In vitro primary cell culture as a physiologically relevant method for preclinical testing of human oncolytic adenovirus

Ad[I/PPT-E1A] is an oncolytic adenovirus that specifically kills prostate cells via restricted replication by a prostate-specific regulatory element. Off-target replication of oncolytic adenoviruses would have serious clinical consequences. As a proposed ex vivo test, we describe the assessment of the specificity of Ad[I/PPT-E1A] viral cytotoxicity and replication in human nonprostate primary cells. Four primary nonprostate cell types were selected to mimic the effects of potential in vivo exposure to Ad[I/PPT-E1A] virus: bronchial epithelial cells, urothelial cells, vascular endothelial cells, and hepatocytes. Primary cells were analyzed for Ad[I/PPT-E1A] viral cytotoxicity in MTS assays, and viral replication was determined by hexon titer immunostaining assays to quantify viral hexon protein. The results revealed that at an extreme multiplicity of infection of 500, unlikely to be achieved in vivo, Ad[I/PPT-E1A] virus showed no significant cytotoxic effects in the nonprostate primary cell types apart from the hepatocytes. Transmission electron microscopy studies revealed high levels of Ad[I/PPT-E1A] sequestered in the cytoplasm of these cells. Adenoviral green fluorescent protein reporter studies showed no evidence for nuclear localization, suggesting that the cytotoxic effects of Ad[I/PPT-E1A] in human primary hepatocytes are related to viral sequestration. Also, hepatocytes had increased amounts of coxsackie adenovirus receptor surface protein. Active viral replication was only observed in the permissive primary prostate cells and LNCaP prostate cell line, and was not evident in any of the other nonprostate cells types tested, confirming the specificity of Ad[I/PPT-E1A]. Thus, using a relevant panel of primary human cells provides a convenient and alternative preclinical assay for examining the specificity of conditionally replicating oncolytic adenoviruses in vivo.

Nagelschmidtite bioceramics with osteostimulation properties : material chemistry activating osteogenic genes and WNT signalling pathway of human bone marrow stromal cells

Bioactive materials with osteostimulation properties are of great importance to promote osteogenic differentiation of human bone marrow stromal cells (hBMSCs) for potential bone regeneration. We have recently synthesized nagelschmidtite (NAGEL, Ca7Si2P2O16) ceramic powders which showed excellent apatite-mineralization ability. The aim of this study was to investigate the interaction of hBMSCs with NAGEL bioceramic bulks and their ionic extracts, and to explore the osteostimulation properties of NAGEL bioceramics and the possible molecular mechanism. The cell attachment, proliferation, bone-related gene expression (ALP, OPN and OCN) and WNT signalling pathways (WNT3a, FZD6, AXIN2 and CTNNB) of hBMSCs cultured on NAGEL bioceramic disks were systematically studied. We further investigated the biological effects of ionic products from NAGEL powders on cell proliferation and osteogenic differentiation of hBMSCs by culturing cells with NAGEL extracts. Furthermore, the effect of NAGEL bioceramics on the osteogenic differentiation in hBMSCs was also investigated with the addition of cardamonin, a WNT inhibitor. The results showed that NAGEL bioceramic disks supported the attachment and proliferation of hBMSCs, and significantly enhanced the bone-related gene expression and WNT signalling pathway of hBMSCs, compared to conventional beta-tricalcium phosphate (β-TCP) bioceramic disks and blank controls. The ionic products from NAGEL powders also significantly promoted the proliferation, bone and WNT-related gene expression of hBMSCs. It was also identified that NAGEL bioceramics could bypass the action of the WNT inhibitor (10 μM) to stimulate the selected osteogenic genes in hBMSCs. Our results suggest that NAGEL bioceramics possess excellent in vitro osteostimulation properties. The possible mechanism for the osteostimulation may be directly related to the released Si, Ca and P-containing ionic products from NAGEL bioceramics which activate bone-related gene expression and WNT signalling pathway of hBMSCs. The present study suggests that NAGEL bioceramics are a potential bone regeneration material with significant osteostimulation capacity.

