Risk factors for alloimmunisation in the general patient population

For hospitals providing services to regional populations, difficulties are associated with transferred patients with poorly communicated medical history and a risk of alloimmunisation. Identification of patients at risk would assist in treatment planning. A retrospective study of alloimmunised patients was undertaken, comparing the demographics and diagnoses of this population with a control patient population. A preponderance of diagnoses of Sepsis, Haematological Malignancy, GIT Bleeds and Renal Failure was demonstrated in the alloimmunised population. Consistent with prior studies, RhD negative patients and female patients were over-represented in the study group, which was also on average significantly older

DNA immunization using a non-viral promoter

AbstractMost DNA vaccines rely on strong viral promoters to optimize levels of transgene expression. Some studies have demonstrated that the potency of viral promoters does not necessarily correlate with DNA vaccine efficacy in vivo. This has partly been attributed to downregulation of these promoters by cytokines such as interferon γ induced by the CpG motives of these vaccines. In an attempt to avoid downregulation of viral promoters by IFN-γ, we tested vaccine vectors driven by the MHC class II promoter. To enhance the activity of this promoter, another plasmid expressing the human MHC class II transactivator driven by a viral promoter, the native IFN-γ inducible CIITA type IV promoter (PIV) or a synthetic promoter containing IFN-γ inducible elements was co-inoculated. Our data show that a non-viral promoter such as the MHC class II promoter tested in this study can indeed be used in DNA vaccines

Vaccine Development for Prescottella Equi

AbstractPrescottella equi (formerly Rhodococcus equi) is an intracellular pathogen that causes pyogranulomatous pneumonia in Thoroughbred foals. There is currently no vaccine available for the prevention of this disease in foals despite years of research. Cell mediated immunity is considered crucial for overcoming an infection caused by this pathogen. The virulence associated protein (VapA) is a well characterized immunogenic protein associated with this pathogen and was used to develop DNA and recombinant protein vaccines in this study. Vaccine candidates and live P. equi based vaccine were tested in BALB/c mice. Mice were challenged with virulent P.equi 2 weeks following the last boost and IgG subtypes and bacterial clearance from spleen and liver determined. The DNA vaccine elicited a significant IgG2a response indicating a Th1 biased immune response. The IFN gamma response from DNA and recombinant VapA vaccinated mice was moderate. The results of the challenge study showed that neither the recombinant VapA protein nor DNA vaccine enhanced clearance of P. equi in this model

Microbial vodcasting – supplementing laboratory time with vodcasts of key microbial skills

First-year microbiology practical classes can be chaotic environments with more than 30 inexperienced laboratory users in close proximity to microbial cultures and flames from bunsen burners. Whilst Charles Sturt University (CSU) prides itself on giving first year students extensive hands-on experience, time constraints and class size can make it difficult to ensure every student receives adequate individual attention as required for the development of competency in key microbiological skills. To address these issues, we provided an introductory microbiology class (N=277) with short videos demonstrating key microbiological skills. The subject serves eight courses: Clinical Science, Forensic Biotechnology, Health Science (Nutrition and Dietetics), Medical Science, Pharmacy, Science and Animal Science. Further, 29 of the 277 students were distance education students who completed the practical component during a four-day residential school. The vodcasts were designed to give each student access to demonstration of key microbiological skills as performed by a highly-skilled individual. Towards the end of the semester, a survey was administered to all students to gauge their use of the vodcasts and their perceptions of how the vodcasts assisted their development of skills or understanding of key concepts. This paper describes the process of developing and delivering the vodcasts and provides an evaluation of their suitability as identified by their consumers, the students

Highly integrated workflows for exploring cardiovascular conditions: Exemplars of precision medicine in Alzheimer's disease and aortic dissection = Processus à haut degré d’intégration pour l’étude de troubles cardiovasculaires : exemples de médecine de précision appliquée à la maladie d’Alzheimer et à la dissection aortique

