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Významnou charakteristikou účetních systémů, která podstatným způsobem určuje vypovídací schopnost účetních informací je způsob ocenění jednotlivých segmentů účetní závěrky. Účetní teorie i praxe vyvinula poměrně širokou škálu možných přístupů k oceňování v účetnictví. Cílem tohoto příspěvku je formulovat základní otázky týkající se volby systému ocenění a na tomto základě diskutovat výhody a nevýhody jednotlivých oceňovacích bází.

The view at an enterprise is changing in connection with the adoption of the concept of sustainable development. An enterprise begins to be perceived as a unity of economic, social, and environmental dimensions. An empirical study which was accomplished within this research confirmed positively that insufficient connrction of information between economic, environmental and social dimensions of business weakens the potential and reliability of presented information. On the other hand the consistent use of multi-dimensional concept demands the interconnection of information on all three dimensions in an annual report. The aim of this paper is to examine whether it is possible to provide complex, multidimensional information about an enterprise and, at the same time, respect the IFRS.

Thyroid tumors are the most common endocrine malignancy. The main genetic changes are point mutations in the RET proto-oncogene (somatic or germ-line) in medullary thyroid carcinoma and point mutations in BRAF and RAS genes or RET/PTC rearrangements in carcinomas developing from follicular cells. Moleculargenetic diagnosis of RET mutations in patients with medullary thyroid carcinoma and their relatives is now the part of clinical approach. Currently, knowledge about genetic causes of thyroid tumors has begun applying into target gene therapy providing new therapeutic drugs and more individualized treatment. This review summarizes main genetic causes of thyroid tumors and their application in target gene therapy.

Purpose: The aim of research study was to evaluate the effect of corneal cross-linking (CXL) in the frame of patients with progressive keratoconus 1 year after treatment., Methods: There were 40 eyes of 35 patients with mean age 28, 45 +/- 9.3 (SD) (15 to 48 years) included in the study. Patients were treated with standard protocol of CXL with abrasion of corneal epithelium. Complete ophthalmological examination included best corrected spectacles visual acuity (BCSVA), slit-lamp microscopic finding, corneal topography and corneal thickness measured with ultrasound method was performed before, on the 5-th day, 1. 6., 12. month after CXL. We divided patients according to the stage of keratoconus into 2 groups (stage I. and stage II.) and according to the age into 3 groups (until 20, from 21 to 39, over 40 years)., Results: In all treated eyes, the CXL was without relevant complications. The only complication was stromal haze of cornea. In the evaluation based on stage of keratoconus, in the first group any patient became a haze of cornea in 1 year after CXL. In the second group 35.7% of patients had a haze of cornea. The average BCSVA 1 year after treatment was improved in the 1. group about 5.38 letter and in the 2. group about 1.25 letter. Topographic analysis showed decrease of simulated keratometry and refraction (1. group--0.1 D, 2. group--0.17 D), maximal keratometry and refraction (1. group--0.67 D, 2. group--0.76 D). Minimal keratometry and refraction in the 1. group decreased (1.17 D) and increased in the 2. group (1.09 D). In the evaluation based on the age was haze monitored in the first group one year after CXL in 12.5% of researched eyes. In the second group was haze of cornea in 20% of eyes and in the third group consisting of patients over 40 years old, in 50% of eyes. The average BCSVA was improved in the 1. group (2.85 letter), and in the 2. group (3.68 letter).The average BCSVA was decreased in the oldest patients in about 1.43 letter. In the 1. and 2. group the topographic analysis showed decrease of simulated keratometry and refraction (1. group--0.12D, 2. group--0.21D), maximal keratometry and refraction (1. group--1.13 D, 2. group--0,68D), minimal keratometry and refraction (1. group--1.17D, 2. group--0,69 D). In the 3. group the topography analysis showed increase of simulated keratometry and refraction (0,8D), maximal keratometry and refraction (0,98D), minimal keratometry and refraction (0,28D). Corneal pachymetry remained stable in all researched groups of patients., Conclusions: CXL is considered as safe procedure to stop progression of keratoconus also for patients until 19 years old. The best effect and minimal complications were by patients until 40 years old and by patients with the I. grade.

Acute promyelocytic leukemia (APL) with atypical breakpoints in the promyelocytic leukemia (PML) and retinoic acid receptor-alpha (RARA) genes represents a rare leukemic event, which occurs preferentially in patients with variant types of the PML/RARA fusion gene. Here we report on a patient with APL with a unique PML/RARA fusion transcript that harbors a short type of this fusion gene, exhibiting unexpected results of standard PCR diagnostics. The detected transcript originates from fusion of PML exon 4 and a truncated form of transcription variant 2 of the RARA gene, with an additional 9 bp insertion. According to our knowledge, this differs from all previously described fusion transcripts.

Chemotherapy in most patients with AML over 80 years of age is not recommended because their median survival is about 1 month. The aim of our study was to identify patients in this age group who might achieve complete remission with standard dose chemotherapy. We report 9 consecutive patients with de novo AML diagnosed and treated in 1992-2008. All bone marrow samples were hypercellular, classified as FAB types M2 in 2 cases, M4 in 6, and M5 in one case. Three patients opted for supportive or palliative therapy and survived 1-4 months. Six patients received standard dose chemotherapy. Two patients with a normal karyotype had resistant AML and survived 1.0 and 2.7 months; one patient with a complex karyotype died of septic shock on the 10th day of therapy. All these three patients exhibited erythroblastic and/or megakaryocytic dysplasia (EMD) at presentation (two in more than 26% erythroblasts, all three in a half or more of megakaryocytes). Three remaining patients with AML M4, a normal karyotype but without EMD, achieved complete remission in spite of co-morbidities and a poor performance status. Two of them survived 18.6 and 28 months on maintenance therapy, the third 16.5 months without it. Very elderly AML patients without EMD appear to represent a favorable prognostic biological category (single-lineage AML) that show a good response to standard dose chemotherapy.