Selective cleavage of human sex hormone-binding globulin by kallikrein-related peptidases and effects on androgen action in LNCaP prostate cancer cells

Stimulation of the androgen receptor via bioavailable androgens, including testosterone and testosterone metabolites, is a key driver of prostate development and the early stages of prostate cancer. Androgens are hydrophobic and as such require carrier proteins, including sex hormone-binding globulin (SHBG), to enable efficient distribution from sites of biosynthesis to target tissues. The similarly hydrophobic corticosteroids also require a carrier protein whose affinity for steroid is modulated by proteolysis. However, proteolytic mechanisms regulating the SHBG/androgen complex have not been reported. Here, we show that the cancer-associated serine proteases, kallikrein-related peptidase (KLK)4 and KLK14, bind strongly to SHBG in glutathione S-transferase interaction analyses. Further, we demonstrate that active KLK4 and KLK14 cleave human SHBG at unique sites and in an androgen-dependent manner. KLK4 separated androgen-free SHBG into its two laminin G-like (LG) domains that were subsequently proteolytically stable even after prolonged digestion, whereas a catalytically equivalent amount of KLK14 reduced SHBG to small peptide fragments over the same period. Conversely, proteolysis of 5α-dihydrotestosterone (DHT)-bound SHBG was similar for both KLKs and left the steroid binding LG4 domain intact. Characterization of this proteolysis fragment by [(3)H]-labeled DHT binding assays revealed that it retained identical affinity for androgen compared with full-length SHBG (dissociation constant = 1.92 nM). Consistent with this, both full-length SHBG and SHBG-LG4 significantly increased DHT-mediated transcriptional activity of the androgen receptor compared with DHT delivered without carrier protein. Collectively, these data provide the first evidence that SHBG is a target for proteolysis and demonstrate that a stable fragment derived from proteolysis of steroid-bound SHBG retains binding function in vitro.

The cultivation of human retinal pigment epithelial cells on Bombyx mori silk fibroin

We have presently evaluated membranes prepared from Bombyx mori silk fibroin (BMSF), for their potential use as a prosthetic Bruch’s membrane and carrier substrate for human retinal pigment epithelial (RPE) cell transplantation. Porous BMSF membranes measuring 3 μm in thickness were prepared from aqueous solutions (3% w/v) containing poly(ethylene oxide) (0.09%). The permeability coefficient for membranes was between 3 and 9 × 10-5 cm/s by using Allura red or 70 kDa FITC-dextran respectively. Average pore size (± sd) was 4.9 ± 2.3 µm and 2.9 ± 1.5 µm for upper and lower membrane surfaces respectively. Optimal attachment of ARPE-19 cells to BMSF membrane was achieved by pre-coating with vitronectin (1 µg/mL). ARPE-19 cultures maintained in low serum on BMSF membranes for approximately 8 weeks, developed a cobble-stoned morphology accompanied by a cortical distribution of F-actin and ZO-1. Similar results were obtained using primary cultures of human RPE cells, but cultures took noticeably longer to establish on BMSF compared with tissue culture plastic. These findings encourage further studies of BMSF as a substrate for RPE cell transplantation.

The role of human factors when evaluating information accountability for eHealth systems

The availability of health information is rapidly increasing; its expansion and proliferation is inevitable. At the same time, breeding of health information silos is an unstoppable and relentless exercise. Information security and privacy concerns are therefore major barriers in the eHealth socio-eco system. We proposed Information Accountability as a measurable human factor that should eliminate and mitigate security concerns. Information accountability measures would be practicable and feasible if legislative requirements are also embedded. In this context, information accountability constitutes a key component for the development of effective information technology requirements for health information system. Our conceptual approach to measuring human factors related to information accountability in eHealth is presented in this paper with some limitations.

