Heating and storage of structured acylglycerols with succinyl-linked stigmasterol residue does not cause negative chemical or biological changes.

Four structured acylglycerols with stigmasterol bonded by a succinyl linker were investigated and their stability were analyzed. Samples were heated to 60 °C and kept at that temperature to simulate storage, and to 180 °C to simulate frying conditions. The degradation of the synthesized compounds and formed derivatives was determined, and their cytotoxicity and genotoxicity on normal human cells from the digestive system was determined. Holding at 180 °C resulted in greater degradation of the compounds than holding at 60 °C. The most stable compound in each sample proved to be one with oleic acid in its structure-1,3-dioleoyl-2-stigmasterylsuccinoyl-sn-glycerol (DO2SSt) at 60 °C and 1,2-dioleoyl-3-stigmasterylsuccinoyl-sn-glycerol (DO3SSt) at 180 °C. These results indicate that the type of fatty acid in the molecule is more important than its position in the glycerol structure. None of the diacylmonostigmasterylsuccinoyl-sn-glycerols (DASStGs) before or after heating exhibited cytotoxic or genotoxic potential to small intestine and colon mucosa cells.

Thermo-oxidative stability and safety of new acylglycerols with stigmasterol residue: Effects of fatty acids saturation and position in the glycerol backbone.

The safety and thermoxidative stability of new diacyl-stigmasterylcarbonoyl-sn-glycerols (DAStGs) with two molecules of palmitic or oleic acids and one molecule of stigmasterol at the sn-2 or sn-3 position were studied. After heating to 60 °C, the compounds with stigmasterol at the sn-2 position were more stable than those with stigmasterol at the sn-3 position. The lowest level of degradation of stigmasterol after heating to 180 °C was detected for both compounds with oleic acid, followed by the samples with palmitic acid. The high content of SOPs, especially triolSt, as well as the high level of dimers showed the most effect on the cytotoxicity of DAStGs heated at both temperatures. DAStGs with oleic acid at sn-1,3 and stigmasterol at sn-2 position were the most stable compounds. Both oleic acid and the location of stigmasterol in the middle of the glycerol molecule play an important role in increasing the thermoxidative stability of stigmasterol.

Differential expression and methylation patterns of NFATC1, NADSYN1 and JAK3 gene in equine chondrocytes expanded in monolayer culture.

Ex vivo expansion of chondrocytes in monolayer (ML) culture for therapeutic purposes is burdened with difficulties related to the loss of cartilaginous phenotype. Epigenetic mechanisms responsible for regulation of gene expression are believed to underlie chondrocyte dedifferentiation. We have inspected the relevance of DNA methylation alterations for passage-related differential expression of NFATC1 gene involved in hard connective tissue turnover and development, NADSYN1 influencing redox metabolism, and JAK3 - an important driver of inflammation. We have assessed relative amount of transcript abundance and performed DNA bisulfite sequencing of upstream located elements. It seems that anabolic-like effects of chondrogenic differentiation were observed in form of NFATC1 and NADSYN1 upregulation in chondrocytes at the earlier stages of passaging whereas JAK3 upregulation at the 11th passage was the sign of chondrocytes dedifferentiation. Summarizing the inversely correlated DNA methylation and expression patterns in NFATC1 and JAK3 locus might be relevant for cellular dedifferentiation during chondrocyte expansion in monolayer. Obtained results are supportive for further studies on the role of encoded proteins in regenerative biology of articular cartilage using in vitro expanded chondrocytes.

Thermo-oxidative stability of asymmetric distigmasterol-modified acylglycerols as novel derivatives of plant sterols.

The study investigated the thermo-oxidative stability of distigmasterol-modified acylglycerols as a new structured acylglycerols. Samples were heated at 60 and 180 °C for 8 h. Their percentage degradation and products formed during heating were compared with free stigmasterol and stigmasteryl esters. The remaining of stigmasterol and fatty acid parts, the formation of stigmasterol oxidation products and the composition of polar and non-polar fractions were analysed using chromatographic methods. The cytotoxicity and genotoxicity were determined with the use of an MTT test and a comet assay, respectively. The highest stability during heating was observed for 2,3-distigmasterylsuccinoyl-1-oleoyl-sn-glycerol (dStigS-OA) and the lowest for 2,3-distigmasterylcarbonoyl-1-oleoyl-sn-glycerol (dStigC-OA). Data showed that the formation of thermo-oxidative degradation products is affected by the temperature and chemical structure of lipids present in the molecule. The dStigMAs bonded by a succinate linker and products formed during their thermo-oxidation showed no cytotoxic or genotoxic activity to normal human cells.

