Leptospirosis is a widespread zoonotic infection that primarily affects residents of tropical regions, but causes infections in animals and humans in temperate regions as well. The agents of leptospirosis comprise several members of the genus Leptospira, which also includes non-pathogenic, saprophytic species. Leptospirosis can vary in severity from a mild, nonspecific illness to severe disease that includes multi-organ failure and widespread endothelial damage and hemorrhage. To begin to investigate how pathogenic leptospires affect endothelial cells, we compared the responses of two endothelial cell lines to infection by pathogenic versus non-pathogenic leptospires. Microarray analyses suggested that pathogenic L. interrogans and non-pathogenic L. biflexa triggered changes in expression of genes whose products are involved in cellular architecture and interactions with the matrix, but that the changes were in opposite directions, with infection by L. biflexa primarily predicted to increase or maintain cell layer integrity, while L. interrogans lead primarily to changes predicted to disrupt cell layer integrity. Neither bacterial strain caused necrosis or apoptosis of the cells even after prolonged incubation. The pathogenic L. interrogans, however, did result in significant disruption of endothelial cell layers as assessed by microscopy and the ability of the bacteria to cross the cell layers. This disruption of endothelial layer integrity was abrogated by addition of the endothelial protective drug lisinopril at physiologically relevant concentrations. These results suggest that, through adhesion of L. interrogans to endothelial cells, the bacteria may disrupt endothelial barrier function, promoting dissemination of the bacteria and contributing to severe disease manifestations. In addition, supplementing antibiotic therapy with lisinopril or derivatives with endothelial protective activities may decrease the severity of leptospirosis.

Objective.Calcium pyrophosphate dihydrate (CPPD) crystals are commonly found in osteoarthritic joints and correlate with a poor prognosis. Intraarticular corticosteroids, such as dexamethasone (Dxm), are commonly used therapies for osteoarthritis with or without CPPD deposition. Dxm has variable effects in mineralization models. We investigated the effects of Dxm on CPPD crystal formation in a well established tissue culture model.Methods.Porcine articular chondrocytes were incubated with ATP to generate CPPD crystals. Chondrocytes incubated with or without ATP were exposed to 1–100 nM Dxm in the presence of45Ca. Mineralization was measured by45Ca uptake in the cell layer. We also investigated the effect of Dxm on mineralization-regulating enzymes such as alkaline phosphatase, nucleoside triphosphate pyrophosphohydrolase (NTPPPH), and transglutaminase.Results.Dxm significantly increased ATP-induced mineralization by articular chondrocytes. While alkaline phosphatase and NTPPPH activities were unchanged by Dxm, transglutaminase activity increased in a dose-responsive manner. Levels of Factor XIIIA mRNA and protein were increased by Dxm, while type II Tgase protein was unchanged. Transglutaminase inhibitors suppressed Dxminduced increases in CPPD crystal formation.Conclusion.These findings suggest a potential for Dxm to contribute to pathologic mineralization in cartilage and reinforce a central role for the transglutaminase enzymes in CPPD crystal formation.

Leptospirosis is a global public health problem, primarily in the tropical developing world. The pathogenic mechanisms of the causative agents, several members of the genus Leptospira, have been underinvestigated. The exception to this trend has been the demonstration of the binding of pathogenic leptospires to the extracellular matrix (ECM) and its components. In this work, interactions of Leptospira interrogans bacteria with mammalian cells, rather than the ECM, were examined. The bacteria bound more efficiently to the cells than to the ECM, and a portion of this cell-binding activity was attributable to attachment to glycosaminoglycan (GAG) chains of proteoglycans (PGs). Chondroitin sulfate B PGs appeared to be the primary targets of L. interrogans attachment, while heparan sulfate PGs were much less important. Inhibition of GAG/PG-mediated attachment resulted in partial inhibition of bacterial attachment, suggesting that additional receptors for L. interrogans await identification. GAG binding may participate in the pathogenesis of leptospirosis within the host animal. In addition, because GAGs are expressed on the luminal aspects of epithelial cells in the proximal tubules of the kidneys, this activity may play a role in targeting the bacteria to this critical site. Because GAGs are shed in the urine, GAG binding may also be important for transmission to new hosts through the environment.

OBJECTIVE:Calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP) crystals occur in up to 60% of osteoarthritic joints and predict an increased severity of arthritis. Articular cartilage vesicles (ACVs) generate CPPD crystals in the presence of ATP and BCP crystals with added beta-glycerophosphate. While ACVs are present in normal articular cartilage, they mineralize primarily in cartilage from osteoarthritic joints. The aim of this study was to explore the hypothesis that ACV mineralization is regulated by components of the surrounding extracellular matrix.METHODS:Porcine ACVs were embedded in agarose gels containing type II and/or type I collagen and/or proteoglycans. Mineralization was measured as (45)Ca accumulation stimulated by ATP or beta-glycerophosphate and reflects both nucleation and growth. Synthetic CPPD and BCP crystals were embedded in similar gels to isolate the effect of matrix components on crystal growth.RESULTS:After establishing baseline responsiveness of ACVs to ATP and beta-glycerophosphate in agarose gels, we examined the ability of ATP and beta-glycerophosphate to stimulate mineral formation in gels containing various matrix components. Type II collagen suppressed the ability of ATP to stimulate mineralization, while a combination of type II plus type I collagen increased the effect of ATP and beta-glycerophosphate on mineralization. Type I collagen affected ACV mineralization in a dose-responsive manner. Neither type of collagen significantly affected crystal growth or levels of mineralization-regulating enzymes. Proteoglycans suppressed mineral formation by ACVs in gels containing both type I and type II collagen.CONCLUSION:Cartilage matrix changes that occur with osteoarthritis, such as increased quantities of type I collagen and reduced proteoglycan levels, may promote ACV mineralization.

