Participation of Endosomes in Toll-Like Receptor 3 Transportation Pathway in Murine Astrocytes.

TLR3 provides immediate type I IFN response following entry of stimulatory PAMPs into the CNS, as it is in HSV infection. The receptor plays a vital role in astrocytes, contributing to rapid infection sensing and suppression of viral replication, precluding the spread of virus beyond neurons. The route of TLR3 mobilization culminating in the receptor activation remains unexplained. In this research, we investigated the involvement of various types of endosomes in the regulation of the TLR3 mobility in C8-D1A murine astrocyte cell line. TLR3 was transported rapidly to early EEA1-positive endosomes as well as LAMP1-lysosomes following stimulation with the poly(I:C). Later, TLR3 largely associated with late Rab7-positive endosomes. Twenty-four hours after stimulation, TLR3 co-localized with LAMP1 abundantly in lysosomes of astrocytes. TLR3 interacted with poly(I:C) intracellularly from 1 min to 8 h following cell stimulation. We detected TLR3 on the surface of astrocytes indicating constitutive expression, which increased after poly(I:C) stimulation. Our findings contribute to the understanding of cellular modulation of TLR3 trafficking. Detailed analysis of the TLR3 transportation pathway is an important component in disclosing the fate of the receptor in HSV-infected CNS and may help in the search for rationale therapeutics to control the replication of neuropathic viruses.

Preliminary Studies on Biodegradable Zinc Oxide Nanoparticles Doped with Fe as a Potential Form of Iron Delivery to the Living Organism.

Iron is the crucial element for living organisms and its deficiency is described as the most common nutritional disorder all over the world. Nowadays, more effective and safe iron supplementation strategies for both humans and animals become one of the most important challenges in the therapy of nutritional deficiencies. Our previous in vivo studies confirmed safety and biodegradability of in-house manufactured zinc oxide-based nanoparticles and their rapid distribution to majority of organs and tissues in the body. In vitro examinations performed on Caco-2 cell line, a model of epithelial cells of the gastrointestinal tract, revealed a low toxicity of studied nanomaterials. In the current study, we investigated biodegradable zinc oxide nanoparticles doped with Fe(III) as a perspective supplementation strategy for iron deficiency. Biodegradable ZnO:Fe nanoparticles were intra-gastrically administered to adult mice and following 24 h, animals were sacrificed with collection of internal organs for further analyses. The iron concentration measured with atomic absorption spectrometry and histological staining (Perl's method) showed a rapid distribution of iron-doped nanoparticles to tissues specifically related with iron homeostasis. Accumulation of iron was also visible within hepatocytes and around blood vessels within the spleen, which might indicate the transfer of Fe-doped nanoparticles from the bloodstream into the tissue. Reassuming, preliminary results obtained in the current study suggest that biodegradable ZnO nanoparticles doped with Fe might be a good carriers of exogenous iron in the living body. Therefore, subsequent investigations focus on determination an exact mechanisms related with an iron deposition in the tissue and influence of nanoparticle carriers on iron metabolism are required.

Transfer of orally administered ZnO:Eu nanoparticles through the blood-testis barrier: the effect on kinetic sperm parameters and apoptosis in mice testes.

Zinc-based nanoparticles are promising materials for various applications, including in biomedicine. The aim of our study was to determine the effect of fluorescent europium-doped zinc oxide nanoparticles (ZnO:Eu NPs) on sperm parameters, cell apoptosis and integrity of the blood-testis barrier (BTB) in mice. Nanostructures were orally administered to adult mice (n = 34). Animals were sacrificed after 3 h, 24 h, 7 d and 14 d following oral administration. Sperm was collected and analysed for viability and kinetic parameters. Collected testes were quantitatively analysed for accumulation of ZnO:Eu NPs. Microscopic evaluation based on immunofluorescence and histopathological studies were also conducted. Results showed that ZnO:Eu NPs were able to overcome the BTB with their subsequent accumulation in the testis. No toxic or pro-apoptotic effects of nanoparticles on the male reproductive system were observed. The results suggested that ZnO:Eu NPs were able to accumulate in the testis with no negative impact on sperm parameters, tissue architecture or the integrity of the BTB.

