Microparticular and Nanoparticular DNA Delivery Systems as Adjuvants for DNA Immunization

In this dissertation different microparticular and nanoparticular DNA carrier systems were developed, with the aim to create an efficient adjuvant system for DNA vaccination. Their suitability was investigated by physico-chemical parameters, such as particle size, z-potential and encapsulation efficiency. Further, the systems were studied in-vitro for DNA stabilization and DNA bioactivity after encapsulation and release, as well as for gene delivery. We investigated modified double emulsion methods and spray drying techniques for DNA microencapsulation. Firstly, DNA was complexed with polyethylenimine (PEI) 25 kDa. We further studied the possibility to encapsulate lyophilized DNA and lyophilized DNA / PEI complexes in the presence of lyoprotectants. The microparticles were formulated using i) a modified double emulsion technique (W/O/W), ii) a solid in oil in water method (S/O/W), iii) a water in oil spray drying technique (W/O) and iv) a solid in oil spray drying technique (S/O). DNA release from particles prepared with double-emulsion methods, in contrast to spray drying techniques, resulted in constant DNA release and relatively low initial burst effects. The complexation with PEI substantially retarded the DNA release for all preparation techniques. In Chapter 4, we adsorbed DNA onto the surface of microparticles. We developed a cationic microparticular system by the incorporation of different amounts of the cationic molecules, PEI or CTAB into the polyester matrix. PEI 10% microparticles exhibited the most promising characteristics, such as a small particle size, a high z-potential of + 47 mV, a high DNA adsorption efficiency for a theoretical loading of 1% over the physiological pH range. The mechanism of gene delivery was studied by confocal microscopy and revealed diffuse fluorescence of DNA and PEI in the cytoplasm of non-phagocytic L929 fibroblasts. This was attributed to polyplex formation after PEI release from the particle. The efficient gene transfer of RG 502H+PEI 10% microparticles was confirmed by luciferase transfection. The challenge experiments with a lethal dose of the pathogen in challenge experiments in mice demonstrated that the formulation had an adjuvant effect. In Chapter 5, a new polymeric system was designed, consisting of poly (vinyl-alcohol) coupled with diamines, such as diethylaminopropylamine (DEAPA), (DMAPA) or (DEAEA). The amphiphilic properties allowed the formulation of DNA nanoparticles by a modified solvent displacement technique without the use of shear forces. DNA nanoparticles exhibited positive z-potentials up to +42 mV. The gene delivery of the nanoparticles was assessed in L929 mouse fibroblasts, which demonstrated high transfection efficiencies, comparable to PEI 25kDa/DNA complexes at a nitrogen to phosphate ratio of 5. In Chapter 6 we chose one representative polymer, P(26)-10, of the new class of amine-modified polyesters to investigate the influence of several process parameters on the nanoparticle formation. In Chapter 7, DNA nanoparticles with amine-modified polyesters were further characterized using two classes of polymers (DEAPA /DEAEA) with different amounts of amine modifications. The nanoparticle x-potentials and sizes were dependent on the N/P ratio. Atomic force microscopy confirmed the small particle sizes. DNA stability during the encapsulation process and release over nine day was demonstrated by electrophoresis, as well as DNA protection from enzyme degradation in dependence of the N/P ratio. The amount of cellular uptake of an efficient candidate P(68)-10, DNA nanoparticles was shown to be dependent on the N/P ratio of the formulation by flow cytometry. The mechanism of cellular uptake was followed by confocal microscopy and exhibited endocytotic uptake of the particles. The very efficient gene delivery of the P(68)-10 polymer was demonstrated by in-vitro transfection assays in four cell lines compared to PEI / DNA complexes at equal N/P ratios

Understanding your digital identity

The term “Digital Identity” is used here to describe the persona a person projects across the internet. Your Digital Identity as perceived by other people is made up of material that you post yourself (for example photographs on Flickr and your own web page) but it also is made up of material other people put there about you (blog posts that mention you, photographs in which you are tagged). The “This is Me” project has developed resources that can be used by students and others to appreciate what their Digital Identity is and how they can control it to help present the persona with the reputation that they want

Microparticular and Nanoparticular DNA Delivery Systems as Adjuvants for DNA Immunization

