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.

Effect of rain on travel demand and traffic accidents

This paper studies the effect of rain on travel demand measured on the Tokyo Metropolitan Expressway (MEX). Rainfall data monitored by the Japan Meteorological Agency's meso-scale network of weather stations are used. This study found that travel demand decreases during rainy days and, in particular, larger reductions occur over the weekend. The effect of rainfall on the number of accidents recorded on 10 routes on the MEX is also analysed. Statistical testing shows that the average frequency of accidents, during periods of rainfall, is significantly different from the average frequency at other times.

The p38 mitogen-activated protein kinase regulates interleukin-1beta-induced IL-8 expression via an effect on the IL-8 promoter in intestinal epithelial cells

Several lines of evidence implicate the p38 mitogen-activated protein kinase (p38 MAPK) in the proinflammatory response to bacterial agents and cytokines. Equally, the transcription factor, nuclear factor (NF)-kappaB, is recognized to be a critical determinant of the inflammatory response in intestinal epithelial cells (IECs). However, the precise inter-relationship between the activation of p38 MAPK and activation of the transcription factor NF-kappaB in the intestinal epithelial cell (IEC) system, remains unknown. Here we show that interleukin (IL)-1beta activates all three MAPKs in Caco-2 cells. The production of IL-8 and monocyte chemotactic protein 1 (MCP-1) was attenuated by 50% when these cells were preincubated with the p38 MAPK inhibitor, SB 203580. Further investigation of the NF-kappaB signalling system revealed that the inhibitory effect was independent of the phosphorylation and degradation of IkappaBalpha, the binding partner of NF-kappaB. This effect was also independent of the DNA binding of the p65 Rel A subunit, as well as transactivation, determined by an NF-kappaB luciferase construct, using both SB 203580 and dominant-negative p38 MAPK. Evaluation of IL-8 and MCP-1 RNA messages by reverse transcription-polymerase chain reaction (RT-PCR) revealed that the inhibitory effect of SB 203580 was associated with a reduction in this parameter. Using an IL-8-luciferase promoter construct, an effect of p38 upon its activation by both pharmacological and dominant-negative p38 construct co-transfection was demonstrated. It is concluded that p38 MAPK influences the expression of chemokines in intestinal epithelial cells, through an effect upon the activation of the chemokine promoter, and does not directly involve the activation of the transcription factor NF-kappaB

Evaluating the effect of a patient education brochure on patients’ expectations and satisfaction with emergency department service

Introduction: The purpose of this study was to assess the capacity of a written intervention, in this case a patient information brochure, to improve patient satisfaction during an Emergency Department (ED) visit. For the purpose of measuring the effect of the intervention the ED journey was conceptualised as a series of distinct areas of service comprising waiting time, service by the triage nurse, care from doctors and nurses and information giving Background of study: Research into patient satisfaction has become a widespread activity endorsed by both governments and hospital administrations. The literature on ED patient satisfaction has consistently indicated three primary areas of patient dissatisfaction: waiting time, nursing care and communication. Recent developments in the literature on patient satisfaction studies however have highlighted the relationship between patients. expectations of a service encounter and their consequent assessment of the experience as dissatisfying or satisfying. Disconfirmation theory posits that the degree to which expectations are confirmed will affect subsequent levels of satisfaction. The conceptual framework utilised in this study is Coye.s (2004) model of disconfirmation. Coye while reiterating satisfaction is a consequence of the degree expectations are either confirmed or disconfirmed also posits that expectations can be modified by interventions. Coye.s work conceptualises these interventions as intra encounter experiences (cues) which function to adjust expectations. Coye suggests some cues are unintended and may have a negative impact which also reinforces the value of planned cues intended to meet or exceed consumer expectations. Consequently the brochure can be characterized as a potentially positive cue, encouraging the patient to understand processes and to orient them in what can be a confronting environment. Only a limited number of studies have examined the effect of written interventions within an ED. No studies could be located which have tested the effect of ED interventions using a conceptual framework which relates the effect of the degree to which expectations are confirmed or disconfirmed in terms of satisfaction with services. Method: Two studies were conducted. Study One used qualitative methods to explore patients. expectations of the ED from the perspective of both patients and health care professionals. Study One was used in part to direct the development of the intervention (brochure) in Study Two. The brochure was an intervention designed to modify patients. expectations thus increasing their satisfaction with the provision of ED service. As there was no existing tools to measure ED patients. expectations and satisfaction a new tool was also developed based on the findings and the literature of Study One. Study Two used a non-randomised, quasi-experimental approach using a non-equivalent post-test only comparison group design used to investigate the effect of the patient education brochure (Stommel and Wills, 2004). The brochure was disseminated to one of two study groups (the intervention group). The effect of the brochure was assessed by comparing the data obtained from both the intervention and control group. These two groups consisted of 150 participants each. It was expected that any differences in the relevant domains selected for examination would indicate the effect of the brochure both on expectation and potentially satisfaction. Results: Study One revealed several areas of common ground between patients and nurses in terms of relevant content for the written intervention, including the need for information on the triage system and waiting times. Areas of difference were also found with patients emphasizing communication issues, whereas focus group members expressed concern that patients were often unable to assimilate verbal information. The findings suggested the potential utility of written material to reinforce verbal communication particularly in terms of the triage process and other ED protocols. This material was synthesized within the final version of the written intervention. Overall the results of Study Two indicated no significant differences between the two groups. The intervention group did indicate a significant number of participants who viewed the brochure of having changed their expectations. The effect of the brochure may have been obscured by a lack of parity between the two groups as the control group presented with statistically significantly higher levels of acuity and experienced significantly shorter waiting times. In terms of disconfirmation theory this would suggest expectations that had been met or exceeded. The results confirmed the correlation of expectations with satisfaction. Several domains also indicated age as a significant predictor with older patients tending to score higher satisfaction results. Other significant predictors of satisfaction established were waiting time and care from nurses, reinforcing the combination of efficient service and positive interpersonal experiences as being valued by patients. Conclusions: Information presented in written form appears to benefit a significant number of ED users in terms of orientation and explaining systems and procedures. The degree to which these effects may interact with other dimensions of satisfaction however is likely to be limited. Waiting time and interpersonal behaviours from staff also provide influential cues in determining satisfaction. Written material is likely to be one element in a series of coordinated strategies to improve patient satisfaction during periods of peak demand.

