Sphingosine-1-phosphate mediates proliferation maintaining the multipotency of human adult bone marrow and adipose tissue-derived stem cells

High renewal and maintenance of multipotency of human adult stem cells (hSCs), are a prerequisite for experimental analysis as well as for potential clinical usages. The most widely used strategy for hSC culture and proliferation is using serum. However, serum is poorly defined and has a considerable degree of inter-batch variation, which makes it difficult for large-scale mesenchymal stem cells (MSCs) expansion in homogeneous culture conditions. Moreover, it is often observed that cells grown in serum-containing media spontaneously differentiate into unknown and/or undesired phenotypes. Another way of maintaining hSC development is using cytokines and/or tissue-specific growth factors; this is a very expensive approach and can lead to early unwanted differentiation. In order to circumvent these issues, we investigated the role of sphingosine-1-phosphate (S1P), in the growth and multipotency maintenance of human bone marrow and adipose tissue-derived MSCs. We show that S1P induces growth, and in combination with reduced serum, or with the growth factors FGF and platelet-derived growth factor-AB, S1P has an enhancing effect on growth. We also show that the MSCs cultured in S1P-supplemented media are able to maintain their differentiation potential for at least as long as that for cells grown in the usual serum-containing media. This is shown by the ability of cells grown in S1P-containing media to be able to undergo osteogenic as well as adipogenic differentiation. This is of interest, since S1P is a relatively inexpensive natural product, which can be obtained in homogeneous high-purity batches: this will minimize costs and potentially reduce the unwanted side effects observed with serum. Taken together, S1P is able to induce proliferation while maintaining the multipotency of different human stem cells, suggesting a potential for S1P in developing serum-free or serum-reduced defined medium for adult stem cell cultures.

In vitro cultivation of adult human chondrocytes : importance of culture system, oxygen and zonal differences

Articular cartilage exhibits limited intrinsic regenerative capacity and focal tissue defects can lead to the development of osteoarthritis (OA), a painful and debilitating loss of cartilage tissue. In Australia, 1.4 million people are affected by OA and its prevalence is increasing in line with current demographics. As treatment options are limited, new therapeutic approaches are being investigated including biological resurfacing of joints with tissue-engineered cartilage. Despite some progress in the field, major challenges remain to be addressed for large scale clinical success. For example, large numbers of chondrogenic cells are required for cartilage formation, but chondrocytes lose their chondrogenic phenotype (dedifferentiate) during in vitro propagation. Additionally, the zonal organization of articular cartilage is critical for normal cartilage function, but development of zonal structure has been largely neglected in cartilage repair strategies. Therefore, we hypothesised that culture conditions for freshly isolated human articular chondrocytes from non-OA and OA sources can be improved by employing microcarrier cultures and a reduced oxygen environment and that oxygen is a critical factor in the maintenance of the zonal chondrocyte phenotype. Microcarriers have successfully been used to cultivate bovine chondrocytes, and offer a potential alternative for clinical expansion of human chondrocytes. We hypothesised that improved yields can be achieved by propagating human chondrocytes on microcarriers. We found that cells on microcarriers acquired a flattened, polygonal morphology and initially proliferated faster than monolayercultivated cells. However, microcarrier cultivation over four weeks did not improve growth rates or the chondrogenic potential of non-OA and OA human articular chondrocytes over conventional monolayer cultivation. Based on these observations, we aimed to optimise culture conditions by modifying oxygen tension, to more closely reflect the in vivo environment. We found that propagation at 5% oxygen tension (moderate hypoxia) did not improve proliferation or redifferentiation capacity of human osteoarthritic chondrocytes. Moderate hypoxia increased the expression of chondrogenic markers during redifferentiation. However, osteoarthritic chondrocytes cultivated on microcarriers exhibited lower expression levels of chondrogenic surface marker proteins and had at best equivalent redifferentiation capacities compared to monolayer-cultured cells. This suggests that monolayer culture with multiple passaging potentially selects for a subpopulation of cells with higher differentiation capacity, which are otherwise rare in osteoarthritic, aged cartilage. However, fibroblastic proteins were found to be highly expressed in all cultures of human osteoarthritic chondrocytes indicating the presence of a high proportion of dedifferentiated, senescent cells with a chondrocytic phenotype that was not rescued by moderate hypoxia. The different zones of cartilage support chondrocyte subpopulations, which exhibit characteristic protein expression and experience varying oxygen tensions. We, therefore, hypothesised that oxygen tension affects the zonal marker expression of human articular chondrocytes isolated from the different cartilage layers. We found that zonal chondrocytes maintained these phenotypic differences during in vitro cultivation. Low oxygen environments favoured the expression of the zonal marker proteoglycan 4 in superficial cells, most likely through the promotion of chondrogenesis. The putative zonal markers clusterin and cartilage intermediate layer protein were found to be expressed by all subpopulations of human osteoarthritic chondrocytes ex vivo and, thus, may not be reliable predictors of in vitro stratification using these clinically relevant cells. The findings in this thesis underline the importance of considering low oxygen conditions and zonal stratification when creating native-like cartilaginous constructs. We have not yet found the right cues to successfully cultivate clinically-relevant human osteoarthritic chondrocytes in vitro. A more thorough understanding of chondrocyte biology and the processes of chondrogenesis are required to ensure the clinical success of cartilage tissue engineering.

