Transcriptomic Characterization of Genes Regulating the Stemness in Porcine Atrial Cardiomyocytes during Primary In Vitro Culture.

Heart failure remains a major cause of death worldwide. There is a need to establish new management options as current treatment is frequently suboptimal. Clinical approaches based on autologous stem cell transplant is potentially a good alternative. The heart was long considered an organ unable to regenerate and renew. However, several reports imply that it may possess modest intrinsic regenerative potential. To allow for detailed characterization of cell cultures, whole transcriptome profiling was performed after 0, 7, 15, and 30 days of in vitro cell cultures (IVC) from the right atrial appendage and right atrial wall utilizing microarray technology. In total, 4239 differentially expressed genes (DEGs) with ratio > abs |2| and adjusted p-value ≤ 0.05 for the right atrial wall and 4662 DEGs for the right atrial appendage were identified. It was shown that a subset of DEGs, which have demonstrated some regulation of expression levels with the duration of the cell culture, were enriched in the following GO BP (Gene Ontology Biological Process) terms: "stem cell population maintenance" and "stem cell proliferation". The results were validated by RT-qPCR. The establishment and detailed characterization of in vitro culture of myocardial cells may be important for future applications of these cells in heart regeneration processes.

Transcriptomic Profile of Genes Regulating the Structural Organization of Porcine Atrial Cardiomyocytes during Primary In Vitro Culture.

Numerous cardiovascular diseases (CVD) eventually lead to severe myocardial dysfunction, which is the most common cause of death worldwide. A better understanding of underlying molecular mechanisms of cardiovascular pathologies seems to be crucial to develop effective therapeutic options. Therefore, a worthwhile endeavor is a detailed molecular characterization of cells extracted from the myocardium. A transcriptomic profile of atrial cardiomyocytes during long-term primary cell culture revealed the expression patterns depending on the duration of the culture and the heart segment of origin (right atrial appendage and right atrium). Differentially expressed genes (DEGs) were classified as involved in ontological groups such as: "cellular component assembly", "cellular component organization", "cellular component biogenesis", and "cytoskeleton organization". Transcriptomic profiling allowed us to indicate the increased expression of COL5A2, COL8A1, and COL12A1, encoding different collagen subunits, pivotal in cardiac extracellular matrix (ECM) structure. Conversely, genes important for cellular architecture, such as ABLIM1, TMOD1, XIRP1, and PHACTR1, were downregulated during in vitro culture. The culture conditions may create a favorable environment for reconstruction of the ECM structures, whereas they may be suboptimal for expression of some pivotal transcripts responsible for the formation of intracellular structures.

Expression Profile of Genes Encoding Proteins Involved in Regulation of Vasculature Development and Heart Muscle Morphogenesis-A Transcriptomic Approach Based on a Porcine Model.

Despite significant advances in treatment of acute coronary syndromes (ACS) many subjects still develop heart failure due to significantly reduced ejection fraction. Currently, there are no commonly available treatment strategies that replace the infarcted/dysfunctional myocardium. Therefore, understanding the mechanisms that control the regeneration of the heart muscle is important. The development of new coronary vessels plays a pivotal role in cardiac regeneration. Employing microarray expression assays and RT-qPCR validation expression pattern of genes in long-term primary cultured cells isolated form the right atrial appendage (RAA) and right atrium (RA) was evaluated. After using DAVID software, it indicated the analysis expression profiles of genes involved in ontological groups such as: "angiogenesis", "blood vessel morphogenesis", "circulatory system development", "regulation of vasculature development", and "vasculature development" associated with the process of creation new blood vessels. The performed transcriptomic comparative analysis between two different compartments of the heart muscle allowed us to indicate the presence of differences in the expression of key transcripts depending on the cell source. Increases in culture intervals significantly increased expression of SFRP2, PRRX1 genes and some other genes involved in inflammatory process, such as: CCL2, IL6, and ROBO1. Moreover, the right atrial appendage gene encoding lysyl oxidase (LOX) showed much higher expression compared to the pre-cultivation state.

