Childhood leukemia in Ukraine after the Chornobyl accident.

This population-based ecological study analyzes the prevalence of childhood leukemia in Ukraine before and after the Chornobyl nuclear power plant accident, based on the contamination status of the territory, time period, gender, and age. Three regions-Zhytomyr, Kyiv (except Kyiv city), and Chernihiv were included as areas contaminated by radioactive 137Cs from 1 to 15 Ci/km2 with annual effective doses exceeding 1.0 mSv, and Sumy region as the control (non-contaminated) area with 137Cs contamination less than 1 Ci/km2 and effective doses less than 0.5 mSv per year. The integrated database of the National Research Centre for Radiation Medicine used in the present study included 1085 childhood leukemia cases. Two aggregated periods were used for analysis: 1980-1986 (pre-accident) and 1987-2000 (post-accident). ICD-9 codes for leukemia (204-208.9) were used to perform analyses according to the extent of leukemic cells maturity (acute, chronic, and maturity unspecified leukemia), leukemic cell lineage (lymphoid, myeloid and lineage unspecified leukemia) and all leukemia cases in different age subgroups (1-4, 5-9, 10-14, and 15-19 years). Standard methods of descriptive epidemiology were used to calculate the prevalence of disease and frequency ratio in regression models. A statistically significant increase in frequency ratio for acute leukemia (1.44; 95% confidence interval (CI), 1.22-1.71), myeloid leukemia (2.93; 95% CI, 1.71-5.40), cell lineage unspecified leukemia (II) (1.48; 95% CI, 1.18-1.87) and all forms of leukemia (1.59; 95% CI, 1.36-1.86) was found for the post-accident period in highly contaminated areas. The results indicate that the frequency of childhood leukemia (and of some of its types) increased in contaminated areas during the post-accident period, suggesting that radiation exposure after the Chornobyl accident might be the cause of the increase. However, further analytical studies, with individual or at least group dose estimates, are needed to confirm a link between childhood leukemia and the Chornobyl accident.

[Clinical, morphological, and prognostic features of papillary thyroid carcinoma with different BRAF mutational status assessed by immunohistochemistry]. / Klinicheskie, morfologicheskie i prognosticheskie osobennosti papilliarnogo raka shchitovidnoi zhelezy s razlichnym statusom BRAF, ustanovlennym immunogistokhimicheskim metodom.

The somatic mutation in BRAFT1799A (BRAFV600E), the data on the prognostic role of which are contradictory, is one of the most common molecular genetic abnormalities in the cells of papillary thyroid carcinoma (PTC). OBJECTIVE: To investigate the association of the immunoexpression of mutant BRAF in PTC with different morphological characteristics and long-term treatment results in patients. MATERIAL AND METHODS: Information on inpatients with PTC was obtained from the database of the Pathology Department, A.F. Tsyb Medical Radiology Research Center (a branch of the National Medical Radiology Research Center, Ministry of Health of the Russia). The paraffin-embedded blocks of surgically removed primary, metastatic, and recurrent PTC tissues were cut and stained with hematoxylin and eosin and anti-mutant BRAF monoclonal antibodies. The results of immunohistochemical tests were assessed and the frequency of BRAF immunoexpression was analyzed in relation to various clinical and morphological parameters of tumor growth, the pattern and volume of treatment in the patients, by taking into account its long-term results (the presence or absence of tumor recurrence and metastases in the postoperative period). RESULTS: The expression of mutant BRAF (BRAF+) was noted in 29 (39.3%) of the 74 cases of PTC. The BRAF+ group differed from the BRAF group by a higher proportion of male patients, older age, advanced clinical stage of carcinoma, a predominance of papillary structure of tumors, and a higher frequency of their recurrence and/or metastases in the postoperative period. CONCLUSION: The immunohistochemical assessment of BRAF status opens up a possibility to predict local recurrence and metastases in the postoperative period in patients treated for PTC.

IL17A gene polymorphisms: relationship to predisposition for chronic viral hepatitis and progression to liver cirrhosis in kazakh population.