Empowerment and role stress in the human interface between the firm and its markets

Frontline employees constitute one of the key interfaces that service organisations have with their markets. Many strategies to enhance the ability of these employees to satisfy the needs of customers have been proposed. Amongst these, empowering employees has been suggested to enhance the customer orientation of the firm and consequently its effectiveness in serving the market. However, the impact of empowerment in service organisations remains somewhat contentious. This paper examines the role of empowerments an organisational service strategy and identifies its consequences for role stress, job satisfaction and the willingness of service employees to serve their customers.

Liking vs. wanting food : importance for human appetite control and weight regulation

Current train of thought in appetite research is favouring an interest in non-homeostatic or hedonic (reward) mechanisms in relation to overconsumption and energy balance. This tendency is supported by advances in neurobiology that precede the emergence of a new conceptual approach to reward where affect and motivation (liking and wanting) can be seen as the major force in guiding human eating behaviour. In this review, current progress in applying processes of liking and wanting to the study of human appetite are examined by discussing the following issues: How can these concepts be operationalised for use in human research to reflect the neural mechanisms by which they may be influenced? Do liking and wanting operate independently to produce functionally significant changes in behaviour? Can liking and wanting be truly experimentally separated or will an expression of one inevitably contain elements of the other? The review contains a re-examination of selected human appetite research before exploring more recent methodological approaches to the study of liking and wanting in appetite control. In addition, some theoretical developments are described in four diverse models that may enhance current understanding of the role of these processes in guiding ingestive behaviour. Finally, the implications of a dual process modulation of food reward for weight gain and obesity are discussed. The review concludes that processes of liking and wanting are likely to have independent roles in characterising susceptibility to weight gain. Further research into the dissociation of liking and wanting through implicit and explicit levels of processing would help to disclose the relative importance of these components of reward for appetite control and weight regulation.

Prandial subcutaneous injections of glucagon-like peptide-1 cause weight loss in obese human subjects

Recombinant glucagon-like peptide-1 (7–36)amide (rGLP-1) was recently shown to cause significant weight loss in type 2 diabetics when administered for 6 weeks as a continuous subcutaneous infusion. The mechanisms responsible for the weight loss are not clarified. In the present study, rGLP-1 was given for 5d by prandial subcutaneous injections (PSI) (76nmol 30min before meals, four times daily; a total of 302·4nmol/24h) or by continuous subcutaneous infusion (CSI) (12·7nmol/h; a total of 304·8nmol/24h). This was performed in nineteen healthy obese subjects (mean age 44·2 (sem 2·5) years; BMI 39·0 (sem 1·2)kg/m2) in a prospective randomised, double-blind, placebo-controlled, cross-over study. Compared with the placebo, rGLP-1 administered as PSI and by CSI generated a 15% reduction in mean food intake per meal (P=0·02) after 5d treatment. A weight loss of 0·55 (sem 0·2) kg (P<0·05) was registered after 5d with PSI of rGLP-1. Gastric emptying rate was reduced during both PSI (P<0·001) and CSI (P<0·05) treatment, but more rapidly and to a greater extent with PSI of rGLP-1. To conclude, a 5d treatment of rGLP-1 at high doses by PSI, but not CSI, promptly slowed gastric emptying as a probable mechanism of action of increased satiety, decreased hunger and, hence, reduced food intake with an ensuing weight loss.

Evaluating sewage associated JCV and BKV polyomaviruses for sourcing human fecal pollution in a coastal river in Southeast Queensland Australia

In this study, the host-sensitivity and -specificity of JCV and BKV polyomaviruses were evaluated by testing wastewater/fecal samples from nine host groups in Southeast Queensland, Australia. The JCV and BKV polyomaviruses were detected in 48 human wastewater samples collected from the primary and secondary effluent suggesting high sensitivity of these viruses in human wastewater. Of the 81 animal wastewater/fecal samples tested, 80 were PCR negative for this marker. Only one sample from pig wastewater was positive. Nonetheless, the overall host-specificity of these viruses to differentiate between human and animal wastewater/fecal samples was 0.99. To our knowledge, this is the first study in Australia that reports the high specificity of JCV and BKV polyomaviruses. To evaluate the field application of these viruses to detect human fecal pollution, 20 environmental samples were collected from a coastal river. Of the 20 samples tested, 15% and 70% samples exceeded the regulatory guidelines for E. coli and enterococci levels for marine waters. In all, 5 (25%) samples were PCR positive for JCV and BKV indicated the presence of human fecal pollution in the studied river. The results suggest that JCV and BKV detection using PCR could be a useful tool for the identification of human sourced fecal pollution in coastal waters.

