The importance of the tissue transglutaminase-fibronectin heterocomplex in the RGD-independent cell adhesion and fibronectin matrix deposition

Tissue transglutaminase (TG2) has been reported as a wound response protein. Once over-expressed by cells under stress such as during wound healing or following tissue damage, TG2 can be secreted and deposited into extracellular matrix, where it forms a heterocomplex (TG-FN) with the abundant matrix protein fibronectin (FN). A further cellular response elicited after tissue damage is that of matrix remodelling leading to the release of the Arg-Gly-Asp (RGD) containing matrix fragments by matrix matelloproteinases (MMPs). These peptides are able to block the interaction between integrin cell surface receptors and ECM proteins, leading to the loss of cell adhesion and ultimately Anoikis. This study provides a mechanism for TG2, as a stress-induced matrix protein, in protecting the cells from the RGD-dependent loss of cell adhesion and rescuing the cells from Anoikis. Mouse fibroblasts were used as a major model for this study, including different types of cell surface receptor knockout mouse embryonic fibroblasts (MEFs) (such as syndecan-4, a5, ß1 or ß3 integrins). In addition specific syndecan-2 targetting siRNAs, ß1 integrin and a4ß1 integrin functional blocking antibodies, and a specific targeting peptide against a5ß1 integrin A5-1 were used to investigate the involvement of these receptors in the RGD-independent cell adhesion on TG-FN. Crucial for TG-FN to compensate the RGD-independent cell adhesion and actin cytoskeleton formation is the direct interaction between the heparan sulfate chains of syndecan-4 and TG2, which elicits the inside-out signalling of a5ß1 integrin and the intracellular activation of syndecan-2 by protein kinase C a (PKCa). By using specific inhibitors, a cell-permeable inhibiting peptide and the detection of the phosphorylation sites for protein kinases and/or the translocation of PKCa via Western blotting, the activation of PKCa, focal adhesion kinase (FAK), ERK1/2 and Rho kinase (ROCK) were confirmed as downstream signalling molecules. Importantly, this study also investigated the influence of TG-FN on matrix turnover and demonstrated that TG-FN can restore the RGD-independent FN deposition process via an a5ß1 integrin and syndecan-4/2 co-signalling pathway linked by PKCa in a transamidating-independent manner. These data provide a novel function for TG2 in wound healing and matrix turnover which is a key event in a number of both physiological and pathological processes.

In vivo tissue diagnosis for myocardial infarction using optical spectroscopy with novel spectral interpretation algorithms

In recent decades, the rapid development of optical spectroscopy for tissue diagnosis has been indicative of its high clinical value. The goal of this research is to prove the feasibility of using diffuse reflectance spectroscopy and fluorescence spectroscopy to assess myocardial infarction (MI) in vivo. The proposed optical technique was designed to be an intra-operative guidance tool that can provide useful information about the condition of an infarct for surgeons and researchers. ^ In order to gain insight into the pathophysiological characteristics of an infarct, two novel spectral analysis algorithms were developed to interpret diffuse reflectance spectra. The algorithms were developed based on the unique absorption properties of hemoglobin for the purpose of retrieving regional hemoglobin oxygenation saturation and concentration data in tissue from diffuse reflectance spectra. The algorithms were evaluated and validated using simulated data and actual experimental data. ^ Finally, the hypothesis of the study was validated using a rabbit model of MI. The mechanism by which the MI was induced was the ligation of a major coronary artery of the left ventricle. Three to four weeks after the MI was induced, the extent of myocardial tissue injury and the evolution of the wound healing process were investigated using the proposed spectroscopic methodology as well as histology. The correlations between spectral alterations and histopathological features of the MI were analyzed statistically. ^ The results of this PhD study demonstrate the applicability of the proposed optical methodology for assessing myocardial tissue damage induced by MI in vivo. The results of the spectral analysis suggest that connective tissue proliferation induced by MI significantly alter the characteristics of diffuse reflectance and fluorescence spectra. The magnitudes of the alterations could be quantitatively related to the severity and extensiveness of connective tissue proliferation.^

