Soluble mediators in platelet concentrates modulate dendritic cell inflammatory responses in an experimental model of transfusion

The transfusion of platelet concentrates (PCs) is widely used to treat thrombocytopenia and severe trauma. Ex vivo storage of PCs is associated with a storage lesion characterized by partial platelet activation and the release of soluble mediators, such as soluble CD40 ligand (sCD40L), RANTES, and interleukin (IL)-8. An in vitro whole blood culture transfusion model was employed to assess whether mediators present in PC supernatants (PC-SNs) modulated dendritic cell (DC)-specific inflammatory responses (intracellular staining) and the overall inflammatory response (cytometric bead array). Lipopolysaccharide (LPS) was included in parallel cultures to model the impact of PC-SNs on cell responses following toll-like receptor-mediated pathogen recognition. The impact of both the PC dose (10%, 25%) and ex vivo storage period was investigated [day 2 (D2), day 5 (D5), day 7 (D7)]. PC-SNs alone had minimal impact on DC-specific inflammatory responses and the overall inflammatory response. However, in the presence of LPS, exposure to PC-SNs resulted in a significant dose associated suppression of the production of DC IL-12, IL-6, IL-1a, tumor necrosis factor-a (TNF-a), and macrophage inflammatory protein (MIP)-1b and storage-associated suppression of the production of DC IL-10, TNF-a, and IL-8. For the overall inflammatory response, IL-6, TNF-a, MIP-1a, MIP-1b, and inflammatory protein (IP)-10 were significantly suppressed and IL-8, IL-10, and IL-1b significantly increased following exposure to PC-SNs in the presence of LPS. These data suggest that soluble mediators present in PCs significantly suppress DC function and modulate the overall inflammatory response, particularly in the presence of an infectious stimulus. Given the central role of DCs in the initiation and regulation of the immune response, these results suggest that modulation of the DC inflammatory profile is a probable mechanism contributing to transfusion-related complications.

Development of a novel experimental model to investigate the influence of mechanics on bone healing

This dissertation proposed a novel experimental model combining a defect configuration with an active instrumented fixation device to investigate the influence of mechanics on bone healing. The proposed defect configuration aimed to minimise physiological loading within an experimental fracture gap and the instrumented fixator was used for the application of controlled displacements and in vivo stiffness monitoring of the healing process. This thesis has provided a novel approach to advance current knowledge and understanding of mechanobiology, which has been limited in previous experimental models.

A new model to study healing of a complex femur fracture with concurrent soft tissue injury in sheep

High energy bone fractures resulting from impact trauma are often accompanied by subcutaneous soft tissue injuries, even if the skin remains intact. There is evidence that such closed soft tissue injuries affect the healing of bone fractures, and vice versa. Despite this knowledge, most impact trauma studies in animals have focussed on bone fractures or soft tissue trauma in isolation. However, given the simultaneous impact on both tissues a better understanding of the interaction between these two injuries is necessary to optimise clinical treatment. The aim of this study was therefore to develop a new experimental model and characterise, for the first time, the healing of a complex fracture with concurrent closed soft tissue trauma in sheep. A pendulum impact device was designed to deliver a defined and standardised impact to the distal thigh of sheep, causing a reproducible contusion injury to the subcutaneous soft tissues. In a subsequent procedure, a reproducible femoral butterfly fracture (AO C3-type) was created at the sheep’s femur, which was initially stabilised for 5 days by an external fixator construct to allow for soft tissue swelling to recede, and ultimately in a bridging construct using locking plates. The combined injuries were applied to twelve sheep and the healing observed for four or eight weeks (six animals per group) until sacrifice. The pendulum impact led to a moderate to severe circumferential soft tissue injury with significant bruising, haematomas and partial muscle disruptions. Posttraumatic measurements showed elevated intra-compartmental pressure and circulatory tissue breakdown markers, with recovery to normal, pre-injury values within four days. Clinically, no neurovascular deficiencies were observed. Bi-weekly radiological analysis of the healing fractures showed progressive callus healing over time, with the average number of callus bridges increasing from 0.4 at two weeks to 4.2 at eight weeks. Biomechanical testing after sacrifice showed increasing torsional stiffness between four and eight weeks healing time from 10% to 100%, and increasing ultimate torsional strength from 10% to 64% (relative to the contralateral control limb). Our results demonstrate the robust healing of a complex femur fracture in the presence of a severe soft tissue contusion injury in sheep and demonstrate the establishment of a clinically relevant experimental model, for research aimed at improving the treatment of bone fractures accompanied by closed soft tissue injuries.

