Assessment of a novel equine tarsocrural experimental joint disease model using recombinant interleukin-1ß and arthroscopic articular sampling of the medial malleolus of the tibia on the standing sedated horse.

Joint disease and osteoarthritis are common problems in the horse and numerous experimental studies have been developed to determine the safety and efficacy of new therapies. Synovitis, a critical component of joint disease, has been experimentally induced using recombinant interleukin-1 beta (reIL-1ß) to investigate new joint therapies in a controlled environment, although the use of reIL-1ß has not been reported in the equine tarsocrural joint. A common consequence of performing controlled experiments is that articular tissue collection typically requires general anesthesia or euthanasia. This report describes a minimally invasive surgical biopsy technique to harvest joint tissues from the tarsocrural joint in standing horses. The aims of the study were to assess subjective and objective pain parameters following reIL-1ß induced synovitis in the tarsocrural joint and to describe the surgical technique including the location and quantity of tissues obtained with this method. Experimental synovitis was induced using reIL-1ß in one equine tarsocrural joint of each horse using a randomised controlled design. The minimally invasive surgical technique provided sufficient amounts of articular cartilage from the medial malleolus of the tibia and synovium to perform viability, biochemical and histological assessments without necessitating general anesthesia. The minimally invasive technique also allowed for lameness assessment that could have been influenced by more invasive methods of tissue collection. No incisional or lameness complications were detected after use. The synovitis model and surgical technique provided ample tissue for laboratory evaluation and avoided general anesthesia or sacrifice of the horse.

Comparison of subjective lameness evaluation, force platforms and an inertial-sensor system to identify mild lameness in an equine osteoarthritis model.

When mild lameness exists, agreement between clinicians is often controversial due to its subjective nature. The goal of the study was to compare subjective and objective methods to identify the presence of mild lameness using an established model of osteoarthritis (OA) in which OA was induced by creating a unilateral carpal osteochondral fragment (OCF) in the middle carpal joint of 16 horses. Subjective lameness evaluations (blinded and unblinded), force platforms (FP), and an inertial-sensor system (ISS) were used to detect forelimb lameness at four time points. Limbs identified as lame by each method were compared as well as compared with the OCF limb at each time point. Spearman correlations were calculated between all outcome parameters. Independent of time, blinded subjective evaluation (54%) and the ISS (60%) identified a higher percentage of horses as lame in the OCF limb compared to FP (40%). Blinded subjective evaluation and the ISS agreed which forelimb was lame more often (50%) compared with blinded subjective evaluation and the FP (38%). Induction of mild lameness within the OCF limb was supported by an increase in the frequency of horses considered lame by both subjective evaluations the ISS and a decrease (3.6%) in mean (among all horses) peak vertical force from baseline to post OCF induction. The percentage of horses identified as lame in the OCF limb, independent of time, was highest with the ISS (60%) followed by blinded subjective evaluation (51%) and the FP (42%). It was concluded that the best agreement was between subjective evaluation and the inertial-sensor system.

Activation of the insulin-signaling pathway in sciatic nerve and hippocampus of type 1 diabetic rats.

Peripheral neuropathy is a major complication associated with diabetes and central neuropathy characterized by Alzheimer's disease-like features in the brain is associated with increased dementia risk for patients with diabetes. Although glucose uptake into the cells of the nervous system is insulin-independent, contribution of impaired insulin support is clearly recognized to play a role, however not yet fully understood, in the development of neuropathy. In this study, we assessed the direct role of insulin on the peripheral nervous system (PNS) and central nervous system (CNS) of insulin-dependent type 1 diabetic rats. Fresh sciatic nerve and hippocampus from control and diabetic rats were incubated with varied ex vivo concentrations of insulin and phosphorylation levels of insulin receptor and glycogen synthase kinase-3 (GSK3ß) were assessed by Western blot analysis. Both the sciatic nerve and hippocampus from type 1 diabetic rats were highly responsive to exogenous insulin with a significantly increased phosphorylation of insulin receptor and GSK3 compared to tissues from control rats. Further, sustained in vivo insulin delivery, not sufficient to restore normal blood glucose, normalized the activation of both insulin receptor and GSK3 in both PNS and CNS tissues. These results suggest that the insulin-signaling pathway is responsive to exogenous insulin in the nervous system of insulin-deficient type 1 diabetic rats and that constant insulin delivery restore normal nerve function and may protect PNS and CNS from damage.