Removal of carbamazepine, diclofenac and trimethoprim by solar driven advanced oxidation processes in a compound triangular collector based reactor: A comparison between homogeneous and heterogeneous processes.

Contaminants of emerging concern (including pharmaceuticals) are not effectively removed by municipal wastewater treatment plants (WWTPs), so particular concern is related to agricultural wastewater reuse due to their possible uptake in crops irrigated with WWTPs effluents. Advanced oxidation processes (AOPs) and solar AOPs have been demonstrated to effectively remove pharmaceuticals from different aqueous matrices. In this study, an heterogeneous photocatalytic process using powdered nitrogen-doped TiO2 immobilized on polystyrene spheres (sunlight/N-TiO2) was compared to the benchmark homogenous AOP sunlight/H2O2 in a compound triangular collector reactor, to evaluate the degradation of three pharmaceuticals (carbamazepine (CBZ), diclofenac (DCF), trimethoprim (TMP)) in water. The degradation of the contaminants by sunlight and sunlight-AOPs well fit the pseudo-first order kinetic model (but for TMP under sunlight). High removal efficiency by solar photolysis was observed for DCF (up to 100%, half-life sunlight cumulative energy QS,1/2 = 2 kJ L-1, half-life time t1/2 = 32 min), while CBZ (32%, QS,1/2 = 28 kJ L-1, t1/2 = 385 min) and TMP (5% removal after 300 min) removal was poor. The degradation rate of CBZ, TMP and DCF was found to be slower during sunlight/H2O2 (QS,1/2 = 5 kJ L-1, t1/2 = 77 min; QS,1/2 = 20 kJ L-1, t1/2 = 128 min; QS,1/2 = 4 kJ L-1, t1/2 = 27 min, respectively) compared to sunlight/N-TiO2 (QS,1/2 = 4 kJ L-1, t1/2 = 55 min; QS,1/2 = 3 kJ L-1, t1/2 = 42 min; QS,1/2 = 2 kJ L-1, t1/2 = 25 min, respectively). These results are promising in terms of solar technology upscale because the faster degradation kinetics observed for sunlight/N-TiO2 process would result in smaller treatment volume, thus possibly perspective compensating the cost of the photocatalyst.

Chondrogenic expression and DNA methylation patterns in prolonged passages of chondrocyte cell lines of the horse.

We investigated the activity of chondrogenic markers and variation of methylation patterns in equine cartilaginous cells cultivated in monolayer. The transcriptional and epigenetic effect of the long-term culture of chondrocytes has been evaluated using several passages of chondrocyte cell-lines derived from equine articular cartilage. Using 3 genes as endogenous control we tested the expression of 7 genes important for different stages of chondrocyte differentiation and maturation. CpG islands in RUNX3 locus were inspected for the evaluation of differential methylation state of passaged cell-lines. The general decline of transcript abundance of marker loci was detected in passage 11 which is the sign of dedifferentiation of cultivated chondrocytes in prolonged monolayer culture. Passages 13 and 14 were characterized by the upregulation of a number of genes, possibly due to the heterogeneity of developed cell lines at this stage of the culture. Instead, gradual increase of methylation percent at particular CpG sites of RUNX3 locus was associated with the growing number of passage. This finding led us to the conclusion that epigenetic alterations better describe the stage of cultivated chondrocytes.

Role of macrophages in the pathogenesis of atherosclerosis and aortocoronary graft disease.

Atherosclerosis and disease of graft implanted to bypass occluded coronary or peripheral arteries are similar processes. Patency of implanted grafts is of paramount importance in respect to long-term outcomes. Although few cell types participate in atherosclerotic plaque formation, macrophages play a crucial role. In this article we review the fate of monocytes that infiltrate vessel wall following endothelium damage, and then undergo transformation to macrophages (identified as CD68 positive cells) and eventually lead to severe stenosis of vessel. Opposing biological activity of two subpopulations of macrophages and their impact on plaque instability and its calcification is also presented. At the end of this paper, a possible clinical significance of pre-existing, CD68 positive cell infiltration of vessel wall, applied as aortocoronary grafts, is discussed.

