Pathogenicity of Plesiomonas shigelloides : interactions with eukaryotic host cells in vitro

Diarrhoea is one of the leading causes of morbidity and mortality in populations in developing countries and is a significant health issue throughout the world. Despite the frequency and the severity of the diarrhoeal disease, mechanisms of pathogenesis for many of the causative agents have been poorly characterised. Although implicated in a number of intestinal and extra-intestinal infections in humans, Plesiomonas shigelloides generally has been dismissed as an enteropathogen due to the lack of clearly demonstrated virulence-associated properties such as production of cytotoxins and enterotoxins or invasive abilities. However, evidence from a number of sources has indicated that this species may be the cause of a number of clinical infections. The work described in this thesis seeks to resolve this discrepancy by investigating the pathogenic potential of P. shigelloides using in vitro cell models. The focus of this research centres on how this organism interacts with human host cells in an experimental model. Very little is known about the pathogenic potential of P. shigel/oides and its mechanisms in human infections and disease. However, disease manifestations mimic those of other related microorganisms. Chapter 2 reviews microbial pathogenesis in general, with an emphasis on understanding the mechanisms resulting from infection with bacterial pathogens and the alterations in host cell biology. In addition, this review analyses the pathogenic status of a poorly-defined enteropathogen, P. shigelloides. Key stages of pathogenicity must occur in order for a bacterial pathogen to cause disease. Such stages include bacterial adherence to host tissue, bacterial entry into host tissues (usually required), multiplication within host tissues, evasion of host defence mechanisms and the causation of damage. In this study, these key strategies in infection and disease were sought to help assess the pathogenic potential of P. shigelloides (Chapter 3). Twelve isolates of P. shigelloides, obtained from clinical cases of gastroenteritis, were used to infect monolayers of human intestinal epithelial cells in vitro. Ultrastructural analysis demonstrated that P. shigelloides was able to adhere to the microvilli at the apical surface of the epithelial cells and also to the plasma membranes of both apical and basal surfaces. Furthermore, it was demonstrated that these isolates were able to enter intestinal epithelial cells. Internalised bacteria often were confined within vacuoles surrounded by single or multiple membranes. Observation of bacteria within membranebound vacuoles suggests that uptake of P. shigelloides into intestinal epithelial cells occurs via a process morphologically comparable to phagocytosis. Bacterial cells also were observed free in the host cell cytoplasm, indicating that P. shige/loides is able to escape from the surrounding vacuolar membrane and exist within the cytosol of the host. Plesiomonas shigelloides has not only been implicated in gastrointestinal infections, but also in a range of non-intestinal infections such as cholecystitis, proctitis, septicaemia and meningitis. The mechanisms by which P. shigelloides causes these infections are not understood. Previous research was unable to ascertain the pathogenic potential of P. shigel/oides using cells of non-intestinal origin (HEp-2 cells derived from a human larynx carcinoma and Hela cells derived from a cervical carcinoma). However, with the recent findings (from this study) that P. shigelloides can adhere to and enter intestinal cells, it was hypothesised, that P. shigel/oides would be able to enter Hela and HEp-2 cells. Six clinical isolates of P. shigelloides, which previously have been shown to be invasive to intestinally derived Caco-2 cells (Chapter 3) were used to study interactions with Hela and HEp-2 cells (Chapter 4). These isolates were shown to adhere to and enter both nonintestinal host cell lines. Plesiomonas shigelloides were observed within vacuoles surrounded by single and multiple membranes, as well as free in the host cell cytosol, similar to infection by P. shigelloides of Caco-2 cells. Comparisons of the number of bacteria adhered to and present intracellularly within Hela, HEp-2 and Caco-2 cells revealed a preference of P. shigelloides for Caco-2 cells. This study conclusively showed for the first time that P. shigelloides is able to enter HEp-2 and Hela cells, demonstrating the potential ability to cause an infection and/or disease of extra-intestinal sites in humans. Further high resolution ultrastructural analysis of the mechanisms involved in P. shigelloides adherence to intestinal epithelial cells (Chapter 5) revealed numerous prominent surface features which appeared to be involved in the binding of P. shige/loides to host cells. These surface structures varied in morphology from small bumps across the bacterial cell surface to much longer filaments. Evidence that flagella might play a role in bacterial adherence also was found. The hypothesis that filamentous appendages are morphologically expressed when in contact with host cells also was tested. Observations of bacteria free in the host cell cytosol suggests that P. shigelloides is able to lyse free from the initial vacuolar compartment. The vacuoles containing P. shigel/oides within host cells have not been characterised and the point at which P. shigelloides escapes from the surrounding vacuolar compartment has not been determined. A cytochemical detection assay for acid phosphatase, an enzymatic marker for lysosomes, was used to analyse the co-localisation of bacteria-containing vacuoles and acid phosphatase activity (Chapter 6). Acid phosphatase activity was not detected in these bacteria-containing vacuoles. However, the surface of many intracellular and extracellular bacteria demonstrated high levels of acid phosphatase activity, leading to the proposal of a new virulence factor for P. shigelloides. For many pathogens, the efficiency with which they adhere to and enter host cells is dependant upon the bacterial phase of growth. Such dependency reflects the timing of expression of particular virulence factors important for bacterial pathogenesis. In previous studies (Chapter 3 to Chapter 6), an overnight culture of P. shigelloides was used to investigate a number of interactions, however, it was unknown whether this allowed expression of bacterial factors to permit efficient P. shigelloides attachment and entry into human cells. In this study (Chapter 7), a number of clinical and environmental P. shigelloides isolates were investigated to determine whether adherence and entry into host cells in vitro was more efficient during exponential-phase or stationary-phase bacterial growth. An increase in the number of adherent and intracellular bacteria was demonstrated when bacteria were inoculated into host cell cultures in exponential phase cultures. This was demonstrated clearly for 3 out of 4 isolates examined. In addition, an increase in the morphological expression of filamentous appendages, a suggested virulence factor for P. shigel/oides, was observed for bacteria in exponential growth phase. These observations suggest that virulence determinants for P. shigel/oides may be more efficiently expressed when bacteria are in exponential growth phase. This study demonstrated also, for the first time, that environmental water isolates of P. shigelloides were able to adhere to and enter human intestinal cells in vitro. These isolates were seen to enter Caco-2 host cells through a process comparable to the clinical isolates examined. These findings support the hypothesis of a water transmission route for P. shigelloides infections. The results presented in this thesis contribute significantly to our understanding of the pathogenic mechanisms involved in P. shigelloides infections and disease. Several of the factors involved in P. shigelloides pathogenesis have homologues in other pathogens of the human intestine, namely Vibrio, Aeromonas, Salmonella, Shigella species and diarrhoeaassociated strains of Escherichia coli. This study emphasises the relevance of research into Plesiomonas as a means of furthering our understanding of bacterial virulence in general. As well it provides tantalising clues on normal and pathogenic host cell mechanisms.

