Macular Thickness as a Potential Biomarker of Mild Alzheimer's Disease

Although several postmortem findings in the retina of patients with Alzheimer's disease (AD) are available, new biomarkers for early diagnosis and follow-up of AD are still lacking. It has been postulated that the defects in the retinal nerve fiber layer (RNFL) may be the earliest sign of AD, even before damage to the hippocampal region that affects memory. This fact may reflect retinal neuronal-ganglion cell death and axonal loss in the optic nerve in addition to aging.

Procalcitonin Biomarker Kinetics to Predict Multiorgan Dysfunction Syndrome in Children With Sepsis and Systemic Inflammatory Response Syndrome

Background: Procalcitonin (PCT) kinetics is a good prognosis marker in infectious diseases, but few studies of children sepsis have been performed. Objectives: The aim of our study was to examine kinetics of procalcitonin, to evaluate its relationship with severity and to analyze its usefulness in the prediction of multiorgan dysfunction syndrome (MODS). Patients and Methods: Prospective observational study in an 8-bed pediatric intensive care unit of a university hospital. Sixty-two children aged 0-19 years with systemic inflammatory response syndrome or septic states. The degree of severity was evaluated according pediatric logistic organ dysfunction (PELOD) score. Blood tests to determine levels of PCT were taken if the patients had the criteria of systemic inflammatory response syndrome or sepsis. The serum to determine levels of PCT in control group has been taken from patients undergoing elective surgery. Results: Higher values of PCT were identified in patients with PELOD score 12 and more compared to those with PELOD < 12 (P = 0.016). Similarly, higher PCT values were found in patients who developed MODS in contrast to those without MODS (P = 0.011). According to ROC analysis cut-off value of 4.05 ng/mL was found to best discriminate patients with PELOD < 12 and PELOD ≥ 12 with AUC = 0.675 (P = 0.035). Effect of procalcitonin levels on mortality was not demonstrated. Conclusions: Levels of procalcitonin from day 1 to day 5 are related to the severity and multiorgan dysfunction syndrome in children.

Climate variability of the last 1000 years in the NW Pacific: high resolution, multi-biomarker records from Lake Toyoni

The East Asian Monsoon (EAM) is an active component of the global climate system and has a profound social and economic impact in East Asia and its surrounding countries. Its impact on regional hydrological processes may influence society through industrial water supplies, food productivity and energy use. In order to predict future rates of climate change, reliable and accurate reconstructions of regional temperature and rainfall are required from all over the world to test climate models and better predict future climate variability. Hokkaido is a region which has limited palaeo-climate data and is sensitive to climate change. Instrumental data show that the climate in Hokkaido is influenced by the East Asian Monsoon (EAM), however, instrumental data is limited to the past ~150 years. Therefore down-core climate reconstructions, prior to instrumental records, are required to provide a better understanding of the long-term behaviour of the climate drivers (e.g. the EAM, Westerlies, and teleconnections) in this region. The present study develops multi-proxy reconstructions to determine past climatic and hydrologic variability in Japan over the past 1000 years and aid in understanding the effects of the EAM and the Westerlies independently and interactively. A 250-cm long sediment core from Lake Toyoni, Hokkaido was retrieved to investigate terrestrial and aquatic input, lake temperature and hydrological changes over the past 1000-years within Lake Toyoni and its catchment using X-Ray Fluorescence (XRF) data, alkenone palaeothermometry, the molecular and hydrogen isotopic composition of higher plant waxes (δD(HPW)). Here, we conducted the first survey for alkenone biomarkers in eight lakes in the Hokkaido, Japan. We detected the occurrence of alkenones within the sediments of Lake Toyoni. We present the first lacustrine alkenone record from Japan, including genetic analysis of the alkenone producer. C37 alkenone concentrations in surface sediments are 18µg C37 g−1 of dry sediment and the dominant alkenone is C37:4. 18S rDNA analysis revealed the presence of a single alkenone producer in Lake Toyoni and thus a single calibration is used for reconstructing lake temperature based on alkenone unsaturation patterns. Temperature reconstructions over the past 1000 years suggest that lake water temperatures varies between 8 and 19°C which is in line with water temperature changes observed in the modern Lake Toyoni. The alkenone-based temperature reconstruction provides evidence for the variability of the EAM over the past 1000 years. The δD(HPW) suggest that the large fluctuations (∼40‰) represent changes in temperature and source precipitation in this region, which is ultimately controlled by the EAM system and therefore a proxy for the EAM system. In order to complement the biomarker reconstructions, the XRF data strengthen the lake temperature and hydrological reconstructions by providing information on past productivity, which is controlled by the East Asian Summer monsoon (EASM) and wind input into Lake Toyoni, which is controlled by the East Asian Winter Monsoon (EAWM) and the Westerlies. By combining the data generated from XRF, alkenone palaeothermometry and the δD(HPW) reconstructions, we provide valuable information on the EAM and the Westerlies, including; the timing of intensification and weakening, the teleconnections influencing them and the relationship between them. During the Medieval Warm Period (MWP), we find that the EASM dominated and the EAWM was suppressed, whereas, during the Little Ice Age (LIA), the influence of the EAWM dominated with time periods of increased EASM and Westerlies intensification. The El Niño Southern Oscillation (ENSO) significantly influenced the EAM; a strong EASM occurred during El Niño conditions and a strong EAWM occurred during La Niña. The North Atlantic Oscillation, on the other hand, was a key driver of the Westerlies intensification; strengthening of the Westerlies during a positive NAO phase and weakening of the Westerlies during a negative NAO phase. A key finding from this study is that our data support an anti-phase relationship between the EASM and the EAWM (e.g. the intensification of the EASM and weakening of the EAWM and vice versa) and that the EAWM and the Westerlies vary independently from each other, rather than coincide as previously suggested in other studies.

