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.

Morphologic and genomic characterisation of the koala Chlamydia pneumoniae strain

Chlamydia pneumoniae is a common human and animal pathogen associated with a wide range of upper and lower respiratory tract infections. In more recent years there has been increasing evidence to suggest a link between C. pneumoniae and chronic diseases in humans, including atherosclerosis, stroke and Alzheimer’s disease. C. pneumoniae human strains show little genetic variation, indicating that the human-derived strain originated from a common ancestor in the recent past. Despite extensive information on the genetics and morphology processes of the human strain, knowledge concerning many other hosts (including marsupials, amphibians, reptiles and equines) remains virtually unexplored. The koala (Phascolarctos cinereus) is a native Australian marsupial under threat due to habitat loss, predation and disease. Koalas are very susceptible to chlamydial infections, most commonly affecting the conjunctiva, urogenital tract and/or respiratory tract. To address this gap in the literature, the present study (i) provides a detailed description of the morphologic and genomic architecture of the C. pneumoniae koala (and human) strain, and shows that the koala strain is microscopically, developmentally and genetically distinct from the C. pneumoniae human strain, and (ii) examines the genetic relationship of geographically diverse C. pneumoniae isolates from human, marsupial, amphibian, reptilian and equine hosts, and identifies two distinct lineages that have arisen from animal-to-human cross species transmissions. Chapter One of this thesis explores the scientific problem and aims of this study, while Chapter Two provides a detailed literature review of the background in this field of work. Chapter Three, the first results chapter, describes the morphology and developmental stages of C. pneumoniae koala isolate LPCoLN, as revealed by fluorescence and transmission electron microscopy. The profile of this isolate, when cultured in HEp-2 human epithelial cells, was quite different to the human AR39 isolate. Koala LPCoLN inclusions were larger; the elementary bodies did not have the characteristic pear-shaped appearance, and the developmental cycle was completed within a shorter period of time (as confirmed by quantitative real-time PCR). These in vitro findings might reflect biological differences between koala LPCoLN and human AR39 in vivo. Chapter Four describes the complete genome sequence of the koala respiratory pathogen, C. pneumoniae LPCoLN. This is the first animal isolate of C. pneumoniae to be fully-sequenced. The genome sequence provides new insights into genomic ‘plasticity’ (organisation), evolution and biology of koala LPCoLN, relative to four complete C. pneumoniae human genomes (AR39, CWL029, J138 and TW183). Koala LPCoLN contains a plasmid that is not shared with any of the human isolates, there is evidence of gene loss in nucleotide salvage pathways, and there are 10 hot spot genomic regions of variation that were previously not identified in the C. pneumoniae human genomes. Sequence (partial-length) from a second, independent, wild koala isolate (EBB) at several gene loci confirmed that the koala LPCoLN isolate was representative of a koala C. pneumoniae strain. The combined sequence data provides evidence that the C. pneumoniae animal (koala LPCoLN) genome is ancestral to the C. pneumoniae human genomes and that human infections may have originated from zoonotic infections. Chapter Five examines key genome components of the five C. pneumoniae genomes in more detail. This analysis reveals genomic features that are shared by and/or contribute to the broad ecological adaptability and evolution of C. pneumoniae. This analysis resulted in the identification of 65 gene sequences for further analysis of intraspecific variation, and revealed some interesting differences, including fragmentation, truncation and gene decay (loss of redundant ancestral traits). This study provides valuable insights into metabolic diversity, adaptation and evolution of C. pneumoniae. Chapter Six utilises a subset of 23 target genes identified from the previous genomic comparisons and makes a significant contribution to our understanding of genetic variability among C. pneumoniae human (11) and animal (6 amphibian, 5 reptilian, 1 equine and 7 marsupial hosts) isolates. It has been shown that the animal isolates are genetically diverse, unlike the human isolates that are virtually clonal. More convincing evidence that C. pneumoniae originated in animals and recently (in the last few hundred thousand years) crossed host species to infect humans is provided in this study. It is proposed that two animal-to-human cross species events have occurred in the context of the results, one evident by the nearly clonal human genotype circulating in the world today, and the other by a more animal-like genotype apparent in Indigenous Australians. Taken together, these data indicate that the C. pneumoniae koala LPCoLN isolate has morphologic and genomic characteristics that are distinct from the human isolates. These differences may affect the survival and activity of the C. pneumoniae koala pathogen in its natural host, in vivo. This study, by utilising the genetic diversity of C. pneumoniae, identified new genetic markers for distinguishing human and animal isolates. However, not all C. pneumoniae isolates were genetically diverse; in fact, several isolates were highly conserved, if not identical in sequence (i.e. Australian marsupials) emphasising that at some stage in the evolution of this pathogen, there has been an adaptation/s to a particular host, providing some stability in the genome. The outcomes of this study by experimental and bioinformatic approaches have significantly enhanced our knowledge of the biology of this pathogen and will advance opportunities for the investigation of novel vaccine targets, antimicrobial therapy, or blocking of pathogenic pathways.