Objective: Of 140 chronic myeloid leukemia patients responding to imatinib with complete cytogenetic remission, 32 exhibited a plateau of BCR-ABL values at >or=0.1% level in a minimum of three subsequent samples (minimal duration, 6 - 9 months). Median follow-up of unchanged BCR-ABL transcript level was 12 months (range, 6 - 64). We tested this group of patient for BCR-ABL mutations to reveal resistance development and to evaluate the risk of disease progression., Materials and Methods: Altogether, 134 samples of peripheral blood of these 32 patients were tested for mutation in BCR-ABL kinase domain., Results: Mutation was detected by direct sequencing in 9 of 32 patients (28%). Loss of complete cytogenetic remission or 1 log rise of BCR-ABL was observed in five of nine patients at a median of 5 months (range, 4-17) since first detection of mutation. One patient with no mutation relapsed 12 months after the start of the BCR-ABL plateau. In 5 of 32 patients without mutation (16%), BCR-ABL level significantly decreased after the first plateau to levels that stayed unchanged for a median of 11 months (range, 7-28)., Conclusion: We show here that the BCR-ABL constant levels >or=0.1% (BCR-ABL plateau) in imatinib-responding patients may indicate mutation analysis. This approach highly reduces the number of examinations for mutation in chronic myeloid leukemia responders and may present cost-effective alternative applicable in clinical practice.

Aim: The aim of the prospective study was to evaluate photopic high-contrast visual acuity, mesopic contrast sensitivity, and high order aberrations, to compare changes and post-operative development of those parameters and to analyze the dependence among aberrations and contrast sensitivity after conventional LASIK treatment., Materials and Methods: The authors followed-up patients treated by means of refractive LASIK treatment during the period from November 2006 to November 2007. The authors analyzed 51 eyes (31 patients). The average age of the group was 28.5 +/- 5.4 years (range, 18 - 41 years), preoperative average spherical equivalent was -4.95 +/- 1.24 D (from -3 to -8,25 D). Before the treatment and 1, 3, 6, and 12 months after LASIK treatment we evaluated the visual acuity (Snellen optotypes), contrast sensitivity under mesopic circumstances (CSV-1000E, VectorVision) and monochromatic aberrations (aberometer Zywave, Bausch & Lomb)., Results: One year after the treatment the average uncorrected visual acuity was 1.07 +/- 0.15, index of effectiveness 0.99, and index of safety 1.02. The contrast sensitivity was in month 12 significantly decreased comparing to the preoperative level at the frequency 12 c/deg, in other already tested frequencies after 3-6 moths did not differed from preoperative values. During the follow-up period the curvature of contrast sensitivity average values was in the upper half of the normal interval range. Conventional LASIK treatment significantly induced the higher order aberration (twice), as well as the spherical aberration (four times).The same level of higher order aberrations root mean square (HOA-RMS), or increased maximally by 0.1 microm was detected by 10 % of cases; the spherical aberration was, compared to the preoperative value, lower, or increased maximally by 0.05 microm in almost one half of the cases. The increase of the higher order aberrations depended directly proportionally to the preoperative value of the spherical equivalent. Before the treatment, the values of total aberrations correlated to the contrast sensitivity of low space frequencies; however, there was not found any correlation between the higher order aberrations and contrast sensitivity. Six months after the LASIK treatment the values of higher order aberrations correlated to the contrast sensitivity except of the lowest frequency tested. The higher order aberrations increased together with decreasing contrast sensitivity. The data from the one-year follow up control did not show statistically significant correlation between the contrast sensitivity and the higher order aberrations. There was not found any correlation between the contrast sensitivity and the spherical aberration at any follow-up control after the surgery., Conclusion: Although after the conventional LASIK treatment the curve of mesopic contrast sensitivity was located in the upper half of the normal range, in the medial space frequency it remained decreased comparing to the preoperative stage. The induction of higher order aberrations was twice as much and was directly correlated to the degree of the laser correction. The spherical aberration was four-times higher comparing to the preoperative values and was independent to the level of the initial refractive error. Significant correlation between the contrast sensitivity and the higher order aberrations was not proven.

Unlabelled: The aim of the prospective study was to evaluate higher order aberrations and contrast sensitivity after photorefractive keratectomy (PRK) using the standard photoablation profile., Materials and Methods: The group consisted of 37 patients (69 eyes), the mean age 27.2 +/- 4.5 years, who underwent PRK with target emetropia during the period January 2007 -December 2007. In 19 cases, it was correction of myopia, in 50 cases myopia with astigmatism.The preoperative spherical equivalent was -3.14 +/- 0.95 D. The PRK was performed by means of excimer laser system Technolas 217 (Bausch & Lomb) with the standard phoptoablation profile (PlanoScan 2000), using the 6.5 mm optical zone. The visual acuity, contrast sensitivity (CS; CSV-1000E, VectorVision) under mesopic circumstances and monochromatic aberrations (Zywave, Bausch & Lomb) were evaluated before the surgery, and 1,3, 6, and 12 months thereafter. The pair t-test, Wilcoxon test, and the Mann - Whitney U test (alpha = 0.05) were used for the statistical analysis., Results: The PRK showed high index of effectiveness and safety (0.98, respectively 1.03 in the first year after the procedure). The contrast sensitivity under mesopic circumstances was not significantly involved after the PRK. The main value of the CS remained during the whole follow-up period within the physiological range in all spatial frequencies. Postoperatively, the part of spherical aberration on the higher order aberrations increased from 13.1% preoperatively to 16.6% one year after the PRK. In one half of the cases, the change of the higher order aberrations was within the range +/- 0.1 microm. In 66% of cases, the change of the spherical aberration was +/- 0.05 microm. The higher order aberrations comparing to the preoperative values decreased or remained unchanged approximately in one third of the cases, and the spherical aberration in one quarter of the cases. There was not established dependence between monochromatic aberrations values and the contrast sensitivity., Conclusion: Although the conventional RPK for low myopia treatment induces higher order aberrations including the spherical aberration, the impact on the contrast sensitivity under mesopic circumstances in our group were not significant. The contrast sensitivity of the most of patients was near the upper limit of the normal range.