Human Granulosa Cells-Stemness Properties, Molecular Cross-Talk and Follicular Angiogenesis.

The ovarian follicle is the basic functional unit of the ovary, comprising theca cells and granulosa cells (GCs). Two different types of GCs, mural GCs and cumulus cells (CCs), serve different functions during folliculogenesis. Mural GCs produce oestrogen during the follicular phase and progesterone after ovulation, while CCs surround the oocyte tightly and form the cumulus oophurus and corona radiata inner cell layer. CCs are also engaged in bi-directional metabolite exchange with the oocyte, as they form gap-junctions, which are crucial for both the oocyte's proper maturation and GC proliferation. However, the function of both GCs and CCs is dependent on proper follicular angiogenesis. Aside from participating in complex molecular interplay with the oocyte, the ovarian follicular cells exhibit stem-like properties, characteristic of mesenchymal stem cells (MSCs). Both GCs and CCs remain under the influence of various miRNAs, and some of them may contribute to polycystic ovary syndrome (PCOS) or premature ovarian insufficiency (POI) occurrence. Considering increasing female fertility problems worldwide, it is of interest to develop new strategies enhancing assisted reproductive techniques. Therefore, it is important to carefully consider GCs as ovarian stem cells in terms of the cellular features and molecular pathways involved in their development and interactions as well as outline their possible application in translational medicine.

Molecular Mechanisms Associated with ROS-Dependent Angiogenesis in Lower Extremity Artery Disease.

Currently, atherosclerosis, which affects the vascular bed of all vital organs and tissues, is considered as a leading cause of death. Most commonly, atherosclerosis involves coronary and peripheral arteries, which results in acute (e.g., myocardial infarction, lower extremities ischemia) or chronic (persistent ischemia leading to severe heart failure) consequences. All of them have a marked unfavorable impact on the quality of life and are associated with increased mortality and morbidity in human populations. Lower extremity artery disease (LEAD, also defined as peripheral artery disease, PAD) refers to atherosclerotic occlusive disease of the lower extremities, where partial or complete obstruction of peripheral arteries is observed. Decreased perfusion can result in ischemic pain, non-healing wounds, and ischemic ulcers, and significantly reduce the quality of life. However, the progressive atherosclerotic changes cause stimulation of tissue response processes, like vessel wall remodeling and neovascularization. These mechanisms of adapting the vascular network to pathological conditions seem to play a key role in reducing the impact of the changes limiting the flow of blood. Neovascularization as a response to ischemia induces sprouting and expansion of the endothelium to repair and grow the vessels of the circulatory system. Neovascularization consists of three different biological processes: vasculogenesis, angiogenesis, and arteriogenesis. Both molecular and environmental factors that may affect the process of development and growth of blood vessels were analyzed. Particular attention was paid to the changes taking place during LEAD. It is important to consider the molecular mechanisms underpinning vessel growth. These mechanisms will also be examined in the context of diseases commonly affecting blood vessel function, or those treatable in part by manipulation of angiogenesis. Furthermore, it may be possible to induce the process of blood vessel development and growth to treat peripheral vascular disease and wound healing. Reactive oxygen species (ROS) play an important role in regulation of essential cellular signaling pathways such as cell differentiation, proliferation, migration and apoptosis. With regard to the repair processes taking place during diseases such as LEAD, prospective therapeutic methods have been described that could significantly improve the treatment of vessel diseases in the future. Summarizing, regenerative medicine holds the potential to transform the therapeutic methods in heart and vessel diseases treatment.

The Proliferation and Differentiation of Adipose-Derived Stem Cells in Neovascularization and Angiogenesis.

Neovascularization and angiogenesis are vital processes in the repair of damaged tissue, creating new blood vessel networks and increasing oxygen and nutrient supply for regeneration. The importance of Adipose-derived Mesenchymal Stem Cells (ASCs) contained in the adipose tissue surrounding blood vessel networks to these processes remains unknown and the exact mechanisms responsible for directing adipogenic cell fate remain to be discovered. As adipose tissue contains a heterogenous population of partially differentiated cells of adipocyte lineage; tissue repair, angiogenesis and neovascularization may be closely linked to the function of ASCs in a complex relationship. This review aims to investigate the link between ASCs and angiogenesis/neovascularization, with references to current studies. The molecular mechanisms of these processes, as well as ASC differentiation and proliferation are described in detail. ASCs may differentiate into endothelial cells during neovascularization; however, recent clinical trials have suggested that ASCs may also stimulate angiogenesis and neovascularization indirectly through the release of paracrine factors.

