New Gene Markers Expressed in Porcine Oviductal Epithelial Cells Cultured Primary In Vitro Are Involved in Ontological Groups Representing Physiological Processes of Porcine Oocytes.

Changes that occur within oviducts after fertilization are dependent on post-ovulation events, including oocyte-oviduct interactions. Although general processes are well-defined, the molecular basis are poorly understood. Recently, new marker genes involved in 'cell development', 'cell growth', 'cell differentiation' and 'cell maturation' processes have been identified in porcine oocytes. The aim of the study was to assess the expression profile of genes in primary in vitro cultured oviductal epithelial cells (OECs), clustered in Gene Ontology groups which enveloped markers also identified in porcine oocytes. OECs (from 45 gilts) were surgically removed and cultured in vitro for ≤ 30 days, and then subjected to molecular analyses. The transcriptomic and proteomic profiles of cells cultured during 7, 15 and 30 days were investigated. Additionally, morphological/histochemical analyzes were performed. The results of genes expression profiles were validated after using RT-qPCR. The results showed a significant upregulation of UNC45B, NOX4, VLDLR, ITGB3, FMOD, SGCE, COL1A2, LOX, LIPG, THY1 and downregulation of SERPINB2, CD274, TXNIP, CELA1, DDX60, CRABP2, SLC5A1, IDO1, ANPEP, FST. Detailed knowledge of the molecular pathways occurring in the OECs and the gametes that contact them may contribute both to developments of basic science of physiology, and new possibilities in advanced biotechnology of assisted reproduction.

Genes regulating hormone stimulus and response to protein signaling revealed differential expression pattern during porcine oocyte in vitro maturation, confirmed by lipid concentration.

Genes influencing oocyte maturation may be valuable for predicting their developmental potential, as well as discerning the mechanistic pathways regulating oocyte development. In the presented research microarray gene expression analysis of immature and in vitro matured porcine oocytes was performed. Two groups of oocytes were compared in the study: before (3 × n = 50) and after in vitro maturation (3 × n = 50). The selection of viable oocytes was performed using the brilliant cresyl blue (BCB) test. Furthermore, microarrays and RT-qPCR was used to analyze the transcriptome of the oocytes before and after IVM. The study focused on the genes undergoing differential expression in two gene-ontology groups: "Cellular response to hormone stimulus" and "Cellular response to unfolded protein", which contain genes that may directly or indirectly be involved in signal transduction during oocyte maturation. Examination of all the genes of interest showed a lower level of their expression after IVM. From the total number of genes in these gene ontologies ten of the highest change in expression were identified: FOS, ID2, BTG2, CYR61, ESR1, AR, TACR3, CCND2, EGR2 and TGFBR3. The successful maturation of the oocytes was additionally confirmed with the use of lipid droplet assay. The genes were briefly described and related to the literature sources, to investigate their potential roles in the process of oocyte maturation. The results of the study may serve as a basic molecular reference for further research aimed at improving the methods of oocyte in vitro maturation, which plays an important role in the procedures of assisted reproduction.

Transcriptomic analysis of expression of genes regulating cell cycle progression in porcine ovarian granulosa cells during short-term in vitro primary culture.