Effect of exogenous butyrate on the gastrointestinal tract of sheep. I. Structure and function of the rumen, omasum, and abomasum.

The aim of this study was to determine the effect of exogenous butyrate on the structure and selected functions of the stomach in sheep. Eighteen rams (30.8 ± 2.1 kg; 12 to 15 mo of age) were allocated to the study and fed a diet for 14 d without (CTRL) or with sodium butyrate (BUT; 36 g/kg of offered DM). Neither DMI nor initial BW differed between treatments (P ≥ 0.61), but final BW was greater for BUT compared with CTRL (P = 0.03). Butyrate concentration in the reticuloruminal fluid and abomasal digesta was greater for BUT compared with CTRL (P ≤ 0.01), but total short-chain fatty acids (SCFA) concentration, as well as concentration of other SCFA, did not differ between treatments (P ≥ 0.07). Relative to BW, reticuloruminal tissue mass tended (P = 0.09) to be greater and omasal digesta was less (P = 0.02) for BUT compared with CTRL. Dietary butyrate did not affect ruminal papillae length, width, and density nor did it affect ruminal epithelium thickness (P ≥ 0.12) in the ventral sac of the rumen. However, the DM of ruminal epithelium (mg/cm2) tended (P = 0.06) to be greater for BUT compared with CTRL. Omasal and abomasal epithelium thicknesses were greater (P ≤ 0.05) for BUT compared with CTRL. Mitosis-to-apoptosis ratio in the abomasal epithelium was less for BUT compared with CTRL (P = 0.04). Finally, the mRNA expression of peptide transporter 1 in the omasal epithelium was less (P = 0.02) and mRNA expression of monocarboxylate transporter 1 in the abomasal epithelium tended (P = 0.07) to be greater for BUT compared with CTRL. It can be concluded that exogenous butyrate supplementation affected not only the rumen but also omasum and abomasum in sheep.

Effect of exogenous butyrate on the gastrointestinal tract of sheep. II. Hydrolytic activity in the rumen and structure and function of the small intestine.

The aim of this study was to determine the effect of exogenous butyrate on the activity of carbohydrate-digesting enzymes in the reticuloruminal digesta and structure and selected functions of the small intestine in sheep. Eighteen rams (30.8 ± 2.1 kg; 12 to 15 mo of age) were fed for 14 d a diet without (CTRL) or with sodium butyrate (BUT; 36 g/kg of offered DM). Butyrate concentration in the reticuloruminal fluid and proximal small intestinal digesta was greater for BUT compared with CTRL (P ≤ 0.05). Amylolytic activity was greater, whereas cellulolytic and xylanolytic activity in the reticuloruminal digesta was less for BUT compared with CTRL (P ≤ 0.04). Relative to BW, small intestinal tissue mass and small intestine length did not differ between treatments (P ≥ 0.15); however, absolute length of the small intestine was greater for BUT compared with CTRL (P = 0.04). In the duodenum, crypt depth tended (P = 0.10) to be greater, whereas in the ileum, crypt depth and muscularis thickness tended (P = 0.10) to be less for BUT compared with CTRL. Mitosis-to-apoptosis ratio in the proximal jejunum was greater for CTRL compared with BUT (P = 0.02). Expression of G-protein-coupled receptor 43 mRNA in the duodenal epithelium was greater for BUT compared with CTRL (P < 0.01). On the other hand, peptide transporter 1 mRNA expression in the distal sections of the small intestine, as well as activity of aminopeptidase A and dipeptidylpeptidase IV, were greater for CTRL (P ≤ 0.05). In summary, exogenous butyrate supplementation in feed affects hydrolytic activity in the rumen, and increased butyrate flow out of the reticulorumen affects both proximal and distal sections of the small intestine in sheep.

