Enhanced chondrogenesis and Wnt signaling in PTH-treated fractures.

Studies have shown that systemic PTH treatment enhanced the rate of bone repair in rodent models. However, the mechanisms through which PTH affects bone repair have not been elucidated. In these studies we show that PTH primarily enhanced the earliest stages of endochondral bone repair by increasing chondrocyte recruitment and rate of differentiation. In coordination with these cellular events, we observed an increased level of canonical Wnt-signaling in PTH-treated bones at multiple time-points across the time-course of fracture repair, supporting the conclusion that PTH responses are at least in part mediated through Wnt signaling. INTRODUCTION: Since FDA approval of PTH [PTH(1-34); Forteo] as a treatment for osteoporosis, there has been interest in its use in other musculoskeletal conditions. Fracture repair is one area in which PTH may have a significant clinical impact. Multiple animal studies have shown that systemic PTH treatment of healing fractures increased both callus volume and return of mechanical competence in models of fracture healing. Whereas the potential for PTH has been established, the mechanism(s) by which PTH produces these effects remain elusive. MATERIALS AND METHODS: Closed femoral fractures were generated in 8-wk-old male C57Bl/6 mice followed by daily systemic injections of either saline (control) or 30 microg/kg PTH(1-34) for 14 days after fracture. Bones were harvested at days 2, 3, 5, 7, 10, 14, 21, and 28 after fracture and analyzed at the tissue level by radiography and histomorphometry and at the molecular and biochemical levels level by RNase protection assay (RPA), real-time PCR, and Western blot analysis. RESULTS: Quantitative muCT analysis showed that PTH treatment induced a larger callus cross-sectional area, length, and total volume compared with controls. Molecular analysis of the expression of extracellular matrix genes associated with chondrogenesis and osteogenesis showed that PTH treated fractures displayed a 3-fold greater increase in chondrogenesis relative to osteogenesis over the course of the repair process. In addition, chondrocyte hypertrophy occurred earlier in the PTH-treated callus tissues. Analysis of the expression of potential mediators of PTH actions showed that PTH treatment significantly induced the expression of Wnts 4, 5a, 5b, and 10b and increased levels of unphosphorylated, nuclear localized beta-catenin protein, a central feature of canonical Wnt signaling. CONCLUSIONS: These results showed that the PTH-mediated enhancement of fracture repair is primarily associated with an amplification of chondrocyte recruitment and maturation in the early fracture callus. Associated with these cellular effects, we observed an increase in canonical Wnt signaling supporting the conclusion that PTH effects on bone repair are mediated at least in part through the activation of Wnt-signaling pathways.     

Mechanisms of immunotherapeutic intervention by anti-CD154 (CD40L) antibody in high-risk corneal transplantation.

This study aimed to determine the effects of anti-CD154 on T cell cytokine profiles and ocular chemokine gene expression after high-risk corneal transplantation and to specifically determine if CD154 blockade is associated with a switch from a Th1 to a Th2 alloimmune response. Mice were used as recipients of syngeneic or multiple minor H or MHC antigen-mismatched corneal grafts. Recipient beds were neovascularized (high-risk). Hosts were randomized to receive either anti-CD154 antibody or control immunoglobulin (Ig) perioperatively. Two weeks after corneal transplantation, allospecific delayed-type hypersensitivity (DTH) was evaluated. Frequencies of interferon-gamma (IFN-gamma)-, interleukin-2 (IL-2)-, IL-4-, and IL-5-secreting T cells in the hosts were measured by enzyme-linked immunospot (ELISPOT) assay. Ocular chemokine gene expression in anti-CD154-treated and control hamster Ig-treated groups was determined using a multiprobe ribonuclease protection assay (RPA). Leukocyte infiltration of corneal grafts was evaluated microscopically. Anti-CD154-treated mice did not exhibit allospecific DTH. The frequencies of Th1 cytokine-producing but not Th2 cytokine-producing T cells were significantly reduced in anti-CD154-treated hosts. Postoperative mRNA levels of RANTES and macrophage inflammatory protein-1beta (MIP-1beta) in anti-CD154-treated eyes were substantially suppressed compared with hamster Ig-treated controls. Leukocyte infiltration was profoundly suppressed in grafts of anti-CD154-treated hosts. These data demonstrate that blockade of the CD40-CD154 costimulatory pathway after corneal transplantation inhibits Th1-mediated responses but does not induce a switch to a Th2-specific response. In addition, anti-CD154 therapy suppresses ocular chemokine gene expression and leukocytic infiltration into allografts.     

Coagulation factor deficiency as a cause of recurrent fetal loss: a red herring!