Background: This study compared the safety, tolerability and switch to oral medication in patients with bipolar disorder or schizophrenia who received intramuscular (IM) olanzapine or other IM antipsychotics for the treatment of acute agitation., Methods: Patients (N = 2011) from 15 countries participated in this prospective, observational, non-interventional study. Inpatients requiring treatment with at least one IM injection of a short-acting antipsychotic were assessed at baseline and within 7 days after the first IM injection. Treatment groups comprised: (i) patients prescribed IM olanzapine at baseline; and (ii) patients prescribed any other IM antipsychotic medication at baseline. Outcome measures included: treatment-emergent adverse events, concomitant psychotropic medication and the time taken to switch to oral medication., Results: Fewer patients in the IM olanzapine group experienced an adverse event than patients in the other IM antipsychotic group (34.4% vs. 46.2%, p < 0.001). The most frequently reported adverse events in both groups were: sedation, Parkinsonism, disturbance in attention, akathisia, dystonia and orthostatic hypotension. Fewer patients in the IM olanzapine group used anticholinergics (13.9% vs. 42.5%, p < 0.001) or anxiolytics/hypnotics (47.6% vs. 51.6%, p = 0.023). Patients in the IM olanzapine group switched to oral medication earlier than patients in the other IM antipsychotic group (median time = 46.5 vs. 48.0 h, p = 0.009)., Conclusions: These findings suggest that IM olanzapine may have a favourable impact on individual patients. However, the high rate of oral concomitant medication used throughout the study limits these findings from being associated with IM olanzapine alone.

In recent years, several independent prognostic factors in cytogenetically normal acute myeloid leukemia (CN-AML) have been reported. Mutations or the expression levels of certain genes have been often used as molecular markers for prediction of a patient's outcome or for evaluation of treatment outcome. One of them, the gene encoding CCAAT/enhanced binding protein alpha (CEBPA), plays an important role in myeloid differentiation and, when mutated, confers a favorable prognosis for patients with CN-AML. Complete mutation screening of the CEBPA gene is therefore beneficial and requires fast, precise, and sensitive diagnostic tools. Thus, for routine diagnostics, we developed a screening method using high-resolution melt curve analysis prior to direct sequencing, where only positive samples (according to reference) are further sequenced. With this approach, all positive and negative patients were successfully distinguished, and the results obtained were in absolute concordance with the direct sequence analysis.

We have retrospectively evaluated a cohort of 144 patients (including 17 pediatric ones) with de novo acute promyelocytic leukemia registered in databases of institutions cooperating within the CELL group (The Czech Leukemia Study Group for Life). The patients were diagnosed according to WHO criteria from 1989 until 2006. The aim was to check how well fared the patients, the majority of whom was not included into clinical trials, in real life. Of 140 evaluable patients, 97 (69.3%) attained complete remission (CR). The projected overall survival (OS) 4 years after diagnosis was 58.9%, and 55.3% at 6 years. In 8 patients (6.0%), no antileukemic therapy at all was given (either they died shortly after admission to the ward or therapy was not feasible due to their clinical status). Of 125 patients with documented commencement of some kind of therapy, 96 (76.8%) achieved CR. Of 102 patients with induction treatment with a combination of anthracycline and tretinoin (ATRA), 84 individuals (82.4%) attained CR (typically, this cohort might have been subjected to clinical trials). This result was better than that of patients treated by chemotherapy only (n = 15; CR 46.7%; P = 0.003) or by ATRA monotherapy (n = 13; CR 62.5%; P = 0.17). Another parameter with a significant impact on attaining CR was the leukocyte (WBC) count at diagnosis: its median values in patients achieving and not achieving CR were 2.1 and 24.0 x 10(9)/l, respectively (P < 0.0001). The WBC counts affected OS as well (P = 0.0001). However, when only patients after attaining CR were evaluated, the initial WBC counts no longer affected OS (P = 0.18). Achieving CR was also influenced by the performance status (PS) 0-1 (P = 0.005), which was in turn closely correlated to WBC counts (P = 0.0006). Additional factors (most likely connected with leukocytosis) influenced attaining CR with borderline statistical significance: e.g. FAB M3v morphology, LDH serum level, fibrinogen level, presence of internal tandem duplication (ITD) of the FLT3 gene (which was strongly associated with leukocytosis and also with the short PML/RARalpha transcript resulting from the bcr3 break in the PML gene). It may be speculated that FLT3-ITD is just one of the possible factors that lead to leukocytosis. The platelet counts at diagnosis had no impact on entering CR. Thus, we have not validated the current PETHEMA risk stratification in distinguishing intertermediate and low risk patients. Our study points to a significant difference of the results obtained in real life and of the results that could be achieved in patients who were fit to enter clinical trials. Among the prognostic factors, the most important one was the WBC count, the PS (which is highly affected by the WBC count), and feasibility of administration of the most potent induction therapy with anthracyclines and ATRA.