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.

Appetite sensations and satiety quotient : predictors of energy intake and weight loss

PURPOSE: The aim of this study was to further evaluate the validity and clinical meaningfulness of appetite sensations to predict overall energy intake as well as body weight loss. METHODS: Men (n=176) and women (n=139) involved in six weight loss studies were selected to participate in this study. Visual analogue scales were used to measure appetite sensations before and after a fixed test meal. Fasting appetite sensations, 1 h post-prandial area under the curve (AUC) and the satiety quotient (SQ) were used as predictors of energy intake and body weight loss. Two separate measures of energy intake were used: a buffet style ad libitum test lunch and a three-day self-report dietary record. RESULTS: One-hour post-prandial AUC for all appetite sensations represented the strongest predictors of ad libitum test lunch energy intake (p0.001). These associations were more consistent and pronounced for women than men. Only SQ for fullness was associated with ad libitum test lunch energy intake in women. Similar but weaker relationships were found between appetite sensations and the 3-day self-reported energy intake. Weight loss was associated with changes in appetite sensations (p0.01) and the best predictors of body weight loss were fasting desire to eat; hunger; and PFC (p0.01). CONCLUSIONS: These results demonstrate that appetite sensations are relatively useful predictors of spontaneous energy intake, free-living total energy intake and body weight loss. They also confirm that SQ for fullness predicts energy intake, at least in women.

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.

Interaction between disinhibition and restraint: Implications for body weight and eating disturbance

An increase in obesity is usually accompanied by an increase in eating disturbances. Susceptibility to these states may arise from different combinations of underlying traits: Three Factor Eating Questionnaire (TFEQ) Restraint and Disinhibition. Two studies were conducted to examine the interaction between these traits; one on-line study (n=351) and one laboratory-based study (n=120). Participants completed a battery of questionnaires and provided self-report measures of body weight and physical activity. A combination of high Disinhibition and high Restraint was associated with a problematic eating behaviour profile (EAT-26), and a higher rate of smoking and alcohol consumption. A combination of high Disinhibition and low Restraint was associated with a higher susceptibility to weight gain and a higher sedentary behaviour. These data show that different combinations of Disinhibition and Restraint are associated with distinct weight and behaviour outcomes.

