Th e role of CDK12 in tumor bio logy.

BACKGROUND: Physiological function of cyclin-dependent kinase 12 (CDK12) is crucial for several cellular processes, including regulation of transcription, RNA splicing, transcription termination and polyadenylation. It is well documented by now that CDK12 controls transcription of the unique set of genes involved in DNA-damage response, replication of DNA and response to cellular stress. Just recently, a key function of CDK12 in the induction of tandem duplication of specific DNA sequences within the metastatic castrate resistant prostate tumors has been documented. Therefore, it is possible to recognize CDK12 as a tumor suppressor; nevertheless, there is a growing body of evidence that CDK12 can support tumor growth under specific circumstances and thus act as a tumor oncogene. CDK12 therefore represents an alternative dia-gnostic approach for breast, ovarian and prostate tumors, especially when conventional treatment is not active and there is a need for more effective approaches, such as concept of synthetic lethality. METHODS: The discussed scientific papers can be reached at the PubMed and Scopus databases before 1th of April 2020. PURPOSE: The aim of the review is to summarize current knowledge relevant to the function of CDK12 as a tumor suppressor or oncogene in various tumors and to discuss the use of specific CDK12 inhibitors for patient treatment. At the end of the article, we discuss the potential use of CDK12 in the treatment of specific tumors by its targeted inhibition in monotherapy or in combination with poly (ADP ribose) polymerase 1 (PARP1) and checkpoint kinase 1 (CHK1) inhibitors. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.

Evaluation of the DOAC-Stop Procedure by LC-MS/MS Assays for Determining the Residual Activity of Dabigatran, Rivaroxaban, and Apixaban.

The effect of direct oral anticoagulants (DOACs) on laboratory tests dependent on the production of their targets, factor IIa and factor Xa (FXa), is a well-known problem and can cause both false positive and negative results. Therefore, the correct interpretation of tests performed in patients receiving DOACs is necessary to avoid misclassification and subsequent clinical consequences. However, even with significant experience, there are situations where it is not possible to assess the influence of some methods. Particularly important is the situation in the diagnosis of lupus anticoagulants using the dilute Russell viper venom timetest, which is based on direct FXa activation. A very promising solution to this situation is offered by the DOAC laboratory balancing procedure DOAC-Stop. For evaluating the effectiveness of this procedure, 60 (20 apixaban, 20 dabigatran, and 20 rivaroxaban) patients treated with DOACs were enrolled. All patient samples were analyzed for the presence of individual DOAC types and subsequently subjected to the DOAC-Stop procedure.We evaluated its effectiveness by our own high-performance liquid chromatography-coupled tandem mass spectrometrymethod, which simultaneously sets all high-sensitivity DOACs. Unlike coagulation tests based on the determination of the residual effects of DOACs on target enzymes, which is complicated by extensive interindividual variation, this methodology is highly specific and sensitive.The DOAC-Stop procedure eliminated dabigatran from 99.5%, rivaroxaban from 97.9%, and apixaban from 97.1% of participants in our group. Residual amounts did not exceed 2.7 ng/mL for dabigatran, 10.9 ng/mL for rivaroxaban, or 13.03 ng/mL for apixaban, which are safe values that do not affect either screening or special coagulation tests.

Hematological findings in non-treated and gamma-irradiated mice deficient for MIC-1/GDF15.

Several members of the TGF-beta family are known to effectively regulate the fate of hematopoietic progenitor cells in a complex and context-dependent manner. Growth differentiation factor-15 (GDF15) is a divergent member of the TGF-beta family. This stress-induced cytokine has been proposed to possess immunomodulatory functions and its high expression is often associated with progression of a variety of pathological conditions. GDF15 is also induced by chemotherapy and irradiation. Very few fundamental studies have been published regarding the effect of GDF15 in hematopoiesis. In this study, we analyzed the hematological status of untreated and gamma-irradiated mice deficient for GDF15 as a result of genetic knock-out (KO), in order to clarify the regulatory role of GDF15 in hematopoiesis. Significant differences between GDF15 KO mice and their pertinent WT controls were found in the parameters of blood monocyte numbers, blood platelet size, and distribution width, as well as in the values of bone marrow granulocyte/macrophage progenitor cells. Different tendencies of some hematological parameters in the GDF15 KO mice in normal conditions and those under exposure of the mice to ionizing radiation were registered. These findings are discussed in the context of the GDF15 gene function and its lack under conditions of radiation-induced damage.