The primary function of ovarian granulosa cells (GCs) is the support of oocytes during maturation and development. Molecular analyses of granulosa cell-associated processes, leading to improvement of understanding of the cell cycle events during the formation of ovarian follicles (folliculogenesis), may be key to improve the in vitro fertilization procedures. Primary in vitro culture of porcine GCs was employed to examine the changes in the transcriptomic profile of genes belonging to "cell cycle", "cell division", "cell cycle process", "cell cycle phase transition", "cell cycle G1/S phase transition", "cell cycle G2/M phase transition" and "cell cycle checkpoint" ontology groups. During the analysis, microarrays were employed to study the transcriptome of GCs, analyzing the total RNA of cells from specific periods of in vitro cultures. This research was based on material obtained from 40 landrace gilts of similar weight, age and the same living conditions. RNA was isolated at specific timeframes: before the culture was established (0 h) and after 48 h, 96 h and 144 h in vitro. Out of 133 differentially expressed genes, we chose the 10 most up-regulated (SFRP2, PDPN, PDE3A, FGFR2, PLK2, THBS1, ETS1, LIF, ANXA1, TGFB1) and the 10 most downregulated (IGF1, NCAPD2, CABLES1, H1FOO, NEK2, PPAT, TXNIP, NUP210, RGS2 and CCNE2). Some of these genes known to play key roles in the regulation of correct cell cycle passage (up-regulated SFRP2, PDE3A, PLK2, LIF and down-regulated CCNE2, TXNIP, NEK2). The data obtained provide a potential reference for studies on the process of mammalian folliculogenesis, as well as suggests possible new genetic markers for cell cycle progress in in vitro cultured porcine granulosa cells.

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.

New Molecular Markers Involved in Regulation of Ovarian Granulosa Cell Morphogenesis, Development and Differentiation during Short-Term Primary In Vitro Culture-Transcriptomic and Histochemical Study Based on Ovaries and Individual Separated Follicles.

Nowadays, science has a lot of knowledge about the physiology of ovarian processes, especially folliculogenesis, hormone production and ovulation. However, the molecular basis for these processes remains largely undiscovered. The cell layer surrounding the growing oocyte-granulosa cells-are characterized by high physiological capabilities (e.g., proliferation, differentiation) and potential for growth in primary cultures, which predisposes them for analysis in the context of possible application of their cultures in advanced methods of assisted reproduction. In this study, we have used standard molecular approaches to analyze markers of these processes in primarily in vitro cultured porcine granulosa, subjected to conditions usually applied to cultures of similar cells. The material for our research came from commercially slaughtered pigs. The cells were obtained by enzymatic digestion of tissues and in vitro culture in appropriate conditions. The obtained genetic material (RNA) was collected at specific time intervals (0 h-before culture; reference, 48, 98, 144 h) and then analyzed using expression microarrays. Genes that showed a fold change greater than |2| and an adjusted p value lower than 0.05 were described as differentially expressed. Three groups of genes: "Cell morphogenesis", "cell differentiation" and "cell development" were analyzed. From 265 differently expressed genes that belong to chosen ontology groups we have selected DAPL1, CXCL10, NEBL, IHH, TGFBR3, SCUBE1, DAB1, ITM2A, MCOLN3, IGF1 which are most downregulated and PDPN, CAV1, TMOD1, TAGLN, IGFBP5, ITGB3, LAMB1, FN1, ITGA2, POSTN genes whose expression is upregulated through the time of culture, on which we focused in downstream analysis. The results were also validated using RT-qPCR. The aim of our work was to conduct primary in vitro culture of granulosa cells, as well as to analyze the expression of gene groups in relation to the proliferation of follicular granulosa cells in the model of primary culture in real time. This knowledge should provide us with a molecular insight into the processes occurring during the in vitro cultures of porcine granulosa cells, serving as a basic molecular entry on the extent of the loss of their physiological properties, as well as gain of new, culture-specific traits.

"Biological Adhesion" is a Significantly Regulated Molecular Process during Long-Term Primary In Vitro Culture of Oviductal Epithelial Cells (Oecs): A Transcriptomic and Proteomic Study.

