Expression of mRNA of lipoprotein receptor related protein 8, low density lipoprotein receptor, and very low density lipoprotein receptor in bovine ovarian cells during follicular development and corpus luteum formation and regression

Lipoproteins in the plasma are the major source of cholesterol obtained by the ovarian theca and granulosa cells for steroidogenesis. In this study, we have identified mRNA expression in bovine theca and granulosa cells of two lipoprotein receptors, low density lipoprotein receptor (LDLr) and very low density lipoprotein receptor (VLDLr) in granulosa cells from small antral follicles through preovulatory follicles and in theca cells from large and medium sized antral follicles. In the corpus luteum (CL) both these receptors were found in the developing and differentiating stages whereas only mRNA for VLDLr was detected in the regression stage. This study also described for the first time, the presence of lipoprotein receptor related protein (LRP8) in granulosa cells from small antral follicles through preovulatory follicles and in theca cells from large and medium sized antral follicles. This may indicate a role of LRP8 in cholesterol delivery to steriodogenic cells. LRP8 was not detected in any of the CL stages. The roles of the LDLr superfamily in lipid transport to ovarian cells and its participation in follicular and CL development and regression is discussed.     

Concanavalin A-binding glycoproteins in the subcommissural and the pineal organ of the sheep (Ovis aries)

Summary Glycoproteins rich in mannosyl or glucosyl residues were analyzed in the subcommissural organ (SCO) and the pineal organ of the sheep (Ovis aries). By use of concanavalin A labelled with fluorescein isothiocyanate, fluorescent material was found both in ependymal and hypendymal cells of the SCO. In the pineal organ, either isolated or grouped parenchymal cells showed a marked fluorescence. These cells may correspond to ependymal elements also called interstitial cells or supporting cells. In addition, scarce slender, fluorescent processes were observed in the pineal parenchyma. The techniques of electrophoresis and electrotransfer on nitrocellulose paper have been applied to analyze the glycopeptide content of the SCO and the pineal organ in comparison to cerebellar and cerebral fractions solubilized by use of Triton X 100. Approximately 30 different concanavalin A-reactive glycopeptides were revealed in each fraction. In the SCO extract four glycopeptides (30, 54, 72, 100 kd) might correspond to subunits of the glycoprotein(s) characteristically stored in the ependymal cells of the SCO. In addition, two glycopeptides (32/33, 115 kd) are specific to the pineal organ extract. The possible similarity of the concanavalin A-reactive material in both organs is discussed and a putative secretory activity of the pineal ependymal cells is postulated.     

Activation of estrogen receptor-beta by a special extract of Rheum rhaponticum (ERr 731), its aglycones and structurally related compounds

The special extract ERr 731^® from the roots of Rheum rhaponticum is the major constituent of Phytoestrol^® N which is used for the treatment of climacteric symptoms in menopausal women. However, the molecular mode of action of ERr 731^® was unknown. For the first time, ERr 731^® and its aglycones trans-rhapontigenin and desoxyrhapontigenin were investigated with regard to the activation of the estrogen receptor- or estrogen receptor-β (ER, ERβ). The related hydroxystilbenes cis-rhapontigenin, resveratrol and piceatannol were studied as comparators. As controls, 17β-estradiol or the selective ER-(propylpyrazoltriol) or ERβ-agonists (diarylpropionitril) were used. Neither in ER-expressing yeast cells, in the ER-responsive Ishikawa cells, nor in human endometrial HEC-1B cells transiently transfected with the ER an activation of ER by ERr 731^® or the other single compounds was detected. Furthermore, an antiestrogenic effect was not observed. In contrast in human endometrial HEC-1B cells transiently transfected with the ERβ, 100 ng/ml ERr 731^® and the single compounds significantly induced the ERβ-coupled luciferase activity in a range comparable to 10⁻⁸ M 17β-estradiol. All effects were abolished with the pure ER antagonist ICI 182780, indicating an ER-specific effect. The ERβ agonistic activity by ERr 731^® could be of importance for its clinical use, as central functions relevant to climacteric complaints are proposed to be mediated via ERβ activation.     

