Abstract: AIM: Plasminogen activator inhibitor type 1 (PAI-1) plays a key role in regulation of fibrinolytic system, cell-associated proteolysis and migration of smooth muscle cells (SMC). This study is focused on the types of PAI-1 expressing cells, quantification of PAI-1 expression in the walls of aneurysmatic abdominal aortas (AAA) and correlation between histological and clinical findings. METHODS: A group of nine patients who underwent surgery for AAA: asymptomatic (aAAA), symptomatic (sAAA) and ruptured (rAAA) and one control specimen (CA) were included in the study. Samples underwent histological processing and immunohistochemistry in comparison with in situ hybridisation. In order to assess the PAI-1 area fraction in histological sections through the aortic wall the Line System module of Ellipse software was used. PAI-1 expressing cells were measured in CA and AAA: endothelium, SMC, and foam cells. Photomicrographs with a total area of 0.7 mm(2) for each specimen were analysed by two independent observers. Mean values of PAI-1 positive components per section area were calculated as average values. RESULTS: The results of both observers are as follows: 28.6% in CA; 18.1% in aAAA; 10.9% in sAAA; 11.0% in rAAA. During the progression of AAA, the SMC (PAI-1 expression was found mainly in them) became less abundant in agreement with the values of PAI-1 area fraction. In rAAA immunohistochemistry detected PAI-1 in necrotic centres of atheromathous plaques. CONCLUSIONS: AAA may be evaluated as the result of gradual changes in regulation of fibrinolysis that is observed as redistribution of cells expressing PAI-1. The area fraction of PAI-1 positive components correlates with clinical classification of AAA.

Abstract: OBJECTIVE: To analyze methodological influences and characterize the concentrations of cell-free fetal DNA (cffDNA) circulating in maternal plasma at different gestational ages in physiological pregnancies. METHODS: We investigated 238 independent samples from single male-bearing pregnancies of different gestation age. In the other 50 pregnancies, the samples were collected three times during pregnancy (at all trimesters) to evaluate the kinetics of cffDNA. The manual and automated DNA extraction methods (Roche) were compared. cffDNA was amplified using real-time PCR method and Y-specific sequences SRY and DYS14. Total cell-free DNA circulating in maternal plasma was determined by the use of the GADPH sequence. RESULTS: The elevation in the concentration of cffDNA during pregnancy with the highest value in the third trimester was observed independently on the DNA extraction method and on the Y-specific amplified sequence. The same is documented for the percentage of fetal DNA in total cell-free DNA in maternal plasma. It increases also in successive trimesters (8.3, 10.7 and 23.2%). CONCLUSIONS: We discuss methodological problems and describe statistical parameters of cffDNA concentrations in maternal plasma during pregnancy as the basic information for comparison with pregnancies having a pathological outcome.

Abstract: The mechanisms of clearance of circulating plasma DNA are not fully understood, and so we aimed to examine it in patients with impaired renal function compared with healthy individuals. We also assessed the effect of peritoneal dialysis and hemodialysis on circulating plasma cell-free DNA (cfDNA) in our treated patients. Overall, 20 healthy volunteers, 20 patients with chronic kidney disease (CKD), 18 patients undergoing peritoneal dialysis (PD), and 17 patients on hemodialysis (HD; high-flux polysulfone membrane) were examined. Cell-free DNA levels were determined using real-time GADPH gene sequence amplification. The levels of cfDNA in all groups of our patients did not differ significantly from those of healthy volunteers. In HD patients, cfDNA levels were significantly increased compared with those of CKD patients (P < 0.05) and PD-treated patients (P < 0.01). In PD-treated patients, cfDNA was detectable in overnight effluent, with its levels correlating inversely with the duration of PD treatment (r=-0.619, Spearman's coefficient, P= 0.008). Factors contributing to these differences may include changes in the quality and quantity of the cell population of the peritoneum, highlighting the need for additional studies clarifying the dynamics of cfDNA during PD. The plasma levels of cfDNA do not seem to be dramatically altered even in CKD patients or those on PD or HD (as long as they are measured prior to the procedure in the latter two). Our data suggest renal elimination is not the main mechanism of circulating cfDNA clearance.

Abstract: Authors present contemporary knowledges, arguments and hypothesis about cancer STEM cells and their relations to current concepts of oncology and surgical oncology. The aim is to introduce new view of carcinogenesis and origin of metastatical process as a diseas of STEM cells to general surgical public that deal with surgical oncology and is confronted with new trends in modern oncological pharmacology. The presented theory of tumour origin by cancer STEM cells is confronted critically with all current theories.

Abstract: Molecular genetic methods that use the polymerase chain reaction (PCR) are due their speed widely employed in diagnostic approaches in microbiology. In this report, the possibilities of application of these methods in medical mycology are discussed with regard not only to species identification, but also for genotyping of strains for epidemiological purposes. Recently, a tendency to exploit molecular genetic methods rather for epidemiological studies than for routine species identification may be observed. With regard to the high inter-species variability, careful standardization using samples of isolates of the tested species from corresponding geographical origin is necessary. Perspectives of future development associated with the explanation of molecular biological relations between human tissues and the pathogen, with the recognition of mechanisms of virulence and resistance to antifungal drugs are discussed.

