Differences in bone turnover markers and injury risks between local and international horses: A Victorian Spring Racing Carnival study.

BACKGROUND: Musculoskeletal injuries (MSI) are common in racehorses and have been of increasing concern in horses travelling internationally to compete. Understanding the differences in bone turnover between local horses and international horses following long-distance air transportation may inform MSI prevention strategies. OBJECTIVES: To understand the differences in bone turnover markers and risk of MSI between local horses and international horses following long-distance air transportation. STUDY DESIGN: Prospective cohort. METHODS: The concentrations of bone turnover markers (OCN and CTXI), markers of stress (cortisol), inflammation (serum amyloid A) and circadian rhythm (melatonin), and bisphosphonates were determined in blood samples collected twice (14-17 days apart), from horses following international travel (n = 69), and from local horses (n = 79). The associations between markers, long-distance travel and MSI were determined using multivariable generalised linear regression models. RESULTS: Within 3-5 days post-transport, concentrations of cortisol in international horses were higher than those of local horses (main effect, Coef. 0.39; 95% CI 0.24, 0.54; p < 0.001) but they decreased and were not different to those of local horses at the second timepoint (interaction effect, Coef. -0.27; 95% CI -0.46, -0.07; p = 0.007). After adjusting for age and sex, OCN and CTXI were not significantly different between international and local horses; however, OCN was lower in international horses at timepoint 2 (interaction effect, Coef. -0.16; 95% CI -0.31, -0.01; p = 0.043). The prevalence of MSI was higher in the international (26%; 95% CI 16, 38%) compared with local horses (8%; 95% CI 3, 16%; p < 0.001), with all severe MSI sustained by the international horses. At the second timepoint compared with the first timepoint post-transport, cortisol remained high or increased (interaction effect, Coef. 0.43; 95% CI 0.24, 0.61; p < 0.001) and OCN increased (interaction effect, Coef. 0.26; 95% CI 0.08, 0.44; p = 0.006) in the horses that sustained severe MSI. MAIN LIMITATIONS: Horse population and racing career parameters differed between groups. Bone turnover markers have low sensitivity to detect local bone changes. CONCLUSIONS: Most horses showed minimal effects of long-distance air transport within 2 weeks relative to local horses as assessed by stress and bone turnover markers. Screening for persistent high cortisol and evidence of net bone formation after long-distance air transportation may help to identify racehorses at high risk of catastrophic MSI.

CONTEXTE: Les blessures musculosquelettiques (MSI) sont communes chez les chevaux de course et demeurent une source d'inquiétude pour les chevaux voyageant à l'international. Comprendre les différences de remodelage osseux entre les chevaux locaux et ceux voyageant suivant un trajet aérien longue distance pourrait aider au développement de stratégies de prévention des dommages musculosquelettiques. OBJECTIFS: Comprendre les différences de marqueurs de remodelage osseux et de risques de MSI entre les chevaux locaux et ceux voyageant à l'international suivant un transport aérien de longue distance. TYPE D'ÉTUDE: Étude de cohorte prospective. MÉTHODES: Les concentrations des marqueurs de remodelage osseux (OCN et CTXI), de stress (cortisol), d'inflammation (serum amyloid A), de rythme circadien (melatonin) et les bisphosphonates ont été mesurés dans des échantillons sanguins à deux reprises (14­17 jours à part) chez des chevaux ayant été à l'international (n = 69) et étant restés localement (n = 79). L'association entre les marqueurs, le transport longue distance et les MSI a été déterminée par modèles de régression linéaire multivarié généralisé. RÉSULTATS: Entre 3 à 5 jours suivant le transport, les concentrations de cortisol chez les chevaux internationaux étaient supérieures aux chevaux locaux (effet primaire, Coef. 0.39; 95% CI 0.24, 0.54; P < 0.001), mais ont diminué par la suite jusqu'à ne plus être différent de ceux des chevaux locaux à la deuxième mesure (effet interaction, Coef. −0.27; 95% CI −0.46, −0.07; P = 0.007). Après ajustement pour l'âge et le sexe, OCN et CTXI n'étaient pas significativement différents entre les chevaux internationaux et locaux. Cependant, OCN était inférieur chez les chevaux internationaux à la deuxième mesure (effet interaction, Coef. −0.16; 95% CI −0.31, −0.01; P = 0.043). La prévalence de MSI était plus élevée chez les chevaux internationaux (26%; 95% CI 16, 38%) comparativement aux chevaux locaux (8%; 95% CI 3, 16%; p < 0.001), avec toutes les MSI sévères subi par les chevaux internationaux. Au moment de la deuxième mesure comparée à la première mesure après le transport, le cortisol est demeuré élevé ou a augmenté (effet interaction, Coef. 0.43; 95% CI 0.24, 0.61; P < 0.001) et l'OCN a augmenté (effet interaction, Coef. 0.26; 95% CI 0.08, 0.44; P = 0.006) chez les chevaux ayant subi une MSI sévère. LIMITES PRINCIPALES: La population équine et leurs paramètres de course diffèrent entre les groupes. Les marqueurs de remodelage osseux ont une faible sensibilité pour la détection de changements osseux localisés. CONCLUSION: En deux semaines, les effets de transport aérien longue distance ont été minimaux pour la majorité des chevaux comparativement aux chevaux locaux, tel que démontré par les marqueurs de stress et de remodelage osseux. La détection de niveau élevé de cortisol de façon persistante et d'évidence d'os néoformé suivant un transport aérien de longue distance pourrait aider à détecter les chevaux de course à haut risque de MSI.