Oviductal epithelial cells (OECs) actively produce stimulating and protecting factors, favoring survival and viability of gametes and early embryos. The oviduct participates in the initial reproductive events, which strongly depends on adhesion. The analysis of differential gene expression in OECs, during long-term in vitro culture, enables recognition of new molecular markers regulating several processes, including "biological adhesion". Porcine oviducts were stained with hematoxylin and eosin, as well as with antibodies against epithelial markers. Then, OECs were long-term in vitro cultured and after 24 h, 7, 15, and 30 days of culture were subjected to transcriptomic and proteomic assays. Microarrays were employed to evaluate gene expression, with Matrix-assisted laser desorption/ionization-time of light (MALDI-TOF) mass spectrometry applied to determine the proteome. The results revealed proper morphology of the oviducts and typical epithelial structure of OECs during the culture. From the set of differentially expressed genes (DEGs), we have selected the 130 that encoded proteins detected by MALDI-TOF MS analysis. From this gene pool, 18 significantly enriched gene ontology biological processes (GO BP) terms were extracted. Among them we focused on genes belonging to "biological adhesion" GO BP. It is suggested that increased expression of studied genes can be attributed to the process of intensive secretion of substances that exhibit favorable influence on oviductal environment, which prime gametes adhesion and viability, fertilization, and early embryo journey.

Transcriptomic Pattern of Genes Regulating Protein Response and Status of Mitochondrial Activity Are Related to Oocyte Maturational Competence-A Transcriptomic Study.

This paper aims to identify and describe new genetic markers involved in the processes of protein expression and modification reflected in the change of mitochondrial activity before and after in vitro maturation of the oocyte. Porcine oocytes collected from the ovaries of slaughtered landrace gilts were subjected to the process of in vitro maturation. Transcriptomic changes in the expression profile of oocyte genes involved in response to hypoxia, the transmembrane protein receptor serine threonine kinase signaling pathway, the "transforming growth factor ß receptor signaling pathway", "response to protein stimulus", and "response to organic substance" were investigated using microarrays. The expression values of these genes in oocytes was analyzed before (immature) and after (mature) in vitro maturation, with significant differences found. All the significantly altered genes showed downregulation after the maturation process. The most changed genes from these gene ontologies, FOS, ID2, VEGFA, BTG2, CYR61, ESR1, AR, TACR3, CCND2, CHRDL1, were chosen to be further validated, described and related to the literature. Additionally, the mitochondrial activity of the analyzed oocytes was measured using specific dyes. We found that the mitochondrial activity was higher before the maturation process. The analysis of these results and the available literature provides a novel insight on the processes that occur during in vitro oocyte maturation. While this knowledge may prove to be useful in further research of the procedures commonly associated with in vitro fertilization procedures, it serves mostly as a basic reference for further proteomic, in vivo, and clinical studies that are necessary to translate it into practical applications.

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.

Microcirculatory Disease in Patients after Heart Transplantation.

Although the treatment and prognosis of patients after heart transplantation have significantly improved, late graft dysfunction remains a critical problem. Two main subtypes of late graft dysfunction are currently described: acute allograft rejection and cardiac allograft vasculopathy, and microvascular dysfunction appears to be the first stage of both. Studies revealed that coronary microcirculation dysfunction, assessed by invasive methods in the early post-transplant period, correlates with a higher risk of late graft dysfunction and death during long-term follow-up. The index of microcirculatory resistance, measured early after heart transplantation, might identify the patients at higher risk of acute cellular rejection and major adverse cardiovascular events. It may also allow optimization and enhancement of post-transplantation management. Moreover, cardiac allograft vasculopathy is an independent prognostic factor for transplant rejection and survival rate. The studies showed that the index of microcirculatory resistance correlates with anatomic changes and reflects the deteriorating physiology of the epicardial arteries. In conclusion, invasive assessment of the coronary microcirculation, including the measurement of the microcirculatory resistance index, is a promising approach to predict graft dysfunction, especially the acute allograft rejection subtype, during the first year after heart transplantation. However, further advanced studies are needed to fully grasp the importance of microcirculatory dysfunction in patients after heart transplantation.

Expression Profile of New Gene Markers Involved in Differentiation of Canine Adipose-Derived Stem Cells into Chondrocytes.

