Effect of polyoxyethylene and polyoxypropylene nonionic block copolymers on performance and recruitment of immune cell subsets in weaned pigs.

BACKGROUND: Because European-wide directives are restricting the non-clinical use of antibiotics as in-feed growth promotors in swine production, there is an intensive search for alternative strategies for control and prevention of losses among young pigs. With the growing knowledge of the porcine immune system and its endogenous modulation, it has been clearly established that exogenous immunomodulation using adjuvants and immune response modifiers (IRMs) represents an important prophylactic/therapeutic approach in the prevention/treatment of both stress- and microbial-induced disorders that accompaning weaning. However, it is essential to select a fully evaluated agent which may act either as a nonspecific IRM or synergistically as an adjuvant with vaccines. The synthetic macromolecules with a long history as adjuvant and IRM are nonionic block copolymers which consist of polyoxyethylene (POE) and polyoxypropylene (POP) molecules. METHODS: The aim of this work was to evaluate the effectiveness of POE-POP given as a single peroral dose on productivity parameters such as body weight gain, feed intake and feed conversion ratio, and systemic and intestinal immune parameters by assessing the proportions of CD45+ lymphoid cells, CD4+ and CD8+ T cells, and CD21+ B cells in the peripheral blood as well as the number of CD45RA+ naive lymphoid cells residing in the ileal mucosa in weaned pigs during a follow-up study 5 weeks after the treatment. RESULTS: Pigs treated with POE-POP had better feed intake (+ 14.57%), higher average body mass at the end of the experiment (20.91 kg vs. 17.61 kg), and higher body weight gain in relation to Day 0 (191.63% vs. 144.58%) as well as in relation to nontreated pigs (+ 18.74%), with a lower feed conversion ratio (- 30.26%) in comparison to the control pigs. A much lower diarrhea severity score (5 vs. 54) was recorded in pigs treated with POE-POP (- 90.74%) than in the control pigs. A higher average diarrhea severity (ADS) was recorded in the control pigs (1.54 vs. 0.14), whereas the treatmant group had much a lower ADS ratio (- 90.91%) after 35 days of the experiment. The pigs that were treated with POE-POP had an increased proportion of CD45+, CD4+ and CD8+ cells at Day 21 (at p < 0.05, p < 0.05 or p < 0.01, respectively), Day 28 (at p < 0.01, respectively) and Day 35 (at p < 0.01, p < 0.05 or p < 0.01, respectively) as well as of CD21+ cells at Day 28 (p < 0.05) and Day 35 of the experiment (p < 0.01). Also, these pigs had more numerous CD45RA+ cells in interfollicular (p < 0.05) and follicular areas (p < 0.01) of the ileal Peyer's patches than did control pigs. CONCLUSION: This property of POE-POP to induce recruitment of circulating and intestinal immune cell subsets in weaned pigs may allow the use of IRM-active block copolymers as adjuvants for vaccines, particularly those orally delivered and targeted to the gut-associated lymphoid tissues that are well known to promote rather tolerogenic than protective immune responses.

Analysis of 8 X-chromosomal markers in the population of central Croatia.

AIM: To analyze 8 X-linked short tandem repeat (STR) markers in the population of central Croatia and to evaluate their forensic efficiency. METHODS: We carried out a statistical analysis of the data from previously performed genetic analyses, collected during routine forensic work by the Forensic Science Centre ''Ivan Vucetic.'' Mentype® Argus X-8 PCR amplification kit was used for typing the data of 99 unrelated healthy women and 78 men from central Croatia. Haplotype frequencies were calculated only in male samples. Arlequin 3.5 software was used to assess Hardy-Weinberg equilibrium (HWE), linkage disequilibrium (LD), observed and expected heterozygosity. Power of discrimination (PD) for men and women, polymorphism information content (PIC), power of exclusion, and mean exclusion chance for deficiency cases, normal trios, and duos were determined using online database ChrX-STR.org. RESULTS: In female samples, deviations from HWE (P=0.006) for each locus were not found. LD test performed both on female and male samples revealed no significant association between markers (P=0.002). DXS10135 was the most polymorphic locus (PIC=0.931). PD varied from 0.692 to 0.935 in male and from 0.845 to 0.992 in female samples. Combined PD reached 99.999999% in men and 99.9999999999% in women. CONCLUSION: Performed analyses revealed that the studied marker set contained polymorphic markers with high power of discrimination. We can conclude that Mentype® Argus X-8 PCR is suitable for application in the population of central Croatia. Results of this study, together with collected allele and haplotype frequencies, are the first step in establishing a national reference X-STR database based on 8 X-STR loci.

