Andrzej Wiczkowski

The Usefulness of Calyculin A for Cytogenetic Prenatal Diagnosis

An increased number of chromosome plates can be obtained by use of calyculin A (CLA). CLA is an inhibitor of protein phosphatases (type 1 and type 2A serine/threonine). Inactivation of these phosphatases leads to premature chromosome condensation (PCC) in all phases of the cell cycle; thus, it is possible to investigate both metaphase and G2-PCC chromosomes. Amniotic fluid (AF) cultures were treated with calyculin A (CLA). GTG banding was obtained. Using this method it is possible to investigate all cell cycle phases, GTG banding, chromosomal breaks, and rates of PCD on the same preparation. Analyses of AF cultures treated with CLA allow complex studies on fetal genetic material. This work presents potential usefulness of CLA for cytogenetic prenatal diagnosis.

sPECAM-1 and sVCAM-1: role in pathogenesis and diagnosis of chronic hepatitis C and association with response to antiviral therapy:

Aim: To analyze the relationship between pretreatment clinical or histological features and the levels of soluble platelet-endothelial cell adhesion molecule-1 (sPECAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1), to determine their serum concentration in responders and nonresponders, to evaluate the behavior under antiviral therapy, to explain their relationship in response to therapy and to assess the association between these two molecules in chronic hepatitis C (CHC). Methods: The study analyzed 65 CHC patients, including 50 patients (Group 1) with marked fibrosis treated with peginterferon plus ribavirin, 15 patients without fibrosis (Group 2) and 13 healthy volunteers (the control group, Group 3). sPECAM-1 and sVCAM-1 levels were assessed by an immunoenzymatic method (ELISA) before and after therapy. Results: sVCAM-1 and sPECAM-1 serum concentrations increased significantly in CHC patients (p<0.001). sPECAM-1 levels corresponded to inflammatory grade (p = 0.03) and fibrosis stage (p = 0.01). sVCAM-1 increased only in advanced fibrosis. After therapy, sPECAM-1 levels decreased significantly (p<0.001) with no difference between responders and nonresponders. sPECAM-1 correlated positively with inflammatory activity (p = 0.02), fibrosis stage (p<0.001), sVCAM-1 (r = 0.56, p<0.001) and alanine aminotransferase activity (r = 0.30, p = 0.05). Receiver operating characteristic curve analysis showed a good discriminant power of serum sPECAM-1 concentrations for detection of liver fibrosis — stage 0 versus stage 1—3, AUC 0.81; cut-off 221.0 ng/ml and a fair discriminant power for distinguishing bridging fibrosis, AUC 0.78; cut-off 237.1 ng/ml. Conclusions: Hepatitis C virus (HCV) infection results in upregulation of sPECAM-1 and sVCAM-1. sPECAM-1 levels are related to necroinflammatory activity and may also identify patients with advanced fibrosis. The sPECAM-1 value was decreased by therapy but its measurement cannot predict therapy outcome and confirm HCV persistence. sPECAM-1 may influence VCAM-1 expression.

Role of nitric oxide in physiology and pathology of the gastrointestinal tract.

In this paper the physiological role of NO and isoforms of NOS in the gastrointestinal tract and the involvement of NO in pathological processes of digestive tract as well as the perspective of therapeutic use of NO-donating drugs and selective inhibitors of phosphodiesterase in the treatment of gastric diseases were presented.

The platelet-to-lymphocyte ratio as a predictor of all-cause mortality in patients with coronary artery disease undergoing elective percutaneous coronary intervention and stent implantation

Background There is no data regarding the association between the platelet-to-lymphocyte ratio (PLR) and long-term mortality in patients with stable coronary artery disease (SCAD). The aim of this study is to evaluate the utility of the pre-procedural PLR for predicting long-term, all-cause mortality in patients with SCAD undergoing percutaneous coronary intervention (PCI) and stent implantation. Methods We analyzed a total of 2959 consecutive patients with SCAD who underwent PCI (balloon angioplasty followed by stent implantation or direct stenting) between July 2006 and December 2011 at our institution. The patients were stratified into tertiles according to their admission PLR. The association between the PLR value and the outcomes was assessed using Cox proportional regression analysis after adjusting for clinical angiographic and laboratory data. Results During median follow-up of 1124 days, mortality was highest in patients with PLR within the 3rd tertile as compared to the 2nd and the 1st tertile (11.0% vs 8.7% vs. 9.6%, respectively, p = 0.03). PLR remained associated with mortality in multivariable analysis including clinical variables, ejection fraction and angiographic parameters HR (per 10 units increase) = 1.02 [95%CI,1.01 ÷ 1.04, p = 0.006]. After adjustment for the eGFR and hemoglobin levels, PLR was however no longer significantly associated with mortality. Conclusion PLR has potential predictive value in patients with SCAD, which has not been reported previously, but statistical significance disappears after adjusting for estimated glomerular filtration rate (eGFR) and hemoglobin levels as a potential confounding variable.

Hepatic Chemerin and Chemokine-Like Receptor 1 Expression in Patients with Chronic Hepatitis C

Introduction. Chemerin seems to be involved in pathogenesis of chronic hepatitis C (CHC). Hepatic expressions of chemerin and its receptor, chemokine receptor-like 1 (CMKLR1), in CHC have not been studied so far. Aim. To evaluate chemerin and CMKLR1 hepatic expression together with serum chemerin concentration in CHC patients and to assess their relationship with metabolic and histopathological abnormalities. Methods. The study included 63 nonobese CHC patients. Transcription of chemerin and CMKLR1 was assessed by quantitative real-time PCR, while serum chemerin was assessed by enzyme-linked immunosorbent assay. Results. Expression of chemerin and CMKLR1 was present in the liver of all CHC patients regardless of sex or age. This expression was not associated with necroinflammatory activity and steatosis grade, fibrosis stage, and metabolic abnormalities. There was a negative association between serum chemerin and chemerin hepatic expression (r = (−0.41), P = 0.006). Conclusion. The study for the first time confirmed a marked expression of chemerin and CMKLR1 in the liver of CHC patients. The study was performed using the homogenates of human liver tissue, so it is not possible to define whether hepatocytes or other cell types which are abundantly represented in the liver constitute the main source of chemerin and CMKLR1 mRNA.