Effects of hyperbaric oxygen and inducible nitric oxide synthase inhibitor treatment on femoral head osteonecrosis in a rat model

Introduction: Apoptosis is the final destiny of many cells in the body, though this process has been observed in some pathological processes. One of these pathological processes is femoral head non-traumatic osteonecrosis. Among many pro/anti-apoptotic factors, nitric oxide has recently been an area of further interest. Osteocyte apoptosis and its relation to pro-apoptotic action invite further research, and the inducible form of nitric oxide synthase (iNOS)—which produces a high concentration of nitric oxide—has been flagged. The aim of this study was to investigate the effect of hyperbaric oxygen (HBO) and inducible NOS suppressor (Aminoguanidine) in prevention of femoral head osteonecrosis in an experimental model of osteonecrosis in spontaneous hypertensive rats (SHRs). Methods: After animal ethic approval 34 SHR rats were divided into four groups. Ten rats were allocated to the control group without any treatment, and eight rats were allocated to three treatment groups namely: HBO, Aminoguanidine (AMG), and the combination of HBO and AMG treatments (HBO+AMG). The HBO group received 250 kPa of oxygen via hyperbaric chamber for 30 days started at their 5th week of life; the AMG group received 1mg/ml of AMG in drinking water from the fifth week till the 17th week of life; and the last group received a combination of these treatments. Rats were sacrificed at the end of the 17th week of life and both femurs were analysed for evidence of osteonecrosis using Micro CT scan and H&E staining. Also, osteocyte apoptosis and the presence of two different forms of NOS (inducible (iNOS) and endothelial (eNOS)) were analysed by immunostaining and apoptosis staining (Hoechst and TUNEL). Results: Bone morphology of metaphyseal and epiphyseal area of all rats were investigated and analysed. Micro CT findings revealed significantly higher mean fractional trabecular bone volume (FBV) of metaphyseal area in untreated SHRs compared with all other treatments (HBO, P<0.05, HBO+AMG, P<0.005, and AMG P<0.001). Bone surface to volume ratio also significantly increased with HBO+AMG and AMG treatments when compared with the control group (18.7 Vs 20.8, P<0.05, and 18.7 Vs 21.1, P<0.05). Epiphyseal mean FBV did not change significantly among groups. In the metaphyseal area, trabecular thickness and numbers significantly decreased with AMG treatment, while trabecular separation significantly increased with both AMG and HBO+AMG treatment. Histological ratio of no ossification and osteonecrosis was 37.5%, 43.7%, 18.7% and 6.2% of control, HBO, HBO+AMG and AMG groups respectively with only significant difference observed between HBO and AMG treatment (P<0.01). High concentration of iNOS was observed in the region of osteonecrosis while there was no evidence of eNOS activity around that region. In comparison with the control group, the ratio of osteocyte apoptosis significantly reduced in AMG treatment (P<0.005). We also observed significantly fewer apoptotic osteocytes in AMG group comparing with HBO treatment (P<0.05). Conclusion: None of our treatments prevents osteonecrosis at the histological or micro CT scan level. High concentration of iNOS in the region of osteonecrosis and significant reduction of osteocyte apoptosis with AMG treatment were supportive of iNOS modulating osteocyte apoptosis in SHRs.

