Christopher M.R. Bax

Stimulation of osteoclastic bone resorption by hydrogen peroxide

The molecular mechanisms underlying the pathophysiology of bone destruction still remain poorly understood. We have found that hydrogen peroxide (H2O2), a reactive oxygen species (ROS), is a potent stimulator of osteoclastic bone resorption and cell motility. A marked enhancement of bone resorption was noted when rat osteoclasts, cultured on devitalised bovine cortical bone, were exposed to 10 nM [H2O2]. Apart from exposing osteoclasts to a low extracellular pH, which is known to enhance osteoclastic bone resorption, we provide first evidence for a molecule that stimulates osteoclastic bone resorption in osteoclast cultures that do not respond to parathyroid hormone and 1, 25 dihydroxyvitamin D3. We envisage that both basic biological and practical clinical implications may eventually follow from these studies.

Culture of syncytiotrophoblast for the study of human placental transfer. Part I: Isolation and purification of cytotrophoblast

Criteria for a successful model for the study of trans-syncytiotrophoblast transfer include isolating substantially pure trophoblast cells from placental villous tissue, and obtaining from them phenotypical villous syncytial syncytiotrophoblast during culture. For studies involving the basal membrane, including overall transfer, basal uptake and output, and controls acting at the basal membrane, a two-sided model is required with a separate compartment of culture medium in contact with the basal cell surface. All current methods of isolating cytotrophoblast, the precursor of syncytiotrophoblast, derive from the original tissue trypsinization method of Thiede (1960), which produces cultures of villous cytotrophoblast cells contaminated with other placental cell types. Lessons learned from successful and unsuccessful development of the model over 35 years are outlined, and recently established methods for purifying the isolated mixed cells discussed. These include sedimentation and centrifugation methods, immunological and receptor binding methods, and more selective release of trophoblast cells from tissue. Immuno flow cytometric cell sorting methods are potentially capable of isolating subpopulations of various phenotypical trophoblast types. We conclude that satisfactory methods are now available for isolating and purifying cytotrophoblast from early or late gestation human placenta.

Ultrastructural changes and immunocytochemical analysis of human placental trophoblast during short-term culture

Trophoblastic cells, of at least 95 per cent purity by immunofluorescence and morphological criteria, were obtained from human term placenta by a simple trypsinisation method without the additional purification steps or complex culture conditions used by others. The differentiation of these cells was followed over four days in culture by fluorescence immunocytochemistry, by scanning and transmission electron microscopy and by light microscopy. The results support the idea that the isolated cells are cytotrophoblast and that these differentiate during this time into cells with characteristics of villous syncytiotrophoblast. This process involved first the formation of a multicellular layer of mononucleated cells, then the development of a syncytium of multinucleated cells and, not necessarily concurrently, functional differentiation. This may be a useful model for the study of syncytiotrophoblast function.

Two-sided culture of human placental trophoblast. Morphology, immunocytochemistry and permeability properties*

We describe the culture of human term placental trophoblast cells on cell-free amniotic membrane, with medium on both sides. Over the course of 2 days, the isolated cells, initially simple, mononucleated and probably cytotrophoblast, form a confluent layer of multinucleated syncytial cells with morphological and immunocytochemical properties of syncytiotrophoblast. This layer becomes polarized with respect to morphology, alkaline phosphatase distribution and hCG secretion. Contamination with amnion cells, and with other cell types that are present in placental tissue, was less than 1 per cent. A preliminary investigation of the permeability properties of the preparation showed that the trophoblast cell layer, rather than the amniotic membrane, was rate-limiting to transtrophoblast transfer, but that possible effects of the supporting membrane should be considered. The transtrophoblast transfer of D-glucose and the non-metabolisable analogue, 3-O-methyl-D-glucose (3OMG), had saturable and non-saturable/leak components in both directions, indicating that carrier-mediated processes were involved. The non-metabolisable amino acid 2-aminoisobutyrate (AIB) was both accumulated within the trophoblast cells, and transferred by saturable and non-saturable processes from the microvillous side, but no saturable accumulation or transfer was observed from the basal side, at the concentrations tried. The results suggest that this model may prove suitable for studies of transtrophoblast transfer.