Biomarker evaluation and toxicological study of essential oils of Foeniculum vulgare and Calamintha nepeta

The aim of this study was to evaluate the toxicological properties of EOs of F. vulgare and C. nepeta, widespread in Mediterranean agrosilvopastoral systems and often used as food condiments in Alentejo. EOs were obtained from aerial part of plants by hydrodistillation and chemical composition was evaluated by GC-FID. Toxicity of essential oils was evaluated by the estimation of LC50 in brine shrimp and LD50 in mice. Oral toxicity assays were performed in mice. Histological analyses and quantification of biomarkers aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma glutamyltransferase (γ-GT), bilirubin, creatinine and urea were performed for monitoring liver and kidney functions. the EOs of C. nepeta and F. vulgare showed very low toxicity suggesting their potential use as food supplement. Additionally, our studies point out the importance of the integration of C. nepeta and F. vugare in silvopastoral agroforestry systems, contributing to the animal health and profitability of livestock.

Biomarker analysis in soils of the Amazon rainforest after vegetation fire.

The objective of this study was to determine the origin of organic matter incorporated in Amazon forest soils subjected to vegetation fire by analyzing the aliphatic biomarkers (n-alkanes) present in lipid extracts of soil samples.

Increased cardio-metabolic risk is associated with increased TV viewing time

Purpose: Television viewing time, independent of leisure-time physical activity, has cross-sectional relationships with the metabolic syndrome and its individual components. We examined whether baseline and five-year changes in self-reported television viewing time are associated with changes in continuous biomarkers of cardio-metabolic risk (waist circumference, triglycerides, high density lipoprotein cholesterol, systolic and diastolic blood pressure, fasting plasma glucose; and a clustered cardio-metabolic risk score) in Australian adults. Methods: AusDiab is a prospective, population-based cohort study with biological, behavioral, and demographic measures collected in 1999–2000 and 2004–2005. Non-institutionalized adults aged ≥ 25 years were measured at baseline (11,247; 55% of those completing an initial household interview); 6,400 took part in the five-year follow-up biomedical examination, and 3,846 met the inclusion criteria for this analysis. Multiple linear regression analysis was used and unstandardized B coefficients (95% CI) are provided. Results: Baseline television viewing time (10 hours/week unit) was not significantly associated with change in any of the biomarkers of cardio-metabolic risk. Increases in television viewing time over five years (10 hours/week unit) were associated with increases in: waist circumference (cm) (men: 0.43 (0.08, 0.78), P = 0.02; women: 0.68 (0.30, 1.05), P <0.001), diastolic blood pressure (mmHg) (women: 0.47 (0.02, 0.92), P = 0.04), and the clustered cardio-metabolic risk score (women: 0.03 (0.01, 0.05), P = 0.007). These associations were independent of baseline television viewing time and baseline and change in physical activity and other potential confounders. Conclusion: These findings indicate that an increase in television viewing time is associated with adverse cardio-metabolic biomarker changes. Further prospective studies using objective measures of several sedentary behaviors are required to confirm causality of the associations found.