Effect of ultraviolet C radiation on biological samples.

AIM: To examine the influence of ultraviolet C (UVC) radiation on blood, saliva, semen, and naked DNA samples for preventing DNA cross-contamination on working surfaces in laboratories. METHODS: Blood, saliva, semen, and DNA isolated from buccal swab samples were obtained from a single male donor and applied to the laboratory working surfaces. UVC radiation was applied to these diluted and undiluted samples with or without previous decontamination of the working surfaces with 10% sodium hypochlorite and 20% ethanol. Genomic DNA was extracted using Chelex. After quantification, DNA was amplified using the AmpFlSTR® NGM™ PCR Amplification Kit. We tested and statistically analyzed DNA concentration, UVC dose, sample volume, radiation time, the number of correctly detected alleles on genetic loci, and the number of correctly detected alleles in four groups in which 16 loci were divided. RESULTS: When working surfaces were not decontaminated and were treated only with UVC radiation in the laboratory, the genetic profile for naked DNA could not be obtained after 2 minutes of UVC radiation and for saliva after 54 hours. For blood and semen, a partial genetic profile was obtained even after 250 hours of UVC radiation in the laminar. When working surfaces were decontaminated with 10% sodium hypochlorite and 20% ethanol, genetic profile could not be obtained for naked DNA after 2 minutes, for saliva after 4 hours, for blood after 16 hours, and for semen after 8 hours of UVC radiation in the laboratory. CONCLUSION: It is recommended to carefully and thoroughly clean working surfaces with 10% sodium hypochlorite and 20% ethanol followed by minimal 16-hour UVC exposure (dose approximately 4380 mJ/cm2) for complete and successful decontamination.

DNA methylation: the future of crime scene investigation?

Proper detection and subsequent analysis of biological evidence is crucial for crime scene reconstruction. The number of different criminal acts is increasing rapidly. Therefore, forensic geneticists are constantly on the battlefield, trying hard to find solutions how to solve them. One of the essential defensive lines in the fight against the invasion of crime is relying on DNA methylation. In this review, the role of DNA methylation in body fluid identification and other DNA methylation applications are discussed. Among other applications of DNA methylation, age determination of the donor of biological evidence, analysis of the parent-of-origin specific DNA methylation markers at imprinted loci for parentage testing and personal identification, differentiation between monozygotic twins due to their different DNA methylation patterns, artificial DNA detection and analyses of DNA methylation patterns in the promoter regions of circadian clock genes are the most important ones. Nevertheless, there are still a lot of open chapters in DNA methylation research that need to be closed before its final implementation in routine forensic casework.

Croatian national reference Y-STR haplotype database.

A reference Y-chromosome short tandem repeat (STR) haplotype database is needed for Y-STR match interpretation as well as for national and regional characterization of populations. The aim of this study was to create a comprehensive Y-STR haplotype database of the Croatian contemporary population and to analyze substructure between the five Croatian regions. We carried out a statistical analysis of the data from previously performed genetic analyses collected during routine forensic work by the Forensic Science Centre "Ivan Vucetic". A total of 1,100 unrelated men from eastern, western, northern, southern and central Croatia were selected for the purpose of this study. Y-STRs were typed using the AmpFISTR Yfiler PCR amplification kit. Analysis of molecular variance calculated with the Y chromosome haplotype reference database online analysis tool included 16 population samples with 20,247 haplotypes. A total of 947 haplotypes were recorded, 848 of which were unique (89.5%). Haplotype diversity was 0.998, with the most frequent haplotype found in 9 of 1,100 men (0.82%). Locus diversity varied from 0.266 for DYS392 to 0.868 for DYS385. Discrimination capacity was 86.1%. Our results suggested high level of similarity among regional subpopulations within Croatia, except for mildly different southern Croatia. Relative resemblance was found with Bosnia and Herzegovina and Serbia. Whit Atheys' Haplogroup Predictor was used to estimate the frequencies of Y-chromosome haplogroups. I2a, R1a, E1b1b and R1b haplogroups were most frequent in all Croatian regions. These results are important in forensics and contribute to the population genetics and genetic background of the contemporary Croatian population.