The interest in stem cell research continuously increased over the last decades, becoming one of the most important trends in the 21st century medicine. Stem cell-based therapies have a potential to become a solution for a range of currently untreatable diseases, such as spinal cord injuries, type I diabetes, Parkinson's disease, heart disease, stroke, and osteoarthritis. Hence, this study, based on canine material, aims to investigate the molecular basis of adipose-derived stem cell (ASC) differentiation into chondrocytes, to serve as a transcriptomic reference for further research aiming to introduce ASC into treatment of bone and cartilage related diseases, such as osteoarthritis in veterinary medicine. Adipose tissue samples were harvested from a canine specimen subjected to a routine ovariohysterecromy procedure at an associated veterinary clinic. The material was treated for ASC isolation and chondrogenic differentiation. RNA samples were isolated at day 1 of culture, day 30 of culture in unsupplemented culture media, and day 30 of culture in chondrogenic differentiation media. The resulting RNA was analyzed using RNAseq assays, with the results validated by RT-qPCR. Between differentiated chondrocytes, early and late cultures, most up- and down-regulated genes in each comparison were selected for further analysis., there are several genes (e.g., MMP12, MPEG1, CHI3L1, and CD36) that could be identified as new markers of chondrogenesis and the influence of long-term culture conditions on ASCs. The results of the study prove the usefulness of the in vitro culture model, providing further molecular insight into the processes associated with ASC culture and differentiation. Furthermore, the knowledge obtained could be used as a molecular reference for future in vivo and clinical studies.

Effects of L-arginine and NG-nitro L-arginine methyl ester (L-NAME) on ischemia/reperfusion injury of skeletal muscle, small and large intestines.

This study analyzed the effects of L-arginine and non-specific nitric oxide (NO) synthase blocker (L-NAME) on structural and metabolic changes in experimental ischemia/reperfusion injury in the rat. Histopathological evaluation of rat tissues after reperfusion was also performed. The animals were divided into four groups: [1] nonischemic control, [2] ischemia 4 hrs/repefusion 30, 60, 120 min, [3] ischemia/reperfusion after L-arginine administration, [4] ischemia/reperfusion, after L-arginine, and L-NAME. L-arginine (500 mg/kg) and L-NAME (75 micromol/rat/day) were administrated orally for 5 days before experiment. Concentrations of free radicals, CD-62P, CD-54 and malonyl dialdehyde (MDA) in tissues, and MDA and NO levels in sera were determined. Free radical levels significantly increased in reperfused skeletal muscle, small and large intestines. In large bowel, reperfusion increased MDA levels and evoked a rise of endotoxin level while NO levels decreased. Histological studies showed an increase in the number of lymphocytes in both intestines. Administration of L-arginine reduced leukocyte adherence associated with ischemia-repefusion injury, decreased the levels of free radicals and MDA in the examined tissues, and inhibited the release of endotoxins into blood. L-arginine-treated animals showed higher serum NO levels and reduced leukocyte bowel infiltration. Concomitant L-NAME administration reduced serum NO and tissue free radical [corrected] levels, but did not affect intestinal leukocyte infiltration. L-arginine could ameliorate intestinal ischemia/reperfusion injury and constitute a possible protective mechanism by decreasing neutrophil-endothelial interactions, stimulating free radical scavenging and reducing lipid peroxidation.

Cytotoxicity, acute and subchronic toxicity of ionic liquid, didecyldimethylammonium saccharinate, in rats.

The aim of this study was to investigate cytotoxicity, acute and subchronic oral toxicity of an ionic liquid didecyldimethylammonium saccharinate [DDA][Sac] in rat. IC(50) values tested on six human cell lines varied from 1.44 microM to 5.47 microM. The compound tested was classified to the 4th toxicity class with a fixed LD(50) cut-off value 500 mg/kg. Organ pathology induced by [DDA][Sac] in an acute experiment included exfoliation of the surface layer of the colon and alveolar septa in lung parenchyma. In a subchronic experiment rats were administered 10, 30 and 100 mg/kg/day [DDA][Sac] for 28 days. Reduced body weight gain and slightly reduced food consumption was observed particularly in high-dose rats. Slight hematology changes were found only in mid-dose females. Statistically significant changes in clinical chemistry parameters included: increases in the ALT, SDH, ALP and GGT activities, and in glucose, blood urea nitrogen and creatinine concentrations. However, these changes did not occur in both sexes and were not dose-related with the exception of ALP in females. No treatment-related microscopic changes were observed in a subchronic experiment. Under the condition of this study the lowest-observed-adverse-effect level of [DDA][Sac] was considered to be 10 mg/kg/day.