8-hydroxy-2′-deoxyguanosine in colorectal adenocarcinoma – is it a result of oxidative stress?

Background 8-hydroxy-2′-deoxyguanosine (8-OHdG) is one of the most abundant oxidatively modified lesions in DNA and is a marker of the oxidative stress. 8-OHdG is a mutagenic lesion and it can mispair with adenine, causing G: C→T: A transversion. Our task was to determine the 8-OHdG level in patients with colorectal adenocarcinoma directly in tumor tissues and corresponding normal mucosa. Material/Methods Samples of tumor tissues and corresponding normal mucosa of 47 patients undergoing surgery for colorectal cancer were analyzed. DNA was isolated from both tumor and normal tissues. Then, DNA was hydrolyzed to nucleotides using nuclease P1 and alkaline phosphatase. The 8-OHdG and 2′-dG (2′-deoxyguanosine) were determined in hydrolysates by high-performance liquid chromatography (HPLC) with electrochemical (EC) and UV detector. Results The levels of 8-OHdG in colorectal adenocarcinoma tissues were higher than in corresponding normal mucosa. No significant differences were shown in 8-OHdG levels in the cancerous and cancer-free tissues between age and sex and stages A/B and C/D of Duke’s classification. Conclusions 8-OHdG reflects the local oxidative stress in colon adenocarcinoma tissue together with ageing processes, but not the intensity of tumorigenesis itself. Because of many factors that could influence the oxidative modification of DNA bases, its role as a diagnostic and/or prognostic factor in colon adenocarcinoma seems to be limited.

Prenatal priming of cord blood T lymphocytes by microbiota in the maternal vagina.

Problem  In the vagina of women at the reproductive age, more than 170 strains of bacteria and yeasts are found. The effect of vaginal flora on neonatal T cells is yet to be investigated.

Functional polymorphism rs710218 in the gene coding GLUT1 protein is associated with in-stent restenosis

AIM To analyze the association between in-stent restenosis (ISR) and polymorphisms in genes coding IGF-1, IGFBP3, ITGB3 and GLUT1, which play an important role in the smooth muscle cell proliferation and extracellular matrix synthesis - the main components of neointima. MATERIALS & METHODS We analyzed 265 patients who underwent bare metal stent implantation. RESULTS The differences in the occurrence of ISR between genotypes of the analyzed polymorphisms in the IGF-1, IGFBP3 and ITGB3 were not statistically significant. The T/T genotype of the rs710218 polymorphism in the GLUT1 (SLC2A1) gene was more common in the ISR group compared with non-ISR patients (81.1 vs 64.8%; p = 0.02). In a multivariable model the A/A and A/T genotype remained correlated with lower occurrence of ISR (odds ratio: 0.45; 95% CI: 0.21-0.97; p = 0.03). CONCLUSION The rs710218 polymorphism in the gene coding GLUT1 protein is a novel risk factor for ISR.

Selected mechanisms of molecular resistance of Candida albicans to azole drugs

A phenomenon of increasing resistance of Candida spp. to azoles has been observed for several years now. One of the mechanisms of lack of sensitivity to azoles is associated with CDR1, CDR2, MRD1 genes (their products are active transport pumps conditioning drug efflux from pathogens cell), and ERG11 gene (encoding lanosterol 14α-demethylase). Test material was 120 strains of Candida albicans (60 resistant and 60 susceptible to azole drugs) obtained from clinical samples. The first stage of experiment assessed the expression of CDR1, CDR2, MDR1 and ERG11 genes by Q-PCR. The impact of ERG11 genes mutations on the expression of this gene was analysed. The final stage of the experiment assessed the level of genome methylation of Candida albicans strains. An increase in the expression of CDR2, MDR1 and ERG11 was observed in azole-resistant strains of Candida albicans in comparison to strains sensitive to this class of drugs. Furthermore, 19 changes in the sequence of ERG11 were detected in tested strains. Four of the discovered mutations: T495A, A530C, G622A and A945C led to the following amino acid substitutions: D116E, K128T, V159I and E266D, respectively. It has also been found that statistically five mutations: T462C, G1309A, C216T, C1257T and A945C affected the expression of ERG11. The applied method of assessing the level of methylation of Candida albicans genome did not confirm its role in the development of resistance to azoles. The results indicate however, that resistance of Candida albicans strains to azole drugs is multifactorial.

Oxidative damage and carcinogenesis

Oxygen is an essential element to conduct life processes but some of the metabolic byproducts e.g. reactive oxygen species (ROS), are toxic for living organisms. Endogenous ROS are produced e.g. reduction of dioxygen; some exogenous sources of radicals also exist, including nicotine and ionizing radiation. Reactive oxygen species include superoxide anion, hydroxyl radical, singlet oxygen, hydrogen peroxide and hypochlorous acid. Carcinogenesis is a multistep process. The exact reasons for the development of cancer are still unknown. Many factors contribute to the development of carcinogenesis, one of which is oxidative stress. Oxidative stress is defined as an imbalance between oxidizing agents (pro-oxidants) and antioxidants, agents that protect biomolecules against injury by pro-oxidants. When reactive oxygen species are overproduced it can damage nucleic acids, proteins and lipids. ROS are considered as a significant class of carcinogens participating in cancer initiation, promotion and progression.