The influence of human rights on land rights and spatial information

This paper reviews the growing influence of human rights issues on land rights, administration, management and tenure. In the last few decades, attention focussed on integrating economic and environmental considerations to achieve sustainable land use. The World Trade Organisation began in 1995. As a condition of membership, nations undertook legislative programmes aimed at reducing price distortions and barriers to international trade. Reducing trade barriers has direct effects on agricultural production as a major land use. Similarly, as signatories to the 1992 Rio Declaration, nations undertook caring for and reporting on the state of the environment. However, quality of life is also an issue in deciding what is sustainable development. The Universal Declaration of Human Rights, proclaimed in 1948, provided a framework for a series of international human rights conventions. These conventions now influence national legislative programmes. The purpose of this paper is to review some of the implications of human rights on rights in land and the production and use of spatial information.

Towards a capabilities database to inform inclusive design : Experimental investigation of effective survey-based predictors of human-product interaction

A key issue in the field of inclusive design is the ability to provide designers with an understanding of people's range of capabilities. Since it is not feasible to assess product interactions with a large sample, this paper assesses a range of proxy measures of design-relevant capabilities. It describes a study that was conducted to identify which measures provide the best prediction of people's abilities to use a range of products. A detailed investigation with 100 respondents aged 50-80 years was undertaken to examine how they manage typical household products. Predictor variables included self-report and performance measures across a variety of capabilities (vision, hearing, dexterity and cognitive function), component activities used in product interactions (e.g. using a remote control, touch screen) and psychological characteristics (e.g. self-efficacy, confidence with using electronic devices). Results showed, as expected, a higher prevalence of visual, hearing, dexterity, cognitive and product interaction difficulties in the 65-80 age group. Regression analyses showed that, in addition to age, performance measures of vision (acuity, contrast sensitivity) and hearing (hearing threshold) and self-report and performance measures of component activities are strong predictors of successful product interactions. These findings will guide the choice of measures to be used in a subsequent national survey of design-relevant capabilities, which will lead to the creation of a capability database. This will be converted into a tool for designers to understand the implications of their design decisions, so that they can design products in a more inclusive way.

Delivery of dimethyloxallyl glycine in mesoporous bioactive glass scaffolds to improve angiogenesis and osteogenesis of human bone marrow stromal cells

Development of hypoxia-mimicking bone tissue engineering scaffolds is of great importance in stimulating angiogenesis for bone regeneration. Dimethyloxallyl glycine (DMOG) is a cell-permeable, competitive inhibitor of hypoxia-inducible factor prolyl hydroxylase (HIF-PH), which can stabilize hypoxia-inducible factor 1α (HIF-1α) expression. The aim of this study was to develop hypoxia-mimicking scaffolds by delivering DMOG in mesoporous bioactive glass (MBG) scaffolds and to investigate whether the delivery of DMOG could induce a hypoxic microenvironment for human bone marrow stromal cells (hBMSC). MBG scaffolds with varied mesoporous structures (e.g. surface area and mesopore volume) were prepared by controlling the contents of mesopore-template agent. The composition, large-pore microstructure and mesoporous properties of MBG scaffolds were characterized. The effect of mesoporous properties on the loading and release of DMOG in MBG scaffolds was investigated. The effects of DMOG delivery on the cell morphology, cell viability, HIF-1α stabilization, vascular endothelial growth factor (VEGF) secretion and bone-related gene expression (alkaline phosphatase, ALP; osteocalcin, OCN; and osteopontin, OPN) of hBMSC in MBG scaffolds were systematically investigated. The results showed that the loading and release of DMOG in MBG scaffolds can be efficiently controlled by regulating their mesoporous properties via the addition of different contents of mesopore-template agent. DMOG delivery in MBG scaffolds had no cytotoxic effect on the viability of hBMSC. DMOG delivery significantly induced HIF-1α stabilization, VEGF secretion and bone-related gene expression of hBMSC in MBG scaffolds in which DMOG counteracted the effect of HIF-PH and stabilized HIF-1α expression under normoxic condition. Furthermore, it was found that MBG scaffolds with slow DMOG release significantly enhanced the expression of bone-related genes more than those with instant DMOG release. The results suggest that the controllable delivery of DMOG in MBG scaffolds can mimic a hypoxic microenvironment, which not only improves the angiogenic capacity of hBMSC, but also enhances their osteogenic differentiation.