Introduction: This work is the first genetic association study of a potential relationship of single nucleotide polymorphisms rs8193036 and rs2275913 located in the IL17A promoter on chromosome 6p12 to chronic viral hepatitis and its progression in Kazakh population. Purpose: Evaluation of the effect of IL17A polymorphism on predisposition for chronic hepatitis B and C and its progression to liver cirrhosis. Materials and Methods: A total of 862 individuals were enrolled in the retrospective case-control association study. Among the participants, 100 patients had chronic hepatitis B and/or C and liver cirrhosis, and 341 patients had chronic viral hepatitis only. Four hundred twenty-one (421) healthy HBV- and HCV-negative donors without liver diseases were recruited as population control. single nucleotide polymorphisms rs8193036[T/C] and rs2275913[G/A] were genotyped by TaqMan assays using genomic DNA extracted from peripheral blood cells. Results. Minor allele frequencies of rs8193036[C] and rs2275913[A] in the groups of patients were very similar to those observed in the control population, 0.4 and 0.3, respectively. Multivariate logistic regression analysis revealed odds ratios close to 1.0 and confidence intervals overlapping with the value of 1.0 and statistical significance p > 0.4 for any groups under comparison in the multiplicative model of inheritance. conclusion: No significant association between two single nucleotide polymorphisms, rs8193036 and rs2275913 in the IL17A promoter, and susceptibility to chronic viral hepatitis C and/or B and disease progression to liver cirrhosis in Kazakh population were found.

Age and gender patterns of thyroid cancer incidence in Ukraine depending on thyroid radiation doses from radioactive iodine exposure after the Chornobyl NPP accident.

UNLABELLED: The objective of this study was to investigate the thyroid cancer incidence in a whole territory of Ukraine and to clear up its age and gender patterns depending on average regional (oblast) thyroid doses from radioactive iodine due to the Chornobyl accident. MATERIALS AND METHODS. On the basis of average accumulated thyroid doses from radioactive iodine the geographical regions of Ukraine with low and high average thyroid doses were identified for a comparative analysis performance. Methods of descriptive epidemiology were used. RESULTS: The level and dynamics of thyroid cancer incidence were analyzed in different gender and age groups (both for attained age and age at the moment of the Chornobyl accident). Results of this study confirmed the radiation excess of thyroid cancer in individuals who were children and adolescents in 1986. Some excess was observed in elder age groups too. Especial situation was observed in female age group 40-49 at the moment of the Chornobyl accident i.e. the age-specific thyroid cancer incidence rates were significantly higher in "high exposure" regions comparing with "low exposure" ones during all years of observation within 1989-2009. CONCLUSIONS: A probable radiation excess of thyroid cancer was suggested not only in children and adolescents but also in adult age groups. In elder age groups this excess was less expressed and manifested after a longer period of time. The origin of the phenomenon in female age group of 40-49 is unclear now. Hypothesis of combined effect of radiation and natural changing of hormonal status in this age should be checked in the future studies.

Effects of low and high concentrations of antitumour drug taxol in anaplastic thyroid cancer cells.

AIM: To study the changes of cell cycle, mitochondrial membrane potential and caspase activation in response to an antitumour drug Taxol in ARO and KTC-2 cell lines of anaplastic thyroid carcinoma. METHODS: Experiments were done on thyroid anaplastic cancer cell lines ARO and KTC-2 using Western blotting, flow cytometry, light and fluorescent microscopy. RESULTS: Taxol significantly activated caspases in ARO cells starting from drug concentration of 5 nM. Maximum activation was observed at 25 nM and further increase of Taxol concentration to 100 nM resulted in a reduction of caspase activation. Concomitant to caspase activation, a loss of mitochondrial membrane potential was observed. At Taxol concentration of 100 nM, most cells lost their mitochondrial membrane potential. Low Taxol concentrations (10 nM) caused changes in the cell cycle that are typical for apoptosis without cell cycle arrest. Higher drug doses starting from 50 nM arrested cell cycle in G2/M phase. In KTC-2 cell line Taxol concentration as low as 1 nM induced apoptosis. 6-15 nM of the drug caused massive (75-83%) cell death. Upon Taxol action, the increase in the number of cells displaying manifestations of accelerated senescence was insignificant. CONCLUSION: Taxol induces bona fide apoptosis in thyroid cancer cell cultures at low (1-25 nM) concentrations. Higher drug doses cause the loss of mitochondrial membrane potential and possibly lead to other types of cell death. No accelerated senescence at different Taxol concentrations was observed. The significance of subG1 and G2/M cell populations at low and high doses of Taxol is discussed.