Assessment of subchondral bone microdamage quantification using contrast-enhanced imaging techniques.

Bone microdamage is common at subchondral bone (SCB) sites subjected to repeated high rate and magnitude of loading in the limbs of athletic animals and humans. Microdamage can affect the biomechanical behaviour of bone under physiological loading conditions. To understand the effects of microdamage on the mechanical properties of SCB, it is important to be able to quantify it. The extent of SCB microdamage had been previously estimated qualitatively using plain microcomputed tomography (µCT) and a radiocontrast quantification method has been used for trabecular bone but this method may not be directly applicable to SCB due to differences in bone structure. In the current study, SCB microdamage detection using lead uranyl acetate (LUA) and quantification by contrast-enhanced µCT and backscattered scanning electron microscopy (SEM) imaging techniques were assessed to determine the specificity of the labels to microdamage and the accuracy of damaged bone volume metrices. SCB specimens from the metacarpus of racehorses, with the hyaline articular cartilage (HAC) removed, were grouped into two with one group subjected to ex vivo uniaxial compression loading to create experimental bone damage. The other group was not loaded to preserve the pre-existing in vivo propagated bone microdamage. A subset of each group was stained with LUA using an established or a modified protocol to determine label penetration into SCB. The µCT and SEM images of stained specimens showed that penetration of LUA into the SCB was better using the modified protocol, and this protocol was repeated in SCB specimens with intact hyaline articular cartilage. The percentage of total label localised to bone microdamage was determined on SEM images, and the estimated labelled bone volume determined by µCT in SCB groups was compared. Label was present around diffuse and linear microdamage as well as oblique linear microcracks present at the articular surface, except in microcracks with high-density mineral infills. Bone surfaces lining pores with recent mineralisation were also labelled. Labelled bone volume fraction (LV/BV) estimated by µCT was higher in the absence of HAC. At least 50% of total labels were localised to bone microdamage when the bone area fraction (B.Ar/T.Ar) of the SCB was greater than 0.85 but less than 30% when B.Ar/T.Ar of the SCB was less than 0.85. To adjust for LUA labels on bone surfaces, a measure of the LV/BV corrected for bone surface area (LV/BV BS-1 ) was used to quantify damaged SCB. In conclusion, removal of HAC and using a modified labelling protocol effectively stained damaged SCB of the metacarpus of racehorses and represents a technique useful for quantifying microdamage in SCB. This method can facilitate future investigations of the effects of microdamage on joint physiology.

Effect of composition, casein genetic variants and glycosylation degree on bovine milk whipping properties.

