a cohort of 364 HIV-infected patients.

J Acquir Immune Defic Syndr 2012 Apr;59(4):360-7

Service des maladies infectieuses et tropicales, Hôpital Saint-Antoine, AP-HP, Paris, France.

Background: In HIV-infected patients, data on immunogenicity of Yellow fever immunization are scarce, and there is conflicting evidence of the influence of CD4 T-cell count and plasma HIV RNA on neutralizing antibody titer (NT) after vaccine injection.

Methods: In this prospective cohort study, NT was measured in all consecutive HIV outpatients who had previously received at least 1 injection of Yellow fever vaccine. Risk factors for vaccine failure (NT < 1:10) and magnitude of NT according to dates of HIV diagnosis and immunization were assessed by logistic regression and general linear models.

Results: Among 364 included patients, 24 (7%) had NT <1:10 after a mean delay of 8.4 years after immunization. Among patients immunized after HIV diagnosis (n = 240), NT <1:10 was associated only with detectable plasma HIV RNA at immunization. Among 79 patients with primary vaccination after diagnosis of HIV infection, higher HIV RNA at immunization was the unique independent risk factor for NT <1:10 [adjusted odds ratio (OR) = 3.73 per log10, 95% confidence interval (CI): 1.14 to 12.28]. Lower values of NT were independently associated with a shorter duration of undetectable plasma HIV RNA (OR = 1.05 per year, 95% CI: 1.005 to 1.09) and higher plasma HIV RNA (OR = 0.91 per log10, 95% CI: 0.84 to 0.99) at immunization.

Conclusions: The key determinant of antibody response was the HIV replication status at immunization. No association was found between antibody response and CD4 T-cell count.
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http://dx.doi.org/10.1097/QAI.0b013e318249de59DOI Listing
April 2012

Functional gene expression profile underlying methotrexate-induced senescence in human colon cancer cells.

Tumour Biol 2011 Oct 16;32(5):965-76. Epub 2011 Jun 16.

Nencki Institute of Experimental Biology, Polish Academy of Sciences, Pasteura 3, 02-093, Warsaw, Poland.

Cellular functions accompanying establishment of premature senescence in methotrexate-treated human colon cancer C85 cells are deciphered in the present study from validated competitive expression microarray data, analyzed with the use of Ingenuity Pathways Analysis (IPA) software. The nitrosative/oxidative stress, inferred from upregulated expression of inducible nitric oxide synthase (iNOS) and mitochondrial dysfunction-associated genes, including monoamine oxidases MAOA and MAOB, β-amyloid precursor protein (APP) and presenilin 1 (PSEN1), is identified as the main determinant of signaling pathways operating during senescence establishment. Activation of p53-signaling pathway is found associated with both apoptotic and autophagic components contributing to this process. Activation of nuclear factor κB (NF-κB), resulting from interferon γ (IFNγ), integrin, interleukin 1β (IL-1β), IL-4, IL-13, IL-22, Toll-like receptors (TLRs) 1, 2 and 3, growth factors and tumor necrosis factor (TNF) superfamily members signaling, is found to underpin inflammatory properties of senescent C85 cells. Upregulation of p21-activated kinases (PAK2 and PAK6), several Rho molecules and myosin regulatory light chains MYL12A and MYL12B, indicates acquisition of motility by those cells. Mitogen-activated protein kinase p38 MAPK β, extracellular signal-regulated kinases ERK2 and ERK5, protein kinase B AKT1, as well as calcium, are identified as factors coordinating signaling pathways in senescent C85 cells.
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http://dx.doi.org/10.1007/s13277-011-0198-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3156317PMC
October 2011

Comparative analysis of the new chronic kidney disease epidemiology collaboration and the modification of diet in renal disease equations for estimation of glomerular filtration rate in HIV type 1-infected subjects.

AIDS Res Hum Retroviruses 2011 Aug 2;27(8):809-13. Epub 2011 Mar 2.

Department of Hepatology and Acquired Immunodeficiences, Warsaw Medical University, Poland.

Recently, a new Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula was presented as a better alternative to the modification of diet in renal disease (MDRD) formula for GFR estimation (eGFR) in patients with relatively well-preserved kidney function. The main objective of our study was to compare the eGFR results arrived by the new CKD-EPI to the older MDRD equation in antiretroviral (ARV)-naive and ARV-treated HIV-1-infected patients. The study was performed in 287 adult HIV-1-infected patients and was an evaluation comparing eGFR results based on age, gender, race, and serum creatinine. The biggest difference in estimated glomerular filtration rate (eGFR) measured by the two formulas was seen in ARV-naive men with well-preserved kidney function (p = 0.001). Moreover, we found a significant negative correlation between mean difference in eGFR measured by the two equations and the age of the studied subjects (r = -0.37, p < 0.001). No correlation was observed between mean difference in eGFR and HIV viral load (r = -0.15, p = 0.2). Independent of the equation used, a significant decrease of eGFR in ARV-treated in comparison to ARV-untreated HIV-1-infected patients was seen (p < 0.001). In conclusion, in HIV-1-infected subjects, especially in ARV-naive men with well-preserved kidney function, eGFR measured by MDRD and CKD-EPI formulas varies strongly following the method used. Such discrepancies may be important in everyday clinical practice and must be confirmed by additional studies using GFR measured with a reference method.
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http://dx.doi.org/10.1089/AID.2010.0233DOI Listing
August 2011

Thyroid dysfunction in antiviral therapy of chronic hepatitis C.

Hepatogastroenterology 2010 Jul-Aug;57(101):826-31

Department of Infectious Diseases and Hepatology, Medical University of Bialystok, 14 Zurawia Str, 15-540 Bialystok, Poland.

Background/aims: Thyroid disorders are common dysfunctions during HCV infection and IFN-therapy. The aim of the study was to evaluate the general risk of thyroid disorders' development in chronic HCV-infected patients and their possible relationship with HCV genotype, type of used IFN-alpha and viral response.

Methodology: A study was performed in 89 patients with chronic hepatitis C (57 males, 32 females) and 25 healthy subjects. All HCV-infected patients were treated by IFN-alpha and RBV for a period depending on the HCV genotype. Hematological parameters, liver and thyroid function tests were analyzed. Serum Tpo-Ab and TG-Ab were detected by semiquantitative ELISA. In statistical analyses U Mann-Whitney and Spearman correlation tests were used. Ap value < 0.05 was considered significant.

Results: Twelve (13.5%) CHC patients developed thyroid disorders during IFN-alpha therapy. Thyroid disorders were more often detected in females, in 3a-genotype-infection and in patients treated with natural leukocyte IFN-alpha. Seven (7.6%) Tpo-Ab and/or TG-Ab positive patients were noticed. Four of them (57.1%) developed hypothyroidism during IFN-alpha therapy.

Conclusion: This study demonstrated low effect of IFN-alpha therapy on thyroid disorders development and on TPO-Ab and TG-Ab serum level in chronic HCV-infected patients.
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January 2011