Culture of syncytiotrophoblast for the study of human placental transfer. Part II: Production, culture and use of syncytiotrophoblast

The conditions necessary for producing syncytical syncytiotrophoblast are examined. Tissue disaggregation conditions, culture media composition, different extracellular matrices and the influence of placental gestational age are all assessed. The importance of evaluating the biochemical and functional differentiational state of the cells is also stressed. Evidence is summarized that syncytiotrophoblast in culture is morphologically and ultrastructurally very similar to syncytiotrophoblast in vivo, and what is so far known biochemically is largely consistent with what is known in vivo. Studies published to date on microvillous membrane uptake and release and relationships with intracellular metabolism using syncytiotrophoblast in conventional culture are outlined from the point of view of the advantages and potential of this model. The present state of development of the two-sided model is assessed, mentioning factors to be considered such as the supporting membrane to be used, accounting for passive diffusion and paracellular leak components of transport and dealing with quantitative effects in kinetic studies of the presence of the supporting membrane. It is concluded that satisfactory methods are now in place for preparing pure villous syncytial syncytiotrophoblast in culture from cytotrophoblast derived from term (but not early) placentae, suitable for studying microvillous membrane transport and relationships with intracellular metabolism. Cytotrophoblast from early gestational age placenta may require different conditions to form true syncytiotrophoblast. A two-sided model for studies of overall transfer, basal transport and basal control mechanisms is now available and possibly with some development should be a good model for such investigations.

Characterisation of the differential expression of marker antigens by normal and malignant endometrial epithelium.

In order to examine the production of marker proteins, a reproducible method has been established for culturing purified epithelial cells from normal and malignant endometrium. We have examined the differential expression of secretory proteins using immunohistochemistry in frozen tissue sections, immunocytochemistry in cell cultures derived from the same specimens and protein assays on the culture supernatants. Placental protein 14 (PP14) was produced by normal premenopausal epithelium but not by the post-menopausal or malignant endometrial epithelium. In contrast, placental alkaline phosphatase (PLAP) was produced by endometrial cancers and the endometrial adenocarcinoma-derived cell line Ishikawa, but not by the normal endometrial epithelium. Other markers such as CA-125, which was produced by both normal and malignant endometrium but not by the cell line, and human chorionic gonadotrophin (beta-hCG), which was produced by Ishikawa cells but not by any of the fresh tissues, were less cancer specific. Placental alkaline phosphatase is a direct product of endometrial cancers that can be readily assayed in serum using this two-site assay to test its clinical usefulness in monitoring patients at risk for endometrial cancer.

Regulation of endometrial cancer cell growth by luteinizing hormone (LH) and follicle stimulating hormone (FSH)

Gonadotrophin releasing hormone analogues (GnRHa) have been used to treat recurrent endometrial cancer. However, the mode of action is uncertain. Our previous studies showed no direct effect of GnRHa on endometrial cancer cell growth in vitro. We have now examined the effect of luteinizing hormone (LH) and follicle stimulating hormone (FSH) on endometrial cancer cell growth. The aim was to determine whether suppression of pituitary LH and FSH by GnRHa could explain the tumour regression seen in up to 44% of patients treated with this drug. We show that recombinant human LH and FSH (rhLH and rhFSH) produce a concentration dependent stimulation of the endometrial cancer cell line HEC-1A, in serum-free medium (maximum increase of 62 and 50% respectively relative to untreated controls). This increase is equivalent to that obtained by addition of 10% newborn calf serum. Growth of the Ishikawa cell line in culture increases in the presence of rhLH (maximum increase of 67%) but not with rhFSH. Using RT-PCR, we show that the Ishikawa cell line intermittently expresses receptor mRNA of LH but not of FSH; there is no expression of either mRNA by HEC-1A. Classically, both LH and FSH act via cAMP linked membrane receptors. However, neither rhLH nor rhFSH elicit cAMP production in either of our endometrial cancer cell lines. Thus, although a growth response to LH and FSH can be shown, and some cells express the LH receptor, stimulation appears to be via a pathway separate from that of the classical gonadotrophin receptor.