Biodegradation of the ZnO:Eu nanoparticles in the tissues of adult mouse after alimentary application.

Biodegradable zinc oxide nanoparticles (ZnO NPs) are considered promising materials for future biomedical applications. To fulfil this potential, biodistribution and elimination patterns of ZnO NPs in the living organism need to be resolved. In order to investigate gastrointestinal absorption of ZnO NPs and their intra-organism distribution, water suspension of ZnO or fluorescent ZnO:Eu (Europium-doped zinc oxide) NPs (10mg/ml; 0.3ml/mouse) was alimentary-administered (IG: intra-gastric) to adult mice. Internal organs collected at key time-points after IG were evaluated by AAS for Zn concentration and analysed by cytometric techniques. We found that Zn-based NPs were readily absorbed and distributed (3 h post IG) in the nanoparticle form throughout the organism. Results suggest, that liver and kidneys were key organs responsible for NPs elimination, while accumulation was observed in the spleen and adipose tissues. We also showed that ZnO/ZnO:Eu NPs were able to cross majority of biological barriers in the organism (including blood-brain-barrier).

Comparison of stem/progenitor cell number and transcriptomic profile in the mammary tissue of dairy and beef breed heifers.

Bovine mammary stem cells (MaSC) are a source of ductal and lobulo-alveolar tissue during the development of the mammary gland and its remodeling in repeating lactation cycles. We hypothesize that the number of MaSC, their molecular properties, and interactions with their niche may be essential in order to determine the mammogenic potential in heifers. To verify this hypothesis, we compared the number of MaSC and the transcriptomic profile in the mammary tissue of 20-month-old, non-pregnant dairy (Holstein-Friesian, HF) and beef (Limousin, LM) heifers. For the identification and quantification of putative stem/progenitor cells in mammary tissue sections, scanning cytometry was used with a combination of MaSC molecular markers: stem cell antigen-1 (Sca-1) and fibronectin type III domain containing 3B (FNDC3B) protein. Cytometric analysis revealed a significantly higher number of Sca-1(pos)FNDC3B(pos) cells in HF (2.94 ± 0.35%) than in LM (1.72 ± 0.20%) heifers. In HF heifers, a higher expression of intramammary hormones, growth factors, cytokines, chemokines, and transcription regulators was observed. The model of mammary microenvironment favorable for MaSC was associated with the regulation of genes involved in MaSC maintenance, self-renewal, proliferation, migration, differentiation, mammary tissue remodeling, angiogenesis, regulation of adipocyte differentiation, lipid metabolism, and steroid and insulin signaling. In conclusion, the mammogenic potential in postpubertal dairy heifers is facilitated by a higher number of MaSC and up-regulation of mammary auto- and paracrine factors representing the MaSC niche.

Intrauterine growth retarded piglet as a model for humans--studies on the perinatal development of the gut structure and function.

The overall acceptance of pig models for human biomedical studies is steadily growing. Results of rodent studies are usually confirmed in pigs before extrapolating them to humans. This applies particularly to gastrointestinal and metabolism research due to similarities between pig and human physiology. In this context, intrauterine growth retarded (IUGR) pig neonate can be regarded as a good model for the better understanding of the IUGR syndrome in humans. In pigs, the induction of IUGR syndrome may include maternal diet intervention, dexamethasone treatment or temporary reduction of blood supply. However, in pigs, like in humans, circa 8% of neonates develop IUGR syndrome spontaneously. Studies on the pig model have shown changes in gut structure, namely a reduced thickness of mucosa and muscle layers, and delayed kinetic of disappearance of vacuolated enterocytes were found in IUGR individuals in comparison with healthy ones. Functional changes include reduced dynamic of gut mucosa rebuilding, decreased activities of main brush border enzymes, and changes in the expression of proteins important for carbohydrate, amino acids, lipid, mineral and vitamin metabolism. Moreover, profiles of intestinal hormones are different in IUGR and non-IUGR piglets. It is suggested that supplementation of the mothers during the gestation and/or the IUGR offspring after birth can help in restoring the development of the gastrointestinal tract. The pig provides presumably the optimal animal model for humans to study gastrointestinal tract structure and function development in IUGR syndrome.