Chilling damage in a changing climate in coastal landscapes of the subtropical zone: a case study from south Florida

Freeze events significantly influence landscape structure and community composition along subtropical coastlines. This is particularly true in south Florida, where such disturbances have historically contributed to patch diversity within the mangrove forest, and have played a part in limiting its inland transgression. With projected increases in mean global temperatures, such instances are likely to become much less frequent in the region, contributing to a reduction in heterogeneity within the mangrove forest itself. To understand the process more clearly, we explored the dynamics of a Dwarf mangrove forest following two chilling events that produced freeze-like symptoms, i.e., leaf browning, desiccation, and mortality, and interpreted the resulting changes within the context of current winter temperatures and projected future scenarios. Structural effects from a 1996 chilling event were dramatic, with mortality and tissue damage concentrated among individuals comprising the Dwarf forest's low canopy. This disturbance promoted understory plant development and provided an opportunity for Laguncularia racemosa to share dominance with Rhizophora mangle. Mortality due to the less severe 2001 event was greatest in the understory, probably because recovery of the protective canopy following the earlier freeze was still incomplete. Stand dynamics were static over the same period in nearby unimpacted sites. The probability of reaching temperatures as low as those recorded at a nearby meteorological station (≤3 °C) under several warming scenarios was simulated by applying 1° incremental temperature increases to a model developed from a 42-year temperature record. According to the model, the frequency of similar chilling events decreased from once every 1.9 years at present to once every 3.4 and 32.5 years with 1 and 4 °C warming, respectively. The large decrease in the frequency of these events would eliminate an important mechanism that maintains Dwarf forest structure, and promotes compositional diversity.

Wounds that heal and wounds that don't - The role of the IL-33/ST2 pathway in tissue repair and tumorigenesis

IL-33 is a member of the IL-1 family of cytokines. IL-33 is predominantly located within the nucleus of cells where it plays a role in gene regulation. Given the right combination of signals and cellular damage, stored IL-33 is released from the cell where it can interact with its receptor ST2, triggering danger-associated responses and act as a cellular "alarmin". Whilst IL-33/ST2 signalling has been shown to induce potent pro-inflammatory responses that can be detrimental in certain disease states, a dichotomous, protective role of IL-33 in promoting wound healing has also emerged in multiple tissues types. This review will explore the current literature concerning this homeostatic role of IL-33/ST2 in tissue repair and also review its role in uncontrolled wound responses as seen in both fibrosis and tumorigenesis.

Inhibition of myeloperoxidase-mediated hypochlorous acid production by nitroxides

Tissue damage resulting from the extracellular production of HOCl (hypochlorous acid) by the MPO (myeloperoxidase)-hydrogen peroxide-chloride system of activated phagocytes is implicated as a key event in the progression of a number of human inflammatory diseases. Consequently, there is considerable interest in the development of therapeutically useful MPO inhibitors. Nitroxides are well established antioxidant compounds of low toxicity that can attenuate oxidative damage in animal models of inflammatory disease. They are believed to exert protective effects principally by acting as superoxide dismutase mimetics or radical scavengers. However, we show here that nitroxides can also potently inhibit MPO-mediated HOCl production, with the nitroxide 4-aminoTEMPO inhibiting HOCl production by MPO and by neutrophils with IC50 values of approx. 1 and 6 μM respectively. Structure–activity relationships were determined for a range of aliphatic and aromatic nitroxides, and inhibition of oxidative damage to two biologically-important protein targets (albumin and perlecan) are demonstrated. Inhibition was shown to involve one-electron oxidation of the nitroxides by the compound I form of MPO and accumulation of compound II. Haem destruction was also observed with some nitroxides. Inhibition of neutrophil HOCl production by nitroxides was antagonized by neutrophil-derived superoxide, with this attributed to superoxide-mediated reduction of compound II. This effect was marginal with 4-aminoTEMPO, probably due to the efficient superoxide dismutase-mimetic activity of this nitroxide. Overall, these data indicate that nitroxides have considerable promise as therapeutic agents for the inhibition of MPO-mediated damage in inflammatory diseases.