Upregulated MT1-MMP/TIMP-2 axis in the TSU-Pr1-B1/B2 model of metastatic progression in transitional cell carcinoma of the bladder

Muscle invasive transitional cell carcinoma (TCC) of the bladder is associated with a high frequency of metastasis, resulting in poor prognosis for patients presenting with this disease. Models that capture and demonstrate step-wise enhancement of elements of the human metastatic cascade on a similar genetic background are useful research tools. We have utilized the transitional cell carcinoma cell line TSU-Pr1 to develop an in vivo experimental model of bladder TCC metastasis. TSU-Pr1 cells were inoculated into the left cardiac ventricle of SCID mice and the development of bone metastases was monitored using high resolution X-ray. Tumor tissue from a single bone lesion was excised and cultured in vitro to generate the TSU-Pr1-B1 subline. This cycle was repeated with the TSU-Pr1-B1 cells to generate the successive subline TSU-Pr1-B2. DNA profiling and karyotype analysis confirmed the genetic relationship of these three cell lines. In vitro, the growth rate of these cell lines was not significantly different. However, following intracardiac inoculation TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2 exhibited increasing metastatic potential with a concomitant decrease in time to the onset of radiologically detectable metastatic bone lesions. Significant elevations in the levels of mRNA expression of the matrix metalloproteases (MMPs) membrane type 1-MMP (MT1-MMP), MT2-MMP and MMP-9, and their inhibitor, tissue inhibitor of metalloprotease-2 (TIMP-2), across the progressively metastatic cell lines, were detected by quantitative PCR. Given the role of MT1-MMP and TIMP-2 in MMP-2 activation, and the upregulation of MMP-9, these data suggest an important role for matrix remodeling, particularly basement membrane, in this progression. The TSU-Pr1-B1/B2 model holds promise for further identification of important molecules.

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 feasibility of vibration analysis as a technique to detect osseointegration of transfemoral implants