Aim of the Study: The main goal of this study was to find the best method for Aspergillus DNA isolation from peripheral blood samples. The method should be very effective but not expensive or time consuming to be suitable for routine diagnostics use., Material and Methods: We compared four different methods for Aspergillus DNA isolation - one method with enzymatic lysis of the fungal cell wall and three methods that combine chemical and mechanical lysis (using high speed cell disruption with glass beads). Peripheral blood samples were inoculated with defined amount of Aspergillus fumigatus suspension and used for DNA isolation. Isolated DNA was then quantitatively analyzed with in-house real-time PCR method using specific probe., Results: Enzymatic method seems not to be useful in a routine diagnostics mainly because of its low efficiency and too long processing time. Better could be the methods using both chemical and mechanical cell disruption that can isolate fungal DNA with high efficiency in a relatively short time., Conclusions: The method using ZR Fungal/Bacterial DNA Kit (Zymo Research, USA) was chosen for routine use in our laboratory because it is cheap, fast and very efficient.

Background: Invasive aspergillosis (IA) is a leading invasive fungal infection in hematooncological patients. The aim of this study was to analyse the incidence, diagnostic procedures and treatment of IA in hematooncological department in large hospital in the Czech Republic., Patients and Methods: A retrospective analysis of medical and laboratory records from patients hospitalised in our department with proven/probable IA between January 2000 and December 2006 was performed., Results: 52 cases of IA in 51 patients were identified (17.3% proven IA/82.7% probable IA). Number of IA cases notably increased during study period (1 case of IA in 2000 vs 21 cases of IA in 2006) and majority of them was of nosocomial origin (61.5%). Pulmonary aspergillosis was diagnosed in 46 cases (88.5%). Patients treated for acute leukemia or undergoing allogeneic stem cell transplantation represent the group at the highest risk of IA (in total 52% of cases). Fever and signs of pulmonary involvement were the most common clinical signs of infection (presented in 92.3% and 69.2 cases respectively). Conventional diagnostic methods including autopsy were able to diagnose only 15 cases of IA (28.8%). In all other cases (71.2%) the diagnosis was done by detection of galactomannan (GM) in serum. Introduction of GM monitoring enabled erlier initiation of antifungal treatment by 4 days. Initial therapy of IA led to the treatment response (partial and complete) in 18 (34.6%) of infections--the highest percentage of response has been seen in voriconazole monotherapy group (42%) and when combination of voriconazole and caspofungin has been used (83%). Salvage therapy was initiated due to the failure of initial treatment in 21 (40.3%) of cases. Patients were treated mostly with combination ofvoriconazole and caspofungin and/or monotherapy with voriconazole has been used with treatment response 55% and 50% respectively. Introduction of new antifungal drugs together with increased number of patients with IA led to the marked increase of total costs spent on treatment of IA per year--from 11,5 thousands CZK in 2000 to 6,2 millions CZK in 2006., Conclusions: IA is the most frequent cause of infection-related mortality in patients with haematological malignancies. Routine use of non-culture base methods in diagnosis of IA together with treatment using new, effective antifungals can improve prognosis of patients with this life threatening infection.

PCR detection of fungal pathogens in clinical samples has been discussed in journals for more than two decades. However, its use for diagnosing invasive aspergillosis is still controversial, despite the fact that molecular methods are routinely used in various fields of modern microbiology. These are e. g. genotyping of bacterial strains resistant to antibiotics, molecular epidemiology or routine detection of viral infections in clinical material. PCR methods have made the diagnostic applications faster, simpler and more accurate. This review deals with issues related to molecular methods for diagnosing invasive fungal infections and the main factors limiting their use in everyday clinical practice.

Unlabelled: Bcl-2/IgH rearrangement is a characteristic molecular rearrangement in patients with follicular lymphoma (FL), yet its prognostic significance is still unclear., Objective: Evaluation of the implications of achieving Bcl-2/IgH negativity for the prognosis of FL patients. Twenty seven patients (54%) were receiving only chemotherapy (CHT), 23 patients (46%) were receiving chemotherapy combined with monoclonal antibody anti/CD20, rituximab (R-CHT)., Results: Molecular genetic remission was achieved in 7 out of 11 patients (64%) after R-CHT, and only in 2 out of 14 patients (14%) after CHT- this difference was statistically significant (p = 0.037). 4 weekly doses of rituximab were administered in a sequence to 17 out of 27 patients who had received only chemotherapy and failed to achieve complete remission. 12 out of 17 patients (71%) on this therapy were Bcl-2/IgH positive prior to treatment. 7 out of 12 (58 %) patients were no longer Bcl-2/IgH positive in a check performed after one month; the remaining 2 out of 5 patients had a negative Bcl-2/IgH record for the interval of 3 months (1 patient) or 6 (1 patient) months, respectively. The following factors were associated with the achievement of Bcl-2/IgH negativity at any point during the treatment: age < 65 years (p = 0.02) and performance status 0 + 1 according to WHO at baseline (p = 0.02). Patients who were Bcl-2/IgH negative after treatment had a lower recurrence/progression risk rate than the Bcl-2/IgH positive group of patients, i.e. 27% vs. 75% (p = 0.03), and a higher chance for progression-free survival, i.e. 81% vs. 38% (p = 0.004), event-free survival, i.e. 74% vs. 38% (p = 0.01), and overall survival, i.e. 87% vs. 74% (p = 0.05) at 2 years., Conclusion: In our experience, achieving Bcl-2/IgH negativity after follicular lymphoma therapy implies a better prognosis.