The Biology, Epidemiology, and Management of Rice Tungro Disease in Asia

Many farmers in South and Southeast Asia describe rice tungro disease as a cancer disease because of the severe damage it causes and the difficulty of controlling it (121). As the most important of the 14 rice viral diseases, tungro was first recognized as a leafhopper-transmitted virus disease in 1963 (88). However, tungro, which means “degenerated growth” in a Filipino dialect, has a much longer history. It is almost certain that tungro was responsible for a disease outbreak that occurred in 1859 in Indonesia, which was referred to at the time as mentek (83). In the past, a variety of names has been given to tungro, including accep na pula in the Philippines, penyakit merah in Malaysia, and yelloworange leaf in Thailand (83).

Mineralized human primary osteoblast matrices as a model system to analyse interactions of prostate cancer cells with the bone microenvironment

Prostate cancer metastasis is reliant on the reciprocal interactions between cancer cells and the bone niche/micro-environment. The production of suitable matrices to study metastasis, carcinogenesis and in particular prostate cancer/bone micro-environment interaction has been limited to specific protein matrices or matrix secreted by immortalised cell lines that may have undergone transformation processes altering signaling pathways and modifying gene or receptor expression. We hypothesize that matrices produced by primary human osteoblasts are a suitable means to develop an in vitro model system for bone metastasis research mimicking in vivo conditions. We have used a decellularized matrix secreted from primary human osteoblasts as a model for prostate cancer function in the bone micro-environment. We show that this collagen I rich matrix is of fibrillar appearance, highly mineralized, and contains proteins, such as osteocalcin, osteonectin and osteopontin, and growth factors characteristic of bone extracellular matrix (ECM). LNCaP and PC3 cells grown on this matrix, adhere strongly, proliferate, and express markers consistent with a loss of epithelial phenotype. Moreover, growth of these cells on the matrix is accompanied by the induction of genes associated with attachment, migration, increased invasive potential, Ca2+ signaling and osteolysis. In summary, we show that growth of prostate cancer cells on matrices produced by primary human osteoblasts mimics key features of prostate cancer bone metastases and thus is a suitable model system to study the tumor/bone micro-environment interaction in this disease.