One of the main causes of above knee or transfemoral amputation (TFA) in the developed world is trauma to the limb. The number of people undergoing TFA due to limb trauma, particularly due to war injuries, has been increasing. Typically the trauma amputee population, including war-related amputees, are otherwise healthy, active and desire to return to employment and their usual lifestyle. Consequently there is a growing need to restore long-term mobility and limb function to this population. Traditionally transfemoral amputees are provided with an artificial or prosthetic leg that consists of a fabricated socket, knee joint mechanism and a prosthetic foot. Amputees have reported several problems related to the socket of their prosthetic limb. These include pain in the residual limb, poor socket fit, discomfort and poor mobility. Removing the socket from the prosthetic limb could eliminate or reduce these problems. A solution to this is the direct attachment of the prosthesis to the residual bone (femur) inside the residual limb. This technique has been used on a small population of transfemoral amputees since 1990. A threaded titanium implant is screwed in to the shaft of the femur and a second component connects between the implant and the prosthesis. A period of time is required to allow the implant to become fully attached to the bone, called osseointegration (OI), and be able to withstand applied load; then the prosthesis can be attached. The advantages of transfemoral osseointegration (TFOI) over conventional prosthetic sockets include better hip mobility, sitting comfort and prosthetic retention and fewer skin problems on the residual limb. However, due to the length of time required for OI to progress and to complete the rehabilitation exercises, it can take up to twelve months after implant insertion for an amputee to be able to load bear and to walk unaided. The long rehabilitation time is a significant disadvantage of TFOI and may be impeding the wider adoption of the technique. There is a need for a non-invasive method of assessing the degree of osseointegration between the bone and the implant. If such a method was capable of determining the progression of TFOI and assessing when the implant was able to withstand physiological load it could reduce the overall rehabilitation time. Vibration analysis has been suggested as a potential technique: it is a non destructive method of assessing the dynamic properties of a structure. Changes in the physical properties of a structure can be identified from changes in its dynamic properties. Consequently vibration analysis, both experimental and computational, has been used to assess bone fracture healing, prosthetic hip loosening and dental implant OI with varying degrees of success. More recently experimental vibration analysis has been used in TFOI. However further work is needed to assess the potential of the technique and fully characterise the femur-implant system. The overall aim of this study was to develop physical and computational models of the TFOI femur-implant system and use these models to investigate the feasibility of vibration analysis to detect the process of OI. Femur-implant physical models were developed and manufactured using synthetic materials to represent four key stages of OI development (identified from a physiological model), simulated using different interface conditions between the implant and femur. Experimental vibration analysis (modal analysis) was then conducted using the physical models. The femur-implant models, representing stage one to stage four of OI development, were excited and the modal parameters obtained over the range 0-5kHz. The results indicated the technique had limited capability in distinguishing between different interface conditions. The fundamental bending mode did not alter with interfacial changes. However higher modes were able to track chronological changes in interface condition by the change in natural frequency, although no one modal parameter could uniquely distinguish between each interface condition. The importance of the model boundary condition (how the model is constrained) was the key finding; variations in the boundary condition altered the modal parameters obtained. Therefore the boundary conditions need to be held constant between tests in order for the detected modal parameter changes to be attributed to interface condition changes. A three dimensional Finite Element (FE) model of the femur-implant model was then developed and used to explore the sensitivity of the modal parameters to more subtle interfacial and boundary condition changes. The FE model was created using the synthetic femur geometry and an approximation of the implant geometry. The natural frequencies of the FE model were found to match the experimental frequencies within 20% and the FE and experimental mode shapes were similar. Therefore the FE model was shown to successfully capture the dynamic response of the physical system. As was found with the experimental modal analysis, the fundamental bending mode of the FE model did not alter due to changes in interface elastic modulus. Axial and torsional modes were identified by the FE model that were not detected experimentally; the torsional mode exhibited the largest frequency change due to interfacial changes (103% between the lower and upper limits of the interface modulus range). Therefore the FE model provided additional information on the dynamic response of the system and was complementary to the experimental model. The small changes in natural frequency over a large range of interface region elastic moduli indicated the method may only be able to distinguish between early and late OI progression. The boundary conditions applied to the FE model influenced the modal parameters to a far greater extent than the interface condition variations. Therefore the FE model, as well as the experimental modal analysis, indicated that the boundary conditions need to be held constant between tests in order for the detected changes in modal parameters to be attributed to interface condition changes alone. The results of this study suggest that in a clinical setting it is unlikely that the in vivo boundary conditions of the amputated femur could be adequately controlled or replicated over time and consequently it is unlikely that any longitudinal change in frequency detected by the modal analysis technique could be attributed exclusively to changes at the femur-implant interface. Therefore further development of the modal analysis technique would require significant consideration of the clinical boundary conditions and investigation of modes other than the bending modes.

Enriching students’ intellectual diet through inquiry based learning

Traditionally, Science education has stressed the importance of teaching students to conduct ‘scientific inquiry’, with the main focus being the experimental model of inquiry used by real world scientists. Current educational approaches using constructivist pedagogy recognise the value of inquiry as a method for promoting the development of deep understanding of discipline content. A recent Information Learning Activity undertaken by a Grade Eight Science class was observed to discover how inquiry based learning is implemented in contemporary Science education. By analysing student responses to questionnaires and assessment task outcomes, the author was able to determine the level of inquiry inherent in the activity and how well the model supported student learning and the development of students’ information literacy skills. Although students achieved well overall, some recommendations are offered that may enable teachers to better exploit the learning opportunities provided by inquiry based learning. Planning interventions at key stages of the inquiry process can assist students to learn more effective strategies for dealing with cognitive and affective challenges. Allowing students greater input into the selection of topic or focus of the activity may encourage students to engage more deeply with the learning task. Students are likely to experience greater learning benefit from access to developmentally appropriate resources, increased time to explore topics and multiple opportunities to undertake information searches throughout the learning activity. Finally, increasing the cognitive challenge can enhance both the depth of students’ learning and their information literacy skills.