Epigenetic Research in Stem Cell Bioengineering-Anti-Cancer Therapy, Regenerative and Reconstructive Medicine in Human Clinical Trials.

The epigenome denotes all the information related to gene expression that is not contained in the DNA sequence but rather results from chemical changes to histones and DNA. Epigenetic modifications act in a cooperative way towards the regulation of gene expression, working at the transcriptional or post-transcriptional level, and play a key role in the determination of phenotypic variations in cells containing the same genotype. Epigenetic modifications are important considerations in relation to anti-cancer therapy and regenerative/reconstructive medicine. Moreover, a range of clinical trials have been performed, exploiting the potential of epigenetics in stem cell engineering towards application in disease treatments and diagnostics. Epigenetic studies will most likely be the basis of future cancer therapies, as epigenetic modifications play major roles in tumour formation, malignancy and metastasis. In fact, a large number of currently designed or tested clinical approaches, based on compounds regulating epigenetic pathways in various types of tumours, employ these mechanisms in stem cell bioengineering.

New markers for regulation of transcription and macromolecule metabolic process in porcine oocytes during in vitro maturation.

Oocyte maturation is essential for proper fertilization, embryo implantation and early development. While the physiological conditions of these processes are relatively well­known, its exact molecular mechanisms remain widely undiscovered. Oocyte growth, differentiation and maturation are therefore the subject of scientific debate. Precious literature has indicated that the oocyte itself serves a regulatory role in the mechanisms underlying these processes. Hence, the present study performed expression microarrays to analyze the complete transcriptome of porcine oocytes during their in vitro maturation (IVM). Pig material was used for experimentation, as it possesses similarities to the reproductive processes and general genetic proximities of Sus scrofa to human. Oocytes, isolated from the ovaries of slaughtered animals were assessed via the Brilliant Cresyl Blue test and directed to IVM. A number of oocytes were left to be analyzed as the 'before IVM' group. Oocyte mRNA was isolated and used for microarray analysis, which was subsequently validated via RT­qPCR. The current study particularly focused on genes belonging to 'positive regulation of transcription, DNA­dependent', 'positive regulation of gene expression', 'positive regulation of macromolecule metabolic process' and 'positive regulation of transcription from RNA polymerase II promoter' ontologies. FOS, VEGFA, ESR1, AR, CCND2, EGR2, ENDRA, GJA1, INHBA, IHH, INSR, APP, WWTR1, SMARCA1, NFAT5, SMAD4, MAP3K1, EGR1, RORA, ECE1, NR5A1, KIT, IKZF2, MEF2C, SH3D19, MITF and PSMB4 were all determined to be significantly altered (fold change, >|2|; P<0.05) among these groups, with their downregulation being observed after IVM. Genes with the most altered expressions were analyzed and considered to be potential markers of maturation associated with transcription regulation and macromolecule metabolism process.

Differences in Expression of Genes Involved in Bone Development and Morphogenesis in the Walls of Internal Thoracic Artery and Saphenous Vein Conduits May Provide Markers Useful for Evaluation Graft Patency.