For precision medicine to be implemented through the lens of in silico technology, it is imperative that biophysical research workflows offer insight into treatments that are specific to a particular illness and to a particular subject. The boundaries of precision medicine can be extended using multiscale, biophysics-centred workflows that consider the fundamental underpinnings of the constituents of cells and tissues and their dynamic environments. Utilising numerical techniques that can capture the broad spectrum of biological flows within complex, deformable and permeable organs and tissues is of paramount importance when considering the core prerequisites of any state-of-the-art precision medicine pipeline. In this work, a succinct breakdown of two precision medicine pipelines developed within two Virtual Physiological Human (VPH) projects are given. The first workflow is targeted on the trajectory of Alzheimer's Disease, and caters for novel hypothesis testing through a multicompartmental poroelastic model which is integrated with a high throughput imaging workflow and subject-specific blood flow variability model. The second workflow gives rise to the patient specific exploration of Aortic Dissections via a multi-scale and compliant model, harnessing imaging, computational fluid-dynamics (CFD) and dynamic boundary conditions. Results relating to the first workflow include some core outputs of the multiporoelastic modelling framework, and the representation of peri-arterial swelling and peri-venous drainage solution fields. The latter solution fields were statistically analysed for a cohort of thirty-five subjects (stratified with respect to disease status, gender and activity level). The second workflow allowed for a better understanding of complex aortic dissection cases utilising both a rigid-wall model informed by minimal and clinically common datasets as well as a moving-wall model informed by rich datasets. / Pour que la médecine actuelle puisse profiter de la technologie in silico, il est impératif que les flux de recherche biophysique offrent un aperçu précis des traitements spécifiques à une maladie particulière et à un sujet particulier. Les limites de la médecine peuvent être repoussées à l’aide de flux de travail multi-échelles, centrés sur la biophysique, qui tiennent compte des constituants fondamentaux des cellules et des tissus, et de leurs environnements dynamiques. L’utilisation de techniques numériques permettant de capter le large spectre des flux biologiques au sein d’organes et de tissus complexes, déformables et perméables est d’une importance capitale lorsqu’il s’agit d’examiner les conditions essentielles de tout pipeline médical de précision de pointe. Dans ce travail, une analyse succinte de deux pipelines de médecine de précision développés dans le cadre de deux projets VPH (Virtual Physiological Human) est donnée. Le premier flux de travail se concentre sur la trajectoire de la maladie d’Alzheimer et permet de tester de nouvelles hypothèses au moyen d’un modèle poroélastique à plusieurs compartiments qui est intégré à un flux de travail d’imagerie à haut débit et à un modèle de variabilité du débit sanguin spécifique au sujet. Le deuxième flux de travail donne lieu à l’exploration spécifique des dissections aortiques chez le patient par le biais d’un modèle multi-échelle conforme, exploitant l’imagerie, la dynamique des fluides computationnelle (CFD) et les conditions limites dynamiques. Les résultats relatifs au premier flux de travail comprennent certains des principaux extrants du cadre de modélisation multiporoélastique et la représentation des zones de gonflement péri-artériel et de solution de drainage périveineux. Ces dernières zones de solutions ont été analysées statistiquement sur une cohorte de trente-cinq sujets (stratifiés en fonction de l’état pathologique, du sexe et du niveau d’activité). Le deuxième flux de travail a permis de mieux comprendre les cas complexes de dissection aortique à l’aide d’un modèle à parois rigides fondé sur des ensembles de données minimales et cliniquement communes et d’un modèle à parois mobiles reposant sur de riches données

“Affimer” synthetic protein scaffolds block oxidized LDL binding to the LOX-1 scavenger receptor and inhibit ERK1/2 activation

In multicellular organisms, a variety of lipid-protein particles control the systemic flow of triacylglycerides, cholesterol, and fatty acids between cells in different tissues. The chemical modification by oxidation of these particles can trigger pathological responses, mediated by a group of membrane proteins termed scavenger receptors. The lectin-like oxidized low-density lipoprotein (LOX-1) scavenger receptor binds to oxidized low-density lipoprotein (oxLDL) and mediates both signaling and trafficking outcomes. Here, we identified five synthetic proteins termed Affimers from a phage display library, each capable of binding recombinant LOX-1 extracellular (oxLDL-binding) domain with high specificity. These Affimers, based on a phytocystatin scaffold with loop regions of variable sequence, were able to bind to the plasma membrane of HEK293T cells exclusively when human LOX-1 was expressed. Binding and uptake of fluorescently labeled oxLDL by the LOX-1-expressing cell model was inhibited with subnanomolar potency by all 5 Affimers. ERK1/2 activation, stimulated by oxLDL binding to LOX-1, was also significantly inhibited (p < 0.01) by preincubation with LOX-1-specific Affimers, but these Affimers had no direct agonistic effect. Molecular modeling indicated that the LOX-1-specific Affimers bound predominantly via their variable loop regions to the surface of the LOX-1 lectin-like domain that contains a distinctive arrangement of arginine residues previously implicated in oxLDL binding, involving interactions with both subunits of the native, stable scavenger receptor homodimer. These data provide a new class of synthetic tools to probe and potentially modulate the oxLDL/LOX-1 interaction that plays an important role in vascular disease