Crosstalk between developmental and tumour-specific signalling pathways : kallikrein-related serine peptidases and nodal in prostrate cancer

Prostate cancer is an important male health issue. The strategies used to diagnose and treat prostate cancer underscore the cell and molecular interactions that promote disease progression. Prostate cancer is histologically defined by increasingly undifferentiated tumour cells and therapeutically targeted by androgen ablation. Even as the normal glandular architecture of the adult prostate is lost, prostate cancer cells remain dependent on the androgen receptor (AR) for growth and survival. This project focused on androgen-regulated gene expression, altered cellular differentiation, and the nexus between these two concepts. The AR controls prostate development, homeostasis and cancer progression by regulating the expression of downstream genes. Kallikrein-related serine peptidases are prominent transcriptional targets of AR in the adult prostate. Kallikrein 3 (KLK3), which is commonly referred to as prostate-specific antigen, is the current serum biomarker for prostate cancer. Other kallikreins are potential adjunct biomarkers. As secreted proteases, kallikreins act through enzyme cascades that may modulate the prostate cancer microenvironment. Both as a panel of biomarkers and cascade of proteases, the roles of kallikreins are interconnected. Yet the expression and regulation of different kallikreins in prostate cancer has not been compared. In this study, a spectrum of prostate cell lines was used to evaluate the expression profile of all 15 members of the kallikrein family. A cluster of genes was co-ordinately expressed in androgenresponsive cell lines. This group of kallikreins included KLK2, 3, 4 and 15, which are located adjacent to one another at the centromeric end of the kallikrein locus. KLK14 was also of interest, because it was ubiquitously expressed among the prostate cell lines. Immunohistochemistry showed that these 5 kallikreins are co-expressed in benign and malignant prostate tissue. The androgen-regulated expression of KLK2 and KLK3 is well-characterised, but has not been compared with other kallikreins. Therefore, KLK2, 3, 4, 14 and 15 expression were all measured in time course and dose response experiments with androgens, AR-antagonist treatments, hormone deprivation experiments and cells transfected with AR siRNA. Collectively, these experiments demonstrated that prostatic kallikreins are specifically and directly regulated by the AR. The data also revealed that kallikrein genes are differentially regulated by androgens; KLK2 and KLK3 were strongly up-regulated, KLK4 and KLK15 were modestly up-regulated, and KLK14 was repressed. Notably, KLK14 is located at the telomeric end of the kallikrein locus, far away from the centromeric cluster of kallikreins that are stimulated by androgens. These results show that the expression of KLK2, 3, 4, 14 and 15 is maintained in prostate cancer, but that these genes exhibit different responses to androgens. This makes the kallikrein locus an ideal model to investigate AR signalling. The increasingly dedifferentiated phenotype of aggressive prostate cancer cells is accompanied by the re-expression of signalling molecules that are usually expressed during embryogenesis and foetal tissue development. The Wnt pathway is one developmental cascade that is reactivated in prostate cancer. The canonical Wnt cascade regulates the intracellular levels of β-catenin, a potent transcriptional co-activator of T-cell factor (TCF) transcription factors. Notably, β-catenin can also bind to the AR and synergistically stimulate androgen-mediated gene expression. This is at the expense of typical Wnt/TCF target genes, because the AR:β-catenin and TCF:β-catenin interactions are mutually exclusive. The effect of β-catenin on kallikrein expression was examined to further investigate the role of β-catenin in prostate cancer. Stable knockdown of β-catenin in LNCaP prostate cancer cells attenuated the androgen-regulated expression of KLK2, 3, 4 and 15, but not KLK14. To test whether KLK14 is instead a TCF:β-catenin target gene, the endogenous levels of β-catenin were increased by inhibiting its degradation. Although KLK14 expression was up-regulated by these treatments, siRNA