The processes of cellular growth, aging, and programmed cell death are involved in lifespan of ovarian granulosa cells during short-term IVC - Study based on animal model.

The oogenesis and folliculogenesis are closely linked and occur simultaneously in the growing ovarian follicles. Biochemical and morphological changes in oocytes (OC) and surrounding granulosa cells (GCs) are highly complex and depend on many factors, including intercellular communication. GCs are cells with many functions, often crucial for the proper viability of the oocyte and subsequent positive fertilization. The purpose of this study was to analyze gene expression in porcine GCs, to define differentially expressed genes belongs to the "cell growth", "aging", "positive regulation of cell death", "apoptotic process", "regulation of cell death", "cell death" and "negative regulation of cell death" ontology groups during the short - term primary in vitro culture. Microarrays were employed to study the transcriptome contained in the total RNA of the cultured GCs. Ovaries were obtained after slaughter, from 40 gilts of swine aged 170 days. The cells were obtained through puncture of the ovaries, collection of follicular fluid, removal of the cumulus - oocyte complexes and centrifugation. The cells were then cultured in vitro. The RNA material was obtained before the culture was established (0h) and then after 48h, 96h and 144h of its course. From 182 differently expressed genes belonging to the these ontology groups, we have selected POSTN, FN1, FMOD, ITGB3, DCN, SERPINB2, SFRP2, IGFBP5, EMP1, and CCL2 which were upregulated, as well as DAPL1, ESR1, IHH, TGFBR3, PPARD, PDK4, TXNIP, IFIT3, CSRNP3, and TNFSF10 genes whose expression was downregulated during the time of in vitro culture of the GCs. The significance of the differential gene expression is to provide new information on the molecular aspects of in vitro granulosa cell culture.

New Gene Markers Involved in Molecular Processes of Tissue Repair, Response to Wounding and Regeneration Are Differently Expressed in Fibroblasts from Porcine Oral Mucosa during Long-Term Primary Culture.

The mechanisms of wound healing and vascularization are crucial steps of the complex morphological process of tissue reconstruction. In addition to epithelial cells, fibroblasts play an important role in this process. They are characterized by dynamic proliferation and they form the stroma for epithelial cells. In this study, we have used primary cultures of oral fibroblasts, obtained from porcine buccal mucosa. Cells were maintained long-term in in vitro conditions, in order to investigate the expression profile of the molecular markers involved in wound healing and vascularization. Based on the Affymetrix assays, we have observed three ontological groups of markers as wound healing group, response to wounding group and vascularization group, represented by different genes characterized by their expression profile during long-term primary in vitro culture (IVC) of porcine oral fibroblasts. Following the analysis of gene expression in three previously identified groups of genes, we have identified that transforming growth factor beta 1 (TGFB1), ITGB3, PDPN, and ETS1 are involved in all three processes, suggesting that these genes could be recognized as markers of repair specific for oral fibroblasts within the porcine mucosal tissue.

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.

Cortical Granule Distribution and Expression Pattern of Genes Regulating Cellular Component Size, Morphogenesis, and Potential to Differentiation are Related to Oocyte Developmental Competence and Maturational Capacity In Vivo and In Vitro.