Gender and age-related variability of macrophage representation in the internal thoracic artery wall: does it matter?

Some recent reports suggested that elderly and female patients did not benefit from implantation of the second internal thoracic artery (ITA) during coronary artery bypass surgery (CABG). Macrophages, among other cells, were described to be involved in both atherosclerosis and aortocoronary grafts failure. The aim of the study was to examine the age and gender association with different distribution of CD68+ cells within the layers of ITA wall. This study involved 158 consecutive patients (95 male and 63 female), with the mean age of 64.5±9.5 years, who underwent elective CABG procedures. During surgery, the surplus distal segments of ITA were harvested for immunohistochemical analysis. The number and distribution of CD68+ cells was calculated and plotted against the age and gender of the study participants. CD68+ cells were present in all of the harvested ITA fragments (median 44), more in women (55) than in men (42) (p less than 0.001). However, this difference was of statistical significance exclusively in the tunica intima. Approximately 70% of macrophages were found in the tunica adventitia. The total number of CD68+ cells the in arterial wall as well as in the tunica intima and adventitia correlated positively with the age of patients (r=0.544, r=501 and r=0.462, respectively). The lack of significant advantages of the use of two thoracic arteries, in elderly patients and women, might have resulted from the larger population of CD68+ cells in their walls, especially the tunica intima. However, this result from immunohistochemical analysis needs validation in long-term clinical research on a larger cohort of patients.

Frequency and predictors of post-stroke delirium in PRospective Observational POLIsh Study (PROPOLIS).

Delirium is the most common and serious neurobehavioral complication in acute hospital admissions. Some patients develop signs of delirium but do not meet all diagnostic criteria. Stroke is a major risk factor for delirium. The aim of this prospective study was to build a predictive model for delirium and subsyndromal post-stroke delirium. Patients with stroke were screened for delirium during the first 7 days after admission. Delirium was diagnosed according to DSM-V criteria. Baseline demographic, biochemical, stroke-related data, medications used, neurological deficit, and premorbid cognitive and functional impairment were assessed. 750 consecutive stroke patients (71.75 ± 13.13 years) were recruited; 203 (27.07%) had delirium. In predictive model for delirium MoCA score and white blood count on admission, neglect, vision deficits, physical impairment, and higher comorbidity prior to stroke had the highest predictive value. Subsyndromal delirium was diagnosed in 60 patients. MoCA score and potassium level on admission, and urinary tract infection during hospitalization had the highest predictive value for its development. Delirium occurs in one-fourth of admissions due to stroke; subsyndromal delirium is less prevalent and affects less than one per ten patients. The hyperactive form is the most rare type of delirium. The factors best predicting delirium are easily assessed in everyday practice and their co-occurrence in patients with stroke should alert the treating physician of high risk of delirium.

Catalytic stereospecific O-glycosylation.

A new simple protocol, using TMSNTf2 or Tf2NH as the catalyst, for the activation of trichloroacetimidate donors is described. This O-glycosylation proceeds with stereospecific inversion of the donor configuration. The scope of the protocol has been investigated using common glycosyl donors reacting with simple alcohols as well as sugar acceptors.

Incorporation of sol-gel bioactive glass into PLGA improves mechanical properties and bioactivity of composite scaffolds and results in their osteoinductive properties.