[High-Resolution MR Examination of the Scapholunate Ligament using a Microscopic Coil: Comparison with Direct MR Arthrography and Arthroscopy Findings]. / MR vysetrení skafolunátního vazu ve vysokém rozlisení s pouzitím mikroskopické cívky: srovnání s prímou MR artrografií a artroskopickými nálezy.

PURPOSE OF THE STUDY Scapholunate dissociation is a clinically most frequently diagnosed form of carpal instability. The aim of this study was to compare high resolution MRI using a microscopic coil with direct MRI arthrography in patients with suspected scapholunate ligament lesions and compare the results with arthroscopy findings and Geissler's arthroscopy classification. MATERIAL AND METHODS A prospective study was carried out in 47 patients (average age, 30.7 years) with clinical symptoms of wrist instability from 2013 to 2014. The patients were examined with the MR device Philips Achieva 1.5T using a microscopic coil and subsequently by direct MR arthrography. The results of examination were evaluated independently by two groups of physicians using a modified arthroscopic classification. The results were verified arthroscopically. For evaluation, an adjusted Geissler's classification was used. The study was approved by the Multicentre Ethics Committee of the Faculty of Medicine in Brno and informed consent was obtained from each patient. RESULTS A total of The MRI examination was evaluated and included in the study in 44 patients (three were excluded for the presence of motion artefacts). Only 20 patients underwent arthroscopy. Examination with a microscopic coil correctly classified 14 of them; an accuracy of 70 % (95 % CI: 45.7 % - 88.1 %) and p = 0.021. Direct MR arthrography correctly classified 16 of 20 injured ligaments, i.e., an accuracy of 80 % (95 % CI: 56.3 % - 94.3 %) and p = 0.002. DISCUSSION Currently, the diagnosis of pathological changes in the wrist is made by routine MRI especially when there is the possibility of using sequences with high spatial resolution. Even though we achieved poorer results by native examination using these techniques, when they were compared with the results of direct MR arthrography, they were still better than those reported in the recent literature. CONCLUSION The optimal method for an examination algorithm of scapholunate ligament lesions is direct MR arthrography. In our study correct findings of direct MR arthrography using Geissler's classification were shown in 80 % of the patients. Key words: scapholunate ligament, scapholunate ligament lesion, direct MR arthrography, microscopic coil, Geissler's classification.

Validation of a New Panel of Automated Chemiluminescence Assays for Anticardiolipin Antibodies in the Screening for Antiphospholipid Syndrome.

BACKGROUND: Antibodies anticardiolipin (aCL) and anti-ß2-glycoprotein I (aß2GPI) are two of three laboratory criteria of antiphospholipid syndrome (APS). All of assays of antiphospholipid antibodies (aPL), coagulation assays as well as ELISAs, show methodological shortcomings, that affect their sensitivity and specificity. Therefore, we decided to validate these parameters for a new chemiluminescent examination (CLIA). METHODS: aCL and aß2GPI antibodies were measured by ELISAs (AIDA, Bad Kreuznach, Germany) and aß2GPI with CLIA kits (Werfen, Barcelona, Spain). RESULTS: When we evaluated both assays, the coefficient of variation for CLIA was slightly lower (9.04 - 12.74%) than for ELISA (11.05 - 15.3%) and the LOD was 0.2 U/L. The dilution series showed significant linearity for all CLIA methods, aCL IgG, aCL IgM, aß2GPI IgG, and aß2GPI IgM (0 - 3000 U/L), and method comparison studies revealed good agreement with the currently used ELISA (Kappa values ranging 0.534 - 0.936) without determination of aß2GPI IgG. The determination aß2GPI IgG by CLIA method shows higher positivity in 31 samples. These new aCL IgG, aCL IgM, aß2GPI IgG, and aß2GPI IgM tests have excellent analytical characteristics and allow fully automated and simultaneous analysis on an analyzer. CONCLUSIONS: Chemiluminescent determination of an automated analyzer can improve the fundamental parameters of tests such as reproducibility between laboratories.