Polyspermia is an adverse phenomenon during mammalian fertilization when more than one sperm fuses with a single oocyte. The egg cell is prepared to prevent polyspermia by, among other ways, producing cortical granules (CGs), which are specialized intracellular structures containing enzymes that aim to harden the zona pellucida and block the fusion of subsequent sperm. This work focused on exploring the expression profile of genes that may be associated with cortical reactions, and evaluated the distribution of CGs in immature oocytes and the peripheral density of CGs in mature oocytes. Oocytes were isolated and then processed for in vitro maturation (IVM). Transcriptomic analysis of genes belonging to five ontological groups has been conducted. Six genes showed increased expression after IVM (ARHGEF2, MAP1B, CXCL12, FN1, DAB2, and SOX9), while the majority of genes decreased expression after IVM. Using CG distribution analysis in immature oocytes, movement towards the cortical zone of the oocyte during meiotic competence acquisition was observed. CGs peripheral density decreased with the rise in meiotic competence during the IVM process. The current results reveal important new insights into the in vitro maturation of oocytes. Our results may serve as a basis for further studies to investigate the cortical reaction of oocytes.

Acute and subacute (28-day) toxicity studies of ionic liquid, didecyldimethyl ammonium acesulfamate, in rats.

The aim of this study was to investigate acute and subacute oral toxicity of an ionic liquid, didecyldimethylammonium acesulfamate [DDA][Ace], in rats. The compound tested was classified to the fourth toxicity class with a fixed LD(50) cut-off value of 500 mg/kg. Organ pathology induced by [DDA][Ace] in acute experiments included exfoliation of the surface layer of the digestive tract and alveolar septa in lung parenchyma. In a subacute experiment, rats were administered 10, 50, and 100 mg/kg/day [DDA][Ace] for 28 days. Reduced body weight gain and reduced food consumption was observed in mid- and high-dose rats. Statistically significant hematology changes were found mostly in high-dose groups of both sexes: increases in hematocrit, mean corpuscular volume, and mean platelet volume. Statistically significant changes in clinical chemistry parameters included increases in the GGT, SDH, and LDH activity and bilirubin concentration, and decreases in triglycerides, glucose, and inorganic phosphorus concentration. No treatment-related microscopic changes were observed. Under the conditions of this study, the lowest-observed-adverse-effect level of [DDA][Ace] was considered to be 10 mg/kg/day.

An increased skin microvessel density is associated with neurovascular complications in type 1 diabetes mellitus.

The aim of this study was to assess the blood vessel density and maturity in the skin of adults with type 1 diabetes in relation to the presence of late neurovascular complications. We included 148 patients (87 men) with a median (interquartile range) age of 41 (31-49) and median diabetes duration of 21 (17-30) years. Microvessel (CD133, CD34, CD31 and von Willebrand factor) markers were evaluated by indirect immunohistochemistry assay in material from a skin biopsy. Diabetic retinopathy was diagnosed using direct ophthalmoscopy, and diabetic kidney disease was estimated in people with increased albuminuria and a 10-year duration of diabetes or evidence of diabetic retinopathy . Diabetic peripheral neuropathy diagnosis was based on Toronto definition, cardiac autonomic neuropathy on validated ProSciCard III program. Microvessel density, assessed by CD34 and CD133, was significantly higher in patients with cardiac autonomic neuropathy [160 (125-175) vs 121 (100-154)/1 mm2, p = 0.001 and 92 (83-104) vs 79 (63-92)/1 mm2, p = 0.007, respectively] and CD34 in patients with diabetic peripheral neuropathy [135 (106-168) vs 121 (95-145)/1 mm2, p = 0.018], as compared with subjects without complications. In multivariate logistic regression, density of CD34 and CD133 positive vessels was associated with presence of cardiac autonomic neuropathy [odds ratio 1.016 (95% confidence interval: 1.002-1.029), p = 0.019 and odds ratio 1.037 (95% confidence interval: 1.008-1.067), p = 0.011, respectively]. It was independent from age, sex, diabetes duration, smoking status, body mass index and HbA1c value. Density of CD34 positive vessels was also associated with diabetic peripheral neuropathy, independently from sex and diabetes duration [odds ratio 1.009 (95% confidence interval: 1.001-1.020), p = 0.037]. Skin microvessel density is increased in adults with clinical evidence of neurovascular complications of type 1 diabetes. This is associated with predominance of the vessels of low maturity.