Randomized trial of effect of bifocal and prismatic bifocal spectacles on myopic progression

Objective: To determine whether bifocal and prismatic bifocal spectacles could control myopia in children with high rates of myopic progression. ---------- Methods: This was a randomized controlled clinical trial. One hundred thirty-five (73 girls and 62 boys) myopic Chinese Canadian children (myopia of 1.00 diopters [D]) with myopic progression of at least 0.50 D in the preceding year were randomly assigned to 1 of 3 treatments: (1) single-vision lenses (n = 41), (2) +1.50-D executive bifocals (n = 48), or (3) +1.50-D executive bifocals with a 3–prism diopters base-in prism in the near segment of each lens (n = 46). ---------- Main Outcome Measures: Myopic progression measured by an automated refractor under cycloplegia and increase in axial length (secondary) measured by ultrasonography at 6-month intervals for 24 months. Only the data of the right eye were used. ---------- Results: Of the 135 children (mean age, 10.29 years [SE, 0.15 years]; mean visual acuity, –3.08 D [SE, 0.10 D]), 131 (97%) completed the trial after 24 months. Myopic progression averaged –1.55 D (SE, 0.12 D) for those who wore single-vision lenses, –0.96 D (SE, 0.09 D) for those who wore bifocals, and –0.70 D (SE, 0.10 D) for those who wore prismatic bifocals. Axial length increased an average of 0.62 mm (SE, 0.04 mm), 0.41 mm (SE, 0.04 mm), and 0.41 mm (SE, 0.05 mm), respectively. The treatment effect of bifocals (0.59 D) and prismatic bifocals (0.85 D) was significant (P < .001) and both bifocal groups had less axial elongation (0.21 mm) than the single-vision lens group (P < .001). ---------- Conclusions: Bifocal lenses can moderately slow myopic progression in children with high rates of progression after 24 months.

A comparative study of the effect of calibration conditions on the water equivalence of a range of gel dosimeters

Gel dosimeters are of increasing interest in the field of radiation oncology as the only truly three-dimensional integrating radiation dosimeter. There are a range of ferrous-sulphate and polymer gel dosimeters. To be of use, they must be water-equivalent. On their own, this relates to their radiological properties as determined by their composition. In the context of calibration of gel dosimeters, there is the added complexity of the calibration geometry; the presence of containment vessels may influence the dose absorbed. Five such methods of calibration are modelled here using the Monte Carlo method. It is found that the Fricke gel best matches water for most of the calibration methods, and that the best calibration method involves the use of a large tub into which multiple fields of different dose are directed. The least accurate calibration method involves the use of a long test tube along which a depth dose curve yields multiple calibration points.

A comparative study of the effect of calibration conditions on the water equivalence of a range of gel dosimeters

Gel dosimeters are of increasing interest in the field of radiation oncology as the only truly three-dimensional integrating radiation dosimeter. There are a range of ferrous-sulphate and polymer gel dosimeters. To be of use, they must be water-equivalent. On their own, this relates to their radiological properties as determined by their composition. In the context of calibration of gel dosimeters, there is the added complexity of the calibration geometry; the presence of containment vessels may influence the dose absorbed. Five such methods of calibration are modelled here using the Monte Carlo method. It is found that the Fricke gel best matches water for most of the calibration methods, and that the best calibration method involves the use of a large tub into which multiple fields of different dose are directed. The least accurate calibration method involves the use of a long test tube along which a depth dose curve yields multiple calibration points.

