Lenka Luptáková

Sex determination of early medieval individuals through nested PCR using a new primer set in the SRY gene

One of the first questions asked about excavated human skeletal remains is the sex. As the morphological sex determination is complicated in cases involving fragmentary bones and in skeletons from infants and children, the development of DNA-based techniques has led to improvements in sex determination. This study is focused on sex determination from ancient DNA obtained from 25 skeletons found in Middle Aged burials in western Slovakia. We performed separate amplifications of DXZ4 repetitive satellite sequences on the X chromosome, and SRY gene - testis determined factor on the Y chromosome, using nested PCR. Our results showed that DXZ4 was amplified in the case of 23 individuals. With newly designed internal and external primer sets for SRY detection with internal PCR products in lengths of 102 bp and 85 bp we succeeded in detecting the SRY locus in 9 samples. Finally, the gender was determined in 23 individuals (14 females and 9 males). In 20 samples, the gender was determined by morphological and molecular methods. Sex determination of 17 samples using nested PCR matched the morphological one, providing evidence of the authenticity and ancient origin of the PCR amplifications. The DXZ4/SRY nested PCR method represents a useful technique in sex determination of medieval human remains and it is a critical addition to anthropological studies.

Power of biomarkers and their relative contributions to metabolic syndrome in Slovak adult women

Background: Metabolic syndrome (MetS) comprises a cluster of risk components which pre-dispose individuals to cardiovascular mortality. Aim: The purpose of this study is to investigate the variability of biochemical and anthropometric characteristics, apolipoprotein E (APOE) and angiotensin converting enzyme (ACE) genes and their contribution to MetS manifestation. Subjects and methods: A total of 438 adult women were recruited from different localities in Slovakia. All data was established by standard anthropometric, biochemical and genetic methods. Results: The logarithm of the ratio of plasma concentration of triglycerides to HDL-cholesterol [log(TG-to-HDL-C)], waist circumference, systolic blood pressure, apolipoprotein A1, glucose and alanin aminotransferase accounted for most of the differences in MetS manifestation. Logistic regression showed that participants with risk values of the atherogenic index log(TG-to-HDL-C) had a 15.62-fold higher risk of MetS compared to those with lower values for this index (95% CI = 8.3–29.1). Women with hyperglycaemia (or formerly diagnosed diabetes mellitus) had an 8.82–times higher risk of MetS (95%CI = 3.22–24.16). Women with hyper-uricaemia had the same risk of MetS incidence as women with abdominal obesity, Exp (B) = 4.05.Hypercholesterolaemia, ACE and APOE genotypes did not influence MetS. Conclusion: MetS may involve many risk factors that can cause serious disorders in multiple organs. However, women with risk values involving plasma atherogenic index log (TG-to-HDL-C) experienced the highest risk of developing MetS.

Association of ACE (I/D) Polymorphism with Metabolic Syndrome and Hypertension in two Ethnic Groups in Slovakia

The objective of this study was to examine the influence of ACE (I/D) genotypes on recognized risk variables for hypertension and Metabolic Syndrome in two ethnic population samples from Slovakia. A total of 150 Romany subjects (68 males and 82 females) and 167 Slovaks (45 males and 122 females) were examined. They were interviewed during a medical examination and they were investigated with respect to a variety of aspects such as medical, anthropometrical and life-style. The studied subjects were defined as hypertensive if the blood pressure was > or = 140/90 mm Hg and Metabolic Syndrome (MS) was defined according to criteria of the National Cholesterol Education Program Adult Treatment Panel III-(NCEP ATPIII). ACE (I/D) polymorphism was subsequently determined by PCR amplification of the ACE gene sequence. In the entire sample, the frequency of the mutant D allele was higher in the Slovak subjects (D = 0.527) than in the Romany subjects (D = 0.447), but the difference was not significant (p = 0.053). Neither the Slovak nor the Romany normotensive and hypertensive subjects differed significantly in the distribution of the three ACE genotypes (Slovak p = 0.169, Romany p = 0.116). In both ethnic samples hypertensive men prevailed (Slovak 51.1% vs. Romany 44.1%). The features of Metabolic Syndrome were identified in both samples; they occurred in 33.3% of Slovak men and 14.8% Slovak women vs. 42.9% of Romany men and 32.4% Romany women. Regression analysis showed no association between ACE genotypes and hypertension nor between ACE genotypes and MS in these Slovak population samples.