Background: Chronic lymphocytic leukemia is a heterogeneous disease manifesting with a variable clinical course. It is evident from many studies, that the division into two main prognostic categories is possible on the basis of mutation status of the immunoglobulin heavy-chain gene. The objective of our work was to identify a presence or absence of IgVH gene mutations in B-CLL patients which are monitored or treated on hematological clinics and to determine the presence of individual D and J, subgenes in malignant population of B-cells., Methods and Results: A nucleotide sequence of IgVH gene of neoplastic cells was analyzed by appropriate molecular-genetic methods. RNA/cDNA was collected from 358 patients and a spectrum of individual subgenes translocations was identified. Our results show that 56.3% of patients manifested an unmutated variable (VH) segment. It is expected from the published data that this group of patients will suffer from aggressive course of the disease and will exhibit a substantially shorter survival in comparison to patients possessing somatic hypermutations. An expanded population of leukemic B-cells showed increased occurrence of clones whose variable segments belong to three different families. VH3 alleles are the ones most frequently used. A frequency of unmutated alleles is prominently shifted into families with V I homology. The preferred "diversity and joining" segments are D3, D2 and JH 4 and JH 6., Conclusions: The analysis of heavy chain immunoglobulin gene after recombinant VH-D-J11 segments translocation belongs to a standard hematooncological investigation. The results are an important prognostic criterion for prediction of expected disease aggressivity and for a minimal residual disease monitoring.

Presented are two cases of systemic mastocytosis in 46- and 63-year-old women, where the correct diagnosis was established in randomly disclosed cervical respectively intraabdominal lymphadenopathy. Both cases lacked characteristic skin and systemic mast-cell mediator symptoms at the time of histologic diagnosis. The first case was classified as a indolent systemic mastocytosis without any proven genetic alteration, the second one met the criteria of aggressive systemic mastocytosis with eosinophilia, where the point mutation asp816val in c-kit gene was confirmed and the patient responded unexpectedly well to Gleevec. Discussed are both conventional morphological differential diagnosis of mastocytosis in lymph nodes and recent advances in genetics of these systemic clonal mast cell proliferations. The latter not only outlines the oncopathogenesis but, in particular, also provides important prognostic and biological implications of this peculiar disease.

Laboratories dealing with human genome, both inherited and acquired changes, dispose with similar methods and technology. The spectrum of genetic tests is relatively broad and the number of mutations or variants tested differs substantially. Also the number of examinations carried out in individual laboratories varies. Data presented in the tables come from the year 2004 and indicate the number of examinations requested and number of positive results. Many laboratories mentioned in the registry CZDDNAL (http://www.uhkt.cz/lab&#95;a&#95;vysetreni/nr lab&#95;dna&#95;diag/dna&#95;lab&#95;db) perform the same tests but there is also a great number of tests carried out by only one laboratory. Reasons of the request, cost-effectiveness and clinical utility of genetic testing is being discussed.

Aim of the Study: The main goal was to explain the discrepancies between two PCR methods used for detection of cytomegalovirus (CMV) in peripheral blood samples of patients of Department of Internal Medicine-Hematooncology, University Hospital, Brno., Materials and Methods: In past we used exon 4 of major immediate-early (MIE) gene as the target for quantitative detection of the CMV in clinical samples, but sometimes this method failed to detect the viral load in samples that were positively tested using less sensitive qualitative method targeting another region (exon 2-4) of the same gene. From January 2004 to January 2005 we totally tested samples from 363 patients. 64 patients were at least once CMV positive using quantitative method, but 20 patients were repeatedly false negative.To find the cause of this discrepancy we performed partial sequence analysis of this region (nt positions 2719-2919, GenBank M21295) and glycoprotein B (gB) genotyping. We sequenced samples from 35 patients-15 giving true positive (both in qualitative and quantitative method) and 20 giving false negative (negative in quantitative but positive in qualitative method) results in several consecutive blood samples., Results: The 15 true positive samples were 100% homological, whereas all 20 false negative samples showed high degree of variation from the laboratory strain AD169. These changes are not random and indicate that the two groups of patients were infected by different CMV genotypes. Moreover, sequence alignment showed similarity to laboratory strains Toledo and Towne. No preferential concordance was observed between clinical context, MIE exon 4 sequence and gB groups., Conclusions: Because of high sequence variability exon 4 of MIE gene can not be used for routine diagnostics. Genetic varibility among the pathogenic strains may seriously affect its proper diagnostics.