In vivo diffusion characteristics following perineural injection of the deep branch of the lateral plantar nerve with mepivacaine or iohexol in horses.

REASONS FOR PERFORMING STUDY: Hindlimb proximal suspensory desmopathy is a common injury of sport horses but diagnosis can be difficult because diagnostic analgesia of the region lacks specificity. Perineural analgesia of the deep branch of the lateral plantar nerve (DBLPN) has been proposed as a more specific method of isolating pain of the proximal aspect of the suspensory ligament but the technique has not been evaluated in vivo. OBJECTIVES: To determine the extent of diffusion of contrast medium and mepivacaine following DBLPN analgesia using a single-needle injection technique and to determine if there is inadvertent involvement of the tarsal sheath and/or tarsometatarsal (TMT) joint using this technique. STUDY DESIGN: In vivo experimental study. METHODS: Perineural injection of the DBLPN was performed in 16 limbs with 3 ml of either mepivacaine hydrochloride or positive contrast medium. Contrast medium-injected limbs were radiographed 5, 15, and 30 min post injection and diffusion characteristics were described. In mepivacaine-injected limbs, synovial fluid from the TMT joint was obtained 10 and 20 min post injection and mepivacaine concentrations were analysed. RESULTS: At 5, 15 and 30 min post injection, the contrast medium extended, on average, 19.6, 20.6 and 21.0 mm proximal and 38.0, 43.5 and 51.9 mm distal to the injection site, respectively. Three of 8 (37.5%) limbs had evidence of contrast medium in the tarsal sheath. Two of 8 (25%) limbs had mepivacaine concentrations within the TMT joint sufficient to produce analgesia (>300 mg/l) at 10 min post injection. CONCLUSIONS: Contrast medium diffused further in a distal direction than in a proximal direction. Analgesia of the DBLPN can result in inadvertent involvement of the tarsal sheath and/or TMT joint.

Human fucosyltransferase 6 enables prostate cancer metastasis to bone.

BACKGROUND: The interaction between human prostate cancer (PCa) cells and bone marrow (BM) endothelium follows a rolling-and-adhesion cascade mediated by E-selectin ligand (ESL): E-selectin. This adhesion is enabled by elevated expression of α-1,3-fucosyltransferases (FTs), enzymes responsible for ESL-mediated bone metastasis in humans. In contrast, the incidence of bone metastasis in mice is rare. METHODS: FT 3, 6 and 7 were overexpressed in mouse PCa cells. The rolling cell number, cell-rolling velocity and transendothelial migration were characterised in vitro. Fucosyltransferases-transduced mouse PCa cells expressing luciferase were inoculated into mice via left ventricle to compare the capability of bone metastasis. Mass spectrometry and immunoprecipitation were utilised for identification of ESLs. RESULTS: Overexpression of FT3, FT6 or FT7 restored ESLs and enabled mouse PCa cells to roll and adhere in E-selectin-functionalised microtubes, similar to trafficking of circulating PCa cells in BM vessels. Following intracardiac inoculation, FT6-transduced cells induced robust bone metastasis in mice. Inhibition of FT6 by a fucose mimetic significantly reduced bone metastasis. Importantly, comparison of FT3, FT6 and FT7 gene expression in existing clinical samples showed significant upregulation of FT6 in PCa-distant metastases. CONCLUSION: FT6 is a key mediator of PCa cells trafficking to the BM. It may serve as a viable drug target in preclinical tests of therapeutics for reduction of PCa bone metastasis.

An immobilized nanoparticle-based platform for efficient gene knockdown of targeted cells in the circulation.