knockdown of β-catenin demonstrated that this effect was independent of β-catenin. These results show that β-catenin is required for maximal expression of KLK2, 3, 4 and 15, but not KLK14. Developmental cells and tumour cells express a similar repertoire of signalling molecules, which means that these different cell types are responsive to one another. Previous reports have shown that stem cells and foetal tissues can reprogram aggressive cancer cells to less aggressive phenotypes by restoring the balance to developmental signalling pathways that are highly dysregulated in cancer. To investigate this phenomenon in prostate cancer, DU145 and PC-3 prostate cancer cells were cultured on matrices pre-conditioned with human embryonic stem cells (hESCs). Soft agar assays showed that prostate cancer cells exposed to hESC conditioned matrices had reduced clonogenicity compared with cells harvested from control matrices. A recent study demonstrated that this effect was partially due to hESC-derived Lefty, an antagonist of Nodal. A member of the transforming growth factor β (TGFβ) superfamily, Nodal regulates embryogenesis and is re-expressed in cancer. The role of Nodal in prostate cancer has not previously been reported. Therefore, the expression and function of the Nodal signalling pathway in prostate cancer was investigated. Western blots confirmed that Nodal is expressed in DU145 and PC-3 cells. Immunohistochemistry revealed greater expression of Nodal in malignant versus benign glands. Notably, the Nodal inhibitor, Lefty, was not expressed at the mRNA level in any prostate cell lines tested. The Nodal signalling pathway is functionally active in prostate cancer cells. Recombinant Nodal treatments triggered downstream phosphorylation of Smad2 in DU145 and LNCaP cells, and stably-transfected Nodal increased the clonogencity of LNCaP cells. Nodal was also found to modulate AR signalling. Nodal reduced the activity of an androgen-regulated KLK3 promoter construct in luciferase assays and attenuated the endogenous expression of AR target genes including prostatic kallikreins. These results demonstrate that Nodal is a novel example of a developmental signalling molecule that is reexpressed in prostate cancer and may have a functional role in prostate cancer progression. In summary, this project clarifies the role of androgens and changing cellular differentiation in prostate cancer by characterising the expression and function of the downstream genes encoding kallikrein-related serine proteases and Nodal. Furthermore, this study emphasises the similarities between prostate cancer and early development, and the crosstalk between developmental signalling pathways and the AR axis. The outcomes of this project also affirm the utility of the kallikrein locus as a model system to monitor tumour progression and the phenotype of prostate cancer cells.

Interactions between human osteoblasts and prostate cancer cells in a novel 3D in vitro model

Cell-cell and cell-matrix interactions play a major role in tumor morphogenesis and cancer metastasis. Therefore, it is crucial to create a model with a biomimetic microenvironment that allows such interactions to fully represent the pathophysiology of a disease for an in vitro study. This is achievable by using three-dimensional (3D) models instead of conventional two-dimensional (2D) cultures with the aid of tissue engineering technology. We are now able to better address the complex intercellular interactions underlying prostate cancer (CaP) bone metastasis through such models. In this study, we assessed the interaction of CaP cells and human osteoblasts (hOBs) within a tissue engineered bone (TEB) construct. Consistent with other in vivo studies, our findings show that intercellular and CaP cell-bone matrix interactions lead to elevated levels of matrix metalloproteinases, steroidogenic enzymes and the CaP biomarker, prostate specific antigen (PSA); all associated with CaP metastasis. Hence, it highlights the physiological relevance of this model. We believe that this model will provide new insights for understanding of the previously poorly understood molecular mechanisms of bone metastasis, which will foster further translational studies, and ultimately offer a potential tool for drug screening. © 2010 Landes Bioscience.