[Statistical methods for the analyses in clinical practice. Part 2. Survival analysis and multivariate statistics].

Statistical analysis is an integral component of clinical studies. The objective of the present paper is to assist clinicians in getting deeper insight into basic principles underlying different methods available for the statistical treatment of medical data without a detailed description of relevant mathematical calculations. The most popular and widely used methods of statistical analysis are considered with special reference to their practical application in clinical and experimental medicine. Part I of the review was devoted to foundations of descriptive statistics and univariate analysis. Part II is focused on the principles of survival analysis and multivariate methods.

Differential effects of low and high doses of Taxol in anaplastic thyroid cancer cells: possible implication of the Pin1 prolyl isomerase.

AIM: To study the molecular mechanisms of dose-dependent effects of an anticancer drug, Taxol, on the cell cycle machinery and apoptosis-related proteins in thyroid anaplastic cancer cell lines ARO and KTC-2. MATERIALS AND METHODS: Western blot analysis was used for the detection of various proteins and of their phosphorylated forms. RESULTS: Low dose of Taxol that cause apoptosis (25 nM) enhanced Rb protein phosphorylation, decreased the expression of cyclin-dependent kinase inhibitors p27(KIP1) and p21(WAF1) , and potentiated the accumulation of phosphorylated p53 and of the prolyl isomerase Pin1. High Taxol doses (100 and 1000 nM) that cause necrosis-like cell death drastically decreased Pin1 level in both cell lines. CONCLUSION: Low doses of Taxol promoted G(1)/S transition, thus exhibiting mitogen-like effect. Drug-induced Pin1 accumulation could probably facilitate this transition and in parallel contribute to apoptosis via the p53/p73-dependent mechanism. At higher doses of Taxol, there was a dramatic decrease of Pin1 levels which may be a reason for G(2)/M cell cycle arrest.

[Novel procedure for preoperative diagnosis of papillary thyroid carcinoma].

Our study has shown that evaluation of marker genes SFTPB and TFF3 expression using fine needle aspiration biopsy of thyroid nodal alterations is an effective means of differential diagnosis of papillary thyroid carcinoma. When used on molecular level, it may detect the disease before clinical signs develop or ultrasound examination is carried out.

[RET and GFRA1 germline polymorphisms in medullary thyroid cancer patients].

The role of RET and GFRA1 germline polymorphisms in predisposition to sporadic medullary thyroid cancer (MTC) and polymorphisms' modulation effect on clinical features of inherited and sporadic MTC were investigated. Blood samples from 67 MTC patients (22 hereditary and 45 sporadic), 3 asymptomatic mutant RET gene carriers and 178 ethnically matched healthy control individuals were tested. Screening of RET exons and portion of introns 1, 8, 10, 13, 14, 15, 16 and GFRA1 5'-UTR was performed by means of direct sequencing and PCR-RFLP. 8 polymorphic variants of RET gene (exons 11, 13, 14, 15 and introns 1, 8, 13, 14) and 4 GFRA1 polymorphisms in GFRA1 were detected. Linkage disequilibrium was found between RET variants G691S and S904S, L769L and IVS8, S836S and IVS13. In sporadic MTCs, allelic frequency of only one polymorphic RET variant, L769L, was significantly decreased versus control group. In hereditary MTCs, a significant over-representation of S836S and under-representation of S904S sequence variants were observed as compared to sporadic MTCs and controls. No co-segregation was found between individual polymorphisms and phenotype of sporadic MTC. In patients with inherited MTC whose genotype was presented with polymorphic L769L and wild-type S836S, disease onset occurred 20 years later than in individuals with polymorphic L769L and S836S or wild-type L769L (p = 0.01) suggestive of a possible protective role of L769L in MTC development and modulating effect of a combination of L769L with wild-type S836S on clinical outcome of inherited MTC.