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 effect of graded levels of exercise on energy intake and balance in free-living men, consuming their normal diet

Objective: To assess the effect of graded increases in exercised-induced energy expenditure (EE) on appetite, energy intake (EI), total daily EE and body weight in men living in their normal environment and consuming their usual diets. Design: Within-subject, repeated measures design. Six men (mean (s.d.) age 31.0 (5.0) y; weight 75.1 (15.96) kg; height 1.79 (0.10) m; body mass index (BMI) 23.3(2.4) kg/m2), were each studied three times during a 9 day protocol, corresponding to prescriptions of no exercise, (control) (Nex; 0 MJ/day), medium exercise level (Mex; ~1.6 MJ/day) and high exercise level (Hex; ~3.2 MJ/day). On days 1-2 subjects were given a medium fat (MF) maintenance diet (1.6 ´ resting metabolic rate (RMR)). Measurements: On days 3-9 subjects self-recorded dietary intake using a food diary and self-weighed intake. EE was assessed by continual heart rate monitoring, using the modified FLEX method. Subjects' HR (heart rate) was individually calibrated against submaximal VO2 during incremental exercise tests at the beginning and end of each 9 day study period. Respiratory exchange was measured by indirect calorimetry. Subjects completed hourly hunger ratings during waking hours to record subjective sensations of hunger and appetite. Body weight was measured daily. Results: EE amounted to 11.7, 12.9 and 16.8 MJ/day (F(2,10)=48.26; P<0.001 (s.e.d=0.55)) on the Nex, Mex and Hex treatments, respectively. The corresponding values for EI were 11.6, 11.8 and 11.8 MJ/day (F(2,10)=0.10; P=0.910 (s.e.d.=0.10)), respectively. There were no treatment effects on hunger, appetite or body weight, but there was evidence of weight loss on the Hex treatment. Conclusion: Increasing EE did not lead to compensation of EI over 7 days. However, total daily EE tended to decrease over time on the two exercise treatments. Lean men appear able to tolerate a considerable negative energy balance, induced by exercise, over 7 days without invoking compensatory increases in EI.

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

Ghrelin and obestatin concentrations during puberty: Relationships with adiposity, nutrition and physical activity

Ghrelin and obestatin are two peptides associated with appetite control and the regulation of energy balance in adults. It is intuitive that they have an important role in growth and development during puberty. Therefore, it is acknowledged that these peptides, in addition to others, form part of the substrate underlying energy homeostasis which in turn will contribute to body weight regulation and could explain changes in energy balance during puberty. Both peptides originate from the stomach; hence, it is intuitive that they are involved in generating signals from tissue stores which influence food intake. This could be manifested via alterations in the drive to eat (i.e. hunger), eating behaviors and appetite regulation. Furthermore, there is some evidence that these peptides might also be associated with physical activity behaviors and metabolism. Anecdotally, children and adolescents experience behavioral and metabolic changes during growth and development which will be associated with physiological changes.

Physical inactivity, appetite regulation and obesity

In a consumerist society obsessed with body image and thinness, obesity levels have reached an all-time high. This multi-faceted book written by a range of experts, explores the social, cultural, clinical and psychological factors that lie behind the Obesity Epidemic . It is required reading for the many healthcare professionals dealing with the effects of obesity and for anyone who wants to know more about the causes of weight gain and the best ways of dealing with it. Fat Matters covers a range of issues from sociology through medicine to technology. This is not a book for the highly specialised expert. Rather it is a book that shows the diversity of approaches to the phenomenon of obesity, tailored to the reader who wants to be up-to-date and well-informed on a subject that is possibly as frequently discussed and as misunderstood as the weather.

Children, Obesity and Exercise: Prevention, Treatment and Management of Childhood and Adolescent Obesity

Throughout the developed world there is an increasing prevalence of childhood obesity. Because of this increase, and awareness of the risks to long term health that childhood obesity presents, the phenomena is now described by many as a global epidemic. Children, Obesity and Exercise provides sport, exercise and medicine students and professionals with an accessible and practical guide to understanding and managing childhood and adolescent obesity. It covers: overweight, obesity and body composition; physical activity, growth and development; psycho-social aspects of childhood obesity; physical activity behaviours; eating behaviours; measuring childrens behaviour; interventions for prevention and management of childhood obesity. Children, Obesity and Exercise addresses the need for authoritative advice and innovative approaches to the prevention and management of this chronic problem.