Coronary artery bypass grafting (CABG) is one of the most efficient procedures for patients with advanced coronary artery disease. From all the blood vessels with the potential to be used in this procedure, the internal thoracic artery (ITA) and the saphenous vein (SV) are the most commonly applied as aortocoronary conduits. Nevertheless, in order to evaluate the graft patency and efficiency effectively, basic knowledge should be constantly expanding at the molecular level as well, as the understanding of predictive factors is still limited. In this study, we have employed the expressive microarray approach, validated with Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR), to analyze the transcriptome of both venous and arterial grafts. Searching for potential molecular factors, we analyzed differentially expressed gene ontologies involved in bone development and morphogenesis, for the possibility of discovery of new markers for the evaluation of ITA and SV segment quality. Among three ontological groups of interest-"endochondral bone morphogenesis", "ossification", and "skeletal system development"-we found six genes common to all of them. BMP6, SHOX2, COL13A1, CSGALNACT1, RUNX2, and STC1 showed differential expression patterns in both analyzed vessels. STC1 and COL13A1 were upregulated in ITA samples, whereas others were upregulated in SV. With regard to the Runx2 protein function in osteogenic phenotype regulation, the RUNX2 gene seems to be of paramount importance in assessing the potential of ITA, SV, and other vessels used in the CABG procedure. Overall, the presented study provided valuable insight into the molecular background of conduit characterization, and thus indicated genes that may be the target of subsequent studies, also at the protein level. Moreover, it has been suggested that RUNX2 may be recognized as a molecular marker of osteogenic changes in human blood vessels.

Genes Involved in the Processes of Cell Proliferation, Migration, Adhesion, and Tissue Development as New Potential Markers of Porcine Granulosa Cellular Processes In Vitro: A Microarray Approach.

Proper course of folliculogenesis and oogenesis have an enormous impact on female fertility. Both processes take place in the ovary and involve not only the maturing germ cell, but also few types of somatic cells that assist the ovarian processes and mediate the dialog with the oocyte. These cells, granulosa and theca, are heavily involved in essential reproductive processes, such as ovulation, fertilization, and embryo implantation. In this study, we have used the expressive microarray approach to analyze the transcriptome of porcine granulosa cells, during short-term in vitro culture. We have further selected differentially expressed gene ontologies, involved in cell proliferation, migration, adhesion, and tissue development, namely, "cell-cell adhesion," "cell motility," "cell proliferation," "tissue development," and "tissue migration" to screen them for the possibility of discovery of new markers of those processes. A total of 303 genes, expression of which varied significantly in different culture periods and belonged to the analyzed ontology groups, were detected, of which 15 that varied the most (between 0 and 48 h of culture) were selected for validation. As the validation confirmed the transcriptomic patterns, 10 genes of biggest changes in expression (CAV1, IGFBP5, ITGB3, FN1, ITGA2, LAMB1, POSTN, FAM83D, KIF14, and CDK1) were analyzed, described, and referred to the context of the study, with the most promising new markers and further proof for the viability of the currently recognized ones detailed. Overall, the study provided valuable insight into the molecular functioning of in vitro granulosa cell cultures.

New Gene Markers of Angiogenesis and Blood Vessels Development in Porcine Ovarian Granulosa Cells during Short-Term Primary Culture In Vitro.

The physiological processes that drive the development of ovarian follicle, as well as the process of oogenesis, are quite well known. Granulosa cells are major players in this occurrence, being the somatic element of the female gamete development. They participate directly in the processes of oogenesis, building the cumulus-oocyte complex surrounding the ovum. In addition to that, they have a further impact on the reproductive processes, being a place of steroid sex hormone synthesis and secretion. It is known that the follicle development creates a major need for angiogenesis and blood vessel development in the ovary. In this study, we use novel molecular approaches to analyze markers of these processes in porcine granulosa cultured primarily in vitro. The cells were recovered from mature sus scrofa specimen after slaughter. They were then subjected to enzymatic digestion and culture primarily for a short term. The RNA was extracted from cultures in specific time periods (0h, 24h, 48h, 96h, and 144h) and analyzed using expression microarrays. The genes that exhibited fold change bigger than |2|, and adjusted p-value lower than 0.05, were considered differentially expressed. From these, we have chosen the members of "angiogenesis," "blood vessel development," "blood vessel morphogenesis," "cardiovascular system development," and "vasculature development" for further selection. CCL2, FGFR2, SFRP2, PDPN, DCN, CAV1, CHI3L1, ITGB3, FN1, and LOX which are upregulated, as well as CXCL10, NEBL, IHH, TGFBR3, SCUBE1, IGF1, EDNRA, RHOB, PPARD, and SLITRK5 genes whose expression is downregulated through the time of culture, were chosen as the potential markers, as their expression varied the most during the time of culture. The fold changes were further validated with RT-qPCR. The genes were described, with special attention to their possible function in GCs during culture. The results broaden the general knowledge about GC's in vitro molecular processes and might serve as a point of reference for further in vivo and clinical studies.