This study investigated the individual and combined effects of ĸ-Casein (ĸ-CN; AA, AB, BB), ß-Casein (ß-CN; A1A1, A1A2, A2A2) and high and low ratios of glycosylated ĸ-CN to total ĸ-CN, referred to as the glycosylation degree (GD), on bovine cream whipping properties. The genetic variants of individual cows were identified using reversed-phase high-performance liquid chromatography (RP-HPLC) and verified through liquid chromatography-mass spectrometry (LC-MS). A previously discovered relationship between days-in-milk and GD was validated and used to obtain high and low GD milk. Whipped creams were created through the mechanical agitation of fat standardised cream from milk of different ĸ-CN, ß-CN, and GD combinations, and whipping properties (the ability to whip, overrun, whipping time and firmness) were evaluated. No significant correlation was measured in whipping properties for cream samples from milks with different ĸ-CN and ß-CN genetic variants. However, 80 % of samples exhibiting good whipping properties (i.e., the production of a stiffened peak) were from milk with low GD suggesting a correlation between whipping properties and levels of glycosylation. Moreover, cream separated from skim milk of larger casein micelle size showed superior whipping properties with shorter whipping times (<5 min), and higher firmness and overrun. Milk fat globule (MFG) size, on the other hand, did not affect whipping properties. Results indicate that the GD of κ-CN and casein micelle size may play a role in MFG adsorption at the protein and air interface of air bubbles formed during whipping; hence, they govern the dynamics of fat network formation and influencing whipping properties.

Reducing the prosthesis modulus by inclusion of an open space lattice improves osteogenic response in a sheep model of extraarticular defect.

Introduction: Stress shielding is a common complication following endoprosthetic reconstruction surgery. The resulting periprosthetic osteopenia often manifests as catastrophic fractures and can significantly limit future treatment options. It has been long known that bone plates with lower elastic moduli are key to reducing the risk of stress shielding in orthopedics. Inclusion of open space lattices in metal endoprostheses is believed to reduce the prosthesis modulus potentially improving stress shielding. However, no in vivo data is currently available to support this assumption in long bone reconstruction. This manuscript aims to address this hypothesis using a sheep model of extraarticular bone defect. Methods: Initially, CT was used to create a virtual resection plan of the distal femoral metaphyses and to custom design endoprostheses specific to each femur. The endoprostheses comprised additively manufactured Ti6Al4V-ELI modules that either had a solid core with a modulus of ∼120 GPa (solid implant group) or an open space lattice core with unit cells that had a modulus of 3-6 GPa (lattice implant group). Osteotomies were performed using computer-assisted navigation followed by implantations. The periprosthetic, interfacial and interstitial regions of interest were evaluated by a combination of micro-CT, back-scattered scanning electron microscopy (BSEM), as well as epifluorescence and brightfield microscopy. Results: In the periprosthetic region, mean pixel intensity (a proxy for tissue mineral density in BSEM) in the caudal cortex was found to be higher in the lattice implant group. This was complemented by BSEM derived porosity being lower in the lattice implant group in both caudal and cranial cortices. In the interfacial and interstitial regions, most pronounced differences were observed in the axial interfacial perimeter where the solid implant group had greater bone coverage. In contrast, the lattice group had a greater coverage in the cranial interfacial region. Conclusion: Our findings suggest that reducing the prosthesis modulus by inclusion of an open-space lattice in its design has a positive effect on bone material and morphological parameters particularly within the periprosthetic regions. Improved mechanics appears to also have a measurable effect on the interfacial osteogenic response and osteointegration.

Effect of a protease-activated receptor-2 antagonist (GB88) on inflammation-related loss of alveolar bone in periodontal disease.