Socioeconomic position, gender, health behaviours and biomarkers of cardiovascular disease and diabetes

Socio-economic gradients in cardiovascular disease (CVD) and diabetes have been found throughout the developed world and there is some evidence to suggest that these gradients may be steeper for women. Research on social gradients in biological risk factors for CVD and diabetes has received less attention and we do not know the extent to which gradients in biomarkers vary for men and women. We examined the associations between two indicators of socio-economic position (education and household income) and biomarkers of diabetes and cardiovascular disease (CVD) for men and women in a national, population-based study of 11,247 Australian adults. Multi-level linear regression was used to assess associations between education and income and glucose tolerance, dyslipidaemia, blood pressure (BP) and waist circumference before and after adjustment for behaviours (diet, smoking, physical activity, TV viewing time, and alcohol use). Measures of glucose tolerance included fasting plasma glucose and insulin and the results of a glucose tolerance test (2 h glucose) with higher levels of each indicating poorer glucose tolerance. Triglycerides and High Density Lipoprotein (HDL) Cholesterol were used as measures of dyslipidaemia with higher levels of the former and lower levels of the later being associated with CVD risk. Lower education and low income were associated with higher levels of fasting insulin, triglycerides and waist circumference in women. Women with low education had higher systolic and diastolic BP and low income women had higher 2 h glucose and lower HDL cholesterol. With only one exception (low income and systolic BP), all of these estimates were reduced by more than 20% when behavioural risk factors were included. Men with lower education had higher fasting plasma glucose, 2 h glucose, waist circumference and systolic BP and, with the exception of waist circumference, all of these estimates were reduced when health behaviours were included in the models. While low income was associated with higher levels of 2-h glucose and triglycerides it was also associated with better biomarker profiles including lower insulin, waist circumference and diastolic BP. We conclude that low socio-economic position is more consistently associated with a worse profile of biomarkers for CVD and diabetes for women.