'Heart development and morphogenesis' is a novel pathway for human ovarian granulosa cell differentiation during long­term in vitro cultivation­a microarray approach.

Granulosa cells (GCs) have many functions in the endocrine system. Most notably, they produce progesterone following ovulation. However, it has recently been proven that GCs can change their properties when subjected to long­term culture. In the present study, GCs were collected from hyper­stimulated ovarian follicles during in vitro fertilization procedures. They were grown in vitro, in a long­term manner. RNA was collected following 1, 7, 15 and 30 days of culture. Expression microarrays were used for analysis, which allowed to identify groups of genes characteristic for particular cellular processes. In addition, reverse transcription­quantitative polymerase chain reaction (RT­qPCR) was performed to validate the obtained results. Two ontological groups characteristic for processes associated with the development and morphogenesis of the heart were identified during the analyses: 'Heart development' and 'heart morphogenesis'. The results of the microarrays revealed that the highest change in expression was demonstrated by the lysyl Oxidase, oxytocin receptor, nexilin F­actin binding protein, and cysteine­rich protein 3 genes. The lowest change was exhibited by odd­skipped related transcription factor 1, plakophilin 2, transcription growth factor­ß receptor 1, and kinesin family member 3A. The direction of changes was confirmed by RT­qPCR results. In the present study, it was suggested that GCs may have the potential to differentiate towards other cell types under long­term in vitro culture conditions. Thus, genes belonging to the presented ontological groups can be considered as novel markers of proliferation and differentiation of GCs towards the heart muscle cells.

Genes responsible for proliferation, differentiation, and junction adhesion are significantly up-regulated in human ovarian granulosa cells during a long-term primary in vitro culture.

The human ovarian granulosa cells (GCs) surround the oocyte and form the proper architecture of the ovarian follicle. The ability of GCs to proliferate and differentiate in the conditions of in vitro culture has been proven. However, there is still a large field for extensive investigation of molecular basics, as well as marker genes, responsible for these processes. This study aimed to find the new marker genes, encoding proteins that regulate human GCs in vitro capability for proliferation and differentiation during long-term primary culture. The human follicular GCs were collected from hyper-stimulated ovarian follicles during IVF procedures and transferred to a long-term in vitro culture. The culture lasted for 30 days, with RNA samples isolated at days 1, 7, 15, 30. Transcriptomic analysis was then performed with the use of Affymetrix microarray. Obtained results were then subjected to bioinformatical evaluation and sorting. After subjecting the datasets to KEGG analysis, three differentially expressed ontology groups "cell differentiation" (GO:0030154), "cell proliferation" (GO:0008283) and "cell-cell junction organization" (GO:0045216) were chosen for further investigation. All three of those ontology groups are involved in human GCs' in vitro lifespan, proliferation potential, and survival capability. Changes in expression of genes of interest belonging to the chosen GOs were validated with the use of RT-qPCR. In this manuscript, we suggest that VCL, PARVA, FZD2, NCS1, and COL5A1 may be recognized as new markers of GC in vitro differentiation, while KAT2B may be a new marker of their proliferation. Additionally, SKI, GLI2, FERMT2, and CDH2 could also be involved in GC in vitro proliferation and differentiation processes. We demonstrated that, in long-term in vitro culture, GCs exhibit markers that suggest their ability to differentiate into different cells types. Therefore, the higher expression profile of these genes may also be associated with the induction of cellular differentiation processes that take place beyond the long-term primary in vitro culture.

The Unique Mechanisms of Cellular Proliferation, Migration and Apoptosis are Regulated through Oocyte Maturational Development-A Complete Transcriptomic and Histochemical Study.