It is well established that specific interaction between adhesion molecules of endothelial cells and receptors on leukocytes can separate and recruit leukocytes from the bloodstream to sites of inflammation and coagulation. Previously, we showed that P-selectin can be absorbed onto the surface of a blood-compatible microrenathane tube, and the P-selectin-coated surface could successfully capture P-selectin receptor-positive stem cells from physiological shear flow in vitro and from the bloodstream in vivo. In this paper, P-selectin was covalently attached to the surface of nanoscale liposomes to create targeting nanoparticles (NPs). Small interfering RNA (siRNA) was encapsulated by these nanoscale liposomes, and the liposomes were stabilized by PEGylation with DSPE-PEG2000. Experiments showed that these P-selectin-, PEGylated-, nanoscale-liposomes (PS-DSPE-PEG NPs) could be absorbed onto the inner surface of microrenathane tubing. The coated surface could specifically capture targeted cells from physiological shear flow, efficiently deliver encapsulated siRNA into adherent cells and dramatically silence the targeted gene neutrophil elastase. With this device, we create a high localized concentration for siRNA delivery in the circulatory system, providing circulating target cells adequate time to interact with therapeutic materials. SiRNA is efficaciously delivered into specific target cells, thereby providing a powerful tool for highly efficient siRNA transfection and other therapeutic materials delivery in circulation. The method should prove especially useful for diseases derived from disorders of blood cells.

The RADAR repository: a resource for studies of infectious agents and their transmissibility by transfusion.

BACKGROUND: An ongoing issue in transfusion medicine is whether newly identified or emerging pathogens can be transmitted by transfusion. One method to study this question is through the use of a contemporary linked donor-recipient repository. STUDY DESIGN AND METHODS: The Retrovirus Epidemiology Donor Study Allogeneic Donor and Recipient (RADAR) repository was established between 2000 and 2003 by seven blood centers and eight collaborating hospitals. Specimens from consented donors were collected, components from their donations were routed to participating hospitals, and recipients of these units gave enrollment and follow-up specimens for long-term storage. The repository was designed to show that zero transmissions to enrolled recipients would indicate with 95 percent confidence that the transfusion transmission rate of an agent with prevalence of 0.05 to 1 percent was lower than 25 percent. RESULTS: The repository contains pre- and posttransfusion specimens from 3,575 cardiac, vascular, and orthopedic surgery patients, linked to 13,201 donation specimens. The mean number of RADAR donation exposures per recipient is 3.85. The distribution of components transfused is 77 percent red cells, 13 percent whole blood-derived platelet concentrates, and 10 percent fresh frozen plasma. A supplementary unlinked donation repository containing 99,906 specimens from 84,339 donors was also established and can be used to evaluate the prevalence of an agent and validate assay(s) performance before accessing the donor-recipient-linked repository. Recipient testing conducted during the establishment of RADAR revealed no transmissions of human immunodeficiency virus, hepatitis C virus, or human T-lymphotropic virus. CONCLUSIONS: RADAR is a contemporary donor-recipient repository that can be accessed to study the transfusion transmissibility of emerging agents.

Rheological analysis and measurement of neutrophil indentation.

Aspects of neutrophil mechanical behavior relevant to the formation of adhesive contacts were assessed by measuring the dependence of the contact area between the cell and a spherical substrate under controlled loading. Micropipettes were used to bring neutrophils into contact with spherical beads under known forces, and the corresponding contact area was measured over time. The neutrophil was modeled as a viscous liquid drop with a constant cortical tension. Both the equilibrium state and the dynamics of the approach to equilibrium were examined. The equilibrium contact area increased monotonically with force in a manner consistent with a cell cortical tension of 16-24 pN/microm. The dynamic response matched predictions based on a model of the cell as a growing drop using published values for the effective viscosity of the cell. The contact pressure between the cell and substrate at equilibrium is predicted to depend on the curvature of the contacting substrate, but to be independent of the impingement force. The approach to equilibrium was rapid, such that the time-averaged stress for a two-second impingement was within 20% of the equilibrium value. These results have implications for the role of mechanical force in the formation of adhesive contacts.

Multiparticle adhesive dynamics: hydrodynamic recruitment of rolling leukocytes.