[Differential diagnosis of the viral etiology of suspected mumps in a population with high vaccine coverage]. / Diferenciální diagnostika virové etiologie suspektních príusnic ve vysoce proockované populaci.

AIM: In this study, buccal swabs from patients with the clinical picture of parotitis epidemica in whom mumps virus (MV) infection was not confirmed by direct detection or serologically were tested. The aim was to detect by molecular methods nucleic acids (NAs) of other respiratory viruses possibly involved in salivary gland swelling. At the same time, paired sera, if available, were tested. MATERIAL AND METHODS: The study group consisted of 72 buccal swabs from patients of the Clinic of infectious, tropical, and parasitic diseases, Na Bulovce Hospital. Paired sera were available from ten patients. Samples were collected in 2013 to 2015. Buccal swabs were tested by PCR for the presence of NAs of adenoviruses (AdV), bocaviruses (hBoV), parainfluenza viruses of types 1-4 (HPIV), human metapneumovirus (hMPV), coronaviruses (HCoV: NL63, OC43, HKU1, and 229E), respiratory syncytial virus (RSV), influenza A virus, influenza B virus, and Epstein-Barr virus (EBV). Paired sera were screened by the complement fixation test (AdV and influenza A and B viruses), hemagglutination inhibition test (HPIV types 2 and 3), ELISA (AdV, EBV), and immunofluorescence (EBV). RESULTS: NAs from viruses other than the mumps virus were detected in 27 of 72 patients with clinical symptoms of parotitis epidemica, and serological tests revealed etiological links with parainfluenza viruses in three more cases. Overall, 30 (41.7%) of 72 patients with suspected mumps tested positive for one or more viruses from the study panel. The most commonly detected viruses were AdV 11/72 (15.3%), EBV 9/72 (12.5%), and HPIV 3/72 (4.2%), but influenza A virus (H3N2) 1/72 (1.4%) was also found. Some patients tested positive for more than one virus: 2/72 (3%) for AdV plus hBoV and 1/72 (1.4%) for HPIV plus HCoV. In addition, examination of paired sera revealed HPIV positivity in three more patients. PCR and serology detected etiological link with HPIV in six (8.3%) of 72 patients tested. CONCLUSION: In our study group, nearly 42% of patients with the clinical picture of parotitis epidemica in whom mumps virus (MV) infection was not confirmed by direct detection or serologically tested positive for viruses other than the mumps virus. Thorough laboratory diagnosis of suspected mumps in vaccinated persons is important not only for the treatment of patients and adoption of isolation and other measures, but also for a better understanding of the epidemiology of the disease and outcomes of the immunisation programmes.

The advantages and limitations of impedance aggregometry in detection of heparin-induced thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) represents a serious complication of heparin treatment. IgG antibodies binding platelet factor 4 (PF4) and heparin trigger the clinical manifestations of HIT. However, only a portion of the antibodies have the ability to activate platelets, and these can be identified by a platelet aggregation test (functional testing). Current methods HIPA and SRA are time-consuming and difficult if HIT is clinically suspected; therefore, numerous new methods have recently been developed. METHODS: To determine HIT, impedance aggregometry using the Multiplate analyzer (MEA) as heparin-induced aggregation techniques and the Technozym HIT Ig ELISA test were used. The MEA method uses sensitization of donor platelets with patient plasma in the presence of heparin at a concentration of 0.5 IU/mL. The results were compared with the ELISA test. RESULTS: We examined 190 patients at clinically intermediate and higher risk of HIT according to the 4T score. All samples were examined by the ELISA test and MEA, with positive samples being further confirmed by high-concentration heparin. The methodology was modified with respect to the dilution for high positive samples and assessment has been extended to an index of inhibition. CONCLUSIONS: In the studied group, we demonstrated that MEA has sufficient sensitivity and higher specificity. In the group of patients, 10.0% showed positive results by MEA as compared with 7.3% determined by ELISA. Unlike the ELISA methods of the same quality, MEA is more suitable for detecting platelet-activating HIT antibodies in practice.