The highs (B1H) and lows (B1L) of human heart B1-adrenoceptors (AR)

The 1AR has two binding sites which can be activated to cause cardiostimulation. The first, termed, 1HAR (high affinity site of 1AR) is activated by noradrenaline and adrenaline and is blocked by relatively low concentrations of β-blockers including carvedilol (Kaumann and Molenaar, 2008). The other, termed, 1LAR (low affinity site of 1AR) has lower affinity for noradrenaline and adrenaline and is activated by some β-blockers including CGP12177 and pindolol, at higher concentrations than those required to block the receptor (Kaumann and Molenaar, 2008). (-)-CGP12177 is a non-conventional partial agonist that causes modest and transient increases of contractile force in human atrial trabeculae (Kaumann and Molenaar, 2008). These effects are markedly increased and maintained by inhibition of phosphodiesterase PDE3. The stimulant effects of (-)-CGP12177 at human β1ARs was verified with recombinant receptors (Kaumann and Molenaar, 2008). However, in a recent report it was proposed that the positive inotropic effects of CGP12177 are mediated through 3ARs in human right atrium (Skeberdis et al 2008). This proposal was not consistent with the lack of blockade of (-)-CGP12177 inotropic effects or increases in L-type Ca2+ current (ICa-L ) by the β3AR blocker 1 μM LY748,337 (Christ et al, 2010). On the otherhand, (-)-CGP12177 increases in inotropic effects and ICa-L were blocked by (-)-bupranolol 1-10 μM (Christ et al, 2010). Chronic infusion of (-)-CGP 12177 (10 mg/Kg/24 hours) for four weeks in an aortic constriction mouse model of heart failure caused an increase in left ventricular wall thickness, fibrosis and inflammation-related left ventricular gene expression levels. Christ T et al (2010) Br J Pharmacol, In press Kaumann A and Molenaar P (2008) Pharmacol Ther 118, 303-336 Skeberdis VA et al (2008) J Clin Invest, 118, 3219-3227

Description and evaluation of a Newton-based electronic appetite rating system for temporal tracking of appetite in human subjects

This study assessed the reliability and validity of a palm-top-based electronic appetite rating system (EARS) in relation to the traditional paper and pen method. Twenty healthy subjects [10 male (M) and 10 female (F)] — mean age M=31 years (S.D.=8), F=27 years (S.D.=5); mean BMI M=24 (S.D.=2), F=21 (S.D.=5) — participated in a 4-day protocol. Measurements were made on days 1 and 4. Subjects were given paper and an EARS to log hourly subjective motivation to eat during waking hours. Food intake and meal times were fixed. Subjects were given a maintenance diet (comprising 40% fat, 47% carbohydrate and 13% protein by energy) calculated at 1.6×Resting Metabolic Rate (RMR), as three isoenergetic meals. Bland and Altman's test for bias between two measurement techniques found significant differences between EARS and paper and pen for two of eight responses (hunger and fullness). Regression analysis confirmed that there were no day, sex or order effects between ratings obtained using either technique. For 15 subjects, there was no significant difference between results, with a linear relationship between the two methods that explained most of the variance (r2 ranged from 62.6 to 98.6). The slope for all subjects was less than 1, which was partly explained by a tendency for bias at the extreme end of results on the EARS technique. These data suggest that the EARS is a useful and reliable technique for real-time data collection in appetite research but that it should not be used interchangeably with paper and pen techniques.

The use of visual analogue scales to assess motivation to eat in human subjects: a review of their reliability and validity with an evaluation of new hand-held computerized stsytems for temporal tracking of appetite ratings.