Genetic polymorphisms of 17 short tandem repeat loci on Y chromosome in central Croatian population.

In forensic casework, Y-chromosome short tandem repeat (STR) haplotyping is used in human identification, paternity testing and sexual assault cases where Y-STRs provide a male-specific DNA profile. The aim of this study was to describe the genetic structure of Y chromosome in a central Croatian population. We carried out a statistical analysis of the data from previously performed genetic analyses collected during routine forensic work by the Forensic Science Centre "Ivan Vucetic". A total of 220 unrelated healthy men from central Croatia were selected for the purpose of this study. Genomic DNA was extracted using a Chelex procedure from FTA(®) cards. Y-chromosomal STRs were determined using the AmpFISTR Yfiler PCR amplification kit. The haplotype frequencies were determined by direct counting and analyzed using Arlequin 3.1 and analysis of molecular variance calculated with the Y chromosome haplotype reference database online analysis tool. A total of 212 haplotypes were identified, 204 of which were unique. Total haplotype diversity was 0.993. Locus diversity varied from 0.325 for DYS392 to 0.786 for DYS385. Discrimination capacity was 92.7%. Allele frequencies diversity was 0.615. Intermediate alleles 17.2, 18.2 and 19.2 were found at DYS458 locus. A comparison with published data for the European minimal haplotype set showed the closest relationship to the Croatian capital of Zagreb and Bosnia and Herzegovina with significant genetic distance from Slovenia and Austria. The central Croatian population is now well characterized in terms of Y-chromosome STRs, thus providing a solid basis for further forensic and genetic epidemiology studies.

Population data for 17 short tandem repeat loci on Y chromosome in northern Croatia.

Human Y-short tandem repeats (STRs) are tandem repeat arrays of two to seven base pair units on non-recombining region (NRY) of the human Y chromosome. Studies on Y-STR are interesting in both population genetics and forensics. The aim of this study was to investigate the population genetic properties of 17 STR loci on Y chromosome in the northern Croatia region. We carried out a statistical analysis of the data from previously performed genetic analysis collected during routine forensic work by the Forensic Science Centre "Ivan Vucetic". A total of 220 unrelated healthy men from northern Croatia were selected for the purpose of this study. Genomic DNA was extracted using Chelex procedure from FTA(®) cards. Y-chromosomal STRs were determined using the AmpFISTR Yfiler PCR amplification kit. The haplotype frequencies were determined by direct counting and analyzed using Arlequin 3.1 and analysis of molecular variance calculated with the Y chromosome haplotype reference database online analysis tool. A total of 210 haplotypes were identified, 200 of which were unique. Total haplotype diversity was 0.995. Locus diversity varied from 0.331 for DYS392 to 0.783 for DYS385 locus. Allele frequencies diversity was 0.662. Discrimination capacity was 95.7%. The use of European minimal haplotype set indicated the most resemblance of this population to the Croatian capital of Zagreb, with modest resemblance to Bosnia and Herzegovina, Serbia and Hungary. This article provides the first overview of the Y chromosome STR variability in northern Croatia, thus providing the referent point for any future forensic and genetic epidemiology efforts in this region.

Dioxins and human toxicity.

The term dioxins usually refers to polychlorinated dibenzo-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). As 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) has the highest toxic potential, the toxic potentials of other PCDDs and PCDFs are defined in comparison with it. Human exposure to dioxins can be environmental (background), occupational, or accidental pollution. In the human body, dioxins are in part metabolised and eliminated, and the rest is stored in body fat. People vary in their capacity to eliminate TCDD, but it is also dose-dependent; the elimination rate is much faster at higher than lower levels. The liver microsomal P4501A1 enzyme oxygenates lipophilic chemicals such as dioxins. It is encoded by the CYP1A1 gene. Cytosolic aryl hydrocarbon receptor (AhR) mediates their carcinogenic action. It binds to dioxin, translocates to nucleus and together with hydrocarbon nuclear translocator (ARNT) and xenobiotic responsive element (XRE) increases the expression of CYP1A1.Dioxins are classified as known human carcinogens, but they also cause noncancerous effects like atherosclerosis, hypertension, and diabetes. Long-term exposures to dioxins cause disruption of the nervous, immune, reproductive, and endocrine system. Short-term exposure to high levels impairs the liver function and causes chloracne. The most sensitive population to dioxin exposure are the foetuses and infants.A large number of health effects have been documented in the scientific literature, and they all place dioxins among the most toxic chemicals known to man.