Chemotherapy and zoledronate sensitize solid tumour cells to Vγ9Vδ2 T cell cytotoxicity

Combinations of cellular immune-based therapies with chemotherapy and other antitumour agents may be of significant clinical benefit in the treatment of many forms of cancer. Gamma delta (γδ) T cells are of particular interest for use in such combined therapies due to their potent antitumour cytotoxicity and relative ease of generation in vitro. Here, we demonstrate high levels of cytotoxicity against solid tumour-derived cell lines with combination treatment utilizing Vγ9Vδ2 T cells, chemotherapeutic agents and the bisphosphonate, zoledronate. Pre-treatment with low concentrations of chemotherapeutic agents or zoledronate sensitized tumour cells to rapid killing by Vγ9Vδ2 T cells with levels of cytotoxicity approaching 90%. In addition, zoledronate enhanced the chemotherapy-induced sensitization of tumour cells to Vγ9Vδ2 T cell cytotoxicity resulting in almost 100% lysis of tumour targets in some cases. Vγ9Vδ2 T cell cytotoxicity was mediated by perforin following TCR-dependent and isoprenoid-mediated recognition of tumour cells. Production of IFN-γ by Vγ9Vδ2 T cells was also induced after exposure to sensitized targets. We conclude that administration of Vγ9Vδ2 T cells at suitable intervals after chemotherapy and zoledronate may substantially increase antitumour activities in a range of malignancies. Financial support and conflicts of interest: This study was supported by grants from Medinet (Japan), and Suncorp Metway and Gallipoli Research Foundation (Australia). No financial or commercial interests arise from this study. Informed consent: This study was approved by Human Research Ethics Committees of the University of Queensland and Greenslopes Private Hospital and informed consent was obtained from all subjects.     

Effect of leukemia inhibitory factor (LIF) on in vitro produced bovine embryos and their outgrowth colonies

In vitro produced (IVP) bovine embryos were subjected to in vitro culture with or without 1000 U/ml human recombinant leukemia inhibitory factor (LIF) added to the culture medium from Days 5 to 8 post insemination (p.i.). Resulting blastocysts were subsequently plated intact on mouse feeder cells in a medium with or without LIF. Significantly more embryos reached the hatched blastocyst stage, and the number of blastocysts with excellent morphology was significantly higher, when LIF was omitted. At Day 8 p.i., total cell count (TCC) and inner cell mass (ICM) cell count was significantly higher in embryos cultured without LIF. In embryos cultured with LIF, cytoplasmic vesicles and lipid droplets were abundant and a decreased expression of both Oct4 and laminin could be observed. Initial hypoblast formation was revealed in almost 1/3 of the LIF-cultured blastocysts whereas this feature was evident in 2/3 of the blastocysts cultured in the absence of LIF. Overall, almost 60% of the blastocysts cultured without LIF formed outgrowth colonies (OCs) when plated on feeders, whereas this phenomenon was only observed in 30% of the blastocysts cultured in the presence of LIF. A tendency for retaining a tightly packed central growth of putative ICM-derived cells was observed, when attachment to the feeder layer was initiated close to the embryonic pole of the blastocyst. At Day 8 of outgrowth culture, approximately 20% of the colonies contained a central core of putative ICM-derived cells appearing large enough for mechanical isolation and further subculture. Immunohistochemical labeling for Oct4 revealed staining of both trophectodermal and ICM-derived cells. The presence of LIF in the outgrowth culture medium did not have any apparent effect on the plating efficiency or colony type. In conclusion, LIF had an adverse effect on in vitro embryonic development when added to the culture medium in the period from Days 5 to 8 p.i., whereas it had no apparent effect on the OCs subsequently formed from such embryos.     