The growth and development of oocyte affect the functional activities of the surrounding somatic cells. These cells are regulated by various types of hormones, proteins, metabolites, and regulatory molecules through gap communication, ultimately leading to the development and maturation of oocytes. The close association between somatic cells and oocytes, which together form the cumulus-oocyte complexes (COCs), and their bi-directional communication are crucial for the acquisition of developmental competences by the oocyte. In this study, oocytes were extracted from the ovaries obtained from crossbred landrace gilts and subjected to in vitro maturation. RNA isolated from those oocytes was used for the subsequent microarray analysis. The data obtained shows, for the first time, variable levels of gene expression (fold changes higher than |2| and adjusted p-value < 0.05) belonging to four ontological groups: regulation of cell proliferation (GO:0042127), regulation of cell migration (GO:0030334), and regulation of programmed cell death (GO:0043067) that can be used together as proliferation, migration or apoptosis markers. We have identified several genes of porcine oocytes (ID2, VEGFA, BTG2, ESR1, CCND2, EDNRA, ANGPTL4, TGFBR3, GJA1, LAMA2, KIT, TPM1, VCP, GRID2, MEF2C, RPS3A, PLD1, BTG3, CD47, MITF), whose expression after in vitro maturation (IVM) is downregulated with different degrees. Our results may be helpful in further elucidating the molecular basis and functional significance of a number of gene markers associated with the processes of migration, proliferation and angiogenesis occurring in COCs.

Expression pattern of new genes regulating female sex differentiation and in vitro maturational status of oocytes in pigs.

The processes underlying maturation of mammalian oocytes are considered crucial for the oocytes ability to undergo monospermic fertilization. The same factors of influence are suggested to impact the development of sex associated characteristics, allowing sex differentiation to progress during embryonic growth. The primary aim of the study was to analyze the gene ontology groups involved in regulation of porcine oocytes' response to endogenous stimuli. The results obtained would indicate potential genes influencing sex differentiation. Additionally, they could help to determine new genetic markers, expression profile of which is substantially regulated during porcine oocytes' in vitro maturation. To achieve that, porcine oocytes were collected for analysis before and after in vitro maturation. Pigs were used as they are a readily available model that presents significant similarity to humans in terms of physiology and anatomy. Microarray analysis of oocytes, before and after in vitro maturation was performed and later validated by RT-qPCR. We have particularly detected and analyzed genes belonging to gene ontology groups associated with hormonal stimulation during maturation of the oocytes, that exhibited significant change in expression (fold change ≥ |2|; p < 0.05) namely "Female sex differentiation" (CCND2, MMP14, VEGFA, FST, INHBA, NR5A1), "Response to endogenous stimulus" (INSR, ESR1, CCND2, TXNIP, TACR3, MMP14, FOS, AR, EGR2, IGFBP7, TGFBR3, BTG2, PLD1, PHIP, UBE2B) and "Response to estrogen stimulus" (INSR, ESR1, CCND2, IHH, TXNIP, TACR3, MMP14). Some of them were characteristic for just one of the described ontologies, while some belonged into multiple ontological terms. The genes were analyzed, with their relation to the processes of interest explained. Overall, the study provides us with a range of genes that might serve as molecular markers of in vitro maturation associated processes of the oocytes. This knowledge might serve as a reference for further studies and, after further validation, as a potentially useful knowledge in assessment of the oocytes during assisted reproduction processes.

"Positive Regulation of RNA Metabolic Process" Ontology Group Highly Regulated in Porcine Oocytes Matured In Vitro: A Microarray Approach.