Abstract: To evaluate the significance of Epstein-Barr virus (EBV) infection in tumorogenesis, we examined ten archival samples of acinic cell carcinomas of salivary glands with lymphoid-rich stroma, four archival samples of lymphoepithelioma-like carcinomas (LELC) of the urinary bladder, ten samples of oncocytic papillary carcinoma of the thyroid (Warthin-like tumors) and one sample of lymphoepithelioma-like carcinoma of the cervix, together 25 paraffin-embedded tumor tissues. Polymerase chain reaction (PCR) and in situ hybridization (ISH) assays were used. The EBV genome was detected by PCR using primers targeting the IR region. ISH was performed using EBER oligonucleotide probes. Each examination was repeated two times. Positive PCR result was obtained in 12% of samples only. However, this result was not confirmed by the subsequent second PCR examination. ISH revealed negative signals in all samples. Our results demonstrate the importance of the diagnostic strategy based on combination at least two independent methods. PCR due to its sensitivity may produce false positive results depending on the degree of infiltration the tumor sample by EBV carrying lymphocytes.

Abstract: Restriction fragment length polymorphism analysis of the 5.8S rRNA gene and the internal transcribed spacers (ITS1 and ITS2) was used for examination of 66 isolates belonging to 19 species. Intraspecies variability was found in the examined region of 11 species (Candida albicans, C. catenulata, C. colliculosa, C. glabrata, C. kefyr, C. melinii, C. parapsilosis, C. guillermondii, C. solanii, C. tropicalis, Saccharomyces cerevisiae). Region of ITS-5.8S rDNA was amplified using the primers ITS1 and ITS4. The amplicons were digested by HaeIII, HinfI and CfoI. The recognized intraspecies variability was confirmed in the second step, in which the shorter fragments of this region were amplified using primers ITS1 and ITS2 and analyzed by capillary electrophoresis. Considerable intraspecific variability renders this method unsuitable for species identification, whereas it can be useful for epidemiological tracing of isolates.

Abstract: AIMS: We report the clinicopathological and immunohistochemical characteristics of 12 cases of a recently recognized entity, oncocytic papillary thyroid carcinoma (PC) with lymphoid stroma (Warthin-like tumour). Methods and results: The cases were retrieved from the surgical pathology files of our departments. There were 11 female patients and one male patient; they ranged in age from 45 to 85 years (mean 64.2 years). The immunohistochemical profile demonstrated positivity of tumour cells for cytokeratins, thyroglobulin, Leu-M1 and anti-mitochondrial antigen. S100 protein-positive stromal dendritic/Langerhans cells were uniformly present. Polymerase chain reaction, in situ hybridization, and immunohistochemistry for Epstein-Barr virus (EBV) detection revealed no significant positive signal. MIB-1 labelling index was low, compatible with that of 'classical' PC. CONCLUSIONS: Warthin-like tumour is a rare variant of PC, occurring predominantly in elderly women. Its histological features are distinct and well recognizable, differentiating this tumour from a more aggressive tall-cell variant of PC. The apparent indolent behaviour seems to be consistent with the presence of dendritic/Langerhans cells and with low proliferative activity. A possible role of EBV in pathogenesis of this lesion was not proven. Further studies are necessary to determine the prognosis and metastatic potential of this neoplasm.

Abstract: Today, polymerase chain reaction is a common part of approaches serving for identification of individuals in legal medicine. This method is easily practicable, however attention must be paid to the optimization of reaction conditions and to the interpretation of results. From the literature, such cases are known, in which during amplification of extremely small amount of DNA (e.g. from one cell) the polymerase chain reaction preferably amplifies only one of two in the template DNA present alleles. If the amplified fragments differ in length, the shorter one is amplified preferably, and it may be cause of false results. In the presented study, DNA from 23 stains of male blood on different fabrics was isolated by two different methods (by treatment with proteinase K and boiling and by treatment with Chelex 100). The obtained DNA samples were amplified using primers, they are complementary to the amelogenin gene sequences. The system is suitable for sex determination, because amplification of the X-chromosomal sequence provides a fragment in length of 632 bp, amplification of the Y-chromosomal one a fragment in length of 443 bp. The isolation method based on proteinase K led in 17.38% of samples to the very intensive preferential amplification of the longer allele, and therefore to a false result. The isolation method based on Chelex 100 provided in all cases correct results with clearly recognizable preferential amplification of the shorter allele. The reported results accentuate the meaning of choice of the appropriate isolation method, the need of accurate PCR optimization, and the careful interpretations of its outputs.

Abstract: During elucidation of crime cases, the need of a sample of DNA of the victim of the violent attack appears very often. Regarding to the obligatory archiving of histopathological preparations from sectioned persons, the paraffin-embedded tissues are easily achievable material suitable for the given purpose. Such a modification of an isolation method was elaborated which allows the yield of the sufficient amount of DNA from one histological section, and its usefulness was tested on tissues of different types and from different persons. The method was used successfully during solving of concrete cases.

Abstract: Polymerase chain reaction is often used for molecular genetic human identification in forensic medicine today. In this study, the possibilities are demonstrated, which are provided by the method in such cases, where it is necessary to determine, if the biological traces are of animal or human origin. The method is rapid and well performed in a molecular genetic laboratory with standard equipment. Eleven samples of DNA isolated from different animals and birds were examined. In all cases, it was possible to distinguish these samples according to the results of reaction from simultaneously examined human DNA.