Whether severe coagulation factor deficiency can cause adverse pregnancy outcomes or recurrent fetal loss is not definitely known. We report here on five women with severe deficiency of coagulation factors (two factor X, one factor XI, one factor VII and one von Willebrand factor) who presented with history of unexplained fetal loss or with adverse pregnancy outcome. Detailed investigations of thrombophilia showed that four patients were positive for antiphospholipid antibodies, one of whom was also homozygous for the plasminogen-activator inhibitor-1 4G/4G polymorphism, and the fifth patient was deficient for protein C. Despite the concomitant presence of both coagulation factor defect and thrombophilia, fetal loss may be attributed to factor defect that in reality is a red herring, with underlying thrombophilia not being evaluated.     

Mefenamic acid-induced bilateral transient myopia, secondary angle closure glaucoma and choroidal detachment

Drug-induced secondary angle closure is quite common and in the majority of cases simply stopping the medication leads to rapid reversal of the condition and resolution of glaucoma. We describe here a patient who presented with secondary angle closure glaucoma and myopia following mefenamic acid ingestion which was managed successfully by stopping the medication, symptomatic treatment and reassurance.     

Effects of a brisk walk on lipoprotein lipase activity and plasma triglyceride concentrations in the fasted and postprandial states

This study aimed to determine whether changes in plasma heparin-releasable lipoprotein lipase (LPL) activity following a brisk walk were associated with decreases in fasting and/or postprandial triglyceride (TG) concentrations. Two groups of pre-menopausal women participated. In one group (fasting study group, n=10), TG concentrations and post-heparin plasma LPL activity were measured in the fasted state on two occasions: ~18 h after a 2-h treadmill walk at 50% maximal oxygen uptake (exercise trial); and after a day of no exercise (control trial). The other group (postprandial study group, n=9) undertook two oral fat tolerance tests (blood samples taken fasting and for 6 h after a high-fat meal), with plasma LPL activity measured 6 h after meal ingestion. Pre-conditions were the same as for the fasting study group (i.e. control and prior exercise). Prior exercise reduced fasting TG concentrations by 23 (7)% (fasting study group) [mean (SEM)] and by 18 (9)% (postprandial study group) (both P<0.05), and the postprandial TG response by 23 (6)% (postprandial study group) (P<0.01). Plasma LPL activity was not significantly increased by exercise in either the fasting or postprandial study groups. However, exercise-induced changes in both fasting and postprandial LPL activity were significantly correlated with the respective exercise-induced changes in fasting TG concentration and the postprandial TG response (r=−0.70 and −0.77 respectively, P<0.05 for both). These data suggest that increased LPL activity may contribute to the hypotriglyceridaemic effect of moderate exercise, although other mechanisms are also likely to be involved. Electronic Publication     

Quantitation of the reconstruction quality of a four-dimensional computed tomography process for lung cancer patients

We have developed a four-dimensional computed tomography (4D CT) technique for mapping breathing motion in radiotherapy treatment planning. A multislice CT scanner (1.5 mm slices) operated in ciné mode was used to acquire 12 contiguous slices in each couch position for 15 consecutive scans (0.5 s rotation, 0.25 s between scans) while the patient underwent simultaneous quantitative spirometry measurements to provide a sorting metric. The spirometry-sorted scans were used to reconstruct a 4D data set. A critical factor for 4D CT is quantifying the reconstructed data set quality which we measure by correlating the metric used relative to internal-object motion. For this study, the internal air content within the lung was used as a surrogate for internal motion measurements. Thresholding and image morphological operations were applied to delineate the air-containing tissues (lungs, trachea) from each CT slice. The Hounsfield values were converted to the internal air content (V). The relationship between the air content and spirometer-measured tidal volume (v) was found to be quite linear throughout the lungs and was used to estimate the overall accuracy and precision of tidal volume-sorted 4D CT. Inspection of the CT-scan air content as a function of tidal volume showed excellent correlations (typically r>0.99) throughout the lung volume. Because of the discovered linear relationship, the ratio of internal air content to tidal volume was indicative of the fraction of air change in each couch position. Theoretically, due to air density differences within the lung and in room, the sum of these ratios would equal 1.11. For 12 patients, the mean value was 1.08 +/- 0.06, indicating the high quality of spirometry-based image sorting. The residual of a first-order fit between v and V was used to estimate the process precision. For all patients, the precision was better than 8%, with a mean value of 5.1% +/- 1.9%. This quantitative analysis highlights the value of using spirometry as the metric in sorting CT scans. The 4D reconstruction provides the CT data required to measure the three-dimensional trajectory of tumor and lung tissue during free breathing.