Mesenchymal stem cells

Cell based therapies as they apply to tissue engineering and regenerative medicine, require cells capable of self renewal and differentiation, and a prerequisite is to be able to prepare an effective dose of ex vivo expanded cells for autologous transplants. The in vivo identification of a source of physiologically relevant cell types suitable for cell therapies therefore figures as an integral part of tissue engineering. Stem cells serve as a reserve for biological repair, having the potential to differentiate into a number of specialised cell types within the body; they therefore represent the most useful candidates for cell based therapies. The primary goal of stem cell research is to produce cells that are both patient specific, as well as having properties suitable for the specific conditions for which they are intended to remedy. From a purely scientific perspective, stem cells allow scientists to gain a deeper understanding of developmental biology and regenerative therapies. Stem cells have acquired a number of uses for applications in regenerative medicine, immunotherapy, gene therapy, but it is in the area of tissue engineering that they generate most excitement, primarily as a result of their capacity for self-renewal and pluripotency. A unique feature of stem cells is their ability to maintain an uncommitted quiescent state in vivo and then, once triggered by conditions such as disease, injury or natural wear or tear, serve as a reservoir and natural support system to replenish lost cells. Although these cells retain the plasticity to differentiate into various tissues, being able to control this differentiation process is still one of the biggest challenges facing stem cell research. In an effort to harness the potential of these cells a number of studies have been conducted using both embryonic/foetal and adult stem cells. The use of embryonic stem cells (ESC) have been hampered by strong ethical and political concerns, this despite their perceived versatility due to their pluripotency. Ethical issues aside, other concerns raised with ESCs relates to the possibility of tumorigenesis, immune rejection and complications with immunosuppressive therapies, all of which adds layers of complications to the application ESC in research and which has led to the search for alternative sources for stem cells. The adult tissues in higher organisms harbours cells, termed adult stem cells, and these cells are reminiscent of unprogrammed stem cells. A number of sources of adult stem cells have been described. Bone marrow is by far the most accessible source of two potent populations of adult stem cells, namely haematopoietic stem cells (HSCs) and bone marrow mesenchymal stem cells (BMSCs). Autologously harvested adult stem cells can, in contrast to embryonic stem cells, readily be used in autografts, since immune rejection is not an issue; and their use in scientific research has not attracted the ethical concerns which have been the case with embryonic stem cells. The major limitation to their use, however, is the fact that adult stem cells are exceedingly rare in most tissues. This fact makes identifying and isolating these cells problematic; bone marrow being perhaps the only notable exception. Unlike the case of HSCs, there are as yet no rigorous criteria for characterizing MSCs. Changing acuity about the pluripotency of MSCs in recent studies has expanded their potential application; however, the underlying molecular pathways which impart the features distinctive to MSCs remain elusive. Furthermore, the sparse in vivo distribution of these cells imposes a clear limitation to their study in vitro. Also, when MSCs are cultured in vitro, there is a loss of the in vivo microenvironment, resulting in a progressive decline in proliferation potential and multipotentiality. This is further exacerbated with increased passage numbers in culture, characterized by the onset of senescence related changes. As a consequence, it is necessary to establish protocols for generating large numbers of MSCs but without affecting their differentiation potential. MSCs are capable of differentiating into mesenchymal tissue lineages, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Recent findings indicate that adult bone marrow may also contain cells that can differentiate into the mature, nonhematopoietic cells of a number of tissues, including cells of the liver, kidney, lung, skin, gastrointestinal tract, and myocytes of heart and skeletal muscle. MSCs can readily be expanded in vitro and can be genetically modified by viral vectors and be induced to differentiate into specific cell lineages by changing the microenvironment–properties which makes these cells ideal vehicles for cellular gene therapy. MSCs can also exert profound immunosuppressive effects via modulation of both cellular and innate immune pathways, and this property allows them to overcome the issue of immune rejection. Despite the many attractive features associated with MSCs, there are still many hurdles to overcome before these cells are readily available for use in clinical applications. The main concern relates to in vivo characterization and identification of MSCs. The lack of a universal biomarker, sparse in vivo distribution, and a steady age related decline in their numbers, makes it an obvious need to decipher the reprogramming pathways and critical molecular players which govern the characteristics unique to MSCs. This book presents a comprehensive insight into the biology of adult stem cells and their utility in current regeneration therapies. The adult stem cell populations reviewed in this book include bone marrow derived MSCs, adipose derived stem cells (ASCs), umbilical cord blood stem cells, and placental stem cells. The features such as MSC circulation and trafficking, neuroprotective properties, and the nurturing roles and differentiation potential of multiple lineages have been discussed in details. In terms of therapeutic applications, the strengths of MSCs have been presented and their roles in disease treatments such as osteoarthritis, Huntington’s disease, periodontal regeneration, and pancreatic islet transplantation have been discussed. An analysis comparing osteoblast differentiation of umbilical cord blood stem cells and MSCs has been reviewed, as has a comparison of human placental stem cells and ASCs, in terms of isolation, identification and therapeutic applications of ASC in bone, cartilage regeneration, as well as myocardial regeneration. It is my sincere hope that this book will update the reader as to the research progress of MSC biology and potential use of these cells in clinical applications. It will be the best reward to all contributors of this book, if their efforts herein may in some way help the readers in any part of their study, research, and career development.

Expression of PSA-RP2, an alternatively spliced variant from the PSA gene, is increased in prostate cancer tissues but the protein is not secreted from prostate cancer cells

PSA-RP2 is a variant transcript expressed from the PSA gene that is conserved in gorillas, chimpanzees and humans suggesting a particular relevance for this transcript in these primates. We demonstrated by qRT-PCR that PSA-RP2 is upregulated in prostate cancer compared with benign prostatic hyperplasia tissues. The PSA-RP2 protein was not detected in seminal fluid and was cytoplasmically localised but not secreted from LNCaP or transfected PC3 prostate cells, despite secretion from transfected Cos-7 and HEK293 kidney cell lines. PSA-RP2-transfected PC3 cells showed slightly decreased proliferation and increased migration towards PC3-conditioned medium that could suggest a functional role in prostate cancer.

