Effects of Ibuprofen and Diclofenac Pre-Treatment on Viability and Apoptosis Processes in Human Dental Pulp Stem Cells.

Background and Objectives: Stem cell-based regeneration strategies have shown therapeutic efficacy in various fields of regenerative medicine. These include bone healing after bone augmentation, often complicated by pain, which is managed by using nonsteroidal anti-inflammatory drugs (NSAIDs). However, information is limited about how NSAIDs affect the therapeutic potential of stem cells. Materials and Methods: We investigated the effects of ibuprofen and diclofenac on the characteristics, morphology, and immunophenotype of human mesenchymal stromal cells isolated from the dental pulp (DPSCs) and cultured in vitro, as well as their effects on the expression of angiogenic growth factors (VEGFA and HGF) and selected genes in apoptosis signalling pathways (BAX, BAK, CASP3, CASP9, and BCL2). Results: Ibuprofen and diclofenac significantly reduced the viability of DPSCs, while the expression of mesenchymal stem cell surface markers was unaffected. Both ibuprofen and diclofenac treatment significantly upregulated the expression of HGF, while the expression of VEGFA remained unchanged. Ibuprofen significantly altered the expression of several apoptosis-related genes, including the upregulation of CASP9 and BCL2, with decreased CASP3 expression. BAK, CASP3, CASP9, and BCL2 expressions were significantly increased in the diclofenac-treated DPSCs, while no difference was demonstrated in BAX expression. Conclusions: Our results suggest that concomitant use of the NSAIDs ibuprofen or diclofenac with stem cell therapy may negatively impact cell viability and alter the expression of apoptosis-related genes, affecting the efficacy of stem cell therapy.

Atorvastatin Treatment Significantly Increased the Concentration of Bone Marrow-Derived Mononuclear Cells and Transcutaneous Oxygen Pressure and Lowered the Pain Scale after Bone Marrow Cells Treatment in Patients with "No-Option" Critical Limb Ischaemia.

BACKGROUND: The present study investigated the outcomes and possible predictive factors of autologous bone marrow cells (BMCs) therapy in patients with "no-option" critical limb ischaemia (CLI). It was focused on exploring the clinical background and prior statin and renin-angiotensin system (RAS)-acting agents pharmacotherapy related to the therapeutic efficacy of BMCs treatment. METHODS: In the present study, we reviewed thirty-three patients (mean age 64.9 ± 10 years; 31 males) with advanced CLI after failed or impossible revascularisation, who were treated with 40 mL of autologous BMCs by local intramuscular application. Patients with limb salvage and wound healing (N = 22) were considered as responders to BMCs therapy, and patients with limb salvage and complete ischemic wound healing (N = 13) were defined as super-responders. Logistic regression models were used to screen and identify the prognostic factors, and a receiver operating characteristics (ROC) curve, a linear regression, and a survival curve were drawn to determine the predictive accuracy, the correlation between the candidate predictors, and the risk of major amputation. RESULTS: Based on the univariate regression analysis, baseline C-reactive protein (CRP) and transcutaneous oxygen pressure (TcPO2) values were identified as prognostic factors of the responders, while CRP value, ankle-brachial index (ABI), and bone marrow-derived mononuclear cells (BM-MNCs) concentration were identified as prognostic factors of the super-responders. An area under the ROC curve of 0.768 indicated good discrimination for CRP > 8.1 mg/L before transplantation as a predictive factor for negative clinical response. Linear regression analysis revealed a significant dependence between the levels of baseline CRP and the concentration of BM-MNCs in transplanted bone marrow. Patients taking atorvastatin before BMCs treatment (N = 22) had significantly improved TcPO2 and reduced pain scale after BMCs transplant, compared to the non-atorvastatin group. Statin treatment was associated with reduced risk for major amputation. However, the difference was not statistically significant. Statin use was also associated with a significantly higher concentration of BM-MNCs in the transplanted bone marrow compared to patients without statin treatment. Patients treated with RAS-acting agents (N = 20) had significantly reduced pain scale after BMCs transplant, compared to the non-RAS-acting agents group. Similar results, reduced pain scale and improved TcPO2, were achieved in patients treated with atorvastatin and RAS-acting agents (N = 17) before BMCs treatment. Results of the Spearman correlation showed a significant positive correlation between CLI regression, responders, and previous therapy before BMCs transplant with RAS-acting agents alone or with atorvastatin. CONCLUSIONS: CRP and TcPO2 were prognostic factors of the responders, while CRP value, ABI, and BM-MNCs concentration were identified as predictive factors of the super-responders. Atorvastatin treatment was associated with a significantly increased concentration of BM-MNCs in bone marrow concentrate and higher TcPO2 and lower pain scale after BMCs treatment in CLI patients. Similarly, reduced pain scales and improved TcPO2 were achieved in patients treated with atorvastatin and RAS-acting agents before BMCs treatment. Positive correlations between responders and previous treatment before BMCs transplant with RAS-acting agents alone or with atorvastatin were significant.