The slow rolling motion of leukocytes along the walls of blood vessels mediated by specific receptor-ligand adhesion is important in inflammation and occurs in postcapillary venules over a wide range of wall shear stresses and vessel diameters. The ability of hydrodynamic collisions between cells to induce capture of free-stream leukocytes to a selectin-bearing surface under shear flow was studied experimentally by using a cell-free assay. It was found that carbohydrate-coated spherical beads, representing model leukocytes, tend to attach to the adhesive wall 4-5 cell diameters up- or downstream of a slowly rolling or stationary adhesive bead. A key feature of such "hydrodynamic recruitment" is that only glancing, indirect collisions occurring close to the plane will result in downstream attachment. A direct numerical simulation of cell capture and rolling that includes multiparticle hydrodynamic interactions is shown to reproduce the observed behavior accurately. The theory predicts that hydrodynamic recruitment will occur in the absence of buoyancy effects and over a range of shear rates, suggesting that the mechanism may be important in vivo. This theory is supported by measurements of leukocyte capture in vivo using the hamster cheek pouch model.

Multiparticle adhesive dynamics. Interactions between stably rolling cells.

A novel numerical simulation of adhesive particles (cells) reversibly interacting with an adhesive surface under flow is presented. Particle--particle and particle--wall hydrodynamic interactions in low Reynolds number Couette flow are calculated using a boundary element method that solves an integral representation of the Stokes equation. Molecular bonds between surfaces are modeled as linear springs and stochastically formed and broken according to postulated descriptions of force-dependent kinetics. The resulting simulation, Multiparticle Adhesive Dynamics, is applied to the problem of selectin-mediated rolling of hard spheres coated with leukocyte adhesion molecules (cell-free system). Simulation results are compared to flow chamber experiments performed with carbohydrate-coated spherical beads rolling on P-selectin. Good agreement is found between theory and experiment, with the main observation being a decrease in rolling velocity with increasing concentration of rolling cells or increasing proximity between rolling cells. Pause times are found to increase and deviation motion is found to decrease as pairs of rolling cells become closer together or align with the flow.

High-risk HPVs and risk of cervical neoplasia: a nested case-control study.

The purpose of this nested case-control study was to estimate the risk of SIL development among a cohort of women providing cervical samples as part of their family planning visit at baseline in 1991-1992. All women had normal cervical cytology (N = 2905) at baseline and provided a cervical sample for subsequent HPV typing. Among this cohort, 426 women developed SIL (22 HSIL and 404 LSIL), 619 developed atypia, and 1860 remained cytologically normal. Two controls per case were sampled from those who remained normal. PCR-based methods with L1 consensus primers were used to assess high-risk HPV positivity. Having an oncogenic HPV type at baseline was associated with an almost fourfold increased risk of HSIL development (relative risk (RR) = 3.8; 95% CI, 1.5--9.0) and a 70% increased risk of LSIL development (RR = 1.7; 95% CI, 1.2--2.3%). The association between HPV positivity and SIL development was strongest in the first year of follow-up (RR = 9.2 for HSIL and 2.5 for LSIL development). The decline in HPV-associated SIL risk may be a function of having only one measure of HPV positivity (at baseline).

Vibrational Branching Ratios and Asymmetry Parameters in the Photoionization of CO2 in the Region Between 650 Å and 840 Å.

The vibrational branching ratios and asymmetry parameters for CO2 have been determined in the wavelength region of 650 Å to near the ionization onset at about 840 Å. The study was performed using synchrotron radiation from the Daresbury storage ring that was dispersed with a 5 m grating monochomator that afforded resolution of 0.1 Å to 0.2 Å. This resolution allowed the study of the branching ratios and asymmetry parameters with enough detail to see the changes in the parameters within the pronounced autoionization structure in CO2 in this wavelength region. While the electron spectrometer resolution was not sufficient to resolve the spin orbit and Renner-Teller splitting in the photoelectron spectra, we are able to fit the data with a model that identifies the major structure in terms of the symmetric stretch and elements of the asymmetric stretch and bending modes. A calculation of the expected relative vibrational excitations based upon the Franck-Condon principle clearly showed non-Franck-Condon behavior in some of the vibrational-electronic transitions.