In this dissertation different microparticular and nanoparticular DNA carrier systems were developed, with the aim to create an efficient adjuvant system for DNA vaccination. Their suitability was investigated by physico-chemical parameters, such as particle size, z-potential and encapsulation efficiency. Further, the systems were studied in-vitro for DNA stabilization and DNA bioactivity after encapsulation and release, as well as for gene delivery. We investigated modified double emulsion methods and spray drying techniques for DNA microencapsulation. Firstly, DNA was complexed with polyethylenimine (PEI) 25 kDa. We further studied the possibility to encapsulate lyophilized DNA and lyophilized DNA / PEI complexes in the presence of lyoprotectants. The microparticles were formulated using i) a modified double emulsion technique (W/O/W), ii) a solid in oil in water method (S/O/W), iii) a water in oil spray drying technique (W/O) and iv) a solid in oil spray drying technique (S/O). DNA release from particles prepared with double-emulsion methods, in contrast to spray drying techniques, resulted in constant DNA release and relatively low initial burst effects. The complexation with PEI substantially retarded the DNA release for all preparation techniques. In Chapter 4, we adsorbed DNA onto the surface of microparticles. We developed a cationic microparticular system by the incorporation of different amounts of the cationic molecules, PEI or CTAB into the polyester matrix. PEI 10% microparticles exhibited the most promising characteristics, such as a small particle size, a high z-potential of + 47 mV, a high DNA adsorption efficiency for a theoretical loading of 1% over the physiological pH range. The mechanism of gene delivery was studied by confocal microscopy and revealed diffuse fluorescence of DNA and PEI in the cytoplasm of non-phagocytic L929 fibroblasts. This was attributed to polyplex formation after PEI release from the particle. The efficient gene transfer of RG 502H+PEI 10% microparticles was confirmed by luciferase transfection. The challenge experiments with a lethal dose of the pathogen in challenge experiments in mice demonstrated that the formulation had an adjuvant effect. In Chapter 5, a new polymeric system was designed, consisting of poly (vinyl-alcohol) coupled with diamines, such as diethylaminopropylamine (DEAPA), (DMAPA) or (DEAEA). The amphiphilic properties allowed the formulation of DNA nanoparticles by a modified solvent displacement technique without the use of shear forces. DNA nanoparticles exhibited positive z-potentials up to +42 mV. The gene delivery of the nanoparticles was assessed in L929 mouse fibroblasts, which demonstrated high transfection efficiencies, comparable to PEI 25kDa/DNA complexes at a nitrogen to phosphate ratio of 5. In Chapter 6 we chose one representative polymer, P(26)-10, of the new class of amine-modified polyesters to investigate the influence of several process parameters on the nanoparticle formation. In Chapter 7, DNA nanoparticles with amine-modified polyesters were further characterized using two classes of polymers (DEAPA /DEAEA) with different amounts of amine modifications. The nanoparticle x-potentials and sizes were dependent on the N/P ratio. Atomic force microscopy confirmed the small particle sizes. DNA stability during the encapsulation process and release over nine day was demonstrated by electrophoresis, as well as DNA protection from enzyme degradation in dependence of the N/P ratio. The amount of cellular uptake of an efficient candidate P(68)-10, DNA nanoparticles was shown to be dependent on the N/P ratio of the formulation by flow cytometry. The mechanism of cellular uptake was followed by confocal microscopy and exhibited endocytotic uptake of the particles. The very efficient gene delivery of the P(68)-10 polymer was demonstrated by in-vitro transfection assays in four cell lines compared to PEI / DNA complexes at equal N/P ratios

E-learning for self-management support: Introducing blended learning for graduate students - A cohort study 13 Education 1303 Specialist Studies in Education 11 Medical and Health Sciences 1117 Public Health and Health Services