Unlabelled: Daunorubicin (DNR) and doxorubicin (DOX) have significant antitumor activity in acute myeloid leukemias (AML) and non-Hodgkin's lymphomas (NHL) but their use is limited by their life-threatening cumulative dose related cardiotoxicity. It is generally recommended not to administer DOX or DNR to patients in doses greater than 500 mg/sqm or 700 mg/sqm, respectively. the aim of the study was to follow up cardiotoxicity and efficacy of DNR or DOX above these limits in the 2nd complete remission (CR) patients pretreated with anthracyclines when they were given 30 minutes after cardioprotective agent dexrazoxane (DRZ) in the ratio 1:10 of DZR., Results: Two patients (54 and 53 years old) with mantle cell or diffuse large cell B-NHL, stage IV, who had relapsed after 6-8 cycles of classical CHOP therapy, reached their 2nd CR after 2-3 cycles of IDEA therapy (ifosfamide 1000 mg/sqm/day x 4, dexamethasone 30 mg/sqm/day x 4, etoposide 75 mg/sqm/day x 4, DOX 30 mg/sqm/day on days 1 and 3). Then they received further 3 cycles IDEA with DRZ 300 mg/sqm before every dose of DOX. After cumulative doses of DOX 600 mg/sqm and 700 mg/sqm these patients survived 12 months in their 2nd CR without significant signs of cardiotoxicity, even after their successful autologous peripheral stem cells transplantation. Their left ventricular ejection fraction (LVEF) remained above 60%. Six patients with AML in their 2nd CR were treated with consolidation cycles consisting of 10 high doses of cytosine arabinoside (2000 mg/sqm/12 hr) plus 2 doses of DNR 45 mg/sqm on the day 4 and 5. Two patients received cumulative doses corresponding to 1300 mg/sqm and 1000 mg/sqm of DNR, the other received DNR doses 550-850 mg/sqm. No signs of significant cardiotoxicity were observed in all 6 patients and their LVEF remained over 50%. One of two patients, transplanted with HLA-identical sibling bone marrow in her 2nd complete remission (CR), is still 8 years in her 2nd CR. Dexrazoxane enables to administer anthracyclines in doses over the recommended cumulative ones in pretreated patients with B-NHL or AML in their 2nd CR with the follow-up of their LVEF.

Background: Telomerase, a ribonucleoprotein complex catalysing synthesis of telomeric DNA, is an essential cellular immortalizing factor whose activation is a critical step in the progression to malignancy. An important agent maintaining the balance between proliferation, differentiation and apoptosis of intestinal epithelial cells in crypts is butyrate, which is formed in the gastrointestinal tract by anaerobic bacterial fermentation. It inhibits cell growth, induces differentiation and triggers apoptosis in neoplastic colonocytes., Materials and Methods: In this study, the responses of adenocarcinoma (HT-29) and fetal (FHC) human colon cells to 5 mM sodium butyrate (NaBt) have been compared., Results: Despite the similar general response of both cell lines to NaBt, i.e., G0/G1 arrest, decrease of growth rate and increase of differentiation (as indicated by alkaline phosphatase activity), they differ in the level and dynamics of the measured parameters. Telomerase activity and the level of mRNA for its catalytic subunit (hTERT) decline significantly after 48 hours, reaching a complete inhibition after 144 hours. While both cell lines show similar kinetics of hTERT transcriptional silencing, the down-regulation of telomerase activity is faster in FHC cells. Correspondingly, we show that a candidate posttranscriptional regulation step, differential splicing of hTERT mRNA, may be involved in the faster loss of telomerase activity in FHC cells., Conclusion: Differences in hTERT mRNA splicing may represent a useful marker of telomere metabolism in normal and malignant colon cells and that these changes may be connected with different cytokinetic patterns of these cells.

Xenopus laevis (African clawed frog) embryos in a 96-h teratogenesis assay (FETAX) were exposed to 0-250 microg/L and 500 microg/L of purified microcystin-LR (MCYST-LR) for the estimation of lethality, as well as to equivalent concentrations of biomass containing MCYST-LR (natural water bloom dominated by Microcystis aeruginosa) and biomass without MCYST-LR (bloom dominated by Microcystis wesenbergii). The highest tested concentrations of purified MCYST-LR caused up to 30% lethality after a 96-h exposure, corresponding to a LC(25) of 380 microg/L. Cyanobacterial biomass containing MCYST-LR caused significant lethality up to 50% at the highest tested concentrations (300 mg/L, i.e., 250 microg/L of MCYST-LR). The estimated 96-h LC(25) values varied from 125 mg/L (biomass containing MCYST-LR) up to 232 mg/L (biomass without MCYST-LR). A statistically significant increase in the number of malformed embryos was observed after exposure to cyanobacterial samples. Purified MCYST-LR at and above 25 microg/L significantly increased the number of malformations, with 53% of surviving embryos malformed in the highest tested concentration, 250 microg/L (EC(25) = 27 microg/L). Exposure to the highest concentration of MCYST-LR containing biomass resulted in more than 60% of the embryos being malformed and an EC(25) of 52 mg/L (i.e., 43 microg of MCYST-LR/L). Cyanobacterial biomass with no natural microcystin also induced substantial malformations-about 50% aberrant embryos at the highest concentration, 300 mg/L (EC(25) = 75 mg/L). External additions of purified MCYST-LR to the biomass that was originally without microcystins resulted in a slight additional increase in the rate of malformations (80% at the highest concentration, 300 mg of biomass plus 250 microg of MCYST-LR per liter). A comparison of lethality and effects on malformations (teratogenic index, TI = LC(25)/EC(25)) showed that all samples had significant teratogenic potential in the FETAX assay (TI(MCYST-LR) = 14; TI for biomass with and without microcystin ranged between 2.4 and 3.1, respectively). We conclude that cyanobacterial water blooms can significantly alter the normal development of amphibian embryos., (Copyright 2002 Wiley Periodicals, Inc.)