[Occurence, etiology and clinical significance of trombocytopenia in pregnancy]. / Incidence, etiologie a klinický význam trombocytopenie v gravidite

OBJECTIVE: The principal objective of the study is to compare results from the experimental group of pregnant women suffering from thrombocytopenia in pregnancy with results from the control group of pregnant women with normal physiologic blood platelet count. SETTING: Department of Obstetrics and Gynaecology of the Tomas Bata Regional Hospital Zlín, Obstetrics and Gynaecology Clinic, Haematology and Oncology Clinic of the Palacky University Teaching Hospital and Medical School in Olomouc, Obstetrics and Gynaecology Clinic of the Ostrava Teaching Hospital. METHODOLOGY: A group of 200 pregnant women suffering from thrombocytopenia underwent thorough medical tests. The level of platelets, presence of anti-platelets agents, liver function (LFT), anti-phospholipid antibodies, complete blood count with differential, specific antibodies for hepatitis B and C, Lyme borreliosis and cytomegalovirus were determined from venous blood using the EIA, ELISA methods. RESULTS: Medical articles and books about thrombocytopenia divide the causes for thrombocytopenia as follows: 79.5% benign gestational thrombocytopenia, 16% preeclampsia, 2.5% HELLP syndrome, 1% immune thrombocytopenia, 1% HVC. The number of women who developed physiological anaemia in pregnancy and were overweight is identical in the experimental group of pregnant women suffering from thrombocytopenia and in the control group of pregnant women with normal physiologic blood platelet count, and the proportion of the different age groups in the two groups of pregnant women is also identical. CONCLUSION: 32% of pregnancies in the experimental group ended in a caesarean section, of which 13.5% in a group of 127 pregnant women suffering from mild thrombocytopenia, 17.5% in a group of 71 pregnant women suffering from moderate thrombocytopenia and 1% in a group of 2 pregnant women suffering from severe thrombocytopenia. 20.5% pregnancies in the control group ended in caesarean section.

[Preeclampsia and thrombin generation test]. / Preeklampsie a trombin generacní test.

OBJECTIVE: Acquiring new information to allow prediction of the development of diseases associated with impaired coagulation. Design effective preventive measures most serious diseases (TEN) in the fields of gynecology and obstetrics. For pregnant women with preeclampsia, hypertension compared with women with normal pregnancies could lead to increased thrombin generation due to the synergistic effect of thrombotic risk factors. Based on the results and found statistically significant differences between the groups among pregnant can select for a higher risk of developing deep vein thrombosis. This risk group could then greatly benefit from more stringent follow-up and possible preventive treatment prophylactic doses of LMWH in reducing maternal and perinatal morbidity and mortality. DESIGN: Prospective study. SETTING: Department of Obstetrics and Gynecology, University Hospital Olomouc. METHODS: In early pregnancy - during pregnancy standard samples (up to the end of the first trimester) patients venous blood was sampled and they completed information questionnaire. A second sampling was carried out between 24 to 28 week, the third sample and between 36th to 40th week. Obtained blood samples were subsequently processed in the coagulation laboratory Hemato-Oncology Clinic and Olomouc. The blood samples were investigated protein C and S, antithrombin, FVIII level, FII, Leiden, and plasma endothelial microparticles, and lupus anticoagulant and APC resistance standardized methodologies. Thrombin generation was determined thrombin generation test. Thrombin generation was measured fully automatically using a kit (Technothrombin TGA, Technoclone, Vienna, Austria) and analyzer Ceveron Alpha (Technoclone, Vienna, Austria) with fully automatic analysis software. As the main parameter is evaluated by the maximum thrombin generation, at the same time, however, was also detected in the total amount of thrombin and the time until the beginning of the formation of thrombin. RESULTS: In the period 2008-2011 were analyzed blood samples of 303 healthy pregnant women. 215 women, ie 71% were nuliparas, 60 women, ie 19.8% were primiparas, 28 women, 9,2% were secundiparas. The average age of pregnant women was 28.6 years(± 3.8 years). The average maternal weight at the beginning of pregnancy was 63.6 kg (± 7.8 kg). Of the 303 women in 18 (6%) developed slight to moderate degree of preeclampsia or HELLP syndrome with varying severity of clinical manifestations. 20 mothers (6.6%) gave birth prematurely terminated before 37 week of pregnancy. 3 pregnancies (0.9%) were discontinued due to genetic indication for fetal birth defect. The complete study protocol (sampling in all three trimesters) thus completed 280 pregnancies. Of the three evaluated, parameter Lag time, ETP and peak we observed significant differences when comparing physiological pregnancies and pregnancies with preeclampsia (Table 3 and Figure 5-7), the statistical level of p < 0.01. In pregnancies with chronic hypertension, these differences were not significant. Comparison of 18 pregnancies, in which the III. trimester developed preeclampsia with other pregnant with physiological pregnancy did not show statistically significant differences in I. and II. trimester. The results suggest the activation of coagulation through the late stages of pregnancy. Results are influenced by strong clinical variability of disease. In severe and early preeclampsia this activation and significant differences begin much earlier. CONCLUSION: We demonstrated significantly higher activation of thrombin generation in women with preeclampsia [10]. Changes in preeclampsia are characterized by increased generation of thrombin in plasma. This fact may explain the partial success of the clinical use of aspirin in preeclampsia. In the third trimester, during the manifestation of the disease, patients with preeclampsia have significantly higher ETP compared to patients with a normal pregnancies. Pregnant women with chronic hypertension also show a slight increase in the activation of thrombin. However, these results are not statistically significant. Examination of coagulation in the first and second trimester in women who later developed preeclampsia, showed no statistically significant differences and thus can not be used in this case as predictive, but only as a diagnostic test.