[Frequency of RET/PTC rearrangement and somatic BRAF mutation in papillary thyroid cancer].

Frequency of RET/PTC rearrangement and somatic BRAF mutation was investigated in patients with papillary thyroid cancer (PTC) vis-a-vis relevant demographic and clinico-pathological features. The study group included 76 patients with a female/male ratio of 4.8:1; mean age - 45.7 +/- 9.7 yrs. BRAF mutation was identified in 49 (65%) (V600E--47, KSRWS600--1 and E585K--1). RET rearrangement was detected in 9 (12%): RET/PTC1--5, RET/PTC3--2, unspecified RET/PTC--1 and delta RET/PTC--1. It was age at diagnosis alone that proved to be consistently associated with BRAF mutations (p = 0.017). Younger tumor patients were mostly prone to RET/PTC rearrangement (p = 0.08). No correlation between mutation and clinico-pathological features was established.

[Molecular analysis of structural abnormalities in papillary thyroid carcinoma gene]. / Molekuliarnyi analiz strukturnykh narushenii genoma papilliarnykh kartsinom shchitovidnoi zhelezy.

Rearrangements of the RET proto-oncogene (RET/PTC) and BRAF gene mutations are the major genetic alterations in the etiopathogenesis of papillary thyroid carcinoma (PTC). We have analyzed a series of 118 benign and malignant follicular cell-derived thyroid tumors for RET/PTC rearrangements and BRAF gene mutations. Oncogenic rearrangements of RET proto-oncogene was revealed by semiquantitative RT-PCR of simultaneously generated fragments corresponding to tyrosine kinase (TK) and extracellular RET domains. The clear quantitative shift toward the TK fragment is indicative for the presence of RET rearrangements. The overall frequency of RET/PTC rearrangements in PTC was 14% (12 of 85), including 7 RET/PTC1, 2 RET/PTC3, 1 deltaRFP/RET and 2 apparently uncharacterized rearrangements. The most common T1796A transversion in BRAF gene was detected in 55 of 91 PTC (60%) using mutant-allele-specific PCR. We also identified two additional mutations: the substitution G1753A (E585K) and a case of 12-bp deletion in BRAF exon 15. Moreover, there was no overlap between PTC harboring BRAF and RET/PTC mutations, which altogether were present in 75.8% of cases (69 of 91). Taken together, our observations are consistent with the notion that BRAF mutations appear to be an alternative pathway to oncogenic MAPK activation in PTCs without RET/PTC activation. Neither RET/PTC rearrangements nor BRAF muta-tions were detected in any of 3 follicular thyroid carcinomas, 11 follicular adenomas and 13 nodular goiters. The high prevalence of BRAF mutations and RET/PTC rearrangements in PTCs and the specificity of these alterations to PTC make them potentially important markers for the preoperative tumor diagnosis.

Molecular mechanisms of the effects of low concentrations of taxol in anaplastic thyroid cancer cells.