Background: Recent findings concerning the role of immunity in the eradication of residual malignant disease after autologous haematopoietic stem cell transplantation have led to extensive studies of T-cell and natural killer (NK) mediated anti-tumour effects. Interleukin 2 (IL-2) activation of autologous bone marrow (BM) or peripheral blood stem cells (PBSC) before transplantation is one of the methods of adoptive cell therapy., Methods: Autologous BM of patients with chronic myelogenous leukaemia (n = 11) and PBSC of patients with multiple myeloma (n = 14) were activated by IL-2 in laboratory conditions with the aim of evaluating the feasibility of this method, the activation of T and NK cells, recovery of active progenitor cells, microbial contamination, and reduction of malignant cell content., Results: Samples of BM (mean 2.6 x 10(6) cells) and PBSC (mean 10.3 x 10(6) cells) were cultured in complete culture medium with IL-2 (6000 Ul/ml) for 24 h. The recovery of CD34+ cells and CFU-GM was 82.5% and 51.5%, respectively, for BM, and 85% and 86%, respectively, for PBSC (mean values). No purging effect was detected by flow cytometry and a small decline in malignant cell contamination was observed by quantitative PCR in BM samples. No microbial contamination occurred during the sample processing., Conclusions: The described in vitro activation of BM and peripheral blood stem cells using IL-2 was evaluated as a safe and reliable method suitable for clinical application.

Background: Recent findings of the role of the immunity in eradication of residual tumour tissue after autologous transplantation rejection leading to extensive studies on T-cell mediated specific antitumor effects or nonspecific NL-cell mediated anticancer effects. We have evaluated on the methods of adoptive cell therapy--IL-2 activation of autologous graft in the preclinical conditions. In laboratory conditions we have manipulated with autologous grafts form patients suffering with chronic myelocytic leukemia and patients suffering with multiple myeloma., Methods and Results: Autologous graft was activated with IL-2 during 24-hours cultivation period in X-Vivo 10 medium with heparine, glutamine and Dnase. Quality of grafts after cultivation, contamination and activation of T and NK cell were evaluated. No significant differences between IL-2 activated graft and control were found. Results of autologous graft quality (CD34+, CFU-GM) were comparable with already published results. Quality of final product allowed starting of clinical experimental trials., Conclusions: We have proved the possibility to use IL-2 activated autologous graft in the clinical conditions. Based on our preclinical results experimental clinical trials have been initiated in patients suffering from chronic myelocytic leukemia and multiple myeloma.

An elevated level of fibroblast growth factor-2 (FGF-2) in peripheral blood is considered to play a role in regulating the growth of leukemia cells. Here, we show that the level of plasma FGF-2 is increased in 54% of B cell chronic lymphocytic leukemias (B-CLL) and in 44% of chronic myeloid leukemias (CML). Notably, white blood cells (WBCs) from B-CLL patients contain 18, 22 and 24 kDa isoforms of FGF-2 whereas WBCs from CML patients contain only the 24 kDa isoform. Furthermore, as cultured B-CLL WBCs release 18 kDa FGF-2 into the medium, they constitute a potential source of FGF-2 in the blood. In a receptor binding assay, 125I-FGF-2 binds weakly to B-CLL WBCs, whereas the ligand binds more strongly to CML WBCs. Correspondingly, FGF-2 is unable to activate mitogen-activated protein kinase kinase (MEK) and its substrate, extracellular signal-regulated kinase (ERK), in B-CLL cells, whereas phosphorylation of both these cell growth-related kinases increases following treatment of CML WBCs. We conclude that B-CLL WBCs secrete FGF-2 with no apparent autocrine actions. In contrast, WBCs in CML bind FGF-2 provided by other FGF-2-hyperproducing cells and activate the MEK/ERK kinase cascade, possibly to modulate cell growth.

Chronic myelogenous leukemia (CML) is associated with a translocation of the protooncogene c-abl from chromosome 9 to chromosome 22, where it fuses to proximal exons of the bcr gene. The expression of the hybrid gene bcr-abl is regulated by the bcr promoter and results in a translation product with high tyrosine kinase activity. In most CML cases, one of two abl promoters (Pa) is nested within the bcr-abl transcription unit, but appears to be usually silent. Recently, de novo methylation of the Pa region and its correlation with disease progression were reported. As these previous studies were limited to the use of methylation-sensitive restriction endonucleases, our aim here was to obtain a complete map of methylcytosines and its variants in CML patients and in model cell lines. To achieve this, bisulfite conversion of cytosines (but not methylcytosines) to uracils in genomic DNA was employed. After modification, the region of interest was PCR-amplified and the products were cloned and sequenced. The results show methylation at a high level and in a homogenous pattern in the BV173 cell line, corresponding to the translocated abl alleles. Variant methylation observed in K562 cells correlates with multiple bcr-abl loci and an intact chromosome 9. Patients that were methylation-positive in restriction analysis showed sporadic and heterogenous occurrence of methylcytosines in bisulfite modification assays. Corresponding results were obtained using a quantitative Southern analysis of the extent of methylation. We conclude that restriction analysis combined with PCR is able to find rare cases of hypermethylation, e. g., for diagnostic purposes, but does not reflect the dominating level of methylation in Ph-positive cells., (Copyright 2000 Academic Press.)

Background: Cytomegalovirus (CMV) represents a serious infectious complication in patients after allogenic haematopoietic stem cell transplantation. Timely initiation of the therapy at the time of incipient active CMV infection (preemptive therapy) is an important prevention of CMV disease. It is not clear, what is the best indicator of this incipient CMV infection., Methods and Results: The significance of the detection of incipient CMV infection by CMV-antigenaemia and DNA-viraemia was compared in this study. DNA-viraemia was detected by the non-nested polymerase chain reaction (PCR). The sensitivity of nested and non-nested PCR was also compared experimentally. We tested 318 blood samples for antigenaemia and DNA-viraemia. Six samples were significantly positive for PCR, no sample was significantly positive for antigenaemia. Timely initiation of therapy resulted in the rapid eradication of the incipient active CMV infection and clearance of DNA-viraemia., Conclusions: Non-nested PCR is a suitable and more reliable method for the monitoring of incipient active CMV infection and it is a good method for timely initiation of preemptive antiviral therapy.

