The Importance of New Generation Sequencing (NGS) HLA Typing in Renal Transplantation-Preliminary Report.

INTRODUCTION: Thanks to new generation sequencing (NGS) and expansion of HLA typing with additional loci, it will be possible to increase the effectiveness of graft survival and to avoid complications related to the immune system. New pharmacogenetic factors are still being researched to develop better immunosuppressive treatment. MATERIAL AND METHODS: The incidence of polymorphic HLA loci variants was established, based on a high-resolution NGS method in kidney graft recipients. Furthermore, haplotypic analysis between examined loci was conducted to type additional loci that may influence the transplantation result. A total of 120 kidney recipients were enrolled in the study. A commercial DNA extraction kit in Tubes (QIAamp DNA Blood Mini Kit Qiagen, Germany) was used to isolate DNA from the blood. Sequencing library preparation was done with TruSight HLA set. The Conexio computer program was used to analyse the results of HLA typing. RESULTS: The patients with alleles A*02:01:01, B*44:02:01, C*03:03:01, C*01:02:01, C*05:01:01, C*07:02:01, DQB1*03:03:02, DQB1*06:04:01, or with haplotypic variation A*25:01:01-B*18:01:01- C*15:01:01 were taking the highest doses of cyclosporine (CsA), in contrast to patients with allele B*18:01:01, DQB1*06:02:01, DQB1*02:02:01, or haplotypic variation A*02:01:01- B*44:02:01-C*01:01:01, who were taking the lowest doses. The highest dose of tacrolimus (TAC) was administered to patients with alleles A*68:01:02, A*29:01:01, B*07:02:01, B*35:02:01, B*38:01:01, DRB1*12:01:01, DQB1*05:03:01, or haplotypic variations A*02:01:01-B*57:01:01-C*07:01:01, A*03:01:01-B*07:02:01-C*13:01:01, A*29:02:01-B*44:03:01- C*07:01:01, and A*01:01:01-B*08:01:01-C*03:01:01. Additionally, it was established that HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DPA1, and HLA-DQA1 show very slight polymorphism, which suggests that there is no need for their typing for transplantation purposes. Moreover, loci HLA-C, HLA-DQB1, and HLA-DPB1, which are not routinely examined in recipient-donor matching, show genetic variability that may increase the risk of transplant rejection or shortened graft life. CONCLUSIONS: Expanding the qualification procedure to include allele genotyping could allow clinicians to establish immunosuppressive treatment schemes that would be optimally suited for recipients' phenotype.

The influence of the tumor necrosis factor-alpa-308G>A polymorphism on the efficacy of immunosuppressive therapy in patients after kidney transplantation.

Cytokines play an important role in the immune response. The calcineurin inhibitors (cyclosporine CsA, tacrolimus TAC) widely used after renal transplantation to prevent allograft rejection are immunosuppressive drugs suppressing the production of cytokines. These drugs are characterized by interindividual variability and require monitoring their blood concentrations to predict their optimal dosage. Therefore, the aim of the study was to determine the correlation between therapeutic effects of immunosuppressants and the tumor necrosis factor-α (TNF-α)-308G>A polymorphism in renal transplant patients. A total of 412 patients receiving TAC and CsA were included in the study. Genotype frequencies were determined using the real-time PCR method. Patients with the GG genotype received higher doses of TAC as compared to carriers of the GA genotype (5.24 mg versus 3.35 mg) and had lower mean drug concentration in blood (5.86 ng/ml versus 6.92 ng/ml). Similar results were also obtained for CsA (GG: 185.33 mg versus GA: 153.30 mg, P < 0.05). The comparison of the TNF-α-308G>A polymorphism with the biochemical parameters did not reveal a potential risk for transplant rejection. These results indicate that the TNF-α-308G>A polymorphism may influence the dosage of immunosuppressive drugs in patients after transplantation as far as individualization of drug therapy is concerned.

Polymorphism of vitamin D3 receptor and its relation to mineral bone density in perimenopausal women.