Understanding the detailed mechanisms of a chemotherapeutic agent action on cancer cells is essential for planning the clinical applications because drug effects are often tissue and cell type specific. This study set out to elucidate the molecular pathways of Taxol effects in human anaplastic thyroid cancer cells using as an experimental model four cell lines, ARO, KTC-2, KTC-3 (anaplastic thyroid cancer), and FRO (undifferentiated follicular cancer), and primary thyrocytes. All cell lines were sensitive to Taxol, although to different extent. In primary thyrocytes the drug displayed substantially lower cytotoxicity. In thyroid cancer cells, Taxol-induced changes characteristic to apoptosis such as poly (ADP-ribose) polymerase and procaspase cleavage and alteration of membrane asymmetry only within a narrow concentration range, from 6 to 50 nm. At higher concentration, other form(s) of cell death perhaps associated with mitochondrial collapse was observed. Low doses of Taxol enhanced Bcl2 phosphorylation and led to its degradation observed on the background of a sustained or increasing Bax level and accumulation of survivin and X-chromosome-linked inhibitor of apoptosis. c-jun-NH(2) terminal kinase activation was essential for the apoptosis in anaplastic thyroid cancer cells, whereas Raf/MAPK kinase/ERK and phosphatidylinositol-3-OH kinase/Akt were likely to comprise main survival mechanisms. Our results suggest an importance of cautious interpreting of biological effects of Taxol in laboratory studies and for determining optimal doses of Taxol to achieve the desired therapeutic effect in anaplastic thyroid cancers.

Determination of somatic mutant frequencies at glycophorin A and T-cell receptor loci for biodosimetry of prolonged irradiation.

PURPOSE: To determine the variant frequencies (VF) at glycophorin A (GPA) and T-cell receptor (TCR) loci in persons exposed to prolonged ionizing radiation at different doses and to assess the significance of the GPA and TCR assays for biodosimetry of prolonged irradiation. MATERIALS AND METHODS: The VF values were determined by means of flow cytometry in 120 persons exposed between 1968-1996. Most exposures were in Chernobyl clean-up workers in 1986-1987. RESULTS: A significant correlation was shown between the NO GPA variant cell frequency and dose (r = 0.61, p < 0.0001). The slope of the linear regression was 6.3 x 10(-6) NO mutant cells/Gy. Dose-dependent increase in the TCR VF was found in the group with recent exposures (slope 2.1 x 10(-4) variant cells/Gy, r = 0.75, p = 0.0002). In the Chernobyl clean up workers who received doses less than 0.25 Gy the TCR VF unlike the GPA VF was significantly higher than in the control non-irradiated individuals (p < 0.01 and p > 0.05 respectively). CONCLUSIONS: The GPA assay has limited potential to be used as a biodosimeter of prolonged irradiation, at least in dose interval up to 2.0 Gy. The TCR assay is likely to have greater potential in estimation of recent radiation exposure than the GPA assay.

[The primary interaction with DNA of "latent high-avidity antibodies" from gamma-globulin preparations, probably, is determined by the presence of a cofactor]. / Pervichnoe vzaimodeistvie s DNK "skrytykh vysokoavidnykh antitel" iz preparatov gamma-globulina, veroiatno, opredeliaetsia prisutstviem kofaktora.

Highly positive by Farr assay, native DNA-binding fraction was derived from commercial normal human gammaglobulin preparations by ion-exchange chromatography. Human serum immunoglobulins of any isotype were not involved into this binding. The existence of nonimmunoglobulin factor primarily binding native DNA and intermediating DNA-Ig interaction is assumed.

Hidden high-avidity anti-DNA antibodies occur in normal human gammaglobulin preparations.

An ability to detect hidden high-avidity DNA-binding protein in human gammaglobulin samples was investigated. Using ion exchange chromatography on QAE-Sephadex A-50 a highly positive Farr assay DNA-binding fraction was reproducibly isolated from several commercial normal human gammaglobulin preparations. The estimated dissociation constant had a value of 1.04 x 10(-11) M thus confirming high avidity protein-DNA complex formation. Anti-DNA antibodies (Ab) ELISA data revealed that immunoglobulin (Ig) G, IgM and IgA participated in the protein-DNA interaction. Inhibitory experiments involving a number of polynucleotides, synthetic and natural polyanions demonstrated that both double-stranded DNA (dsDNA) and heat-denatured DNA but not RNA inhibited the protein-3H-DNA binding. Generally, the inhibiting effect was more pronounced when purine base-containing polynucleotides and polydeoxy- rather than polyribonucleotides were used. Synthetic polyanions and normal human sera (NHS) also markedly reduced the binding. The presence of hidden high-avidity DNA-binding antibodies in normal gammaglobulin preparations was suggested.