The role of human capital in economic growth revisited

Benhabib and Spiegel (1994) examine the role of human capital in the development process empirically using a theory-driven specification rather than the standard production function approach. While they find evidence of a positive impact of human capital on income growth, their result is not robust to the inclusion of inequality as an additional covariate. Using an alternate dataset and different measures of inequality, we find robust support for the hypothesis that human capital matters even when we account for the adverse effect of income inequality on growth.

The effect of MC1R variants and sunscreen on the response of human melanocytes in vivo to ultraviolet radiation and implications for melanoma.

We conducted a clinical trial to compare the molecular and cellular responses of human melanocytes and keratinocytes in vivo to solar-simulated ultraviolet radiation (SSUVR) in 57 Caucasian participants grouped according to MC1R genotype. We found that, on average, the density of epidermal melanocytes 14 days after exposure to 2 minimal erythemal dose (MED) SSUVR was twofold higher than baseline (unirradiated) skin. However, the change in epidermal melanocyte counts among people carrying germline MC1R variants (97% increase) was significantly less than those with wild-type MC1R (164% increase; P = 0.01). We also found that sunscreen applied to the skin before exposure to 2 MED SSUVR completely blocked the effects of DNA damage, p53 induction, and cellular proliferation in both melanocytes and keratinocytes.

Regional chromosomal assignment of human renin gene to 1q12→qter and use in linkage studies in Charcot-Marie-Tooth disease

The gene for renin, previously mapped to human chromosome 1, was further localized to 1q12 → qter using human-mouse somatic cell hybrid DNAs. The renin DNA probe used (λ HR5) could detect a HindIII restriction fragment length polymorphism. When used in studies of 12 informative families, no linkage could be found between the renin and Charcot-Marie-Tooth disease. Furthermore, an association of any renin allele with hypertension was not apparent.

Immunosuppressive properties of mesenchymal stromal cell cultures derived from the limbus of human and rabbit corneas

Background aims Mesenchymal stromal cells (MSCs) cultivated from the corneal limbus (L-MSCs) provide a potential source of cells for corneal repair. In the present study, we investigated the immunosuppressive properties of human L-MSCs and putative rabbit L-MSCs to develop an allogeneic therapy and animal model of L-MSC transplantation. Methods MSC-like cultures were established from the limbal stroma of human and rabbit (New Zealand white) corneas using either serum-supplemented medium or a commercial serum-free MSC medium (MesenCult-XF Culture Kit; Stem Cell Technologies, Melbourne, Australia). L-MSC phenotype was examined by flow cytometry. The immunosuppressive properties of L-MSC cultures were assessed using mixed leukocyte reactions. L-MSC cultures were also tested for their ability to support colony formation by primary limbal epithelial (LE) cells. Results Human L-MSC cultures were typically CD34−, CD45− and HLA-DR− and CD73+, CD90+, CD105+ and HLA-ABC+. High levels (>80%) of CD146 expression were observed for L-MSC cultures grown in serum-supplemented medium but not cultures grown in MesenCult-XF (approximately 1%). Rabbit L-MSCs were approximately 95% positive for major histocompatibility complex class I and expressed lower levels of major histocompatibility complex class II (approximately 10%), CD45 (approximately 20%), CD105 (approximately 60%) and CD90 (<10%). Human L-MSCs and rabbit L-MSCs suppressed human T-cell proliferation by up to 75%. Conversely, L-MSCs from either species stimulated a 2-fold to 3-fold increase in LE cell colony formation. Conclusions L-MSCs display immunosuppressive qualities in addition to their established non-immunogenic profile and stimulate LE cell growth in vitro across species boundaries. These results support the potential use of allogeneic L-MSCs in the treatment of corneal disorders and suggest that the rabbit would provide a useful pre-clinical model.