A biomechanical investigation of vertebral staples for fusionless scoliosis correction

Background: Fusionless scoliosis surgery is an early-stage treatment for idiopathic scoliosis which claims potential advantages over current fusion-based surgical procedures. Anterior vertebral stapling using a shape memory alloy staple is one such approach. Despite increasing interest in this technique, little is known about the effects on the spine following insertion, or the mechanism of action of the staple. The purpose of this study was to investigate the biomechanical consequences of staple insertion in the anterior thoracic spine, using in vitro experiments on an immature bovine model. Methods: Individual calf spine thoracic motion segments were tested in flexion, extension, lateral bending and axial rotation. Changes in motion segment rotational stiffness following staple insertion were measured on a series of 14 specimens. Strain gauges were attached to three of the staples in the series to measure forces transmitted through the staple during loading. A micro-CT scan of a single specimen was performed after loading to qualitatively examine damage to the vertebral bone caused by the staple. Findings: Small but statistically significant decreases in bending stiffness occurred in flexion,extension, lateral bending away from the staple, and axial rotation away from the staple. Each strain-gauged staple showed a baseline compressive loading following insertion which was seen to gradually decrease during testing. Post-test micro-CT showed substantial bone and growth plate damage near the staple. Interpretation: Based on our findings it is possible that growth modulation following staple insertion is due to tissue damage rather than sustained mechanical compression of the motion segment.

Study of mechanical deformations on tough skinned vegetables during mechanical peeling process

Peeling is an essential phase of post harvesting and processing industry; however undesirable processing losses are unavoidable and always have been the main concern of food processing sector. There are three methods of peeling fruits and vegetables including mechanical, chemical and thermal, depending on the class and type of fruit. By comparison, the mechanical methods are the most preferred; mechanical peeling methods do not create any harmful effects on the tissue and they keep edible portions of produce fresh. The main disadvantage of mechanical peeling is the rate of material loss and deformations. Obviously reducing material losses and increasing the quality of the process has a direct effect on the whole efficiency of food processing industry, this needs more study on technological aspects of these operations. In order to enhance the effectiveness of food industrial practices it is essential to have a clear understanding of material properties and behaviour of tissues under industrial processes. This paper presents the scheme of research that seeks to examine tissue damage of tough skinned vegetables under mechanical peeling process by developing a novel FE model of the process using explicit dynamic finite element analysis approach. A computer model of mechanical peeling process will be developed in this study to stimulate the energy consumption and stress strain interactions of cutter and tissue. The available Finite Element softwares and methods will be applied to establish the model. Improving the knowledge of interactions and involves variables in food operation particularly in peeling process is the main objectives of the proposed study. Understanding of these interrelationships will help researchers and designer of food processing equipments to develop new and more efficient technologies. Presented work intends to review available literature and previous works has been done in this area of research and identify current gap in modelling and simulation of food processes.

Neutrophil activation, antioxidant supplements and exercise-induced oxidative stress