The effect of rhBMP-2 on canine osteoblasts seeded onto 3D bioactive polycaprolactone scaffolds

Our strategy entails investigating the influence of varied concentrations (0, 10, 100 and 1000 ng/ml) of human recombinant bone morphogenetic protein-2 (rhBMP-2) on the osteogenic expression of canine osteoblasts, seeded onto poly-caprolactone 20% tricalcium phosphate (PCL-TCP) scaffolds in vitro. Biochemical assay revealed that groups with rhBMP-2 displayed an initial burst in cell growth that was not dose-dependent. However, after 13 days, cell growth declined to a value similar to control. Significantly less cell growth was observed for construct with 1000 ng/ml of rhBMP-2 from 20 days onwards. Confocal microscopy confirmed viability of osteoblasts and at day 20, groups seeded with rhBMP-2 displayed heightened cell death as compared to control. Phase contrast and scanning electron microscopy revealed that osteoblasts heavily colonized surfaces, rods and pores of the PCL-TCP scaffolds. This was consistent for all groups. Finally, Von Kossa and osteocalcin assays demonstrated that cells from all groups maintained their osteogenic phenotype throughout the experiment. Calcification was observed as early as four days after stimulation for groups seeded with rhBMP-2. In conclusion, rhBMP-2 seems to enhance the differentiated function of canine osteoblasts in a non-dose dependent manner. This resulted in accelerated mineralization, followed by death of osteoblasts as they underwent terminal differentiation. Notably, PCL-TCP scaffolds seeded only with canine osteoblasts could sustain excellent osteogenic expression in vitro. Hence, the synergy of PCL with bioactive TCP and rhBMP-2 in a novel composite scaffold, could offer an exciting approach for bone regeneration.

Indentation size effect and strain rate sensitivity of nanocrystalline Mg-Al alloys

Deformation Behaviour of microcrystalline (mc) and nanocrystalline (nc) Mg-5%Al alloys produced by hot extrusion of ball-milled powders were investigated using instrumented indentation tests. The hardness values of the mc and nc metals exhibited indentation size effect (ISE), with nc alloys showing weaker ISE. The highly localized dislocation activities resulted in a small activation volume, hence enhanced strain rate sensitivity. Relative higher strain rate sensitivity and the negative Hall-Petch Relationship suggested the increasingly important role of grain boundary mediated mechanisms when the grain size decreased to nanometer region.

Raman spectroscopic study of the uranyl titanate mineral betafite (Ca,U)2(Ti,Nb)2O6(OH) –Effect of Metamictization

Raman spectra of the uranyl titanate mineral betafite were obtained and related to the mineral structure. A comparison is made with the spectra of uranyl oxyhydroxide hydrates. Observed bands are attributed to the (UO2)2+ stretching and bending vibrations, U-OH bending vibrations, H2O and (OH)- stretching, bending and libration modes. U-O bond lengths in uranyls and O-H…O bond lengths are calculated from the wavenumbers assigned to the stretching vibrations. Raman bands of betafite are comparable with those of the uranyl oxyhydroxides. The mineral betafite is metamict as is evidenced by the intensity of the UO stretching and bending modes being of lower intensity than expected and with bands that are significantly broader.

The impact of gender diversity on turnover : the moderating effect of human resource policies and practices

Demography theory suggests that high gender diversity leads to high turnover. As turnover is costly for organizations, we examined whether HR policies and practices influence the expected gender diversity-turnover relationship. Survey data were collected from 198 HR decision makers at publicly listed organizations. We found that HR policies and practices that are supportive of diversity moderate the gender diversity-turnover relationship, such that high gender diversity leads to low turnover in organizations with many diversity supportive policies and practices. Results suggest that organizations can avoid the negative consequences of high gender diversity by implementing diversity supportive HR polices and practices.

Effect of the Summer Months on Body Weight and Composition in College Women

BACKGROUND AND OBJECTIVES: College students and young adults are experiencing the greatest increases in rates of obesity, and 20% of college students are classified as obese. The objective of this study was to compare changes and rates of change in body weight and body composition between the freshman academic year and the summer after the freshman year among female college students. METHODS: Participants were recruited early in their freshman year of college to participate in a prospective longitudinal study examining changes in body weight and composition over the college years. Height and weight were measured, and body composition was assessed using dual energy x-ray absorptiometry (DEXA) at the beginning and end of the freshman year. Upon return from the summer for their sophomore year, participants returned to have all measurements repeated. Sixty-nine female participants completed all three visits. RESULTS: Body weight increased 1.3 kg during the academic period and an additional 0.1 kg during the summer period. Body mass index (BMI) increased between the first two visits but did not change between the last two visits. However, percent fat increased at each visit. Fat-free mass significantly increased 0.5 kg over the academic year but decreased by 1.1 kg over the summer (p<0.05). Greater rates of change were detected in percent fat, fat-free mass, and BMI during the summer compared with the academic year (p<0.05). CONCLUSIONS: Differences in body composition between the academic and summer periods may reflect changes in living situations between these periods. Unfavorable changes during the summer suggest the need to promote healthy lifestyles to freshman women before they leave campus for the summer