A fragment of the LG3 peptide of endorepellin is present in the urine of physically active mining workers : a potential marker of physical activity

Biomarker analysis has been implemented in sports research in an attempt to monitor the effects of exertion and fatigue in athletes. This study proposed that while such biomarkers may be useful for monitoring injury risk in workers, proteomic approaches might also be utilised to identify novel exertion or injury markers. We found that urinary urea and cortisol levels were significantly elevated in mining workers following a 12 hour overnight shift. These levels failed to return to baseline over 24h in the more active maintenance crew compared to truck drivers (operators) suggesting a lack of recovery between shifts. Use of a SELDI-TOF MS approach to detect novel exertion or injury markers revealed a spectral feature which was associated with workers in both work categories who were engaged in higher levels of physical activity. This feature was identified as the LG3 peptide, a C-terminal fragment of the anti-angiogenic / anti-tumourigenic protein endorepellin. This finding suggests that urinary LG3 peptide may be a biomarker of physical activity. It is also possible that the activity mediated release of LG3 / endorepellin into the circulation may represent a biological mechanism for the known inverse association between physical activity and cancer risk / survival.

The expression, regulation and function of kallikrein 4 in prostate cancer

Prostate cancer is a significant health problem faced by aging men. Currently, diagnostic strategies for the detection of prostate cancer are either unreliable, yielding high numbers of false positive results, or too invasive to be used widely as screening tests. Furthermore, the current therapeutic strategies for the treatment of the disease carry considerable side effects. Although organ confined prostate cancer can be curable, most detectable clinical symptoms occur in advanced disease when primary tumour cells have metastasised to distant sites - usually lymph nodes and bone. Many growth factors and steroids assist the continued growth and maintenance of prostatic tumour cells. Of these mitogens, androgens are important in the development of the normal prostate but are also required to sustain the growth of prostate cancer cells in the early stage of the disease. Not only are androgens required in the early stage of disease, but also many other growth factors and hormones interact to cause uncontrolled proliferation of malignant cells. The early, androgen sensitive phase of disease is followed by an androgen insensitive phase, whereby androgens are no longer required to stimulate the growth of the tumour cells. Growth factors such as transforming growth factor  and  (TGF/), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factors (IGFs), Vitamin D and thyroid hormone have been suggested to be important at this stage of disease. Interestingly, some of the kallikrein family of genes, including prostate specific antigen (PSA), the current serum diagnostic marker for prostate cancer, are regulated by androgens and many of the aforementioned growth factors. The kallikrein gene family is a group of serine proteases that are involved in a diverse range of physiological processes: regulation of local blood flow, angiogenesis, tissue invasion and mitogenesis. The earliest members of the kallikrein gene family (KLK1-KLK3) have been strongly associated with general disease states, such as hypertension, inflammation, pancreatitis and renal disease, but are also linked to various cancers. Recently, this family was extended to include 15 genes (KLK1-15). Several newer members of the kallikrein family have been implicated in the carcinogenesis and tumour metastasis of hormone-dependent cancers such as prostate, breast, endometrial and ovarian cancer. The aims of this project were to investigate the expression of the newly identified kallikrein, KLK4, in benign and malignant prostate tissues, and prostate cancer cell lines. This thesis has demonstrated the elevated expression of KLK4 mRNA transcripts in malignant prostate tissue compared to benign prostates. Additionally, expression of the full length KLK4 transcript was detected in the androgen dependent prostate cancer cell line, LNCaP. Based on the above finding, the LNCaP cell line was chosen to assess the potential regulation of full length KLK4 by androgen, thyroid hormone and epidermal growth factor. KLK4 mRNA and protein was found to be up-regulated by androgen and a combination of androgen and thyroid hormone. Thyroid hormone alone produced no significant change in KLK4 mRNA or protein over the control. Epidermal growth factor treatment also resulted in elevated expression levels of KLK4 mRNA and protein. To assess the potential functional role(s) of KLK4/hK4 in processes associated with tumour progression, full length KLK4 was transfected into PC-3 cells - a prostate cancer cell line originally derived from a secondary bone lesion. The KLK4/hK4 over-expressing cells were assessed for their proliferation, migration, invasion and attachment properties. The KLK4 over-expressing clones exhibited a marked change in morphology, indicative of a more aggressive phenotype. The KLK4 clones were irregularly shaped with compromised adhesion to the growth surface. In contrast, the control cell lines (parent PC-3 and empty vector clones) retained a rounded morphology with obvious cell to cell adhesion, as well as significant adhesion to their growth surface. The KLK4 clones exhibited significantly greater attachment to Collagen I and IV than native PC-3s and empty vector controls. Over a 12 hour period, in comparison to the control cells, the KLK4 clones displayed an increase in migration towards PC-3 native conditioned media, a 3 fold increase towards conditioned media from an osteoblastic cell line (Saos-2) and no change in migration towards conditioned media from neonatal foreskin fibroblast cells or 20% foetal bovine serum. Furthermore, the increase in migration exhibited by the KLK4 clones was partially blocked by the serine protease inhibitor, aprotinin. The data presented in this thesis suggests that KLK4/hK4 is important in prostate carcinogenesis due to its over-expression in malignant prostate tissues, its regulation by hormones and growth factors associated with prostate disease and the functional consequences of over-expression of KLK4/hK4 in the PC-3 cell line. These results indicate that KLK4/hK4 may play an important role in tumour invasion and bone metastasis via increased attachment to the bone matrix protein, Collagen I, and enhanced migration due to soluble factors produced by osteoblast cells. This suggestion is further supported by the morphological changes displayed by the KLK4 over-expressing cells. Overall, this data suggests that KLK4/hK4 should be further studied to more fully investigate the potential value of KLK4/hK4 as a diagnostic/prognostic biomarker or in therapeutic applications.