BACKGROUND AND OBJECTIVE: Protease-activated receptor-2 (PAR2 ), a pro-inflammatory G-protein coupled receptor, has been associated with pathogenesis of periodontitis and the resulting bone loss caused by oral pathogens, including the keystone pathogen Porphyromonas gingivalis (P. gingivalis). We hypothesised that administration of a PAR2 antagonist, GB88, might prevent inflammation and subsequent alveolar bone resorption in a mouse model of periodontal disease. METHODS: Periodontitis was induced in mice by oral inoculations with P. gingivalis for a total of eight times over 24 days. The infected mice were treated with either GB88 or vehicle for the duration of the trial. Following euthanasia on day 56, serum was collected and used for the detection of mast cell tryptase. The right maxillae were defleshed and stained with methylene blue to measure the exposed cementum in molar teeth. The left maxillae were prepared for cryosections followed by staining for tartrate-resistant acid phosphatase to identify osteoclasts or with toluidine blue to identify mast cells. Reverse transcription quantitative PCR (RT-qPCR) was used to quantify the expression of inflammatory cytokines in the gingival tissue. Supernatants of T-lymphocyte cultures isolated from the regional lymph nodes were assayed using a cytometric bead array to measure the Th1/Th2/Th17 cytokine levels. RESULTS: Measurement of the exposed cementum showed that GB88 reduced P. gingivalis-induced alveolar bone loss by up to 69%. GB88 also prevented the increase in osteoclast numbers observed in the infected mice. Serum tryptase levels were significantly elevated in both the infected groups, and not altered by treatment. RT-qPCR showed that GB88 prevented the upregulation of Il1b, Il6, Ifng and Cd11b. In T-lymphocyte supernatants, only IFNγ and IL-17A levels were increased in response to infection, but this was prevented by GB88 treatment. CONCLUSIONS: GB88 significantly reduced osteoclastic alveolar bone loss in mice infected with P. gingivalis, seemingly by preventing the upregulation of several inflammatory cytokines. PAR2 antagonism may be an effective treatment strategy for periodontal disease.

The role of glycosylation in amyloid fibril formation of bovine κ-casein.

In order to explore the functions of glycosylation of κ-Casein (κ-CN) in bovine milk, unglycosylated (UG) and twice glycosylated (2G) forms of κ-CN B were purified by selective precipitation followed by anion exchange chromatography from κ-CN BB milk and tested for their amyloid fibril formation and morphology, oligomerisation states and protein structure. The diameter of self-assembled κ-CN B aggregates of both glyco-form were shown for the first time to be in the same 26.0-28.7 nm range for a 1 mg mL-1 solution. The presence of two bound glycans in the protein structure of 2G κ-CN B led to a greater increase in the maximum amyloid fibril formation rate with increasing protein concentration and a difference in both length (82.0 ± 29.9 vs 50.3 ± 13.7 nm) and width (8.6 ± 2.1 vs 13.9 ± 2.5 nm) for fibril morphology compared to UG κ-CN B. The present results suggest that amyloid fibril formation proceeds at a slow but steady rate via the self-assembly of dissociated, monomeric κ-CN B proteins at concentrations of 0.22-0.44 mg mL-1. However amyloid fibril formation proceeds more rapidly via the assembly of either aggregated κ-CN present in a micelle-like form or dissociated monomeric κ-CN, packed into reorganised formational structures above the critical micellar concentration to form fibrils of differing width. The degree of glycosylation has no effect on the polarity of the adjacent environment, nor non-covalent and disulphide interactions between protein molecules when in the native form. Yet glycosylation can influence protein folding patterns of κ-CN B leading to a reduced tryptophan intrinsic fluorescence intensity for 2G compared to UG κ-CN B. These results demonstrate that glycosylation plays an important role in the modulation of aggregation states of κ-CN and contributes to a better understanding of the role of glycosylation in the formation of amyloid fibrils from intrinsically disordered proteins.

Protease-activated receptor-2 dependent and independent responses of bone cells to prostate cancer cell secretory products.