[Y chromosome: from evolution to forensics--an overview]. / Kromosom Y: od evolucije do forenzike.

About 300 million years ago, chromosomes X and Y were true homologues, similar in size and gene content. Over time, deletions in the Y chromosome resulted in its size reduction to approximately 60 Mb. Significant homology in sequence with the X chromosome is still present. Y chromosome contains the fewest number of genes of any chromosome and is mostly composed of heterochromatin. The genes that are present on the Y chromosome are critically important in sexual development (sex-determining region on the Y gene, SRY, which only determines male sex). Y chromosome contains two pseudoautosomal regions at both ends of the chromosome, where possible recombination with the X chromosome occurs during spermatogenesis. Euchromatin contains functional genes and transcription inert heterochromatin forming a non-recombining region on Y chromosome, which comprises 95% of the chromosome. The same is only present in male, is inherited unchanged from father to son and is rich in polymorphic repetitive elements, microsatellite and minisatellite DNA. Short tandem repeat (STR) loci are located on the non-recombining region of the Y chromosome and are inherited as a block of linked haplotypes. Y-STR haplotyping is of great importance for forensic applications, such as identification of unknown persons, paternity testing, detecting male DNA profile in mixtures and of azoospermic individuals, and verification of amelogenin deficient males.

Population genetic analysis of haplotypes based on 17 short tandem repeat loci on Y chromosome in population sample from eastern Croatia.

AIM: To investigate the population genetics of 17 short tandem repeat (STR) loci on the Y chromosome in the population of eastern Croatia. METHODS: We carried out a statistical analysis of the data from previously performed genetic analysis collected during routine forensic work by the Forensic Science Centre "Ivan Vucetic". A total of 220 unrelated healthy men from eastern Croatia were selected for the purpose of this study. Genomic DNA was extracted by Chelex from FTA cards. Y-chromosomal STRs were determined using the AmpFISTR Yfiler PCR amplification kit. The haplotype frequencies were determined by direct counting and analyzed using Arlequin 3.1 and analysis of molecular variance calculated with the Y-chromosome haplotype reference database online analysis tool. RESULTS: A total of 207 haplotypes were recorded, 197 of which were unique (90%). Haplotype diversity was 0.9993, with the most frequent haplotype found in 4 of 220 men (1.8%). Average locus diversity was 0.600, and it ranged from 0.256 for DYS392 to 0.780 for DYS458. Our results were compared with the pattern of Y-chromosome variability in publicly available population samples based on a minimal European haplotype set of 9 STRs and the greatest resemblance was found with samples from the Croatian capital of Zagreb, from Bosnia and Herzegovina, and from Serbia. CONCLUSION: This is the first description of Y chromosome haplotyping of the population of eastern Croatia, which may serve as a basis for genetic epidemiology and forensic studies. Further studies are needed for characterization of the genetic structure of the Y-chromosome in the modern Croatian population.

C-reactive protein gene polymorphisms affect plasma CRP and homocysteine concentrations in subjects with and without angiographically confirmed coronary artery disease.

Human C-reactive protein (CRP) is a reactant involved in the acute phase response and one of the many molecular factors involved in pathogenesis of coronary artery disease (CAD). CRP gene variants potentially mediate CRP plasma concentrations and the development of CAD. 220 Croatian subjects with angiographically confirmed CAD and 132 control subjects were included in the study. CRP gene polymorphisms 1059G/C and -717G/A were determined by RFLPs, using MaeIII and KspI endonuclease, respectively. Plasma concentrations of CRP and homocysteine were determined by immunoturbidimetry and FPIA, respectively. CRP 1059G/C gene variants were significantly associated with CAD (OR = 0.50; 95% CI = 0.27, 0.94; P = 0.032). Wild GG genotype and rare allele C carrier genotypes were 184 and 22 in CAD(+) group, and 101 and 24 in CAD(-) group, respectively. Multivariate analysis with age, gender, BMI, smoking status, hypertension and diabetes as covariates showed that 1059C carriers had lower CRP concentrations in CAD(-) (P = 0.010) and CAD(+) subjects (P = 0.028). This allele was also significantly associated with lower plasma homocysteine concentrations in both groups (P = 0.018 for CAD(-) and 0.002 for CAD(+). There was no significant difference between CAD(+) and CAD(-) subjects in absolute frequencies for CRP -717A/G gene variant, but multivariate analysis showed that carriers of the rarer G allele had significantly higher CRP plasma concentrations in CAD(-) subjects (P = 0.031) and higher homocysteine concentrations in CAD(+) group (P < 0.001). Atherosclerosis is an inflammatory disease resulting from different genetic and environmental factors. Results presented here support the contribution of CRP genetic variations in the development of CAD.