In this study, 3D porous bioactive composite scaffolds were produced and evaluated for their physico-chemical and biological properties. Polymer poly-L-lactide-co-glycolide (PLGA) matrix scaffolds were modified with sol-gel-derived bioactive glasses (SBGs) of CaO-SiO2-P2O5 systems. We hypothesized that SBG incorporation into PLGA matrix would improve the chemical and biological activity of composite materials as well as their mechanical properties. We applied two bioactive glasses, designated as S2 or A2, differing in the content of SiO2 and CaO (i.e. 80 mol% SiO2, 16 mol% CaO for S2 and 40 mol% SiO2, 52 mol% CaO for A2). The composites were characterized for their porosity, bioactivity, microstructure and mechanical properties. The osteoinductive properties of these composites were evaluated in human bone marrow stromal cell (hBMSC) cultures grown in either standard growth medium or treated with recombinant human bone morphogenetic protein-2 (rhBMP-2) or dexamethasone (Dex). After incubation in simulated body fluid, calcium phosphate precipitates formed inside the pores of both A2-PLGA and S2-PLGA scaffolds. The compressive strength of the latter was increased slightly compared to PLGA. Both composites promoted superior hBMSC attachment to the material surface and stimulated the expression of several osteogenic markers in hBMSC compared to cells grown on unmodified PLGA. There were also marked differences in the response of hBMSC to composite scaffolds, depending on chemical compositions of the scaffolds and culture treatments. Compared to silica-rich S2-PLGA, hBMSC grown on calcium-rich A2-PLGA were overall less responsive to rhBMP-2 or Dex and the osteoinductive properties of these A2-PLGA scaffolds seemed partially dependent on their ability to induce BMP signaling in untreated hBMSC. Thus, beyond the ability of currently studied composites to enhance hBMSC osteogenesis, it may become possible to modulate the osteogenic response of hBMSC, depending on the chemistry of SBGs incorporated into polymer matrix.

Treatment of MAS with PPHN using combined therapy: SLL, bolus surfactant and iNO.

The objective of the study was to compare the effectiveness of surfactant treatment either by bolus or surfactant lung lavage followed by inhaled nitric oxide (iNO) therapy in infants with meconium aspiration syndrome (MAS) complicated by persistent pulmonary hypertension (PPHN). In this study, thirteen infants with diagnosis of MAS and PPHN were first treated with conventional respiratory support. Then between 2 and 22h of life they were randomized either to bolus surfactant treatment (n=6) or surfactant lung lavage (SLL, n=7) treatment. Then all infants were treated with iNO therapy. The groups were compared with regard to their clinical course: changes in PaO(2), FiO(2), MAP, OI, A-a oxygen gradient, duration of iNO therapy, length of ventilation and hospitalization. Complications and mortality were also compared. The results showed that infants treated with SLL had significant improvements in oxygenation, decreases in MAP and A-a gradients. But there were no significant differences in duration of ventilation, iNO treatment, length of hospitalization or complications. In conclusion these data show no advantage of SLL therapy over bolus surfactant treatment in infants with MAS complicated by PPHN.

Pulmonary function after surfactant lung lavage followed by surfactant administration in infants with severe meconium aspiration syndrome.

OBJECTIVE: The effect of surfactant lung lavage (SLL) on pulmonary function was examined in neonates with severe form of meconium aspiration syndrome (MAS) (mean +/- standard deviation: birth weight, 3178.1 +/- 237.6 g; gestational age, 37.7 +/- 1.8 weeks). METHODS: The infants were on mechanical ventilation and were subjected to SLL with Survanta. Dynamic compliance (Cdyn), airway resistance (Raw) and mean airway pressure (MAP) were obtained with use of the pneumotachometric method. Measurements of Cdyn, Raw, MAP and time constant (Tc) were compared in the study and control groups and between the groups before SLL and 24 and 48 h after SLL. RESULTS: A significant increase in Cdyn (from 1.06 +/- 0.23 to 2.12 +/- 0.99 (ml/cmH(2)O)/kg) (p < 0.05) and a drop in both Raw (from 264.7 +/- 41.5 to 146.6 +/- 39.4 (cmH(2)O/l)/s) and MAP (from 12.4 +/- 3.6 to 5.4 +/- 2.1 cmH(2)O) were observed 48 hours after SLL. CONCLUSIONS: These data suggest that SLL is associated with a rapid and significant improvement in pulmonary mechanics, together with an improvement in oxygenation, in newborns with severe MAS. We found that the beneficial effects of SLL on pulmonary mechanics persisted for at least 48 h after introduction of the procedure.

Stages of the rat thymic medulla development in foetal period.

Development of thymic medulla was examined on consecutive gestational days (GD) in Wistar rats. Medullary thymic epithelial cells (TEC) were identified by immunocytochemical localisation of neuron-specific enolase (NSE). Organisation of thymic medullary architecture was determined by interaction of thymocytes with NSE-positive TEC, that led to formation of lymphoepithelial complexes (GD 19), in which the cells exhibited proliferative activity or traits of apoptosis. The studies indicated that differentiation events and organisation of thymic medulla require stage-specific interactions between TEC and thymocytes.