Neutrophils produce free radicals known as reactive oxygen species (ROS), which assist in the clearance of damaged host tissue. Tissue damage may occur during exercise due to muscle damage, thermal stress and ischaemia/reperfusion. When produced in excess, neutrophil-derived ROS may overwhelm the body's endogenous antioxidant defence mechanisms, and this can lead to oxidative stress. There is increasing evidence for links between oxidative stress and a variety of pathological disorders such as cardiovascular diseases, cancer, chronic inflammatory diseases and post-ischaemic organ injury. A small number of studies have investigated whether there is a link between neutrophil activation and oxidative stress during exercise. In this review, we have summarised the findings of these studies. Exercise promotes the release of neutrophils into the circulation, and some evidence suggests that neutrophils mobilised after exercise have an enhanced capacity to generate some forms of ROS when stimulated in vitro. Neutrophil activation during exercise may challenge endogenous antioxidant defence mechanisms, but does not appear to increase lipid markers of oxidative stress to any significant degree, at least in the circulation. Antioxidant supplements such as N-acetylcysteine are effective at attenuating increases in the capacity of neutrophils to generate ROS when stimulated in vitro, whereas vitamin E reduces tissue infiltration of neutrophils during exercise. Free radicals generated during intense exercise may lead to DNA damage in leukocytes, but it is unknown if this damage is the result of neutrophil activation. Exercise enhances the expression of inducible haem (heme)-oxygenase (HO-1) in neutrophils after exercise, however, it is uncertain whether oxidative stress is the stimulus for this response.

Transcriptome analysis of neutrophils after endurance exercise reveals novel signaling mechanisms in the immune response to physiological stress

Neutrophils serve as an intriguing model for the study of innate immune cellular activity induced by physiological stress. We measured changes in the transcriptome of circulating neutrophils following an experimental exercise trial (EXTRI) consisting of 1 h of intense cycling immediately followed by 1 h of intense running. Blood samples were taken at baseline, 3 h, 48 h, and 96 h post-EXTRI from eight healthy, endurance-trained, male subjects. RNA was extracted from isolated neutrophils. Differential gene expression was evaluated using Illumina microarrays and validated with quantitative PCR. Gene set enrichment analysis identified enriched molecular signatures chosen from the Molecular Signatures Database. Blood concentrations of muscle damage indexes, neutrophils, interleukin (IL)-6 and IL-10 were increased (P < 0.05) 3 h post-EXTRI. Upregulated groups of functionally related genes 3 h post-EXTRI included gene sets associated with the recognition of tissue damage, the IL-1 receptor, and Toll-like receptor (TLR) pathways (familywise error rate, P value < 0.05). The core enrichment for these pathways included TLRs, low-affinity immunoglobulin receptors, S100 calcium binding protein A12, and negative regulators of innate immunity, e.g., IL-1 receptor antagonist, and IL-1 receptor associated kinase-3. Plasma myoglobin changes correlated with neutrophil TLR4 gene expression (r = 0.74; P < 0.05). Neutrophils had returned to their nonactivated state 48 h post-EXTRI, indicating that their initial proinflammatory response was transient and rapidly counterregulated. This study provides novel insight into the signaling mechanisms underlying the neutrophil responses to endurance exercise, suggesting that their transcriptional activity was particularly induced by damage-associated molecule patterns, hypothetically originating from the leakage of muscle components into the circulation.

Metal ion levels post unilateral primary Exeter total hip replacement

There has been much discussion and controversy in the media recently regarding metal toxicity following large head metal on metal (MoM) total hip replacement (THR). Patients have been reported as having hugely elevated levels of metal ions with, at times, devastating systemic, neurolgical and/or orthopaedic sequelae. However, no direct correlation between metal ion level and severity of metallosis has yet been defined. Normative levels of metal ions in well functioning, non Cobalt-Chrome hips have also not been defined to date. The Exeter total hip replacement contains no Cobalt-Chrome (Co-Cr) as it is made entirely from stainless steel. However, small levels of these metals may be present in the modular head of the prosthesis, and their effect on metal ion levels in the well functioning patient has not been investigated. We proposed to define the “normal” levels of metal ions detected by blood test in 20 well functioning patients at a minimum 1 year post primary Exeter total hip replacement, where the patient had had only one joint replaced. Presently, accepted normal levels of blood Chromium are 10–100 nmol/L and plasma Cobalt are 0–20 nmol/L. The UK Modern Humanities Research Association (MHRA) has suggested that levels of either Cobalt or Chromium above 7 ppb (equivalent to 135 nmol/L for Chromium and 120 nmol/L for Cobalt) may be significant. Below this level it is indicated that significant soft tissue reaction and tissue damage is less likely and the risk of implant failure is reduced. Hips were a mixture of cemented and hybrid procedures performed by two experienced orthopaedic consultants. Seventy percent were female, with a mixture of head sizes used. In our cohort, there were no cases where the blood Chromium levels were above the normal range, and in more than 70% of cases, levels were below recordable levels. There were also no cases of elevated plasma Cobalt levels, and in 35% of cases, levels were negligible. We conclude that the implantation with an Exeter total hip replacement does not lead to elevation of blood metal ion levels.