An oxidative stress-mediated positive-feedback iron uptake loop in neuronal cells

Intracellular reactive iron is a source of free radicals and a possible cause of cell damage. In this study, we analyzed the changes in iron homeostasis generated by iron accumulation in neuroblastoma (N2A) cells and hippocampal neurons. Increasing concentrations of iron in the culture medium elicited increasing amounts of intracellular iron and of the reactive iron pool. The cells had both IRP1 and IRP2 activities, being IRP1 activity quantitatively predominant. When iron in the culture medium increased from 1 to 40 microm, IRP2 activity decreased to nil. In contrast, IRP1 activity decreased when iron increased up to 20 microm, and then, unexpectedly, increased. IRP1 activity at iron concentrations above 20 microm was functional as it correlated with increased (55) Fe uptake. The increase in IRP1 activity was mediated by oxidative-stress as it was largely abolished by N-acetyl-L-cysteine. Culturing cells with iron resulted in proteins and DNA modifications. In summary, iron uptake by N2A cells and hippocampus neurons did not shut off at high iron concentrations in the culture media. As a consequence, iron accumulated and generated oxidative damage. This behavior is probably a consequence of the paradoxical activation of IRP1 at high iron concentrations, a condition that may underlie some processes associated with neuronal degeneration and death.     

Switching roles: the functional plasticity of adult tissue stem cells

Adult organisms have to adapt to survive, and the same is true for their tissues. Rates and types of cell production must be rapidly and reversibly adjusted to meet tissue demands in response to both local and systemic challenges. Recent work reveals how stem cell (SC) populations meet these requirements by switching between functional states tuned to homoeostasis or regeneration. This plasticity extends to differentiating cells, which are capable of reverting to SCs after injury. The concept of the niche, the micro‐environment that sustains and regulates stem cells, is broadening, with a new appreciation of the role of physical factors and hormonal signals. Here, we review different functions of SCs, the cellular mechanisms that underlie them and the signals that bias the fate of SCs as they switch between roles.     

Expression of cytokeratin 20 in urine cytology smears: a potential marker for the detection of urothelial carcinoma

BACKGROUND: Urine cytomorphology is one of the oldest methods for screening and monitoring patients with transitional cell carcinoma (TCC). Sensitivity of urine cytology is relatively low. Ancillary techniques on urine sample may increase the sensitivity. AIM: To explore the utility of cytokeratin 20 (CK20) immunostaining in identifying malignant cells in urine cytology smears. MATERIALS AND METHODS: Fourteen cases each of confirmed TCC and benign urinary cytology along with five cases of atypical cells in urine were immunostained with a monoclonal CK20 antibody. Of 14 cases of TCC, 12 showed strong positive staining with the antibody. All benign cases were negative except for a few cases in which the umbrella cells were weakly to moderately positive. In all five cases of atypical urine cytology the atypical cells stained positive with the antibody. These cases were later confirmed as TCC on histopathology of bladder wall biopsy. CONCLUSION: CK20 is an important biomarker that can be used to identify TCC in urine cytology smears. It is particularly useful in those cases where malignancy cannot be confirmed by morphology alone.     

Immune response to stem cells and strategies to induce tolerance

Although recent progress in cardiovascular tissue engineering has generated great expectations for the exploitation of stem cells to restore cardiac form and function, the prospects of a common mass-produced cell resource for clinically viable engineered tissues and organs remain problematic. The refinement of stem cell culture protocols to increase induction of the cardiomyocyte phenotype and the assembly of transplantable vascularized tissue are areas of intense current research, but the problem of immune rejection of heterologous cell type poses perhaps the most significant hurdle to overcome. This article focuses on the potential advantages and problems encountered with various stem cell sources for reconstruction of the damaged or failing myocardium or heart valves and also discusses the need for integrating advances in developmental and stem cell biology, immunology and tissue engineering to achieve the full potential of cardiac tissue engineering. The ultimate goal is to produce 'off-the-shelf' cells and tissues capable of inducing specific immune tolerance.