Cyclosporine and cremaphor modulate von Willebrand factor release from cultured human endothelial cells.

Cyclosporine has been associated with microangiopathic hemolysis (MAHA) and other thrombotic complications of bone marrow and renal transplantation. MAHA is characterized by intravascular platelet aggregation, which, in some situations, is thought to be mediated by hyperactive high molecular weight von Willebrand factor (vWF). We have hypothesized that transplant-related MAHA may be caused by CsA-mediated release of von Willebrand factor from endothelial cells. This hypothesis was tested by studying vWF release from human umbilical vein endothelial cells primed with either CsA or cremophor EL. CsA and cremophor alone did not increase vWF release until toxic concentrations were reached (50-100 micrograms/ml). However, at therapeutic concentrations (0.1-5 micrograms/ml) vWF release by cells stimulated with thrombin, histamine, PMA, and the calcium ionophore A23187 was enhanced by both CsA and cremophor in a concentration-dependent manner. In single isolated endothelial cells, the thrombin-induced increase in cytosolic free calcium was enhanced by both CsA and cremophor. Preincubation for 24 hr with CsA but not cremophor suppressed vWF release after thrombin stimulation. These observations were mirrored by a concentration-dependent suppression of [3H]thymidine uptake by CsA. We conclude that CsA vehicle, cremophor, enhances stimulated vWF release in vitro, probably by processes dependent upon increased cytosolic free calcium. This suggests a possible mechanism for thrombotic transplant complications.

Enhancing learning through formative assessment

The student cohort on the University Science Extended Degree (SED) course is diverse in terms of educational experience. One of the key facets of teaching at this level is to engage and prepare students for higher levels of education in the sciences. The purpose of this evaluation is to relate a specific virtual framework, designed for students participating on biology modules contained in the SED course, with levels of student engagement. Central to this is the use of weekly on-line formative tests that students are expected to complete in their own time. Whilst over-all module pass rates remain stable, results indicate that a substantial proportion of students completed all of these assessments, and this appears to be directly linked to attainment of higher grades. Student feedback indicated that over 80% of responding students found the tests helpful. This model relating to learning and teaching encourages students to take responsibility for their own learning experience.

Human fetal endothelial cells acquire Zinc(II) from both the protein bound and nonprotein bound pools in serum

To help determine physiologically important routes by which zinc (Zn) is acquired by human fetal vascular endothelium, the authors incubated cultured umbilical vein endothelial cells with65Zn(II)-tracer labeled human fetal whole serum, ultrafiltrate (containing low molecular mass serum zinc complexes), and dialyzed serum (containing protein-bound zinc). Zinc from whole serum and from both serum fractions entered a rapidly labeled cellular compartment removable by edetic acid (EDTA), representing Zn bound to the outside cell surface, and accumulatively, an EDTA-resistant compartment’probably largely internalized Zn. Entry of Zn into the EDTA-resistant pool from both serum fractions was strongly temperature-dependent, and was not via the EDTA-sensitive pool. Entry from the ultrafiltrate was resolvable into high affinity saturable, and non-(or hardly-) saturable components. Transfer from the dialyzed serum fraction was not significantly saturable, but only partially accounted for by nonspecific pinocytosis. Thus, Zn is obtained by fetal vascular endothelium partly from low molecular mass serum species, probably through at least one carrier-mediated membrane transport system; but also from Zn complexed with serum protein, via at least one metabolism-related route.