In silico design and biological evaluation of a dual specificity kinase inhibitor targeting cell cycle progression and angiogenesis

Methodology: We have utilized a rational in silico-based approach to demonstrate the design and study of a novel compound that acts as a dual inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2) and cyclin-dependent kinase 1 (CDK1). This compound acts by simultaneously inhibiting pro-Angiogenic signal transduction and cell cycle progression in primary endothelial cells. JK-31 displays potent in vitro activity against recombinant VEGFR2 and CDK1/cyclin B proteins comparable to previously characterized inhibitors. Dual inhibition of the vascular endothelial growth factor A (VEGF-A)-mediated signaling response and CDK1-mediated mitotic entry elicits anti-Angiogenic activity both in an endothelial-fibroblast co-culture model and a murine ex vivo model of angiogenesis

VEGF-A isoforms differentially regulate ATF-2-dependent VCAM-1 gene expression and endothelial-leukocyte interactions

Vascular endothelial growth factor A (VEGF-A) regulates many aspects of vascular physiology. VEGF-A stimulates signal transduction pathways that modulate endothelial outputs such as cell migration, proliferation, tubulogenesis, and cell-cell interactions. Multiple VEGF-A isoforms exist, but the biological significance of this is unclear. Here we analyzed VEGF-A isoform-specific stimulation of VCAM-1 gene expression, which controls endothelial-leukocyte interactions, and show that this is dependent on both ERK1/2 and activating transcription factor-2 (ATF-2). VEGF-A isoforms showed differential ERK1/2 and p38 MAPK phosphorylation kinetics. A key feature of VEGF-A isoform-specific ERK1/2 activation and nuclear translocation was increased phosphorylation of ATF-2 on threonine residue 71 (T71). Using reverse genetics, we showed ATF-2 to be functionally required for VEGF-A-stimulated endothelial VCAM-1 gene expression. ATF-2 knockdown blocked VEGF-A-stimulated VCAM-1 expression and endothelial-leukocyte interactions. ATF-2 was also required for other endothelial cell outputs, such as cell migration and tubulogenesis. In contrast, VCAM-1 was essential only for promoting endothelial-leukocyte interactions. This work presents a new paradigm for understanding how soluble growth factor isoforms program complex cellular outputs and responses by modulating signal transduction pathways

Highly integrated workflows for exploring cardiovascular conditions: Exemplars of precision medicine in Alzheimer's disease and aortic dissection

For precision medicine to be implemented through the lens of in silico technology, it is imperative that biophysical research workflows offer insight into treatments that are specific to a particular illness and to a particular subject. The boundaries of precision medicine can be extended using multiscale, biophysics-centred workflows that consider the fundamental underpinnings of the constituents of cells and tissues and their dynamic environments. Utilising numerical techniques that can capture the broad spectrum of biological flows within complex, deformable and permeable organs and tissues is of paramount importance when considering the core prerequisites of any state-of-the-art precision medicine pipeline. In this work, a succinct breakdown of two precision medicine pipelines developed within two Virtual Physiological Human (VPH) projects are given. The first workflow is targeted on the trajectory of Alzheimer's Disease, and caters for novel hypothesis testing through a multicompartmental poroelastic model which is integrated with a high throughput imaging workflow and subject-specific blood flow variability model. The second workflow gives rise to the patient specific exploration of Aortic Dissections via a multi-scale and compliant model, harnessing imaging, computational fluid-dynamics (CFD) and dynamic boundary conditions. Results relating to the first workflow include some core outputs of the multiporoelastic modelling framework, and the representation of peri-arterial swelling and peri-venous drainage solution fields. The latter solution fields were statistically analysed for a cohort of thirty-five subjects (stratified with respect to disease status, gender and activity level). The second workflow allowed for a better understanding of complex aortic dissection cases utilising both a rigid-wall model informed by minimal and clinically common datasets as well as a moving-wall model informed by rich datasets