This present paper reviews the reliability and validity of visual analogue scales (VAS) in terms of (1) their ability to predict feeding behaviour, (2) their sensitivity to experimental manipulations, and (3) their reproducibility. VAS correlate with, but do not reliably predict, energy intake to the extent that they could be used as a proxy of energy intake. They do predict meal initiation in subjects eating their normal diets in their normal environment. Under laboratory conditions, subjectively rated motivation to eat using VAS is sensitive to experimental manipulations and has been found to be reproducible in relation to those experimental regimens. Other work has found them not to be reproducible in relation to repeated protocols. On balance, it would appear, in as much as it is possible to quantify, that VAS exhibit a good degree of within-subject reliability and validity in that they predict with reasonable certainty, meal initiation and amount eaten, and are sensitive to experimental manipulations. This reliability and validity appears more pronounced under the controlled (but more arti®cial) conditions of the laboratory where the signal : noise ratio in experiments appears to be elevated relative to real life. It appears that VAS are best used in within-subject, repeated-measures designs where the effect of different treatments can be compared under similar circumstances. They are best used in conjunction with other measures (e.g. feeding behaviour, changes in plasma metabolites) rather than as proxies for these variables. New hand-held electronic appetite rating systems (EARS) have been developed to increase reliability of data capture and decrease investigator workload. Recent studies have compared these with traditional pen and paper (P&P) VAS. The EARS have been found to be sensitive to experimental manipulations and reproducible relative to P&P. However, subjects appear to exhibit a signi®cantly more constrained use of the scale when using the EARS relative to the P&P. For this reason it is recommended that the two techniques are not used interchangeably

To err (on the road) is human? An on-road study of driver errors

Human error, its causes and consequences, and the ways in which it can be prevented, remain of great interest to road safety practitioners. This paper presents the findings derived from an on-road study of driver errors in which 25 participants drove a pre-determined route using MUARC's On-Road Test Vehicle (ORTeV). In-vehicle observers recorded the different errors made, and a range of other data was collected, including driver verbal protocols, forward, cockpit and driver video, and vehicle data (speed, braking, steering wheel angle, lane tracking etc). Participants also completed a post trial cognitive task analysis interview. The drivers tested made a range of different errors, with speeding violations, both intentional and unintentional, being the most common. Further more detailed analysis of a sub-set of specific error types indicates that driver errors have various causes, including failures in the wider road 'system' such as poor roadway design, infrastructure failures and unclear road rules. In closing, a range of potential error prevention strategies, including intelligent speed adaptation and road infrastructure design, are discussed.

Labour rights as human rights : workers' safety at work in Australian-based supply chains

The increase of buyer-driven supply chains, outsourcing and other forms of non-traditional employment has resulted in challenges for labour market regulation. One business model which has created substantial regulatory challenges is supply chains. The supply chain model involves retailers purchasing products from brand corporations who then outsource the manufacturing of the work to traders who contract with factories or outworkers who actually manufacture the clothing and textiles. This business model results in time and cost pressures being pushed down the supply chain which has resulted in sweatshops where workers systematically have their labour rights violated. Literally millions of workers work in dangerous workplaces where thousands are killed or permanently disabled every year. This thesis has analysed possible regulatory responses to provide workers a right to safety and health in supply chains which provide products for Australian retailers. This thesis will use a human rights standard to determine whether Australia is discharging its human rights obligations in its approach to combating domestic and foreign labour abuses. It is beyond this thesis to analyse Occupational Health and Safety (OHS) laws in every jurisdiction. Accordingly, this thesis will focus upon Australian domestic laws and laws in one of Australia’s major trading partners, the Peoples’ Republic of China (China). It is hypothesised that Australia is currently breaching its human rights obligations through failing to adequately regulate employees’ safety at work in Australian-based supply chains. To prove this hypothesis, this thesis will adopt a three- phase approach to analysing Australia’s regulatory responses. Phase 1 will identify the standard by which Australia’s regulatory approach to employees’ health and safety in supply chains can be judged. This phase will focus on analysing how workers’ rights to safety as a human right imposes a moral obligation on Australia to take reasonablely practicable steps regulate Australian-based supply chains. This will form a human rights standard against which Australia’s conduct can be judged. Phase 2 focuses upon the current regulatory environment. If existing regulatory vehicles adequately protect the health and safety of employees, then Australia will have discharged its obligations through simply maintaining the status quo. Australia currently regulates OHS through a combination of ‘hard law’ and ‘soft law’ regulatory vehicles. The first part of phase 2 analyses the effectiveness of traditional OHS laws in Australia and in China. The final part of phase 2 then analyses the effectiveness of the major soft law vehicle ‘Corporate Social Responsibility’ (CSR). The fact that employees are working in unsafe working conditions does not mean Australia is breaching its human rights obligations. Australia is only required to take reasonably practicable steps to ensure human rights are realized. Phase 3 identifies four regulatory vehicles to determine whether they would assist Australia in discharging its human rights obligations. Phase 3 then analyses whether Australia could unilaterally introduce supply chain regulation to regulate domestic and extraterritorial supply chains. Phase 3 also analyses three public international law regulatory vehicles. This chapter considers the ability of the United Nations Global Compact, the ILO’s Better Factory Project and a bilateral agreement to improve the detection and enforcement of workers’ right to safety and health.