Acquisition of estrogen independence and antiestrogen resistance in breast cancer : association with the invasive and metastatic phenotype

A significant percentage of human breast cancer (HBC) is dependent upon the ovarian hormone estrogen for its onset and progression. The presence or lack of estrogen receptors (ERs) in human breast cancer is an important determinant both of prognosis and of choice of treatment - a poorer prognosis being associated with ER–ve disease. Cell lines established from human breast cancer provide models for breast cancer in various stages of progression (Engel & Young 1978). When grown as tumors in athymic nude mice, these lines represent the major in vivo experimental model for HBC studies (Brünner et al 1987). The ease of both in vitro and in vivo maintenance, the human derivation of the tissue, and the similarities in plasma estrogen levels between ovariectomized nude mice and postmenopausal women (Seibert et al. 1983, Brünner et al. 1986), make the growth of human breast cancer cell lines in nude mice an attractive...

The effect of cryotherapy on the vascular regeneration following closed soft tissue trauma

INTRODUCTION Icing (cryotherapy) is being widely used for the treatment of closed soft tissue trauma (CSTT), such as those resulting from sport injuries. It is believed that cryotherapy induces vasoconstriction and through this mechanism reduces inflammation [1]. However, the impact of this technique on the healing of impaired vasculature and muscle injuries following trauma remains controversial. Recent evidence suggests that the muscle regeneration is delayed after cryotherapy [2]. Consequently, we aimed to investigate the effect of cryotherapy on the vascular morphology following CSTT using an experimental model in rats by contrast-enhanced micro-CT imaging. METHODS Fifty four rats were divided into three main groups: control (no injury, n=6), sham (CSTT but no icing treatment, n=24) and icing (CSTT, treated with one session of ice block massaged directly on the injured muscle for 20 minutes, n=24). The CSTT was induced to the left thigh (Biceps Femoris) of anaesthetised rats (Male, Wistar) to create a standardized and reproducible vascular and muscle injury using an impact device [3]. Following trauma, animals were euthanized after 1, 3, 7, and 28 days healing time (n=6 for each time point). For a three-dimensional vascular morphological assessment, the blood vessels of euthanised rats were flushed with heparinised saline and then perfused with a radio-opaque contrast agent (Microfil, MV 122, Flowtech, USA) using an infusion pump. Both hind-limbs were dissected, and then the injured and non-injured limbs were imaged using a micro-CT scanner (µCT 40, Scanco Medical, Switzerland) and total volume of the perfused blood vessels (TVV) was calculated. More detailed morphological parameters such as vessel volume (VV), diameter (VD), spacing (VSp), number (VN) and connectivity (VConn) were quantified through high resolution (6 µm), micro-CT-scanned biopsy samples (diameter: 8mm) taken directly from the region of the injured muscles. The biopsies were then analysed histologically to confirm the results derived from contrast-enhanced micro-CT imaging. RESULTS AND DISCUSSION The TVV was significantly higher in the injured legs compared to the non-injured legs at day 1 and 7 in the sham group and at day 28 in both sham and icing groups. The biopsies from the injured legs of the icing group showed a significant reduction in VV, VN, VD, VConn and an increase in VSp compared to those in the sham and control groups at days 1, 3 and 7, post injury. While the injured legs of the sham group exhibited a decrease in VN and VConn 28 days post trauma, indicating a return to the original values prior to trauma, these parameters had increased in the icing group (Figure 1). Also, at day 1 post injury, VV and VD of the injured legs were significantly higher in the sham group compared to the icing group, which may be attributed to the effect of vasoconstriction induced by icing. Further histomorphological evaluation of day 1 post injury, indicated that although cryotherapy significantly reduced the injury size and influx of inflammatory cells, including macrophages and neutrophils, a delay in vascular and muscle fiber regeneration was found at later time points confirming other reports from the literature [2]. CONCLUSIONS We have demonstrated using micro-CT imaging that the vascular morphology changes after CSTT, and that its recovery is affected by therapeutic modalities such as icing. This may be useful for the development of future clinical monitoring, diagnosis and treatment of CSTT. While icing reduces the swelling after trauma, our results suggest that it may delay the recovery of the vasculature in the injured tissue.