The cumulus-oocyte complexes (COCs) growth and development during folliculogenesis and oogenesis are accompanied by changes involving synthesis and accumulation of large amount of RNA and proteins. In this study, the transcriptomic profile of genes involved in "oocytes RNA synthesis" in relation to in vitro maturation in pigs was investigated for the first time. The RNA was isolated from oocytes before and after in vitro maturation (IVM). Interactions between differentially expressed genes/proteins belonging to "positive regulation of RNA metabolic process" ontology group were investigated by STRING10 software. Using microarray assays, we found expression of 12258 porcine transcripts. Genes with fold change higher than |2| and with corrected p value lower than 0.05 were considered as differentially expressed. The ontology group "positive regulation of RNA metabolic process" involved differential expression of AR, INHBA, WWTR1, FOS, MEF2C, VEGFA, IKZF2, IHH, RORA, MAP3K1, NFAT5, SMARCA1, EGR1, EGR2, MITF, SMAD4, APP, and NR5A1 transcripts. Since all of the presented genes were downregulated after IVM, we suggested that they might be significantly involved in regulation of RNA synthesis before reaching oocyte MII stage. Higher expression of "RNA metabolic process" related genes before IVM indicated that they might be recognized as important markers and specific "transcriptomic fingerprint" of RNA template accumulation and storage for further porcine embryos growth and development.

Exposure to hepatitis E virus, hepatitis A virus and Borrelia spp. infections in forest rangers from a single forest district in western Poland.

BACKGROUND: Hepatitis E virus (HEV) infection is an emerging problem in developed countries. At least 2 zoonotic genotypes of the virus (HEV-3 and HEV-4) infect human beings. There are some data suggesting that forest rangers (FRs) can be at a higher risk of contact with HEV. OBJECTIVES: The aim of this study was to assess the prevalence of HEV exposure markers in FRs from a single forest district in Greater Poland in relation to anti-HAV (hepatitis A virus) IgG, and anti-Borrelia spp. IgM and IgG antibodies. MATERIAL AND METHODS: In total, 138 participants (48 FRs and 90 blood donors - BDs) were tested for anti-HEV IgM and IgG (EUROIMMUN Medizinische Labordiagnostika AG, Luebeck, Germany) and 96 individuals (48 FRs and 48 BDs) were tested for anti-HAV IgG (ARCHITECT immunoassays, Abbott Laboratories, Wiesbaden, Germany); anti-Borrelia IgM and IgG (EUROIMMUN kits) were assessed in FRs only. RESULTS: Anti-HEV markers were detected in 3 participants (2.2%; IgM in 1 FR, IgG in 2 BDs), less frequently than anti-HAV (16 out of 96 individuals, about 17%; FRs 19% vs BDs 15%) or anti-Borrelia antibodies (18 out of 48 individuals, 37.5%) (p < 0.0001 for both). Older study participants (≥45 years of age) were more frequently HAV-seropositive (29% vs 4% of the younger individuals; p = 0.0012). CONCLUSIONS: We failed to unequivocally prove HEV exposure in FRs. The HAV seroprevalence in this study paralleled the situation in the general population. Exposure to Borrelia spp. in FRs was common.

New Gene Markers for Metabolic Processes and Homeostasis in Porcine Buccal Pouch Mucosa during Cells Long Term-Cultivation-A Primary Culture Approach.

The oral mucosal tissue is a compound structure composed of morphologically and physiologically different cell types. The morphological modification involves genetically determined lifespan, which may be recognized as the balance between cell survival and apoptosis. Although the biochemical processes and pathways in oral mucosa, with special regards to drug transport, delivery, and metabolism, are well known, the cellular physiological homeostasis in this tissue requires further investigation. The porcine buccal pouch mucosal cells (BPMCs) collected from 20 pubertal crossbred Landrace gilts, were used in this study. Immediately after recovery, the oral mucosa was separated micro-surgically, and treated enzymatically. The dispersed cells were transferred into primary in vitro culture systems for a long-term cultivation of 30 days. After each step of in vitro culture (IVC), the cells were collected for isolation of total RNA at 24 h, 7, 15, and 30 days of IVC. While the expression was analyzed for days 7, 15, and 30, the 24th hour was used as a reference for outcome calibration. The gene expression profile was determined using Affymetrix microarray assays and necessary procedures. In results, we observed significant up-regulation of SCARB1, PTGS2, DUSP5, ITGB3, PLK2, CCL2, TGFB1, CCL8, RFC4, LYN, ETS1, REL, LIF, SPP1, and FGER1G genes, belonging to two ontological groups, namely "positive regulation of metabolic process", and "regulation of homeostatic process" at 7 day of IVC as compared to down-regulation at days 15 and 30. These findings suggest that the metabolic processes and homeostatic regulations are much more intense in porcine mucosal cells at day 7 of IVC. Moreover, the increased expression of marker genes, for both of these ontological groups, may suggest the existence of not only "morphological lifespan" during tissue keratinization, but also "physiological checkpoint" dedicated to metabolic processes in oral mucosa. This knowledge may be useful for preclinical experiments with drugs delivery and metabolism in both animals and humans.