UNLABELLED: Postmenopausal osteoporosis is the most common metabolic bone disease with important genetic factors. We evaluated the frequency of polymorphism 283G/A of the vitamin D3 VDR gene receptor. The study included 800 women at the postmenopausal (505) and reproductive (295) age. Statistically significant changes, depending on the genotype, were shown. INTRODUCTION: Postmenopausal osteoporosis is the most common metabolic bone disease of strong genetic origin with population variability determined by the interaction of genetic and environmental factors. Recognition of different genetic variants underlying development of osteoporosis would make it possible to administer individual symptomatic treatment as well as early prophylactics of osteoporosis. METHODS: The aim of the study was to evaluate the frequency of polymorphism 283G/A of the vitamin D3 VDR gene receptor and assessment of its relations with the clinical parameters of osseous turnover and degree of postmenopausal osteoporosis. The study included 800 women at the postmenopausal (505) and reproductive (295) age throughout the Wielkopolska region in Poland. The postmenopausal group included women with osteoporosis and osteopenia and the healthy ones. Women at the reproductive age were healthy. Frequency of the tested gene polymorphism was evaluated in the group where bone mineral density (BMD) was marked and in the control group. RESULTS: The obtained test results pointed to correlation of polymorphism VDR 283G/A with the BMD scores for the lumbar vertebrae in women with osteopenia and osteoporosis, therefore the ones at risk of fractures. Vitamin D receptor (VDR) polymorphism correlated with reduced BMD values. CONCLUSIONS: Polymorphism 283G/A of the vitamin D3 receptor gene has been proved to be the genetic factor of postmenopausal osteoporosis. The polymorphism mentioned above has been proved to be a factor of mineral bone density changes of women.

Involvement of the different extracts from roots of Salvia miltiorrhiza Bunge on acute hypobaric hypoxia-induced cardiovascular effects in rats--preliminary report.

The present study was carried out to investigate the protective effects of roots of Salvia miltiorrhiza Bunge on hypobaric hypoxia. Two extracts of S. miltiorrhiza (extract 1: ethanol : water - 50 : 50; extract 2: 96% ethanol) were used. The experiments were performed after 7 consecutive days of administration of the extracts (200 mg/kg b.w., intragastrically) to male Wistar rats. Next, after placing animals for 60 min in the controlled acute hypobaric hypoxia (500 mm Hg) the systolic arterial blood pressure (SAP) in conscious rats, bioelectric heart activity in unconscious rats and analysis of oxidative stress parameters in the blood of rats: malonyldialdehyde (MDA) and lipid peroxidase (LPO) concentration, activity of superoxide dismutase (SOD) or glutathione peroxidase (GPX) were assayed. It was found out that the extract 1 augmented the lowering of SAP shown in hypoxia affected control rats. On the contrary the extract 2 reversed SAP to values obtained in control animals. Moreover, both extracts led to the normalization of hypoxia-induced tachycardia and levels of MDA, LPO and SOD. It seems that the above-mentioned effects are coupled with different active compounds content in the extracts, however more studies are needed to confirm this hypothesis.

The effect of standardized Echinacea purpurea extract on rat cytochrome P450 expression level.

It is claimed that application of botanical supplements or herbal medicinal products with synthetic drugs that are cytochrome P450 enzymes substrates may induce significant herb-drug interactions and may alter pharmacotherapy. Echinacea preparations are one of the best selling products in the Europe and their medicinal use is still increasing but data about interactions of Echinacea extract with CYP enzymes are limited. In this study, we have investigated potential influence of standardized Echinacea purpurea extract containing 3.7% polyphenolic compounds on the mRNA expression level of major CYP450 enzymes using animal model. Total RNA was isolated from the rat liver tissue according to the manufacturer's protocol. Complementary DNA was synthesized from a mature mRNA template using reverse transcription. The level of mRNA expression in liver was analyzed by real-time quantitative PCR using specific target primers for CYP450 genes. In this study, it was demonstrated a significant increase of rat CYP2D1 and CYP1A1 expression level by 40% (p = 0.007) and 80% (p = 0.01), respectively. A weak inductory effect of the extract was observed for CYP1A2 by 16% (p > 0.05) compared with the control group. The levels of rat CYP3A1 and CYP3A2 mRNA were reduced by 41% (p < 0.05) and 25% (p = 0.001), respectively. A weak inhibitory effect was observed for CYP2D2 by 15% (p = 0.008) and CYP2C6 by 18% (p = 0.004) after long application of the Echinacea ethanolic extract. CYP2D2 and CYP2C6 activities were also inhibited by extract but in a lesser degree than CYP3A1 activity. Moreover, very little or no inhibition was noted for CYP2E1 both after 3 and 10 days of treatment. Our in vivo data indicate that the Echinacea ethanolic extract can potently inhibit the expression of CYP3A1/2 and can also induce of CYP1A1, CYP2D1. These findings suggest that Echinacea extract may influence the P450-mediated metabolism of different drugs and may initiate chemical carcinogenesis by activation of some compounds to their carcinogenic metabolites.