The atherogenic effect of lupus sera: systemic lupus erythematosus-derived immune complexes stimulate the accumulation of cholesterol in cultured smooth muscle cells from human aorta.

The influence of systemic lupus erythematosus (SLE) patients' sera on lipid accumulation in the cultured smooth muscle cells (SMC) from unaffected human aortic intima was examined. It was demonstrated that the cholesterol uptake in the SMC cultured in the presence of SLE sera is 1.5- to 6-fold higher than in the cells cultured with normal human sera (NHS) obtained from healthy donors. Incubation of the SMC with circulating immune complexes (CIC) isolated from lupus sera by precipitation with 2.5% polyethylene glycol 6000 (PEG) caused a 3- to 4-fold rise in the intracellular cholesterol level. The atherogenic effect of lupus sera, as well as isolated CIC, strongly correlated (r = 0.98) with the low density lipoprotein (LDL) content in the PEG-precipitated CIC. The cholesterol level in cultured SMC also increased 2- to 3-fold when growth medium was supplemented with LDL, DNA, and anti-DNA autoantibodies (IgG) affinity isolated from lupus sera. Using immunofluorescent staining, it was shown that the addition of a DNA-anti-DNA IgG mixture to the growth medium, together with NHS, stimulated LDL incorporation in the SMC. The results of double-label staining suggest the formation of LDL-DNA-IgG complexes which seem to be entrapped in cells more actively than free LDL. The composition of PEG-precipitated CIC was studied by electrophoresis and immunoblotting. Significant amounts of apolipoprotein B, as well as low molecular weight DNA and immunoglobulins, were found in SLE-derived CIC. The data obtained suggest that the atherogenic effect of human lupus sera in vitro is generally due to the appearance of LDL-containing immune complexes. Different mechanisms possibly involved in the lupus atherogenesis are discussed.

LDL-mediated interaction of DNA and DNA-anti-DNA immune complexes with cell surface.

The effect of low-density lipoprotein (LDL) on the binding of DNA and DNA-anti-DNA immune complexes to cultured human skin fibroblasts was examined. Using radioisotope analysis, ELISA and indirect immunofluorescent staining, a correlation between plasma membrane-bound DNA or DNA-anti-DNA immune complexes and cell-associated LDL was established. It was demonstrated that cytotoxicity and internalization of DNA-anti-DNA immune complexes may be LDL mediated. The data obtained suggest that the binding of the major part of DNA and immune complexes bound to surface of normal skin fibroblasts is due to the formation of a DNA-LDL-LDL receptor linkage. A possible role of LDL-containing immune complexes in the pathogenesis of systemic lupus erythematosus is discussed.

Polyanions inhibit binding of human autoantibodies to certain cellular proteins.

The effect of polyanions on the reactivity of human autoantibodies with cellular proteins was studied. The results of immunoblotting revealed that dextran sulfate (DS), heparin, single-stranded (ss) DNA, and polyinosinic acid (poly I) inhibit interaction between immunoglobulins (Ig) from human autoimmune sera and many polypeptides with various molecular mass. These proteins were suggested to belong to a new subclass of autoantigens, the immunoreactivity of which is sensitive to the presence of polyanions. For some of these antigens, molecular mass, intracellular localization and frequency of appearance of positive sera were determined.

[Polyanions inhibit the binding of autoantibodies with some cellular proteins]. / Polianiony ingibiruiut sviazyvanie autoantitel s nekotorymi kletochnymi belkami.

Effect of polyanions on reaction of the Ig from sera of patients with systemic lupus erythematosus and scleroderma with cellular proteins was studied by immunoblotting. It has been shown that dextran sulfate, heparin, denatured DNA and poly I inhibited the binding of autoantibodies with some polypeptides. The molecular weight of these antigens was determined. The molecular mechanism of immunological reactions studied and it's role in pathogenesis of autoimmune diseases is discussed.