Population genetic analysis of 12 X-STR markers in Slovakia.

Background: During the last 20 years, X-chromosomal STR markers have become widely used in forensic genetics and paternity testing. Nevertheless, to exploit their full potential in any given population, a reliable reference dataset needs to be established. Since no relevant studies concerning these markers have been performed on the Slovak population so far, we decided to analyse several commonly used markers in this population.Aim: To create an informative set of Slovak population data concerning X-STR markers.Subjects and methods: We genotyped 378 individuals and analysed 12 loci (DXS10148, DX10135, DXS8378, DXS7132, DXS10079, DXS10074, DXS10103, HPRTB, DXS10101, DXS10146, DXS10134 and DXS742) localised in four distinct linkage groups.Results: Our analysis showed that the most informative marker is DXS10135 (PIC = 0,927) and the most informative linkage group (LG) is LG1 with 149 different haplotypes. This analysis also confirmed linkage disequilibrium for two pairs of markers (DX10101-DX10103 and DX10101-HPRTB) within LG3 in female samples. No statistically significant departure from HWE was observed for any locus. Moreover, the interpopulation comparison of 8 European populations based on haplotype frequencies showed no statistically significant FST values in any LG, except for LG2 in comparison with the German population.Conclusion: We created a haplotype database for forensic analyses and kinship testing in Slovakia, as well as the CE dataset which can be used to further increase the decision power in similar analyses in the future.

Effect of Atorvastatin on Angiogenesis-Related Genes VEGF-A, HGF and IGF-1 and the Modulation of PI3K/AKT/mTOR Transcripts in Bone-Marrow-Derived Mesenchymal Stem Cells.

Stem cell transplantation represents a unique therapeutic tool in tissue engineering and regenerative medicine. However, it was shown that the post-injection survival of stem cells is poor, warranting a more comprehensive understanding of activated regenerative pathways. Numerous studies indicate that statins improve the therapeutic efficacy of stem cells in regenerative medicine. In the present study, we investigated the effect of the most widely prescribed statin, atorvastatin, on the characteristics and properties of bone-marrow-derived mesenchymal stem cells (BM-MSCs) cultured in vitro. We found that atorvastatin did not decrease the viability of BM-MSCs, nor did it change the expression of MSC cell surface markers. Atorvastatin upregulated the mRNA expression levels of VEGF-A and HGF, whereas the mRNA expression level of IGF-1 was decreased. In addition, the PI3K/AKT signaling pathway was modulated by atorvastatin as indicated by the high mRNA expression levels of PI3K and AKT. Moreover, our data revealed the upregulation of mTOR mRNA levels; however, no change was observed in the BAX and BCL-2 transcripts. We propose that atorvastatin benefits BM-MSC treatment due to its ability to upregulate angiogenesis-related genes expression and transcripts of the PI3K/AKT/mTOR pathway.