The development of an effective vaccine against Chlamydia : utilisation of a non-toxic mucosal adjuvant to generate a protective mucosal response

Chlamydia is responsible for a wide range of diseases with enormous global economic and health burden. As the majority of chlamydial infections are asymptomatic, a vaccine has greatest potential to reduce infection and disease prevalence. Protective immunity against Chlamydia requires the induction of a mucosal immune response, ideally, at the multiple sites in the body where an infection can be established. Mucosal immunity is most effectively stimulated by targeting vaccination to the epithelium, which is best accomplished by direct vaccine application to mucosal surfaces rather than by injection. The efficacy of needle-free vaccines however is reliant on a powerful adjuvant to overcome mucosal tolerance. As very few adjuvants have proven able to elicit mucosal immunity without harmful side effects, there is a need to develop non-toxic adjuvants or safer ways to administered pre-existing toxic adjuvants. In the present study we investigated the novel non-toxic mucosal adjuvant CTA1-DD. The immunogenicity of CTA1-DD was compared to our "gold-standard" mucosal adjuvant combination of cholera toxin (CT) and cytosine-phosphate-guanosine oligodeoxynucleotide (CpG-ODN). We also utilised different needle-free immunisation routes, intranasal (IN), sublingual (SL) and transcutaneous (TC), to stimulate the induction of immunity at multiple mucosal surfaces in the body where Chlamydia are known to infect. Moreover, administering each adjuvant by different routes may also limit the toxicity of the CT/CpG adjuvant, currently restricted from use in humans. Mice were immunised with either adjuvant together with the chlamydial major outer membrane protein (MOMP) to evaluate vaccine safety and quantify the induction of antigen-specific mucosal immune responses. The level of protection against infection and disease was also assessed in vaccinated animals following a live genital or respiratory tract infectious challenge. The non-toxic CTA1-DD was found to be safe and immunogenic when delivered via the IN route in mice, inducing a comparable mucosal response and level of protective immunity against chlamydial challenge to its toxic CT/CpG counterpart administered by the same route. The utilisation of different routes of immunisation strongly influenced the distribution of antigen-specific responses to distant mucosal surfaces and also abrogated the toxicity of CT/CpG. The CT/CpG-adjuvanted vaccine was safe when administered by the SL and TC routes and conferred partial immunity against infection and pathology in both challenge models. This protection was attributed to the induction of antigen-specific pro-inflammatory cellular responses in the lymph nodes regional to the site of infection and rather than in the spleen. Development of non-toxic adjuvants and effective ways to reduce the side effects of toxic adjuvants has profound implications for vaccine development, particularly against mucosal pathogens like Chlamydia. Interestingly, we also identified two contrasting vaccines in both infection models capable of preventing infection or pathology exclusively. This indicated that the development of pathology following an infection of vaccinated animals was independent of bacterial load and was instead the result of immunopathology, potentially driven by the adaptive immune response generated following immunisation. While both vaccines expressed high levels of interleukin (IL)-17 cytokines, the pathology protected group displayed significantly reduced expression of corresponding IL-17 receptors and hence an inhibition of signalling. This indicated that the balance of IL-17-mediated responses defines the degree of protection against infection and tissue damage generated following vaccination. This study has enabled us to better understand the immune basis of pathology and protection, necessary to design more effective vaccines.