Molecular characterization and first report of Cryptosporidium genotypes in human population in the Slovak Republic

In our study, we examined 91 fecal samples from five different groups of people containing HIV patients, hemodialysis patients, kidney transplant recipients, immunocompetent humans without clinical signs, and humans with suspected cryptosporidiosis. The purpose of our study was to determine species and genotype composition of representatives of Cryptosporidium spp. using PCR analysis of small subunit ribosomal RNA gene and 60‐kDa glycoprotein gene and examine their phylogenetic relationship. In HIV‐positive/AIDS‐infected group of patients and in hemodialysis patients, no presence of Cryptosporidium species was detected. In two kidney transplant recipients, we detected species/genotypes Cryptosporidium parvum IIaA13G1T1R1 (KT355488) and Cryptosporidium hominis IaA11G2R8 (KT355489) and in two immunocompetent patients with clinical symptoms, we identified Cryptosporidium muris and C. hominis IbA10G2T1 (KT355490). In the group of healthy immunocompetent individuals without clinical signs, we identified species/genotype C. hominis IbA11G2 (KT355491) in one sample.

First report of Enterocytozoon bieneusi and Encephalitozoon intestinalis infection of wild mice in Slovakia

Increased risk of zoonotic transmission of the potential human pathogenic species Enterocytozoon bieneusi, Encephalitozoon intestinalis and Encephalitozoon cuniculi was detected in wild immunocompetent mice (Mus musculus musculus; n=280). Analysis was conducted with the use of PMP1/PMP2 primers and SYBR Green RT-PCR. Using Real Time PCR and comparing the sequences with sequences in the GenBank, E. bieneusi was detected in 3 samples (1.07 %), E. cuniculi in 1 sample (0.35 %) and E. intestinalis in 1 sample (0.35 %). The results of this report document the low host specificity of detected microsporidia species, and imply the importance of synanthropic rodents as a potential source of human microsporidial infection.

Rodents as a reservoir of infection caused by multiple zoonotic species/genotypes of C. parvum, C. hominis, C. suis, C. scrofarum, and the first evidence of C. muskrat genotypes I and II of rodents in Europe

Cryptosporidium spp. is an important causative agent of intestinal parasitoses-induced diarrhoea in humans and animals worldwide. Rodents (small mammals), the main reservoir of infections, are globally expanded and overpopulated, which increases the risk of transfer of human and zoonotic pathogens from the genus Cryptosporidium. In this study, Cryptosporidium was detected in wild immunocompetent asymptomatic small mammals. Altogether 262 fecal samples were collected from five areas in Eastern Slovakia from four different rodent species (Myodes glareolus, Apodemus agrarius, Apodemus flavicollis, Rattus norvegicus), eight samples originated from two insectivore species (Sorex araneus, Crocidura suaveolens), and two sample from a carnivore Mustela nivalis. The samples were examined using a method modified in our laboratory, based on the use of specific primers on a small subunit rRNA (18S rRNA) gene for species identification, and amplification of GP60 gene coding 60-kDa glycoprotein for genotype determination. The following species were identified: Cryptosporidium parvum (n=15), genotypes IIaA18G3R1 (n=11; KU311673), IIaA10G1R1 (n=1; KU311670), IIcA5G3a (n=1; KU311669), IIiA10 (n=2; KU311672); Cryptosporidium suis (n=4; KU311671); Cryptosporidium scrofarum (n=28); Cryptosporidium environment sp. (n=12; KU311677); Cryptosporidium muskrat genotype I (n=3; KU311675); Cryptosporidium muskrat genotype II (n=3; KU311676). From one of the rodent, the species Cryptosporidium hominis genotype IbA10G2 (KU311668) was identified for the first time. The results of this study indicate low host specificity of the detected Cryptosporidium species and imply the importance of free-living small mammals in urban and suburban habitats as a potential source of human cryptosporidiosis.

Changes of the immunological and haematological parameters in rabbits after bendiocarbamate application

The effect of bendiocarbamate application (5 mg/kg b.w.) on the haematological and immunological parameters in rabbits was evaluated. Total leukocyte cell count, erythrocyte cell count, differential cell count were determined during the period of three months of bendiocarbamate application and compared with those in healthy animals. The immunotoxic effect was evaluated by the test of ingestion ability of phagocytes (phagocytic activity and index of phagocytic activity) and proliferation activity of lymphocytes after mitogen stimulation. The significant decrease of total leukocytes, lymphocytosis and neutropaenia were found after bendiocarbamate application. The functional activities of phagocytes (expressed as phagocytic activity) and lymphocytes (proliferative activity) were significantly suppressed in rabbits treated with bendiocarbamate compared with those in control groups and values before the experiment.

Variant in the FTO gene and biomarkers related to health in mature Slovak women.