Age-related changes in rat thymic epithelial cells.

We investigated age-related changes in immunocytochemical localisation of cytokeratin 16 (CK16) in thymuses of female Wistar rats at various stages of adult life (months 1, 3, 6, 12). Within the 1 st month of life, distribution of CK typical for individual subsets of thymic epithelial cells (TEC) was observed. The most numerous CK16+ TEC were observed in the outer region of medulla, in the outer cells of Hassall's corpuscles and in the superficial epithelial layer neighbouring the connective tissue of the capsule, septa and vessels of the thymus. In the 3rd month of life, increased intensity of CK16 reaction in superficial TEC was accompanied by increased numbers of CK 16+ TEC in the outer region of the medulla. Age-related alterations in the distribution of the studied markers were evident beginning from the 6th month of life and involved increased expression of CK16 in the superficial layer of TEC, which at the interface with the septa formed stratified epithelium. In parallel, decreased numbers of CK16+ TEC were observed in the outer region of the medulla. Changes in CK16+ TEC distribution of a similar type developed in 12-month old rats and they probably reflected altered functions of some TEC populations and decreased or increased biological activity of other TEC populations.

Lympho-epithelial interactions in rat thymus during pregnancy.

Alterations in the thymic epithelial cell activity were analysed during pregnancy and lactation in Wistar rats by examining the presence and in situ distribution of lymphoepithelial complexes formed by thymic nurse cells (TNC). TNC were identified in paraffin sections by their expression of MHC class II antigens, CD54 molecule and a neuromarker, protein gene product 9.5 (PGP9.5). On the first days of pregnancy (gestational days, GD) the number of PGP9.5+ TNC was found to decrease abruptly. On GD 14, a transient increase was noted in the number of PGP9.5+, MHC+, CD54+ TNC. Another increase was observed in the course of lactation, when the weight of the thymus reached the lowest values. While the increase in TNC numbers during lactation may be linked to the process of reconstruction of the thymic lymphoid population, the augmented activity of lymphoepithelial interactions on GD14 may be associated with thymic engagement in pregnancy-induced immune processes.

Gel-derived materials of a CaO-P(2)O(5)-SiO(2) system modified by boron, sodium, magnesium, aluminum, and fluorine compounds.

Bioactive glass-ceramic materials of the CaO-P(2)O(5)-SiO(2) system modified by adding boron, magnesium, sodium, fluorine, and aluminum were obtained using the sol-gel method. Gel-derived materials were produced in the pellet form obtained by compression of powders as well as in coatings on glass slides. The materials obtained were examined in vitro with regard to the ability of calcium phosphate layer to form on the material surface as the result of contact with simulated body fluid (SBF). SBF pH changes and calcium solubility in this solution were determined and scanning electron microscopy, energy-dispersive X-ray analysis, and infrared spectroscopy studies were conducted before and after contact of the materials with SBF. The gels modified by aluminum were amorphous, whereas the sodium and fluorine additives promoted the bulk crystallization of gel-derived materials. The ability of calcium phosphates to crystallize on the surface of gel-derived materials depended only slightly on the types of additives applied, and the character of this dependence was different from that observed in melted glasses. Moreover, to estimate the biocompatibility of gel-derived coatings, we examined the proliferation, collagen synthesis, adhesion, and morphology of fibroblasts (NRK cells) cultured in the presence of gel-derived materials. The results of these experiments showed that none of the tested materials significantly reduced any cell function.

Evidence for the presence of 5'-deiodinase in mammalian seminal plasma and for the increase in enzyme activity in the prepubertal testis.