[Myopia or hyperopia?]. / Myopie nebo hypermetropie?

INTRODUCTION: The study describes cases of patients screened for worse vision and headaches. We are trying to point out we can measure minus diopters even at latent hypemetropes. These patients come to a doctor for a variety of problems that may be caused by inadequate correction of ametropia. It is necessary to know about this possibility, and rather perform cycloplegia in sporadic cases. METHODS: Patients were measured at autorefractometer without mydriasis, and then after using UNITROPIC 1% or CYCLOGYL 1%. Both of these substances induce cycloplegia. Visual acuity with the best correction was tested with and without cycloplegia. RESULTS: After cycloplegia, a significant change in both objective and subjective refraction was detected in most of the selected patients. This change was within the meaning of a shift to hyperopia. Subsequent adjustment correction led to resolving of problems. CONCLUSION: The work should highlight the necessity of an individual approach of prescription of the best correction. Not always an autorefractometer gives correct information, the real-needed correction is completely different in some cases.

[Endothelial dysfunction in pregnancies with chronic hypertension]. / Dysfunkce endotelu u tehotných s chronickou formou hypertenze.

AIM OF THE STUDY: To establish endothelial activation markers which could uncover endothelial damage during hysiological pregnancy and pregnacies with chronic hypertension. SETTINGS: Department of Hemato-oncology, Obstetrics and Gynecology and Department of Medical Genetics and Fetal Medicine Medical Faculty of Palacký University Olomouc, Department of Obstetrics and Gynecology Medical Faculty Ostrava. METHODS: We examined 298 pregnant women with a physiological pregnancy. Venous blood samples were collected from the women in both arms at the beginning of the pregnancy, a second sample was collected in the interval 24-28 weeks gestation, the third sample was colected about the 36 weeks of gestation. Parameters were examined using methods: t-PA - ELISA, PAI-1 - ELISA, vWF - EIA, ePCR - ELISA, MMP-2,9 - ELISA with fluorogenic detection, TIMP-2 - ELISA, endothelial microparticles - flow cytometry. RESULTS: In accordance with the literature, we have observed in our study significantly increased endothelial activatition in hypertensive pregnancies compared with women with physiological pregnancy, as evidenced by significant increases in vWF activity and antigen, thrombomodulin and PAI-1 in all trimesters. In the other investigated parameters statistically significant changes were not observed. CONCLUSION: We have found significant signs of endothelial dysfunction in the group of women with pre-existing hypertension, a wide range of parameters examined markers indicating an significantly increased endothelial activation pregnant women with pre-existing hypertension, confirming the need for strict follow-up of pregnant women with hypertensive disease.

[Bleeding disorders in pregnancy]. / Problematika krvácivých chorob v porodnictví

UNLABELLED: Bleeding disorders in pregnancy represent heterogenous and often serious group of diseases. Solving of this problems and proper treatment of the patiens requires close cooperation between obstetricians and hematologists. There are disorders specific for pregnancy (HELLP syndrom) and disorders occurring independently in pregnancy, but these can be modified in pregnancy as well. Bleeding and postpartum haemorrhage still remain the leading cause of maternal mortality. KEYWORDS: bleeding - pregnancy - thrombocytopenia - coagulopathy.