Expression Profile of Genes Regulating Steroid Biosynthesis and Metabolism in Human Ovarian Granulosa Cells-A Primary Culture Approach.

Because of the deep involvement of granulosa cells in the processes surrounding the cycles of menstruation and reproduction, there is a great need for a deeper understanding of the ways in which they function during the various stages of those cycles. One of the main ways in which the granulosa cells influence the numerous sex associated processes is hormonal interaction. Expression of steroid sex hormones influences a range of both primary and secondary sexual characteristics, as well as regulate the processes of oogenesis, folliculogenesis, ovulation, and pregnancy. Understanding of the exact molecular mechanisms underlying those processes could not only provide us with deep insight into the regulation of the reproductive cycle, but also create new clinical advantages in detection and treatment of various diseases associated with sex hormone abnormalities. We have used the microarray approach validated by RT-qPCR, to analyze the patterns of gene expression in primary cultures of human granulosa cells at days 1, 7, 15, and 30 of said cultures. We have especially focused on genes belonging to ontology groups associated with steroid biosynthesis and metabolism, namely "Regulation of steroid biosynthesis process" and "Regulation of steroid metabolic process". Eleven genes have been chosen, as they exhibited major change under a culture condition. Out of those, ten genes, namely STAR, SCAP, POR, SREBF1, GFI1, SEC14L2, STARD4, INSIG1, DHCR7, and IL1B, belong to both groups. Patterns of expression of those genes were analyzed, along with brief description of their functions. That analysis helped us achieve a better understanding of the exact molecular processes underlying steroid biosynthesis and metabolism in human granulosa cells.

Decreased mRNA expression levels of DNA methyltransferases type 1 and 3A in systemic lupus erythematosus.

OBJECTIVES: Systemic lupus erythematosus (SLE) is a chronic relapsing autoimmune disease characterized by the presence of autoantibodies directed against nuclear antigens and by chronic inflammation. Although the etiology of SLE remains unclear, the influence of environment factors, which is largely reflected by the epigenetic mechanisms, with DNA methylation changes in particular, is generally considered as main players in the pathogenesis of SLE. We studied DNA methyltransferases' (DNMTs) type 1, 3A and 3B transcript levels in peripheral blood mononuclear cells from patients diagnosed with systemic lupus erythematosus and from the healthy control subjects. Furthermore, the association of DNMT1, DNMT3A, and DNMT3B mRNA levels with gender, age, and major clinical manifestations was analyzed. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from 32 SLE patients and 40 healthy controls. Reverse transcription and real-time quantitative polymerase chain reaction (RT-qPCR) analyses were used to determine DNMT1, DNMT3A, and DNMT3B mRNA expression levels. RESULTS: Significantly lower DNMT1 (p = 0.015543) and DNMT3A (p = 0.003652) transcript levels in SLE patients were observed compared with healthy controls. Nevertheless, the DNMT3B mRNA expression levels were markedly lower compared with DNMT1 and DNMT3A, both in PBMCs from affected patients and those from control subjects. Furthermore, the DNMT1 transcript levels were positively correlated with SLE disease activity index (SLEDAI) (r s = 0.4087, p = 0.020224), while the DNMT3A transcript levels were negatively correlated with patients age (r s = -0.3765, p = 0.03369). CONCLUSIONS: Our analyses confirmed the importance of epigenetic alterations in SLE etiology. Moreover, our results suggest that the presence of some clinical manifestations, such as phototosensitivity and arthritis, might be associated with the dysregulation of DNA methyltransferases' mRNA expression levels.