Assessment of the direct effects of DDAH I on tumour angiogenesis in vivo

Nitric oxide (NO) has been strongly implicated in glioma progression and angiogenesis. The endogenous inhibitors of NO synthesis, asymmetric dimethylarginine (ADMA) and N-monomethyl-l-arginine (l-NMMA), are metabolized by dimethylarginine dimethylaminohydrolase (DDAH), and hence, DDAH is an intracellular factor that regulates NO. However, DDAH may also have an NO-independent action. We aimed to investigate whether DDAH I has any direct role in tumour vascular development and growth independent of its NO-mediated effects, in order to establish the future potential of DDAH inhibition as an anti-angiogenic treatment strategy. A clone of rat C6 glioma cells deficient in NO production expressing a pTet Off regulatable element was identified and engineered to overexpress DDAH I in the absence of doxycycline. Xenografts derived from these cells were propagated in the presence or absence of doxycycline and susceptibility magnetic resonance imaging used to assess functional vasculature in vivo. Pathological correlates of tumour vascular density, maturation and function were also sought. In the absence of doxycycline, tumours exhibited high DDAH I expression and activity, which was suppressed in its presence. However, overexpression of DDAH I had no measurable effect on tumour growth, vessel density, function or maturation. These data suggest that in C6 gliomas DDAH has no NO-independent effects on tumour growth and angiogenesis, and that the therapeutic potential of targeting DDAH in gliomas should only be considered in the context of NO regulation

Surface-Associated Plasminogen Binding of Cryptococcus neoformans Promotes Extracellular Matrix Invasion

BACKGROUND:The fungal pathogen Cryptococcus neoformans is a leading cause of illness and death in persons with predisposing factors, including: malignancies, solid organ transplants, and corticosteroid use. C. neoformans is ubiquitous in the environment and enters into the lungs via inhalation, where it can disseminate through the bloodstream and penetrate the central nervous system (CNS), resulting in a difficult to treat and often-fatal infection of the brain, called meningoencephalitis. Plasminogen is a highly abundant protein found in the plasma component of blood and is necessary for the degradation of fibrin, collagen, and other structural components of tissues. This fibrinolytic system is utilized by cancer cells during metastasis and several pathogenic species of bacteria have been found to manipulate the host plasminogen system to facilitate invasion of tissues during infection by modifying the activation of this process through the binding of plasminogen at their surface. METHODOLOGY:The invasion of the brain and the central nervous system by penetration of the protective blood-brain barrier is a prerequisite to the establishment of meningoencephalitis by the opportunistic fungal pathogen C. neoformans. In this study, we examined the ability of C. neoformans to subvert the host plasminogen system to facilitate tissue barrier invasion. Through a combination of biochemical, cell biology, and proteomic approaches, we have shown that C. neoformans utilizes the host plasminogen system to cross tissue barriers, providing support for the hypothesis that plasminogen-binding may contribute to the invasion of the blood-brain barrier by penetration of the brain endothelial cells and underlying matrix. In addition, we have identified the cell wall-associated proteins that serve as plasminogen receptors and characterized both the plasminogen-binding and plasmin-activation potential for this significant human pathogen. CONCLUSIONS:The results of this study provide evidence for the cooperative role of multiple virulence determinants in C. neoformans pathogenesis and suggest new avenues for the development of anti-infective agents in the prevention of fungal tissue invasion

Occlusion of the Lumbar Spine Canal During High-Rate Axial Compression

BACKGROUND CONTEXT: While burst fracture is a well-known cause of spinal canal occlusion with dynamic, axial spinal compression, it is unclear how such loading mechanisms might cause occlusion without fracture. PURPOSE: To determine how spinal canal occlusion during dynamic compression of the lumbar spine is differentially caused by fracture or mechanisms without fracture and to examine the influence of spinal level on occlusion. STUDY DESIGN: A cadaveric biomechanical study. METHODS: Twenty sets of three-vertebrae specimens from all spinal levels between T12 and S1 were subjected to dynamic compression using a hydraulic loading apparatus up to a peak velocity between 0.1 and 0.9 m/s. The presence of canal occlusion was measured optically with a high-speed camera. This was repeated with incremental increases of 4% compressive strain until a vertebral fracture was detected using acoustic emission measurements and computed tomographic imaging. RESULTS: For axial compression without fracture, the peak occlusion (Omax) was 29.9±10.0%, which was deduced to be the result of posterior bulging of the intervertebral disc into the spinal canal. Omax correlated significantly with lumbar spinal level (p<0.001), the compressive displacement (p<0.001) and the cross-sectional area of the vertebra (p=0.031). CONCLUSIONS: Spinal canal occlusion observed without vertebral fracture involves intervertebral disc bulging. The lower lumbar spine tended to be more severely occluded than more proximal levels. CLINICAL SIGNIFICANCE: Clinically, intermittent canal occlusion from disc bulging during dynamic compression may not show any radiographic features. The lower lumbar spine should be a focus of injury prevention intervention in cases of high-rate axial compression

Cortical and Trabecular Bone Fracture Characterisation in the Vertebral Body Using Acoustic Emission

The ability to rapidly detect localised fractures of cortical and/or trabecular bone sustained by the vertebral body would enhance the analysis of vertebral fracture initiation and propagation during dynamic loading. In this study, high rate axial compression tests were performed on twenty sets of three-vertebra lumbar spine specimens. Acoustic Emission (AE) sensor measurements of sound wave pressure were used to classify isolated trabecular fractures and severe compressive fractures of vertebral body cortical and trabecular bone. Fracture detection using standard AE parameters was compared to that of traditional mechanical parameters obtained from load cell and displacement readings. Results indicated that the AE parameters achieved slightly enhanced classification of isolated trabecular fractures, whereas the mechanical parameters better identified combined fractures of cortical and trabecular bone. These findings demonstrate that AE may be used to promptly and accurately identify localised fractures of trabecular bone, whereas more extensive fractures of the vertebral body are best identified by load cell readings due to the considerable loss in compressive resistance. The discrimination thresholds corresponding to the AE parameters were based on calibrated measurements of AE wave pressure and may ultimately be used to examine the onset and progression of vertebral fracture in other loading scenarios

Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease

Genes and mechanisms involved in common complex diseases, such as the autoimmune disorders that affect approximately 5% of the population, remain obscure. Here we identify polymorphisms of the cytotoxic T lymphocyte antigen 4 gene (CTLA4)—which encodes a vital negative regulatory molecule of the immune system—as candidates for primary determinants of risk of the common autoimmune disorders Graves' disease, autoimmune hypothyroidism and type 1 diabetes. In humans, disease susceptibility was mapped to a non-coding 6.1 kb 3' region of CTLA4, the common allelic variation of which was correlated with lower messenger RNA levels of the soluble alternative splice form of CTLA4. In the mouse model of type 1 diabetes, susceptibility was also associated with variation in CTLA-4 gene splicing with reduced production of a splice form encoding a molecule lacking the CD80/CD86 ligand-binding domain. Genetic mapping of variants conferring a small disease risk can identify pathways in complex disorders, as exemplified by our discovery of inherited, quantitative alterations of CTLA4 contributing to autoimmune tissue destruction.<br/