Post-translational modifications regulate signalling by Ror1.

AIM: In this study, we analysed the post-translational modification of receptor tyrosine kinase-like orphan receptor (Ror1). Ror1 is highly upregulated in B cells of patients with chronic lymphocytic leukaemia (CLL). Molecularly, Ror1 acts as the Wnt receptor in the non-canonical Wnt pathway. METHODS: The level of Ror1 glycosylation in HEK293 cells and in primary human CLL cells was analysed by treatment of inhibitors interfering with different steps of glycosylation process and by direct treatment of cell lysates with N-glycosidase. Ror1 ubiquitination was determined by ubiquitination assay. Functional consequences of post-translational modifications were analysed by immunohistochemistry and by analysis of cell surface proteins. Differences in Ror1 glycosylation were confirmed by analysis of 14 samples of B cells from CLL patients. RESULTS: We demonstrate that Ror1 is extensively modified by N-linked glycosylation. Glycosylation produces several variants of Ror1 with electrophoretic migration of approx. 100, 115 and 130 kDa. Inhibition of glycosylation interferes with cell surface localization of the 130-kDa variant of Ror1 and prevents Ror1-induced formation of filopodia. Moreover, we show that 130-kDa Ror1 is mono-ubiquitinated. Furthermore, individual CLL patients show striking differences in the electrophoretic migration of Ror1, which correspond to the level of glycosylation. CONCLUSION: Our data show that Ror1 undergoes complex post-translational modifications by glycosylation and mono-ubiquitination. These modifications regulate Ror1 localization and signalling, and are highly variable among individual CLL patients. These may suggest that Ror1 signals only in a subset of CLL patients despite Ror1 levels are ubiquitously high in all CLL patients.

Leukaemia inhibitory factor inhibits cardiomyogenesis of mouse embryonic stem cells via STAT3 activation.

The leukaemia inhibitory factor is a cytokine that exhibits pleiotropic activities in a wide range of cell types. There are evidences that leukaemia inhibitory factor-regulated signalling pathways are involved in cardiomyogesis and maintenance of cardiomyocytes. In the present work we studied the effect of leukaemia inhibitory factor on cardiomyogenesis of embryonic stem cells together with the role of serum-born factors. We showed that leukaemia inhibitory factor had an inhibitory effect during both the induction and progression phases of cardiomyogenesis of embryonic stem cells. The leukaemia inhibitory factor-mediated inhibition of cardiomyogenesis was abolished by inhibitors of STAT3 activity. These results suggest that leukaemia inhibitory factor- activated STAT3 is responsible for the inhibition of cardiomyogenesis in embryonic stem cells.

[Course and delivery outcomes of 34 pregnancies complicated by HELLP syndrom]. / Prubeh a výsledky 34 tehotenství komplikovaných syndromem HELLP.

OBJECTIVE: To analyze the course and delivery outcomes of pregnancies complicated by HELLP syndrom and compare them to previously published data. DESIGN: Retrospective cohort study. SUBJECT AND METHOD: We analyzed pregnancy outcomes of 34 pregnancies complicated by HELLP syndrom of women who delivered at our hospital between years 2004 and 2009. SETTING: Department of Obstetrics and Gynecology, University Hospital Ostrava. RESULTS: There were 34 pregnancies and deliveries complicated by HELLP syndrom between the years 2004 and 2009 (64 months). The average age of mothers was 28.5 years, the average gestational age at delivery time was 33 weeks and 4 days. Only 2 women delivered vaginaly, the other 32 delivered by cesarean section and the average weight of the newborn was 1930 g. There were 3 twin pregnacies. Twenty two (65%) patients had laboratory abnormalities in coagulation parameters but clinical signs of coagulation disorder were noted in 8 patients only (23.5%). Six patients recevied blood transfusions (18 units in total) and 9 patients received plasma transfusions (20 units in total). The average length of stay of mothers at intensive care unit was 3.2 days. There was no maternal mortality. One fetus was stillborn and 12 newborns (33.3%) had their early postnatal period complicated by severe morbidity. CONCLUSION: Early diagnosis and active management is a major factor for the prevention of severe forms of HELLP syndrom which still remains one of the most severe complications of pregnancy.