Sodium ascorbate and basic fibroblast growth factor protect muscle-derived cells from H2O2-induced oxidative stress.

Engraftment of muscle-derived cells (MDCs) into the urethral sphincter may cure urinary incontinence. However, poor cell survival after injection prompted us to evaluate whether 24-h preincubation with sodium ascorbate (ASC) or basic fibroblast growth factor (bFGF) prior to cell transfer improves the survival of MDCs facing oxidative stress in vitro. We examined MDCs isolated from female rats for the presence of myogenic markers and for the ability to differentiate and respond to growth factors. Isolated cells were positive for desmin, M-cadherin, and myogenin. The fusion index exceeded 29%, and Akt kinase was phosphorylated at Ser473 residue upon exposure to insulin-like growth factor 1 (100 ng/ml). We then autologously transplanted MDCs transfected with lacZ marker gene into urethral wall of the rats; 2 wk later, the urethra and caudal wall of the urinary bladder were harvested from these animals. Serial cryosections revealed that transplanted cells formed multinuclear clusters at injection sites. In addition, we found that the viability of MDCs exposed to a cytotoxic concentration of H2O2 was higher after preincubation with 0.1 mM ASC (2.6-fold), 10 ng/ml bFGF (2.9-fold), or 20 ng/ml bFGF (3.5-fold) than that after exposure to H2O2 only. We conclude that preincubation with ASC or bFGF increases the resistance of MDCs to oxidative stress in vitro. Pretreatment with either agent might be used to enhance survival of MDCs after transplantation.

Autophagy is the dominant type of programmed cell death in breast cancer MCF-7 cells exposed to AGS 115 and EFDAC, new sesquiterpene analogs of paclitaxel.

The molecular mechanism of cell death induced by AGS 115 and EFDAC, sesquiterpene analogs of paclitaxel, was investigated in human breast cancer MCF-7 cells. The study was carried out using laser scanning cytometry, homeostatic confocal microscopy, atomic force microscopy and electron microscopy. AGS 115 and EFDAC exhibited a microtubule-stabilizing effect as confirmed by a significant increase in alpha-tubulin aggregation. Both paclitaxel analogs also induced death in MCF-7 cells. Evaluation of biochemical and morphological features suggested that the major form of programmed cell death induced by AGS 115 and EFDAC was autophagy. This was confirmed by MAP I LC3 expression and the ultrastructural pattern revealed by electron microscopy. Surface images of cells undergoing autophagy showed that, unlike during apoptosis, the dimensions remained unchanged, but the surface of the cell was deformed. The occurrence of apoptosis was confirmed by the efflux of Smac/DIABLO from mitochondria, caspase-7 activation and DNA loss, and did not exceed 9.7%. Therefore, AGS 115 and EFDAC appear to be promising candidates for further investigation in anti-cancer therapy.

Helicobacter pylori VacA cytotoxin interacts with fibronectin and alters HeLa cell adhesion and cytoskeletal organization in vitro.

Helicobacter pylori vacuolating cytotoxin VacA causes multiple effects on epithelial cell function and morphology, but the effects of VacA on signal transduction pathways and the cytoskeleton have not been investigated in detail. In this study, we analyzed the effects of native VacA on HeLa and AGS cell adhesion to fibronectin and laminin under serum-free conditions. Confocal microscopic examination revealed increased number of cells with rounded morphology and inhibition of actin fiber formation, in the presence of VacA. VacA binds to fibronectin in vitro in a dose-dependent manner. This interaction was partly inhibited by a peptide containing an arginine-glycine-aspartic acid motif. The adhesion of HeLa cells to fibronectin, but not to laminin, was decreased in the presence of VacA. Thus, VacA may interact with fibronectin and influence integrin receptor-induced cell signaling and cytoskeleton-dependent cell functions.