A 2-h diagnostic protocol to assess patients with chest pain symptoms in the Asia-Pacific region (ASPECT): a prospective observational validation study

Background: Patients with chest pain contribute substantially to emergency department attendances, lengthy hospital stay, and inpatient admissions. A reliable, reproducible, and fast process to identify patients presenting with chest pain who have a low short-term risk of a major adverse cardiac event is needed to facilitate early discharge. We aimed to prospectively validate the safety of a predefined 2-h accelerated diagnostic protocol (ADP) to assess patients presenting to the emergency department with chest pain symptoms suggestive of acute coronary syndrome. Methods: This observational study was undertaken in 14 emergency departments in nine countries in the Asia-Pacific region, in patients aged 18 years and older with at least 5 min of chest pain. The ADP included use of a structured pre-test probability scoring method (Thrombolysis in Myocardial Infarction [TIMI] score), electrocardiograph, and point-of-care biomarker panel of troponin, creatine kinase MB, and myoglobin. The primary endpoint was major adverse cardiac events within 30 days after initial presentation (including initial hospital attendance). This trial is registered with the Australia-New Zealand Clinical Trials Registry, number ACTRN12609000283279. Findings: 3582 consecutive patients were recruited and completed 30-day follow-up. 421 (11•8%) patients had a major adverse cardiac event. The ADP classified 352 (9•8%) patients as low risk and potentially suitable for early discharge. A major adverse cardiac event occurred in three (0•9%) of these patients, giving the ADP a sensitivity of 99•3% (95% CI 97•9–99•8), a negative predictive value of 99•1% (97•3–99•8), and a specificity of 11•0% (10•0–12•2). Interpretation: This novel ADP identifies patients at very low risk of a short-term major adverse cardiac event who might be suitable for early discharge. Such an approach could be used to decrease the overall observation periods and admissions for chest pain. The components needed for the implementation of this strategy are widely available. The ADP has the potential to affect health-service delivery worldwide.