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.

Adherence to fish oil intervention in patients with chronic kidney disease

Objective: With growing recognition of the role of inflammation in the development of chronic and acute disease, fish oil is increasingly used as a therapeutic agent, but the nature of the intervention may pose barriers to adherence in clinical populations. Our objective was to investigate the feasibility of using a fish oil supplement in hemodialysis patients. ---------- Design: This was a nonrandomized intervention study.---------- Setting: Eligible patients were recruited at the Hemodialysis Unit of Wesley Hospital, Brisbane, Queensland, Australia. Patients The sample included 28 maintenance hemodialysis patients out of 43 eligible patients in the unit. Exclusion criteria included patients regularly taking a fish oil supplement at baseline, receiving hemodialysis for less than 3 months, or being unable to give informed consent.---------- Intervention: Eicosapentaenoic acid (EPA) was administered at 2000 mg/day (4 capsules) for 12 weeks. Adherence was measured at baseline and weekly throughout the study according to changes in plasma EPA, and was further measured subjectively by self-report.---------- Results: Twenty patients (74%) adhered to the prescription based on changes in plasma EPA, whereas an additional two patients self-reported good adherence. There was a positive relationship between fish oil intake and change in plasma EPA. Most patients did not report problems with taking the fish oil. Using the baseline data, it was not possible to characterize adherent patients.---------- Conclusions: Despite potential barriers, including the need to take a large number of prescribed medications already, 74% of hemodialysis patients adhered to the intervention. This study demonstrated the feasibility of using fish oil in a clinical population.

Predicting dry eye using non-invasive techniques of tear film surface assessment

PURPOSE. To measure tear film surface quality in healthy and dry eye subjects using three noninvasive techniques of tear film quality assessment and to establish the ability of these noninvasive techniques to predict dry eye. METHODS. Thirty four subjects participated in the study, and were classified as dry eye or normal, based on standard clinical assessments. Three non-invasive techniques were applied for measurement of tear film surface quality: dynamic-area high-speed videokeratoscopy (HSV), wavefront sensing (DWS) and lateral shearing interferometry (LSI). The measurements were performed in both natural blinking conditions (NBC) and in suppressed blinking conditions (SBC). RESULTS. In order to investigate the capability of each method to discriminate dry eye subjects from normal subjects, the receiver operating curve (ROC) was calculated and then the area under the curve (AUC) was extracted. The best result was obtained for the LSI technique (AUC=0.80 in SBC and AUC=0.73 in NBC), which was followed by HSV (AUC=0.72 in SBC and AUC=0.71 in NBC). The best result for DWS was AUC=0.64 obtained for changes in vertical coma in suppressed blinking conditions, while for normal blinking conditions the results were poorer. CONCLUSIONS. Non-invasive techniques of tear film surface assessment can be used for predicting dry eye and this can be achieved in natural blinking as well as suppressed blinking conditions. In this study, LSI showed the best detection performance, closely followed by the dynamic-area HSV. The wavefront sensing technique was less powerful, particularly in natural blinking conditions.

Compliance, peritoneal dialysis and chronic kidney disease : lessons from the literature

Poor patient compliance with peritoneal dialysis (PD) has significant adverse effects on morbidity and mortality rates in individuals with chronic kidney disease (CKD). It also adds to the resource burdens of healthcare services and providers. This paper explores the notion of PD compliance in patients with CKD with reference to the relevant published literature. The analysis of the literature reveals that ‘PD compliance’ is a complex and challenging construct for both patients and health professionals. There is no universal definition of compliance that is widely adopted in practice and research, and therefore a lack of consensus on how to determine ‘compliant’ patient outcomes. There are also multiple and interconnected determinants of PD compliance that are context-bound, which healthcare professionals must be aware of, and which makes producing consensus of measuring PD compliance difficult. The complexity of the interventions required to produce even a modest improvement in PD compliance, which are described in this paper, are significant. Compliance with PD and other treatments for CKD is a multidimensional, context-bound concept, that to date has tended to efface the role and needs of the renal patient. We conclude the paper with the implications for contemporary practice.