Disturbed microtubule function and induction of micronuclei by chelate complexes of mercury(II)

Interactions of mercury(II) with the microtubule network of cells may lead to genotoxicity. Complexation of mercury(II) with EDTA is currently being discussed for its employment in detoxification processes of polluted sites. This prompted us to re-evaluate the effects of such complexing agents on certain aspects of mercury toxicity, by examining the influences of mercury(II) complexes on tubulin assembly and kinesin-driven motility of microtubules. The genotoxic effects were studied using the micronucleus assay in V79 Chinese hamster fibroblasts. Mercury(II) complexes with EDTA and related chelators interfered dose-dependently with tubulin assembly and microtubule motility in vitro. The no-effect-concentration for assembly inhibition was 1 μM of complexed Hg(II), and for inhibition of motility it was 0.05 μM, respectively. These findings are supported on the genotoxicity level by the results of the micronucleus assay, with micronuclei being induced dose-dependently starting at concentrations of about 0.05 μM of complexed Hg(II). Generally, the no-effect-concentrations for complexed mercury(II) found in the cell-free systems and in cellular assays (including the micronucleus test) were identical with or similar to results for mercury tested in the absence of chelators. This indicates that mercury(II) has a much higher affinity to sulfhydryls of cytoskeletal proteins than to this type of complexing agents. Therefore, the suitability of EDTA and related compounds for remediation of environmental mercury contamination or for other detoxification purposes involving mercury has to be questioned.

Altered immunoglobulin E diversity and regulation of allergic inflammation in asthma

The clinical efficacy of anti-immunoglobulin E (IgE) therapy indicates a central role for IgE in perpetuation of allergic inflammatory diseases. Omalizumab is now uti- lized in treatment of a wide variety of allergic conditions including severe asthma, allergic rhinitis, atopic dermati- tis, food allergy and urticaria either alone or adjunct with other therapies such as steroid administration or allergen- specific immunotherapy [1, 2]. Current research activity is focused on the cellular and molecular mechanisms by which IgE influences the immunopathogenesis of allergic disease [3]. Increased knowledge of how IgE exerts its effects will underpin effective clinical use of anti-IgE treatment. In this issue Kerzel et al. [4] investigate the effects of altered antibo dy repertoire on the outcomes of an experimental model of allergic asthma.

Flow evaluation and hemolysis analysis of BVAD centrifugal blood pump by computational fluids dynamics

Computational fluid dynamics (CFD) and particle image velocimetry (PIV) are commonly used techniques to evaluate the flow characteristics in the development stage of blood pumps. CFD technique allows rapid change to pump parameters to optimize the pump performance without having to construct a costly prototype model. These techniques are used in the construction of a bi-ventricular assist device (BVAD) which combines the functions of LVAD and RVAD in a compact unit. The BVAD construction consists of two separate chambers with similar impellers, volutes, inlet and output sections. To achieve the required flow characteristics of an average flow rate of 5 l/min and different pressure heads (left – 100mmHg and right – 20mmHg), the impellers were set at different rotating speeds. From the CFD results, a six-blade impeller design was adopted for the development of the BVAD. It was also observed that the fluid can flow smoothly through the pump with minimum shear stress and area of stagnation which are related to haemolysis and thrombosis. Based on the compatible Reynolds number the flow through the model was calculated for the left and the right pumps. As it was not possible to have both the left and right chambers in the experimental model, the left and right pumps were tested separately.