The purpose of this study was to investigate whether variant (rs178 17449, G/T) in the first intron of the fat mass and obesity-associated gene (FTO) was related to different obesity parameters and blood pressure in mature women from Slovakia. A total of 419 Slovak women (241 premenopausal and 178 postmenopausal) ranging in age from 39 to 65 years were recruited from different parts of Slovakia. The subgroups were categorized based on the WHO (1996) criteria. All participants gave written informed consent for participation in this study. Anthropometric parameters were measured using standard methods. Fat mass was examined by bioimpedance and blood pressure was measured in the morning during the medical examination. Genomic DNA was extracted from blood or saliva samples by the JET-QUICK Tissue DNA spine kit. The FTO variant was determined by PCR and restriction analysis according to the methodology of Hubacek et al. (2008). The obtained data were statistically analyzed by SPSS 17.0 for Windows. The FTO genotype and allele frequencies in the entire sample and in subgroups according to their menopausal and blood pressure status fell within the Hardy-Weinberg equilibrium. In postmenopausal women the FTO (rs178 17449) genotype was significantly associated with systolic blood pressure (SBP) (p = 0.024) in the dominant GG/GT vs.TT model and with diastolic blood pressure (DBP) (p = 0.030) in the recessive GG vs. GT/TT and the additive model (p = 0.043), respectively. In these postmenopausal women regression analysis showed a statistically significant effect of age, BMI and FTO dominant model on SBP, and of BMI on DBP among the other variables capable of inducing blood pressure differences. This study demonstrates that the SNP rs178 17449 in the FTO gene is associated with systolic and diastolic blood pressure but not with BMI and obesity variables, as already replicated in several populations throughout the world.

Bioelectrical Impedance Vector Analysis (BIVA) in Slovak population: application in a clinical sample

The purpose of this study is to provide new data on body composition in the Slovak population, particularly impedance vector components according to sex and age, relevant for bioelectrical impedance vector analysis (BIVA) in a clinical sample. The reference sample consisted of 1543 apparently healthy individuals (1007 females and 536 males), aged from 18 to 92 years and of 60 patients with Parkinson’s disease (PD) (26 females and 34 males), aged from 40 to 81 years. Bioelectrical parameters of resistance (R) and reactance (Xc) were measured with a monofrequency analyser (BIA 101). BIVA was used to analyse tissue electric properties in control subjects and patients with PD. The mean vector position differed significantly between PD patients and healthy controls in males of age subgroups 60–69 years and 70–79 years, respectively. These results were conterminous with significant Hotelling’s T2-test; 60–69 y T2=7.8, P=0.024 and 70–79 y T2=7.6, P=0.026. In the RXc-score graph three patients had values outside the 95% ellipse. Altered tissue electric properties were present in 23.5% of males and 15.4% of females. Distribution of impedance vector components in different age categories of healthy Slovak subjects are relevant to comparative population studies and to clinical practice.

The association of cytochrome P450 1B1 Leu432Val polymorphism with biological markers of health and menopausal symptoms in Slovak midlife women.

ObjectiveIn this study, the CYP1B1 polymorphism was examined in relationship to recognized risk factors for cardiovascular disease. In particular, this study focused on plasma lipid levels, atherogenic indices, and body composition. Furthermore, this polymorphism was analyzed as a predisposing factor for menopausal symptoms among women during midlife, subdivided according to their menopause status. MethodsA total of 399 women aged from 39 to 60 years were examined. They were recruited from the western and middle parts of Slovakia. Participants were interviewed during their medical examination, and they were investigated with respect to a variety of aspects such as anthropometric and medical aspects, and a menopause-specific questionnaire was included. The participants provided a saliva or blood sample for DNA genotyping and a blood sample for biochemical analysis. ResultsThe Leu432Val genotype demonstrated statistically significant associations with triglycerides, with the ratio of total cholesterol to high-density lipoprotein cholesterol, and with the logarithm of the ratio of plasma concentration of triglycerides to high-density lipoprotein cholesterol in women in their reproductive period. The mean values were significantly lower in women carrying the Val/Val genotype. Four atherogenic indices showed a decreasing trend in relationship to the CYP1B1 genotypes in women during their reproductive period (in the following order of magnitude: Leu/Leu + Leu/Val vs Val/Val) and an increasing trend among postmenopausal women in the same order. Furthermore, the Val/Val genotype diminished experiences of bloated stomach, of vaginal dryness in perimenopausal and postmenopausal women, and of palpitations in premenopausal women. ConclusionsThe Leu432Val polymorphism may be associated with the lipid profile in midlife women. Moreover, this polymorphism may influence the risk of some menopausal symptoms.