Background: E-learning allows delivery of education in many diverse settings and researchers have demonstrated it can be as effective as learning conducted in traditional face-to-face settings. However, there are particular practices and skills needed in the area of providing patient self-management support (SMS), that may not be achievable online. The aim of this study was to compare three approaches in the training of university students regarding the preparation of a Chronic Condition Self-Management Care Plan: 1) traditional face-to-face delivery of SMS training, 2) an e-learning approach and 3) a blended approach (combining e-learning and face-to-face teaching). Methods: Graduate entry physiotherapy students and medical students at Flinders University were recruited. Depending on the cohort, students were either exposed to traditional face-to-face training, e-learning or a blended model. Outcomes were compared between the three groups. We measured adherence to care plan processes in the preparation of an assessment piece using the Flinders Program Chronic Care Self Management tools. A total of 183 care plans were included (102 traditional, 52 blended, 29 e-learning,). All students submitted the Flinders Program Chronic Care Plan for university assessment and these were later assessed for quality by researchers. The submission was also assigned a consumer engagement score and a global competence score as these are integral to successful delivery of SMS and represent the patient perspective. Results: The blended group performed significantly better than the traditional group in quality use of the Flinders Program tools: Problem and Goals (P < 0.0001). They also performed significantly better in the total care plan score (P < 0.0001) and engagement score (P < 0.0001). There was no significant difference between the groups for the Partners in Health tool. Conclusions: In this pilot study, the blended learning model was a more effective method for teaching self-management skills than the traditional group, as assessed in the development of a chronic condition self-management care plan. We anticipate that future research with identical groups of students would yield similar results but in the meantime, academics can have confidence that blended learning is at least as effective as traditional learning methods

Serum anticholinergic activity and cerebral cholinergic dysfunction: An EEG study in frail elderly with and without delirium

<p>Abstract</p> <p>Background</p> <p>Delirium increases morbidity, mortality and healthcare costs especially in the elderly. Serum anticholinergic activity (SAA) is a suggested biomarker for anticholinergic burden and delirium risk, but the association with cerebral cholinergic function remains unclear. To clarify this relationship, we prospectively assessed the correlation of SAA with quantitative electroencephalography (qEEG) power, delirium occurrence, functional and cognitive measures in a cross-sectional sample of acutely hospitalized elderly (> 80 y) with high dementia and delirium prevalence.</p> <p>Methods</p> <p>61 consecutively admitted patients over 80 years underwent an extensive clinical and neuropsychological evaluation. SAA was determined by using radio receptor assay as developed by Tune, and standard as well as quantitative EEGs were obtained.</p> <p>Results</p> <p>15 patients had dementia with additional delirium (DD) according to expert consensus using DSM-IV criteria, 31 suffered from dementia without delirium (D), 15 were cognitively unimpaired (CU). SAA was clearly detectable in all patients but one (mean 10.9 ± 7.1 pmol/ml), but was not associated with expert-panel approved delirium diagnosis or cognitive functions. Delirium-associated EEG abnormalities included occipital slowing, peak power and alpha decrease, delta and theta power increase and slow wave ratio increase during active delirious states. EEG measures correlated significantly with cognitive performance and delirium severity, but not with SAA levels.</p> <p>Conclusion</p> <p>In elderly with acute disease, EEG parameters reliable indicate delirium, but SAA does not seem to reflect cerebral cholinergic function as measured by EEG and is not related to delirium diagnosis.</p

Leadership Reconsidered: Engaging Higher Education in Social Change

Colleges and universities can provide effective environments for the development of future leaders. This book addresses the application of transformative leadership to higher education, identifies resources to use in the process, and..

Early Identification and Prevention of the Spread of Ebola - United States

In response to the 2014-2016 Ebola virus disease (Ebola) epidemic in West Africa, CDC prepared for the potential introduction of Ebola into the United States. The immediate goals were to rapidly identify and isolate any cases of Ebola, prevent transmission, and promote timely treatment of affected patients. CDC\u27s technical expertise and the collaboration of multiple partners in state, local, and municipal public health departments; health care facilities; emergency medical services; and U.S. government agencies were essential to the domestic preparedness and response to the Ebola epidemic and relied on longstanding partnerships. CDC established a comprehensive response that included two new strategies: 1) active monitoring of travelers arriving from countries affected by Ebola and other persons at risk for Ebola and 2) a tiered system of hospital facility preparedness that enabled prioritization of training. CDC rapidly deployed a diagnostic assay for Ebola virus (EBOV) to public health laboratories. Guidance was developed to assist in evaluation of patients possibly infected with EBOV, for appropriate infection control, to support emergency responders, and for handling of infectious waste. CDC rapid response teams were formed to provide assistance within 24 hours to a health care facility managing a patient with Ebola. As a result of the collaborations to rapidly identify, isolate, and manage Ebola patients and the extensive preparations to prevent spread of EBOV, the United States is now better prepared to address the next global infectious disease threat.The activities summarized in this report would not have been possible without collaboration with many U.S. and international partners (http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/partners.html)