The effect of artichoke (Cynara scolymus L.) extract on respiratory chain system activity in rat liver mitochondria.

The effect of artichoke extract on mitochondrial respiratory chain (MRC) activity in isolated rat liver mitochondria (including reaction kinetics) was studied. The effect of the extract on the activity of isolated cytochrome oxidase was also studied. Extract in the range of 0.68-2.72 microg/ml demonstrated potent and concentration-dependent inhibitory activity. Concentrations > or =5.4 microg/ml entirely inhibited MRC activity. The succinate oxidase system (MRC complexes II-IV) was the most potently inhibited, its activity at an extract concentration of 1.36 microg/ml being reduced by 63.3% compared with the control (p < 0.05). The results suggest a complex inhibitory mechanism of the extract. Inhibition of the succinate oxidase system was competitive (K(i) = 0.23 microg/ml), whereas isolated cytochrome oxidase was inhibited noncompetitively (K(i) = 126 microg/ml). The results of this study suggest that the salubrious effects of artichoke extracts may rely in part on the effects of their active compounds on the activity of the mitochondrial respiratory chain system.

An optimization of hematopoietic stem and progenitor cell isolation for scientific and clinical purposes by the application of a new parameter determining the hematopoietic graft efficacy.

The transplantation of hematopoietic stem and progenitor cells (HSPC) is an established lifesaving therapy. Bone marrow (BM), harvested from heparinized cadaveric organ donors, peripheral blood (PB) and cord blood (CB), are important sources of hematopoietic stem cells. HSPCs, which are used for transplantation purposes, are routinely evaluated in terms of number of mononuclear cells (MNCs), CD34+ MNCs count and viability. The efficacy of grafting is determined additionally in clonogenic tests in vitro. These tests deliver important information about the number of HSPCs and their proliferative potential. Unfortunately, they do not give a possibility to evaluate the functional HSPC chemotactic reactivity in the SDF-1 gradient, which is probably the key phenomenon for HSPC homing after transplantation procedure. Thus, the aim of our study was to optimize HSPC isolation according to their chemotactic reactivity in SDF-1 gradient. Using multiparameter cell sorter (FACS Aria, BD) we examined the HSPCs attracted by SDF-1 on a single cell level. The population of cells which participated in the chemotactic process was highly enriched in CXCR4+lin-AC133+CD45+ cells (referred as hematopoietic stem cells) and to our surprise in CXCR4+lin-AC133+CD45- cells (referred as pluripotent stem cells) in quantitative amounts. Since reactivity of HSPCs may depend on various factors involved in the protocol of their isolation and short-term storage, we tested the most commonly used anticoagulants (ACD, CPDA-1, EDTA and Heparin) and culture media (DME, IMDM, RPMI). HSPCs, harvested from CB, PB and BM, were subsequently investigated for clonogenic growth of CFU-GM in methylcellulose cultures and for the level of apoptosis by employing annexin V staining. Evaluating clonogenic potential, ability of chemotactic reactivity in SDF-1 gradient and intensification of apoptosis of HSPC as the most safe anticoagulant and medium were selected. This study has proved that chemotactic reactivity of HSPCs is a new but very important parameter which should be included in the procedure of their isolation.

The effect of artichoke (Cynara scolymus L.) extract on ROS generation in HUVEC cells.

The effect of an artichoke extract on induced reactive oxygen species (ROS) generation in cultured human umbilical endothelial cells (HUVECs) and its reductive properties were evaluated. Preincubation of HUVEC cells with the artichoke extract at concentrations of 25-100 microg/mL for 24 h abolished ROS generation induced by LPS and oxyLDL as evaluated by the fluorescence intensity of 2',7'-dichlorofluorescein (DCF). Potent, concentration-dependent reductive properties of the artichoke extract were demonstrated by the reduction kinetics of cytochrome c in reference to ascorbate were also revealed. The results of the present study the warrant application of artichoke extracts as endothelium protecting agents.

Low-density lipoprotein receptor-related protein-associated protein (LRPAP1) gene IVS5 insertion/deletion polymorphism is not a risk factor for gallstone disease in a Polish population.