Mutational analysis of 16 STR markers in the Slovak population.

BACKGROUND: Short tandem repeats (STRs) are genetic markers frequently used for human identification and paternity testing. They are highly mutable, which may occasionally lead to inconsistencies between the genotypes of parents and their children. As the mutation rates of individual STR markers can vary among populations, population-specific data are of high importance. AIM: To investigate the mutation rates of 16 STR markers in the Slovak population. SUBJECTS AND METHODS: In this study, we analysed the germline mutation rates of 16 STR markers (TH01, D3S1358, vWA, D21S11, D16S539, D1S1656, D19S433, SE33, D10S1248, D22S1045, D12S391, D8S1179, D2S1338, D2S441, D18S51 and FGA) in the Slovak population. At these loci, we analysed 42 096 allelic transfers and identified 61 mutation events. RESULTS: The loci with the highest overall mutation rates were SE33 and FGA, while no mutations were identified in TH01, D19S433 and D22S1045. The average paternal mutation rate was higher than the maternal mutation rate. All but one mutation consisted of gains or losses of a single repeat unit and the overall mutation rate was estimated to be 1.45 x10-3 per meiosis. CONCLUSION: This study provides data which can be used to further strengthen the correct paternity index calculations and reliability of paternity testing in Slovakia.

Normalization strategy for selection of reference genes for RT-qPCR analysis in left ventricles of failing human hearts.

BACKGROUND: Quantitative RT-PCR is a valuable tool for assessing the gene expression in different human tissues, particularly due to its exceptional sensitivity, accuracy and reliability. However, the choice of adequate control for normalization is a crucial step, greatly affecting the results of all subsequent analyses. So far, only a few studies were focused on the selection of optimal reference genes in left ventricles of failing human hearts, leading to several disparities in experimental results focused on differential gene expression in this area. Therefore, the main objective of this study was to identify a set of suitable reference genes in normal and failing left ventricle tissues, which could increase the reliability of RT-qPCR-based studies in the future. METHODS: We analyzed the expression of 15 commonly used housekeeping genes (ACTB, B2M, GAPDH, GUSB, HMBS, HPRT1, IPO8, PGK1, POLR2A, PPIA, RPLP0, TBP, TFRC, UBC and YWHAZ) in left ventricles of normal and failed hearts with two-step approach. In the first step, we excluded genes which are variantly expressed using ANOVA-based statistical method. Afterwards, the remaining genes were analyzed using geNorm, NormFinder and BestKeeper algorithms, together with delta Cq method. Finally, the geometric mean of gene rankings across all methods was calculated. RESULTS: Our analysis identified IPO8 and POLR2A as the most stably expressed genes, whereas ACTB and B2M were found to be expressed variantly, suggesting a potential role of these genes in the pathophysiological processes in failing human hearts. DISCUSSION/CONCLUSION: Using our two-step approach, we identified and validated two reference genes expressed invariantly in left ventricles of both healthy and failing human hearts, as well as provided a guideline for the selection of reference genes in studies comparing gene expression in these types of tissues.

Population and forensic analysis of 9 non-CODIS markers in Slovak population.

Genetic analyses of STR markers provide data for several important applications, including criminal caseworks, kinship analyses and population studies. In this study, we describe the parameters of nine STR markers (D2S1360, D3S1744, D4S2366, D5S2500, D6S474, D7S1517, D8S1132, D10S2325, D21S2055) in Slovak population and compare them with several European and worldwide populations. Since these markers are not commonly used in forensic practice, our data might provide subsidiary discriminatory power in the most demanding criminal examinations, where additional markers need to be analyzed. Moreover, we performed the MDS analysis and constructed a phylogenetic tree representing genetic relationships between several European populations based on Nei's DA distance. We show that the analysis of just 9 markers is sufficient for reliable clustering of different European populations and their separation from the populations from other geographical areas. Taken together, our data provide a reliable dataset characterizing the Slovak population, which might serve as a basis for criminal, population and kinship studies.