Unified Methods in Collecting, Preserving, and Archiving Coral Bleaching and Restoration Specimens to Increase Sample Utility and Interdisciplinary Collaboration

Coral reefs are declining worldwide primarily because of bleaching and subsequent mortality resulting from thermal stress. Currently, extensive efforts to engage in more holistic research and restoration endeavors have considerably expanded the techniques applied to examine coral samples. Despite such advances, coral bleaching and restoration studies are often conducted within a specific disciplinary focus, where specimens are collected, preserved, and archived in ways that are not always conducive to further downstream analyses by specialists in other disciplines. This approach may prevent the full utilization of unexpended specimens, leading to siloed research, duplicative efforts, unnecessary loss of additional corals to research endeavors, and overall increased costs. A recent US National Science Foundation-sponsored workshop set out to consolidate our collective knowledge across the disciplines of Omics, Physiology, and Microscopy and Imaging regarding the methods used for coral sample collection, preservation, and archiving. Here, we highlight knowledge gaps and propose some simple steps for collecting, preserving, and archiving coral-bleaching specimens that can increase the impact of individual coral bleaching and restoration studies, as well as foster additional analyses and future discoveries through collaboration. Rapid freezing of samples in liquid nitrogen or placing at −80 °C to −20 °C is optimal for most Omics and Physiology studies with a few exceptions; however, freezing samples removes the potential for many Microscopy and Imaging-based analyses due to the alteration of tissue integrity during freezing. For Microscopy and Imaging, samples are best stored in aldehydes. The use of sterile gloves and receptacles during collection supports the downstream analysis of host-associated bacterial and viral communities which are particularly germane to disease and restoration efforts. Across all disciplines, the use of aseptic techniques during collection, preservation, and archiving maximizes the research potential of coral specimens and allows for the greatest number of possible downstream analyses

Breast cancer stroma frequently recruits fetal derived cells during pregnancy

Breast carcinomas associated with pregnancy display a high frequency of inflammatory types, multifocal lesions and lymph node metastasis. Because pregnancy results in transfer to mothers of foetal stem cells that can migrate and differentiate into various tissues, we addressed the issue of whether such cells are present in breast carcinoma associated with pregnancy

Divergent Immune Responses in Behaviorally-Inhibited vs. Non-Inhibited Male Rats

Stable behavioral traits (temperament, personality) often predict health outcomes. Temperament-specific differences in immune function could explain temperament-specific health outcomes, however, we have limited information on whether immune function varies by personality. In the present study, we examined the relationship between a basic behavioral trait (behavioral-inhibition vs. non-inhibition) and two immune responses (innate inflammation and delayed-type hypersensitivity, DTH) in a rodent model. In humans, behavioral inhibition (fearful temperament) is associated with altered stress physiology and allergies. In laboratory rats, the trait is associated with elevated glucocorticoid production. We hypothesized that behavioral inhibition is associated with glucocorticoid resistance and dampened T-helper 1 cell responses often associated with chronic stress and allergies. Further, this immune profile would predict poorly-regulated innate inflammation and dampened DTH. In male Sprague-Dawley rats, we quantified consistent behavioral phenotypes by measuring latency to contact two kinds of novelty (object vs. social), then measured lipopolysaccharide(LPS)-induced innate inflammation or keyhole limpet hemocyanin(KLH)-induced DTH. Behaviorally-inhibited rats had heightened glucocorticoid and interleukin-6 responses to a low/moderate dose of LPS and reduced DTH swelling to KLH re-exposure compared to non-inhibited rats. These results suggest that behavioral inhibition is associated with a glucocorticoid resistant state with poorly regulated innate inflammation and dampened cell-mediated immune responses. This immune profile may be associated with exaggerated T-helper 2 responses, which could set the stage for an allergic/asthmatic/atopic predisposition in inhibited individuals. Human and animal models of temperament-specific immune responses represent an area for further exploration of mechanisms involved in individual differences in health