There is an increasing demand, both from customers and regulatory sources, for safer and more energy efficient products. Manufacturers are having to look to their design and development processes to service these demands. Traditional approaches have been to use prototype testing and only delve more deeply into specific aspects of the performance when issues arise. In this work the complex flow within the cooling circuit of the door of a pyrolytic oven is studied. A combination of Computational Fluid Dynamics (CFD) and experimental techniques is used. It will be shown that CFD can help with the achievement of an optimal solution, with the understanding of the flow behaviour and that there is a synergy between the numerical and experimental techniques. Using only one of these techniques would limit the understanding of the flow behaviour and could lead to a less than optimal solution to the design problem. This work aims to explore this particular complex industrial fluid flow situation to:understand the flow around the oven door's cooling circuitdemonstrate the synergy of CFD and experimental work within development of a complex productexplore the role of CFD within the product development process.

OBJECTIVE Basic calcium phosphate (BCP) crystals are common components of osteoarthritis (OA) synovial fluid. Progress in understanding the role of these bioactive particles in clinical OA has been hampered by difficulties in their identification. Tetracyclines stain calcium phosphate mineral in bone. The aim of this study was to investigate whether tetracycline staining might be an additional or alternative method for identifying BCP crystals in synovial fluid. METHODS A drop of oxytetracycline was mixed with a drop of fluid containing synthetic or native BCP, calcium pyrophosphate dihydrate (CPPD), or monosodium urate (MSU) crystals and placed on a microscope slide. Stained and unstained crystals were examined by light microscopy, with and without a portable broad-spectrum ultraviolet (UV) pen light. A small set of characterized synovial fluid samples were compared by staining with alizarin red S and oxytetracycline. Synthetic BCP crystals in synovial fluid were quantified fluorimetrically using oxytetracycline. RESULTS After oxytetracycline staining, synthetic and native BCP crystals appeared as fluorescent amorphous aggregates under UV light. Oxytetracycline did not stain CPPD or MSU crystals or other particulates. Oxytetracycline staining had fewer false-positive test results than did alizarin red S staining and could provide estimates of the quantities of synthetic BCP crystals in synovial fluid. CONCLUSION With further validation, oxytetracycline staining may prove to be a useful adjunct or alternative to currently available methods for identifying BCP crystals in synovial fluid.

Several high-risk factors lead to stress fractures. They include excessive training in athletes leading to overuse injuries, nutritional deficiencies, and endocrine disorders. While stress fractures are common, bilateral stress fractures are rarely seen. Few cases have been reported of bilateral femoral stress fractures in young athletes. This article presents a case of a 14-year-old cross country runner with a bilateral femoral supracondylar stress fracture. He presented with bilateral supracondylar stress fractures from running. The patient followed a strict vegan diet, but his parents stated that, to their knowledge, he was getting adequate protein and calcium. Treatment consisted of decreased activity to pain-free levels with acetaminophen for pain. Low-impact conditioning such as swimming and bicycling was allowed. Hamstring and quadricep stretching was suggested. Nutritional consultation was obtained to ensure appropriate nutrition on a vegan diet. At 1-month follow-up, he was pain free and allowed to proceed with a gradual return to running activities. In this case, the onset of a new workout routine was intolerable for this patient's low bone density, causing insufficiency fractures. Appropriate vegan diets were not associated with stress fracture in our literature review. He may have had an inadequate diet prior to this injury. As in this case, full recovery can be made after this rest period, and the patient may return to his or her original activity safely. In young athletes, diet and nutrition must be kept in mind.

Impinging jets have been fascinating experimentalists and numerical modellers for many years with seemingly simple geometries revealing complex flow characteristics. Due to their high heat and mass transfer rates, impinging jets find wide use in engineering and industrial applications. Not only is the impinging jet interesting in isolation but it is often found as part of a more complex flow situation within an industrial setting. The dilemma for analysts under commercial pressure is always what level of detail is required for the results to be helpful but available in a reasonable time. The aims of this paper are: To understand a horizontal confined heated impinging jet using a combination of visualisation, experimental and numerical techniques. To understand the significance numerical modelling decisions can have in the context of an industrial setting. To make informed decisions about turbulence modelling dependent on the context of the flow problem.

Calcific tendonitis is a common clinical condition associated with high rates of tendon rupture, prolonged symptoms, and poor response to therapy. Little is known about the pathogenesis of calcifications in tendons and consequently few effective therapies are available. We hypothesized that tendon calcification, like pathologic calcification in other sites, was generated by extracellular organelles known as matrix vesicles and that isolated matrix vesicles would constitute the basis for a useful model of this process. Tendon matrix vesicles were isolated from adult porcine patellar tendons using enzymatic digestion and differential centrifugation. Vesicle morphology was examined with electron microscopy. Levels of calcium, phosphate, pyrophosphate, ATP, and mineralization-associated enzymes were measured and compared with articular cartilage vesicles from porcine articular cartilage. Vesicles were embedded in agarose gels with or without type I collagen or dermatan sulfate and incubated in calcifying salt solution trace labeled with (45)calcium. (45)Calcium in the vesicle fraction was measured after 5-7 days. The type of mineral formed was determined by micro-x-ray diffraction. Matrix vesicles isolated from adult porcine tendon were similar morphologically to those obtained from articular cartilage. They contained mineralization-related enzymes and formed hydroxyapatite mineral in vitro. Mineralization was suppressed by levamisole and modulated by extracellular matrix components. Matrix vesicles isolated from tendons mineralize in vitro. This model may aid in the study of the pathogenesis of calcific tendonitis as well as serve as a means to identify effective therapies for this common disorder.