BACKGROUND: There is growing evidence that gallstone formation may be genetically determined. It was recently presented that a common polymorphism in the LRPAP1 gene might be associated with gallstone disease. AIM: Since reproducibility of data is important in genetic association studies, a case control study was designed to find out whether LRPAP1 gene polymorphism is associated with gallstone disease in a Polish population. SUBJECTS: Two hundred eighty-nine Polish Caucasian gallstone disease patients and 251 healthy controls participated in the study. METHODS: A 37-bp insertion/deletion polymorphism in intron 5 of LRPAP1 (rs11267919) was determined by means of polymerase chain reaction assay. RESULTS: The frequencies and distribution of the insertion/deletion alleles did not differ significantly between gallstone disease patients and controls. No significant gender-related differences in allele frequencies or distributions were noted. CONCLUSION: The LRPAP1 insertion/deletion polymorphism is not associated with gallstone disease in a Polish population.

Interleukin-18 promoter polymorphism in patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease in which interleukin (IL)-18 plays an important role. However, there are controversial reports on IL-18 promoter polymorphism as an independent marker of RA susceptibility. The aim of present study was to examine the IL-18 promoter polymorphism in patients with RA, and its association with disease susceptibility, activity and severity. We examined 309 patients with RA from a Polish population diagnosed according to the criteria of American College of Rheumatology. An allele-specific polymerase chain reaction was used for analysis of the polymorphisms in positions - 137 and - 607 in promoter region of IL-18 gene. A significantly decreased number of subjects with AC/AC and AG/AG diplotypes was observed among RA patients as compared with healthy controls (OR - 0.51, 95%CI 0.28-0.95, P = 0.045) and (OR - 0.12, 95% CI 0.02-0.97, P = 0.042), respectively. Nevertheless, there was no significant association with disease activity, joint erosions, extra-articular manifestations, rheumatoid factor. Above results suggest that IL-18-137 and - 607 promoter polymorphisms are not the significant factors influencing RA course and severity in a Polish population.

Frequencies of the common promoter polymorphisms in cytokine genes in a Polish population.

Common polymorphisms in cytokine genes, widely distributed in many populations, may affect gene transcription, leading to individual variations in cytokine levels. Some of those polymorphisms were associated with the risk of graft rejection or autoimmune and parasitic diseases development as well as treatment outcome. In the current study the frequencies of promoter polymorphisms in genes encoding interleukin-2 (-330 G/C), interleukin-4 (-590 C/T), interleukin-6 (-174 G/C), interleukin-10 (-1082 G/A, -592 C/A) and TNFalpha (-307 G/A) have been studied in a sample of 205 unrelated healthy Polish subjects of Caucasian origin. The frequencies of the determined cytokine alleles were as follows: interleukin-2 (-330 G/C): T -64.9%, G -35.1%; interleukin-4 (-590 C/T): C -79.8%, T -20.2%; interleukin-6 (-174 G/C): G -53.4%, C -46.6%; interleukin-10 (-1082 G/A, -819 G/T, -592 C/A): GCC -44.1%, ACC -32.0, ATA -23.9%; TNFalpha (-307 G/A): G -85.1%, A -14.9%. We also compared our results with other populations studied to date, describing some significant differences.

[Effect of quercetin on the course of chronic poisoning with fluorine compounds in rats]. / Wplyw kwercytyny na przebieg przewleklego zatrucia zwiazkami fluoru u szczurów.

Biochemical examinations in serum, homogenates and microsomal fraction of the liver, as well as biochemical and morphological examinations of liver tissue were performed in rats of Wistar strain exposed chronically for 6 months to ammonium fluoride vapours. A part of animals was protected with a mixture of sodium salts of quercetin synthetized in Inorganic Chemistry Department of the Technical University at Rzeszów, and added to the standard feed in a dose of 5 and 20 mg/kg body weight. It has been disclosed that NH4F causes disturbances in the activities of enzymes (cholinesterases, transaminases, alkaline and acid phosphatase), a rise of bilirubin concentration in serum, as well as disturbances in lipid metabolism, an increase in the content of total lipids, and disorders of lipid metabolism--increase in the content of total lipids, cholesterol and triglycerides, with phospholipids being decreased. The biochemical changes are accompanied by the hepatic tissue lesion. The use of quercetin alleviates, to a considerable degree, the biochemical and morphological disturbances due to protracted exposure to ammonium fluoride vapours.

A search for new derivatives of chiral amino alcohols with an antiarrhythmic activity.

Twelve new (R) and (S) Schiff bases and twelve new (R) and (S) 2-benzylamino-1-butanols have been obtained. Pharmacological tests showed a weak antiarrhythmic and hypotensive activity of the screened chiral 2-benzylamino-1-butanols.