Genotype frequencies of UDP-glucuronosyltransferase 1A1 promoter gene polymorphism in the population of healthy Croatian pre-scholars.

Increased serum bilirubin levels in patients with Gilbert syndrome (GS) are caused by reduction of hepatic activity of bilirubin glucuronosyltranferase to about 30% of normal. UGT1A1 genetic polymorphism with absent or very low bilirubin UDP-glucuronosyltransferase (B-UGT) activity is associated with Gilbert's syndrome (GS) and other hyperbilirubinemias. The genetic basis of GS is the insertion of two additional TA nucleotides (resulting in seven repeats of TA) in the TATAA box, present in proximal promoter of UGT1A1 gene. This study included 323 Croatian pre-scholars, including 164 boys and 159 girls. Statistical analysis showed significant difference for total bilirubin concentration between different genotypes (p < 0.001). Also, statistically significant difference for total bilirubin concentration was emphasized between genotypes 6/6 and 7/7 (p < 0.001) as well as 6/7 and 7/7 (p < 0.001). Higher total plasma bilirubin concentrations are significantly correlated with 7/7 genotype which is present in 9.8% of population studied.

[Influence of gene polymorphisms in adhesion molecules and inflammation mediators as risk factors for coronary heart disease and myocardial infarction--an overview]. / Utjecaj polimorfizama gena za adhezijske molekule i medijatore upale kao cimbenika rizika za koronarnu bolest srca i infarkt miokarda.

Cardiovascular diseases are influenced by inheritance and environmental factors. There is a growing number of genetic variants, which may be included in the genesis and development of coronary artery disease (CAD). CAD or ischemic heart disease is a set of clinical symptoms caused by inadequate transport of oxygen because of changes in coronary circulation leading to myocardial ischemia. The most common cause of CAD is atherosclerosis of coronary arteries, which primarily narrows or occludes the lumen of coronary arteries or stimulates thrombosis. In this review, the role of the most important polymorphisms in adhesion molecules, intracellular adhesion molecule-1, integrins alpha2beta1 and beta3, E-selectin as well as of inflammation mediators, tumor necrosis factors alpha and beta, in the development of CAD risk and myocardial infarction is discussed. A review of different genotyping results provides an insight into the mechanisms responsible for the disease risk and helps detect the key sets of predictive markers that are clinically informative.

Association of two genetic variations of lipoprotein lipase, S447X and Hind III, with coronary artery disease and hypertriglyceridemia.

This study was performed to assess the effect of the S447X and Hind III lipoprotein lipase gene polymorphisms on development of coronary artery disease and hypertriglyceridemia. The study included 132 patients and 98 healthy control subjects of Croatian descent. The lipoprotein lipase S447X polymorphism was associated with coronary artery disease and hypertriglyceridemia, as indicated by the lower frequency of S447 allele in the patient group (p = 0.005) and odds ratio (O.R = 0.40, p = 0.006). The patient and control groups also showed a significant difference in the distribution of Hind III/S447X genotype combinations (p = 0.013). There were no significant associations with lipid parameters for any genotype or genotype combination in the patient group. Frequencies of the S447X polymorphism and S447X/Hind III combinations differed between the CAD/TG and control group, thus these polymorphisms may be associated with CAD and hypertriglyceridemia.

Expression of hyaluronan synthase genes in umbilical cord blood stem/progenitor cells.