Time course-dependent changes in the transcriptome of human skeletal muscle during recovery from endurance exercise: From inflammation to adaptive remodeling

Re-programming of gene expression is fundamental for skeletal muscle adaptations in response to endurance exercise. This study investigated the time-course dependent changes in the muscular transcriptome following an endurance exercise trial consisting of 1 h of intense cycling immediately followed by 1 h of intense running. Skeletal muscle samples were taken at baseline, 3 h, 48 h, and 96 h post-exercise from eight healthy, endurance-trained, male individuals. RNA was extracted from muscle. Differential gene expression was evaluated using Illumina microarrays and validated with qPCR. Gene set enrichment analysis identified enriched molecular signatures chosen from the Molecular Signatures Database. Three h post-exercise, 102 gene sets were up-regulated [family wise error rate (FWER), P < 0.05]; including groups of genes related with leukocyte migration, immune and chaperone activation, and cyclic AMP responsive element binding protein (CREB) 1-signaling. Forty-eight h post-exercise, among 19 enriched gene sets (FWER, P < 0.05), two gene sets related to actin cytoskeleton remodeling were up-regulated. Ninety-six h post-exercise, 83 gene sets were enriched (FWER, P < 0.05), 80 of which were up-regulated; including gene groups related to chemokine signaling, cell stress management, and extracellular matrix remodeling. These data provide comprehensive insights into the molecular pathways involved in acute stress, recovery, and adaptive muscular responses to endurance exercise. The novel 96 h post-exercise transcriptome indicates substantial transcriptional activity, potentially associated with the prolonged presence of leukocytes in the muscles. This suggests that muscular recovery, from a transcriptional perspective, is incomplete 96 h after endurance exercise involving muscle damage.

An extracellular signal-regulated kinase 2 survival pathway mediates resistance of human mesothelioma cells to asbestos-induced injury

We hypothesized that normal human mesothelial cells acquire resistance to asbestos-induced toxicity via induction of one or more epidermal growth factor receptor (EGFR) - linked survival pathways (phosphoinositol-3-kinase/AKT/ mammalian target of rapamycin and extracellular signal - regulated kinase [ERK] 1/2) during simian virus 40 (SV40) transformation and carcinogenesis. Both isolated HKNM-2 mesothelial cells and a telomerase-immortalized mesothelial line (LP9/TERT-1) were more sensitive to crocidolite asbestos toxicity than an SV40 Tag-immortalized mesothelial line (MET5A) and malignant mesothelioma cell lines (HMESO and PPM Mill). Whereas increases in phosphorylation of AKT (pAKT) were observed in MET5A cells in response to asbestos, LP9/TERT-1 cells exhibited dose-related decreases in pAKT levels. Pretreatment with an EGFR phosphorylation or mitogen-activated protein kinase kinase 1/2 inhibitor abrogated asbestos-induced phosphorylated ERK (pERK) 1/2 levels in both LP9/TERT-1 and MET5A cells as well as increases in pAKT levels in MET5A cells. Transient transfection of small interfering RNAs targeting ERK1, ERK2, or AKT revealed that ERK1/2 pathways were involved in cell death by asbestos in both cell lines. Asbestos-resistant HMESO or PPM Mill cells with high endogenous levels of ERKs or AKT did not show dose-responsive increases in pERK1/ERK1, pERK2/ERK2, or pAKT/AKT levels by asbestos. However, small hairpin ERK2 stable cell lines created from both malignant mesothelioma lines were more sensitive to asbestos toxicity than shERK1 and shControl lines, and exhibited unique, tumor-specific changes in endogenous cell death - related gene expression. Our results suggest that EGFR phosphorylation is causally linkedto pERK and pAKT activation by asbestos in normal and SV40 Tag - immortalized human mesothelial cells. They also indicate that ERK2 plays a role in modulating asbestos toxicity by regulating genes critical to cell injury and survival that are differentially expressed in human mesotheliomas.

Robust recognition of human behaviour in challenging environments

Novel techniques have been developed for the automatic recognition of human behaviour in challenging environments using information from visual and infra-red camera feeds. The techniques have been applied to two interesting scenarios: Recognise drivers' speech using lip movements and recognising audience behaviour, while watching a movie, using facial features and body movements. Outcome of the research in these two areas will be useful in the improving the performance of voice recognition in automobiles for voice based control and for obtaining accurate movie interest ratings based on live audience response analysis.