Thyroid hormones are critical for structural and functional development of the testis and Sertoli cells are considered true target cells for triiodothyronine (T3). However, the role of thyroid hormones in the adult testis seems to be minimal and the mechanism by which they affect testicular function is not known. Due to the existing blood-testis barrier the concentration of thyroid hormones in seminal plasma is kept lower than in blood plasma. We have found that T3 may reach the testis not only from the circulation but also from local enzymic conversion of thyroxine to T3. The presence of the enzymic activity responsible for thyroxine 5'deiodination and for generating T3 locally was also found in boar's seminal plasma. The seminal plasma 5'-deiodinase (5'-D) appeared to be predominantly the propylthiouracil (PTU)-insensitive type II isoenzyme found, so far, in tissues where it plays a role in paracrine signalling. It contains selenocysteine in its molecule (inhibition by aurothioglucose), and has an apparent Km for reverse-T3 as substrate of 0.36 nM and a Vmax 23.8 fmol I-/mg protein/min. Because the seminal plasma 5'-D is partially, but uncompetitively, inhibited by PTU, the presence in seminal plasma of two 5'-D isoenzymes (type I and II) cannot be excluded. The 5'-D activity in testes increased significantly between week 3 and 4, and this increase was concomitant with increase in testicular size. The relationship between testicular weight gain and age showed a similar characteristic change and corresponded to the change in 5'-D activity. Unlike in rodents, the testis of the prepubertal pig has thyroid hormone receptors in Sertoli cells, and suggests that in growing piglets, testicular 5'-D is a key factor regulating local supply of biologically active T3, and is an essential factor in testicular paracrine function. The present results are the first demonstration and characterization of the 5'-deiodinase in seminal plasma.

Triiodothyronine (T3), insulin and characteristics of 5'-monodeiodinase (5'-MD) in mare's milk from parturition to 21 days post-partum.

It is generally accepted that hormones and tissue growth factors are supplied from mother to neonate via mammary secretion. Among the protein hormones, insulin and prolactin are considered as the most important milk components for neonates. The significance of the thyroid hormones, namely triiodothyronine (T3) generated locally by 5'-monodeiodinase (5'-MD) in the mammary tissues, for the mammary gland itself and for suckling neonates is still under consideration. In the present study the activity of the 5'-MD and the concentrations of T3 and insulin in mare's colostrum and milk during the first 21 days of lactation were measured. Post partum, T3 increased to its highest concentration around day 4 (1.14+/-0.08 nmol/L), then progressively decreased until day 7, reaching a relatively stable concentration of 0.71+/-0.06 nmol/L (overall mean for days 7-21). The colostral insulin concentration, highest on the day of parturition (401.0 +/-24.9 microU/mL), decreased to a nadir value (25.0+/-3.4 microU/mL) on day 5, after which it tended to increase. The mare's milk showed the presence of PTU-sensitive (type I) and PTU-insensitive (type II) 5'-monodeiodinases (5'-MD). Contrary to the classical type II 5'-MD, the mare's milk isoenzyme was inhibited non-competitively by aurothioglucose. A significant relationship (r=0.962, P < 0.01 ) between T3 concentration and 5'-MD activity, from the I st to the 6th lactational day was found, which may indicate a dependence of T3 concentration on the milk 5'-MD activity. The presence of 5'-MD of type II suggests that intra-mammary T3 generation may play a paracrine role supporting lactogenesis. Estimating that 1.8 microg of colostral T3 (0.456 microg/L) is consumed daily by a suckling foal, the T3 hormone action within the intestinal tract cannot be ruled out. This is the first paper to provide evidence of T3 and insulin concentrations, and of T4-5'-monodeiodinases activity in colostrum and milk of the mare.

Adhesion of Pseudomonas aeruginosa to collagen biomaterials: effect of amikacin and ciprofloxacin on the colonization and survival of the adherent organisms.

The adherence of P. aeruginosa to collagen membrane, sponge, and to a new anti-infective COLL dressing and the susceptibility of the organisms attached to the biomaterials to amikacin were investigated in vitro. After 17 h of attachment, the bacteria demonstrated an increased resistance to amikacin compared with their free-floating counterparts. Amikacin, even at a concentration exceeding 150 times the minimal bactericidal concentration (MBC) for the strain tested, did not eradicate the attached bacteria from the surface of collagen membrane. However, when the drug at a high concentration (over 16 times the minimal inhibitory concentration, MIC) was present in the incubation medium before it had been inoculated with P. aeruginosa, a reduction of 2 log10 units in the organisms adherent to the surface of collagen membrane was observed. We conclude that slow release of the antibiotic from the COLL dressing could control the bacterial colonization on the surface. In fact, the released amikacin at the final concentration of 32 times the MBC reduced the number of adherent bacteria by 6 log10 units. In contrast, ciprofloxacin at the same final bactericidal concentration completely eradicated the bacteria from the surface of COLL dressing. However, as ciprofloxacin is not recommended for use as a topical antimicrobial agent, a further search is needed to find an agent with a similar anticolonization activity.