Diagnosis of mycotic infections is despite the immense effort devoted to this problem still very inaccurate. A new and promising method is the polymerase chain reaction, PCR, which theoretically can detect a single cell. In their original study the authors decided to develop a new method for the detection of fungi by PCR and to compare this examination with post-mortem findings. Thus it was possible to determine sufficiently reliably the sensitivity and specificity of the method. For the detection of fungi the authors selected the sequence coding for a small subunit of ribosomal RNA (18S rDNA). The method is able to detect the amount of DNA from some 10-100 cells. The sensitivity was 90% and the specificity 92%. The method is so far too laborious for common practice, its simplification would be however very useful.

A detailed study of the mutant phenylalanine hydroxylase (PAH) gene from the eastern part of the Czech Republic (Moravia) is reported. A total of 190 mutant alleles from 95 phenylketonuria (PKU) families were analyzed for 21 prevalent Caucasian mutations and restriction fragment length polymorphism/variable number of tandem repeats (RFLP/VNTR) haplotypes. Eighty per cent of all mutant alleles were found to carry 11 mutations. The most common molecular defect was the mutation R408W (55.3%), with a very high degree of homozygosity (34.6%). Each of four other mutations (R158Q, R243X, G272X, IVS12nt1) accounted for more than 3% of PKU alleles. Rarely present were mutations IVS10nt546 (2.6%), R252W (2.6%), L48S (2.1%), R261Q (1.6%), Y414C (1.0%) and 165T (0.5%). Mutations that have been predominantly described in southern Europe (IVS7nt1, A259V, Y277D, R241H, T278N) were not detected. A total of 14 different mutant haplotypes were observed. Three unusual genotype-haplotype associations were identified (R158Q on haplotypes 2.3 and 7.8 and R252W on haplotype 69.3). There was a strong association between the mutation R408W and haplotype 2.3 (54.7%). Heterogeneity was found at mutations R408W (haplotypes 2.3 and 5.9), R158Q (haplotypes 4.3, 2.3 and 7.8) and IVS10nt546 (haplotypes 6.7 and 34.7). The molecular basis of PKU in the Moravian area appears to be relatively homogeneous in comparison with other southern and western European populations, thus providing a good starting point for prenatal diagnosis and early clinical classification.

Background: Phenylketonuria is as regards the genotype a very heterogenous disease. Successful prenatal and postnatal DNA diagnosis calls for knowledge of different mutations in a given population. The objective of the investigation was to introduce direct detection of 21 mutations in the gene for phenylalanine hydroxylase and to find the distribution and frequency of these mutations in the population of northern and southern Moravia., Methods and Results: The authors analyzed a group of 95 patients where according to phenotypic classification classical phenylketonuria was involved which comprised 190 mutant alleles. The presence of mutations was assessed by means of a polymerase chain reaction of a Perkin Elmer DNA Thermal Cycler 480. From the total number of 21 mutations which were sought, 11 were identified in our population, which accounts for 80% of all mutations. It was revealed that mutation R408W is found in 55.3% of our patients. Twenty per cent of the mutations are still unknown., Conclusions: This investigation laid the foundations for direct DNA diagnosis of phenylketonuria in the Czech Republic. The results assembled in the Moravian region suggest that our population is as regards genotypes relatively homogenous. This gives great hope of successful prenatal diagnosis and postnatal genotype classification.

The possibility of detecting chlamydia by the direct immunofluorescence method was tested in the impression preparation of experimentally infected mice, chick embryos and naturally infected domestic mammals. In comparison with slight-microscope detection, this method was found to be sensitive and expedient. The greatest amount of antigen could be detected in the lungs of mice after intranasal infection already within 6 to 12 hours. In the other organs the findings were less ample; the visualization of the antigen had a good fluorescence brilliance if log 10 KEID50 was more than 3.0. In the tissues of chick embryos, the antigen could be detected in the impression preparations of the allanto-amnionic fluid, embryonal muscle, yolk sac, and chorio-allantoid membrane the fifth day after infection. After the intratracheal infection of ducklings with 1 000 000 infection doses, only individual specifically radiating cells with a low brilliance of fluorescence were detected in the organ preparations of ducklings killed 22 t0 57 days after infection. The method proved to be suitable for the detection of chlamydia in the organs of naturally infected domestic mammals in which a great amount of specifically fluorescing high-brilliance cells were found.

The diagnostic value of laboratory examination methods was assessed in the diagnosis of avian encephalomyelitis in seven to twenty-two days old chickens. Histological examination and immunofluorescent test are expedient methods meeting the requirements for timely application of infection-control measures. The agreement of histological examination with the over-all laboratory examination is 70 to 81% and the agreement of the immunofluorescence test is 76%. Bioassay on chickens followed by a histological examination appears to be the best method, providing results with the highest significance. However, it is lengthy, applicable to the necessary demonstration of a virus or to cases when the histological or immunofluorescence examinations are dubious. With the over-all laboratory evaluation the method shows agreement in 95 to 100% of cases. The diagnostic value of the method of virus isolation in chick embryos is reduced by the fact that intercurrent viruses (adenoviruses, IB and others) may be passaged at the same time.