Urinary biomolecular indicators of exercise-induced over exertion injury

Poor health and injury represent major obstacles to the future economic security of Australia. The national economic cost of work-related injury is estimated at $57.5 billion p/a. Since exposure to high physical demands is a major risk factor for musculoskeletal injury, monitoring and managing such physical activity levels in workers is a potentially important injury prevention strategy. Current injury monitoring practices are inadequate for the provision of clinically valuable information about the tissue specific responses to physical exertion. Injury of various soft tissue structures can manifest over time through accumulation of micro-trauma. Such micro-trauma has a propensity to increase the risk of acute injuries to soft-tissue structures such as muscle or tendon. As such, the capacity to monitor biomarkers that result from the disruption of these tissues offers a means of assisting the pre-emptive management of subclinical injury prior to acute failure or for evaluation of recovery processes. Here we have adopted an in-vivo exercise induced muscle damage model allowing the application of laboratory controlled conditions to assist in uncovering biochemical indicators associated with soft-tissue trauma and recovery. Importantly, urine was utilised as the diagnostic medium since it is non-invasive to collect, more acceptable to workers and less costly to employers. Moreover, it is our hypothesis that exercise induced tissue degradation products enter the circulation and are subsequently filtered by the kidney and pass through to the urine. To test this hypothesis a range of metabolomic and proteomic discovery-phase techniques were used, along with targeted approaches. Several small molecules relating to tissue damage were identified along with a series of skeletal muscle-specific protein fragments resulting from exercise induced soft-tissue damage. Each of the potential biomolecular markers appeared to be temporally present within urine. Moreover, the regulation of abundance seemed to be associated with functional recovery following the injury. This discovery may have important clinical applications for monitoring of a variety of inflammatory myopathies as well as novel applications in monitoring of the musculoskeletal health status of workers, professional athletes and/or military personnel to reduce the onset of potentially debilitating musculoskeletal injuries within these professions.

First aid treatment of burn injuries

The recommendations for the first aid treatment of burn injuries have previously been based upon conflicting published studies and as a result the recommendations have been vague with respect to optimal first aid treatment modality, temperature, duration and delay after which treatment is still effective. The public have also continued to use treatments such as ice and alternative therapies, however there is little evidence to support their use. Recently there have been several studies conducted by burn researchers in Australia which have enabled the recommendations to be clarified. First aid should consist of cool running water (2-15°C), applied for 20 minutes duration, as soon as possible but for up to 3 hours after the burn injury has occurred. Ice should not be used and alternative therapies should only be used to relieve pain as an adjunct to cold water treatment. Optimal first aid treatment significantly reduces tissue damage, hastens wound re-epithelialisation and reduces scarring and should be promoted widely to the public.

Different in vitro activation methods for latent transforming growth factors (TGF)–β : considerable exogenous factors to promote higher mesenchymal-origin cell proliferation in a bioprocessing platform

Regenerative medicine includes two efficient techniques, namely tissue-engineering and cell-based therapy in order to repair tissue damage efficiently. Most importantly, huge numbers of autologous cells are required to deal these practices. Nevertheless, primary cells, from autologous tissue, grow very slowly while culturing in vitro; moreover, they lose their natural characteristics over prolonged culturing period. Transforming growth factors-beta (TGF-β) is a ubiquitous protein found biologically in its latent form, which prevents it from eliciting a response until conversion to its active form. In active form, TGF-β acts as a proliferative agent in many cell lines of mesenchymal origin in vitro. This article reviews on some of the important activation methods-physiochemical, enzyme-mediated, non-specific protein interaction mediated, and drug-induced- of TGF-β, which may be established as exogenous factors to be used in culturing medium to obtain extensive proliferation of primary cells.