Exploring retinal and functional markers of diabetic neuropathy

Diabetic peripheral neuropathy (DPN) is one of the most debilitating complications of diabetes. DPN is a major cause of foot ulceration and lower limb amputation. Early diagnosis and management is a key factor in reducing morbidity and mortality. Current techniques for clinical assessment of DPN are relatively insensitive for detecting early disease or involve invasive procedures such as skin biopsies. There is a need for less painful, non-invasive and safe evaluation methods. Eye care professionals already play an important role in the management of diabetic retinopathy; however recent studies have indicated that the eye may also be an important site for the diagnosis and monitoring of neuropathy. Corneal nerve morphology has been shown to be a promising marker of diabetic neuropathy occurring elsewhere in the body, and emerging evidence tentatively suggests that retinal anatomical markers and a range of functional visual indicators could similarly provide useful information regarding neural damage in diabetes – although this line of research is, as yet, less well established. This review outlines the growing body of evidence supporting a potential diagnostic role for retinal structure and visual functional markers in the diagnosis and monitoring of peripheral neuropathy in diabetes.

Effect of surgical approach on bone vascularisation, fracture and soft tissue healing : comparison of less invasive to open approach

Over the past ten years, minimally invasive plate osteosynthesis (MIPO) for the fixation of long bone fractures has become a clinically accepted method with good outcomes, when compared to the conventional open surgical approach (open reduction internal fixation, ORIF). However, while MIPO offers some advantages over ORIF, it also has some significant drawbacks, such as a more demanding surgical technique and increased radiation exposure. No clinical or experimental study to date has shown a difference between the healing outcomes in fractures treated with the two surgical approaches. Therefore, a novel, standardised severe trauma model in sheep has been developed and validated in this project to examine the effect of the two surgical approaches on soft tissue and fracture healing. Twenty four sheep were subjected to severe soft tissue damage and a complex distal femur fracture. The fractures were initially stabilised with an external fixator. After five days of soft tissue recovery, internal fixation with a plate was applied, randomised to either MIPO or ORIF. Within the first fourteen days, the soft tissue damage was monitored locally with a compartment pressure sensor and systemically by blood tests. The fracture progress was assessed fortnightly by x-rays. The sheep were sacrificed in two groups after four and eight weeks, and CT scans and mechanical testing performed. Soft tissue monitoring showed significantly higher postoperative Creatine Kinase and Lactate Dehydrogenase values in the ORIF group compared to MIPO. After four weeks, the torsional stiffness was significantly higher in the MIPO group (p=0.018) compared to the ORIF group. The torsional strength also showed increased values for the MIPO technique (p=0.11). The measured total mineralised callus volumes were slightly higher in the ORIF group. However, a newly developed morphological callus bridging score showed significantly higher values for the MIPO technique (p=0.007), with a high correlation to the mechanical properties (R2=0.79). After eight weeks, the same trends continued, but without statistical significance. In summary, this clinically relevant study, using the newly developed severe trauma model in sheep, clearly demonstrates that the minimally invasive technique minimises additional soft tissue damage and improves fracture healing in the early stage compared to the open surgical approach method.

Chronic disease, geographic location and socioeconomic disadvantage as obstacles to equitable access to e-health

Despite recent public attention to e-health as a solution to rising healthcare costs and an ageingpopulation, there have been relatively few studies examining the geographical pattern of e-health usage. This paper argues for an equitable approach to e-health and attention to the way in which e-health initiatives can produce locational health inequalities, particularly in socioeconomically disadvantaged areas. In this paper, we use a case study to demonstrate geographical variation in Internet accessibility, Internet status and prevalence of chronic diseases within a small district. There are signifi cant disparities in access to health information within socioeconomically disadvantaged areas. The most vulnerable people in these areas are likely to have limited availability of, or access to Internet healthcare resources. They are also more likely to have complex chronic diseases and, therefore, be in greatest need of these resources. This case study demonstrates the importance of an equitable approach to e-health information technologies and telecommunications infrastructure.