Scientific progress reveals an ever-expanding role of hyaluronan (HA) in diverse biological functions. It has become increasingly clear that HA might also be essential for certain functions of stem cells. CD133+ cells isolated from umbilical cord blood (UCB) seem to represent an alternative to CD34+ cells as a source of transplantable haematopoietic progenitor cells. The aim of this study was to investigate expression patterns of hyaluronan synthases (HAS) genes in freshly isolated and cultured UCB progenitor cells and to compare HAS mRNA levels to those found in non-progenitor cells. CD133+ stem cells were isolated from UCB using an immunomagnetic procedure. Investigation of HAS mRNA expression patterns in CD133+ and CD133- cells by RT-PCR was performed immediately after isolation as well as after cultivation towards myelomonocytic lineage. In addition, activation patterns of mitogen activated protein kinases (MAPK) were analyzed by Western blot experiments. mRNA for HAS1 is undetectable but HAS3 mRNA can be readily detected in freshly isolated CD133+ as well as in CD133- UCB cells. More importantly, our data demonstrate that mRNA for HAS2 can only be detected in CD133+ progenitor cells. In addition, while MAPK are slightly activated in CD133- UCB cells, no significant phosphorylation of MAPK could be observed in CD133+ cells, excluding a role of these kinases in the regulation of HAS2. HAS2 is expressed only in freshly isolated CD133+ cells and quickly diminishes during differentiation. Because of this, HAS2 gene expression might be suitable as a new marker for CD133+ UCB-derived stem cells.

[The frequencies of apolipoprotein E genotypes in health and disease in the Croatian population--an overview of expectations and real results]. / Ucestalost genotipova apolipoproteina e u zdravlju i bolesti: ocekivanja i rezultati u hrvatskih ispitanika.

The aim of the overview is to show the distribution of common apolipoprotein E (APOE) genotypes in the Croatian population, and to test whether it could serve as a new molecular biomarker in some clinical entities. The study included the following groups: patients with angiographically confirmed coronary artery disease, myocardial infarction, Alzheimer's dementia, vascular dementia, hyperlipidemias, diabetes mellitus, pancreatitis, and healthy subjects. Group comparisons of different clinical entities and control group were performed using Pearson's Chi2-test. There was no difference in APOE genotype frequencies between coronary artery disease neither myocardial infarction and control group. The ApoE genotype frequencies in patients with Alzheimer's disease were significantly different from those in the control group. APOE-4 allele tends to be a risk factor for the development of Alzheimer's disease. The frequencies were only marginally different in vascular dementia. Patients with hypercholesterolemia, those with inherited familial hypercholesterolemia, children with diabetes mellitus, and patients with pancreatitis of different etiology showed distributions of APOE genotypes that differed from the control group. It is concluded that the frequencies of APOE genotypes yielded no statistically significant result to confirm the association between APOE genotypes and any specific disease with the exception of Alzheimer's disease; APO-epsilon4 allell has become one of the important biomarkers in diagnosis of Alzheimer's dementia.

Differentiation of human umbilical cord blood CD133+ stem cells towards myelo-monocytic lineage.

BACKGROUND: Characterisation of stem cells by flow cytometry, their expansion and differentiation are presently of major interest for cell engineering as the basis of a therapeutic concept for transplantation. Haematopoietic stem cells (HSC) express CD34, the adhesion structure which binds 2L-selectin, CD117, a receptor for stem cell factor (SCF; c-kit ligand), and CD133, a transmembrane protein belonging to the family of mucoproteins. METHODS: The aim of the present investigation was the systematic investigation of proliferation and differentiation characteristics of umbilical cord blood stem cells (UCBSC) isolated by an immmunomagnetic separation system using CD133 antibody-coated microbeads and to evaluate the effects of different sera and various concentrations, as well as the effects of IL-3 and IL-6 on total cell expansion and differentiation of isolated CD133+ cells. Differentiation patterns were measured by flow cytometry. RESULTS: After the immmunomagnetic separation the yield of CD133+ cells was 0.45+/-0.17 x 10(6) cells/ml; the purity of isolated CD133+ cells was 95.79+/-1.86%. The majority of CD133+ cells coexpressed CD117. The most pronounced expansion during cultivation of 2 weeks was achieved in media supplemented with 12.5% horse serum plus 12.5% fetal calf serum (FCS) with stem cell factor and interleukine 3; the fold-expansion was 16.67+/-6.20. During the cultivation period, UCBSC were constantly loosing stem cell markers and differentiated towards myelo-monocyte lineage (granulocytes and/or monocytes). CONCLUSIONS: These in vitro results demonstrate that thorough investigation of various cultivation conditions is needed for successful expansion and differentiation of stem cells towards different lineages to be used therapeutically for replacement of damaged cells.