[Monitoring of endothelial activation markers during physiological pregnancy]. / Monitorování hladin markeru aktivace endotelu behem fyziologické gravidity.

AIM OF THE STUDY: To establish endothelial activation markers which could uncover endothelial damage during physiological pregnancy. TYPE OF STUDY: Prospective study. METHOD: We examined 403 pregnant women with a physiological pregnancy. Venous blood samples were collected from the women at the beginning of the pregnancy, a second sample was collected in the interval 24-28 weeks gestation. Parameters were examined using methods: t-PA--ELISA, PAI-1--ELISA, vWF Ag--EIA ePCR--ELISA, MMP-2,9--ELISA with fluorogenic detection, TIMP-2--ELISA, endothelial microparticles - flow cytometry. RESULTS: The level of vWF antigen increased during the entire course of pregnancy (in the I. trimester the average level was 152.32%, in the II. and III. trimester 173.34% and 216.20% respectively). At the same time, vWf activity also increased (I. trimester average level 130.20%, II. and III. trimester 150.09% and 181.91% respectively). The level of thrombomodulin significantly increased during pregnancy (I. trimester average level 19.05 ng/ml, II. and III. trimester 28.47 ng/ml and 39.86 ng/ml respectively). The level of soluble form of EPCR increased during pregnancy (I. trimester average level 201.76 ng/ml, II. and III. trimester 274.68 ng/ml and 324.07 ng/ml respectively). The level of PAI-1 increased during the entire course of pregnancy (I. trimester average level 36.14 ng/ml, II. and III. trimester 50.07 ng/ml and 60.12 ng/ml respectively). The level of t- PA did not change significantly during the course of pregnancy (I. trimester average level 2.48 ng/ml, II. and III. trimester 2.97 and 3.34 ng/ml respectively). The levels of MMP-2 (I. trimester average level 9043.76 RFU, II. and III. trimester 9315.38 and 8800.27 RFU respectively), MMP-9 (I. trimester average level 8371.90, II. and III. trimester 8290.81 and 7470.50 respectively), TIMP-2 (I. trimester average level 92.5 ng/ml, II. and III. trimester 98.5 and 96.5 ng/ml respectively) or endothelial microparticles (I. trimester average level 3838.38 particles/microl, II. and III. trimester 3836.59 and 3650.59 particles/microl respectively) did not change significantly throughout the individual trimesters. CONCLUSION: We confirmed the hypothesis regarding the significant influence pregnancy has on changes in levels of these markers.

[Antithrombotic therapy in pregnancy]. / Antitrombotická terapie v gravidite.

Despite the fact of low prevalence of maternal death, deep venous thrombosis remains one of the most serious complication in pregnancy and puerperium. Virchows triad--vascular stasis, hypercoagulability, and vascular trauma plays the main role in the pathogenesis of deep vein thrombosis in pregnancy. Low molecular weight heparins and unfractionated heparins are the best treatment option. The aim of the treatment is to be effective in extension of thrombus and prevention of the postthrombotic syndrome and pulmonary embolism. Management of pregnant women with increased risk of venous thromboembolism can be stratified by determining whether the prior episode VTE was unprovoked or associated with a transient risk factor and the presence or absence of an inherited thrombophilia.

2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) induces degradation of adherens junction proteins and inhibits beta-catenin-dependent transcription in liver epithelial cells.