Early Progression of Xanthogranulomatous Pyelonephritis in Children Might Be Dependent on Vimentin Expression.

BACKGROUND Xanthogranulomatous pyelonephritis (XP) is an extremely rare, severe, atypical form of chronic renal parenchymal inflammation accompanied by hydronephrosis and/or urolithiasis. The pathomechanism of XP is not yet fully understood. Microscopically, XP is indicated by the presence of multinucleated giant cells and lipid-laden macrophages, as well as inflammatory infiltration and intensive renal fibrosis. The lipid accumulation in kidney parenchyma may be secondary to the altered flow of low-density lipoprotein (LDL)-derived cholesterol particles inside the affected cells. Physiologically, the process of LDL-derived cholesterol transport from lysosomes to the sites of its esterification is dependent on vimentin, which is a molecule comprising the cytoskeleton in mesenchymal cells. CASE REPORT A 7-year old girl was hospitalized because of the finding of unexplained kidney lesions on an abdominal ultrasound examination (an enlarged and deformed collecting system of the right kidney with hyperechogenic, solid, staghorn lesions in the calyces). Three months earlier, the patient had experienced recurrent urinary tract infection. Based on the subsequent laboratory and imaging diagnostics, the final diagnosis of XP was established and the girl was qualified for right-sided nephrectomy Microscopic examination revealed numerous foci of granuloma formations with no evident exponents of dysplastic or neoplastic abnormalities. Significant CD68-positive cell infiltrations and scattered foam cells arranging the numerous foci of granuloma inflammation were noticed. Renal parenchyma, adjacent to granuloma lesions, presented a vimentin expression. CONCLUSIONS Vimentin expression in XP may confirm a focal character of chronic granuloma formation and may suggest the complexity of XP pathogenesis involving not only macrophage and fibroblast activation but also local lipid deregulation and fibrosis.

Tissue distribution of pneumadin immunoreactivity in the rat.

Pneumadin (PNM) is a decapeptide, originally isolated from mammalian lungs, which exerts a potent stimulating effect on arginine-vasopressin (AVP) release, thereby evoking an antidiuretic effect. We have established a specific radioimmunoassay (RIA) method for rat PNM determination, the sensitivity of which is sufficient for measuring tissue content of the peptide. Moreover, raised antibodies have been used for the immunocytochemical detection of PNM in several rat organs. As expected, high concentrations of PNM were detected by RIA in newborn and adult rat lungs and immunocytochemistry (ICC) localized PNM immunoreactivity (IR) in the bronchial and bronchiolar epithelium. Very high concentrations of PNM were measured by RIA in the prostate, and ICC showed that PNM-IR is contained in the epithelial cells. RIA and ICC demonstrated the presence of low amounts of PNM in the thymus. The highest content of radioimmunoassayable PNM was found in the kidneys and intestinal tract, but dilution test suggested the presence of some interfering substances in these tissues. Accordingly, ICC-detectable PNM-IR was absent in the kidneys and present only in the duodenal criptae and Brunner's glands of the intestinal tract. RIA did not measure sizeable PNM concentrations in the thyroid gland, but ICC showed PNM-IR in C-cells. RIA and ICC did not detected PNM in testes, seminal vesicles, ovaries, uterus, pancreas, liver, spleen, adrenal glands, and heart. Taken together, our findings suggest that PNM, in addition to its role as hypothalamo-pituitary AVP secretagogue, may be involved in the autocrine-paracrine functional regulation of other peripheral organs, like lungs and prostate and perhaps duodenum, thymus and thyroid gland.