Cell cycle analysis of the CD133+ and CD133- cells isolated from umbilical cord blood.

BACKGROUND: Umbilical cord blood cells (stem/progenitor cells) exhibit high proliferative capacities leading to a large expansion of cells in appropriate cell culture conditions. The aim of this study was to evaluate by flow cytometry the cycling status of CD133+ and CD133- cells depending on various culture conditions, such as sera, stem cell factor (SCF), interleukin 3 (IL-3) and interleukin 6 (IL-6). METHODS: An immunomagnetic system was used for cell separation. CD133+ and CD133- cells were seeded in Iscove's Modified Dulbecco's Medium (IMDM) with different serum concentrations and were stimulated with SCF (100 ng/ml), IL-3 (50 ng/ml) and IL-6 (50 ng/ml). RESULTS: Our experiments demonstrated that immediately after separation, 96.75+/-0.58% of CD133+ cells and 97.04+/-1.76% of CD133- cells were in G0/G1-phase, while 2.02+/-0.38% and 0.88+/-0.52% were in the S-phase, respectively. Our data documented that CD133+ cells are more active than CD133- cells after the first week of cultivation (p<0.01). Statistically significant difference was found for CD133+ cells vs. CD133- cells after second week of cultivation in G0/G1- and S-phases under all tested conditions. A combination of 12.5% FCS+12.5% HS yielded the highest cell expansion for CD133+ cells; this was concomitant with highest percentage of S-phase and G2M-phase. Our data show that the medium with 25% HS was the best for cell expansion and cycling of the CD133- cells for the first week, followed by the 12.5% FCS+12.5% HS. After 2 weeks of cultivation, obviously 12.5% HS and 12.5% FCS+12.5% HS exhibited similar S-phase amounts in CD133- cells. A decrease of HS concentrations seemed to stimulate CD133- cells' S-phase after the second week. CONCLUSIONS: Our data indicate that the source and the concentration of the serum used for cultivation have an impact on both cell populations: CD133+ cells are most comfortable with a combination of FCS and HS; CD133- cells prefer media-containing HS. Cell cycle status may be an important factor for defining cultivation strategies for stem cell expansion.

Lipoprotein lipase gene polymorphisms in Croatian patients with coronary artery disease.

Modifications in lipoprotein lipase levels lead to elevated triglycerides and reduced high density lipoprotein (HDL), both of which are risk factors for coronary artery disease (CAD). Hence, we examined the influence of the -93T/G, D9N, N291S, and S447X polymorphisms in the lipoprotein lipase (LPL) gene on CAD risk and lipid levels in Croatian patients with and without angiographically confirmed CAD. The N291S polymorphism was significantly associated with CAD (OR = 0.36; 95% CI = 0.13, 0.99; p = 0.048). This association was only moderately affected by adjusting for various lipids (OR = 0.36; 95% CI = 0.12, 1.08; p = 0.068). HDL2-cholesterol and apolipoprotein A-I levels were significantly higher in non-carriers of the -93T/G and D9N polymorphisms in the CAD group (p = 0.017 and 0.028, respectively). The N291S genetic variant did not show any significant difference between carriers and non-carriers in either group studied for any of the lipids. Lower triglyceride and higher HDL2-cholesterol levels in the control group were associated with carriers of the S447X mutation (p = 0.043 and 0.056, respectively). LPL gene polymorphisms might be involved in predisposition to CAD and determination of lipid profiles.

Mutation analysis of the MPZ and PMP22 genes in Croatian patients.

We used single-strand conformation polymorphism analysis for mutational screening in two candidate genes, MPZ and PMP22, which have an important role in the pathogenesis of Charcot-Marie-Tooth disease (CMT) and related peripheral neuropathies. A novel Ser8Ser polymorphism was found in exon 1 of the MPZ gene in two heterozygous subjects, in a father with mild CMT2 phenotype and his daughter with normal clinical data. Thr118Met polymorphism was found in exon 5 of the PMP22 gene. The patient heterozygous for 118Met allele had CMT1 disease. We can conclude that the occurrence of the 118Met allele does not usually cause CMT1 and that it is not a clinically relevant disease marker.