The toxic modes of action of non-dioxin-like polychlorinated biphenyls (PCBs) in liver cells are still only partially understood. Several recent studies have indicated that PCBs may interfere with cell membrane protein functions. Therefore, we analyzed in the present study the effects of di-ortho-substituted 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) on proteins involved in the formation of adherens junctions in a model of rat liver progenitor cells - WB-F344 cell line. PCB 153, at micromolar concentrations, induced a gradual degradation of E-cadherin, beta-catenin or plakoglobin (gamma-catenin) proteins. This effect was not due to changes in gene expression, as PCB 153 had no effect on mRNA levels of the above mentioned proteins. Moreover, apart from the reduction of total beta-catenin pool, PCB 153 also decreased levels of the active beta-catenin form, dephosphorylated at residues Ser37 and Thr41, which is the key co-activator of Wnt-induced TCF/LEF-dependent gene expression. Therefore, we also evaluated the impact of PCB 153 on expression of Axin2, a known transcriptional target of canonical Wnt signaling. PCB 153 reduced basal Axin2 mRNA levels and it inhibited induction of Axin2 expression by recombinant mouse Wnt3a. Nevertheless, PCB 153 had no effect on phosphorylation of glycogen synthase kinase-3beta (GSK-3beta), which is supposed to target beta-catenin for its proteasomal degradation. This suggested that GSK-3beta activity is not modulated by PCB 153 and, consequently, not involved in the observed PCB 153-induced decrease of both total and active beta-catenin levels. Protein levels of E-cadherin and beta-catenin were partially restored with lysosomal inhibitor leupeptin, thus suggesting a possible role of lysosomes in the observed degradation of adherens junction proteins. Taken together, the present data suggest that PCB 153 may interfere with functions of adherens junction proteins involved in both cell-to-cell communication and intracellular signaling. Such mechanisms might be involved in the effects of non-dioxin-like PCBs contributing to liver tumor promotion.

[Molecular methods in thrombophilic states diagnostics]. / Molekulární metody v diagnostice trombofilních stavu.

Molecular genetic methods passed into the field of investigation of thrombophilic states in 90th years of last century, along with the first discoveries of coagulation inhibitors (AT III, protein C and protein S). They have acquired a widespread use above all with the detection of the molecular basis of activated protein C (APC) resistance in 1994 by prof. Bertina. At the present time, a wide range of molecular genetic markers, linked with a clearly documented increased risk of thrombophilia are adapted. They include mutations of factor V Leiden 506R/Q, of protrombin 20210G/A, MTHFR 677C/T in homozygous form, mutation of PAI-1 4G/5G, mutations of different coagulation inhibitors and finally a range of polymorphisms with still not precisely defined increased risk for thrombophilia (F XIII Val34leu, platelets glycopeproteins, endothelial protein C receptor and trombomodulin). From the methodological viewpoint, all these techniques are based on the principle polymerase chain reaction (PCR). In the last period of time, however there was a rapid evolution, allowing a significant improvement in their laboriousness. Nowadays, splitting with the aid of restriction endonucleases, real time PCR or allel specific primers for PCR. The second, where molecular genetic methods are currently under use, is pathophysiological investigation of the single coagulation processes. Here, in a fact, most significant progress has been in the field of APC resistance made elucidation. Although still in the 90th years of the past century the genetical cause of these coagulation disturbance was unequivocally documented its clinically heterozygous appears not yet fully understood at the moment. Similarly, in prothrombin mutation, only the latest investigations have outlined the probable mechanism of expression. Concerning the future evolution of molecular genetic methods, there can be observed a clear cut tendency to better understanding the pathophysiologic cause of thrombophilia in comparison with the searching for new coagulation defects which consecutively bear lesser a relative risk of thrombosis.

[Cataract surgery without preoperative mydriasis]. / Operace katarakty bez predoperacní mydriázy.

Preoperative mydriasis is usually obtained by means of using combinations of sympatomimetics and parasympatolytics. The disadvantages of this method are: longer preoperative preparing time, toxic effect of eye drops and adverse systemic effects in some patients. The alternative to the standard dilating drops is use of intracameral lidocaine during the surgery. In our study, we used intracameral lidocaine 1% to achieve the mydriasis during the cataract surgery. By using this method, we operated on 38 eyes of 33 patients, 12 men and 21 women. The age ranged between 54 years and 85 years (mean, 72.2 years). The mean pupil diameter before dilation was 3.04 +/- 0.62 mm. The time necessary to dilate the pupil was 8.82 +/- 2.54 sec and the mean pupil diameter after the dilation was 7.11 +/- 0.74 mm. The pupil enlarged even more after the instillation of the ophthalmic viscoelastic material. At the end of the surgery, the diameter of the pupil was 7.12 +/- 0.62 mm. The mydriatic effect of intracameral lidocaine application is adequate and longstanding enough. This surgical method is wholly safe.