BACKGROUND: Metastatic prostate cancer lesions in the skeleton are frequently characterized by excessive formation of bone. Prostate cancer cells secrete factors, including serine proteases, that are capable of influencing the behavior of surrounding cells. Some of these proteases activate protease-activated receptor-2 (PAR2 ), which is expressed by osteoblasts (bone-forming cells) and precursors of osteoclasts (bone-resorbing cells). The aim of the current study was to investigate a possible role for PAR2 in regulating the behavior of bone cells exposed to metastatic prostate cancer cells. METHODS: The effect of medium conditioned by the PC3, DU145, and MDA-PCa-2b prostate cancer cell lines was investigated in assays of bone cell function using cells isolated from wildtype and PAR2 -null mice. Osteoclast differentiation was assessed by counting tartrate-resistant acid phosphatase-positive multinucleate cells in bone marrow cultured in osteoclastogenic medium. Osteoblasts were isolated from calvariae of neonatal mice, and BrdU incorporation was used to assess their proliferation. Assays of alkaline phosphatase activity and quantitative PCR analysis of osteoblastic gene expression were used to assess osteoblast differentiation. Responses of osteoblasts to medium conditioned by MDA-PCa-2b cells were analyzed by RNAseq. RESULTS: Conditioned medium (CM) from all three cell lines inhibited osteoclast differentiation independently of PAR2 . Media from PC3 and DU145 cells had no effect on assays of osteoblast function. Medium conditioned by MDA-PCa-2b cells stimulated BrdU incorporation in both wildtype and PAR2 -null osteoblasts but increased alkaline phosphatase activity and Runx2 and Col1a1 expression in wildtype but not PAR2 -null cells. Functional enrichment analysis of RNAseq data identified enrichment of multiple gene ontology terms associated with lysosomal function in both wildtype and PAR2 -null cells in response to MDA-PCa-2b-CM. Analysis of individual genes identified osteogenesis-associated genes that were either upregulated by MDA-PCa-2b-CM selectively in wildtype cells or downregulated selectively in PAR2 -null cells. CONCLUSIONS: Factors secreted by prostate cancer cells influence bone cell behavior through both PAR2 -dependent and -independent mechanisms. Both PAR2 -independent suppression of osteoclast differentiation and PAR2 -dependent stimulation of osteogenesis are likely to determine the nature of prostate cancer metastases in bone.

Tetrathiomolybdate Treatment Attenuates Bleomycin-Induced Angiogenesis and Lung Pathology in a Sheep Model of Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive chronic lung disease characterized by excessive extracellular matrix (ECM) deposition in the parenchyma of the lung. Accompanying the fibrotic remodeling, dysregulated angiogenesis has been observed and implicated in the development and progression of pulmonary fibrosis. Copper is known to be required for key processes involved in fibrosis and angiogenesis. We therefore hypothesized that lowering bioavailable serum copper with tetrathiomolybdate could be of therapeutic value for treating pulmonary fibrosis. This study aimed to investigate the effect of tetrathiomolybdate on angiogenesis and fibrosis induced in sheep lung segments infused with bleomycin. Twenty sheep received two fortnightly infusions of either bleomycin (3U), or saline (control) into two spatially separate lung segments. A week after the final bleomycin/saline infusions, sheep were randomly assigned into two groups (n = 10 per group) and received twice-weekly intravenous administrations of either 50 mg tetrathiomolybdate, or sterile saline (vehicle control), for 6 weeks. Vascular density, expressed as the percentage of capillary area to the total area of parenchyma, was determined in lung tissue sections immuno-stained with antibodies against CD34 and collagen type IV. The degree of fibrosis was assessed by histopathology scoring of H&E stained sections and collagen content using Masson's trichrome staining. Lung compliance was measured via a wedged bronchoscope procedure prior to and 7 weeks following final bleomycin infusion. In this large animal model, we show that copper lowering by tetrathiomolybdate chelation attenuates both bleomycin-induced angiogenesis and pulmonary fibrosis. Moreover, tetrathiomolybdate treatment downregulates vascular endothelial growth factor (VEGF) expression, and improved lung function in bleomycin-induced pulmonary fibrosis. Tetrathiomolybdate also suppressed the accumulation of inflammatory cells in bronchoalveolar lavage fluid 2 weeks after bleomycin injury. The molecular mechanism(s) underpinning copper modulation of fibrotic pathways is an important area for future investigation, and it represents a potential therapeutic target for pulmonary fibrosis.

Small airway remodeling in a sheep model of bleomycin-induced pulmonary fibrosis.

BACKGROUND: Although IPF is described traditionally as a disease affecting lung parenchyma, there is renewed interest in the alterations in the structure and function of the small airways in both IPF patients, and animal models of pulmonary fibrosis. Small airway remodeling may contribute to the pathophysiology of pulmonary fibrosis. Given the dearth of knowledge of small airway changes in pulmonary fibrosis, this study aims to assess the structural remodeling, as well as functional changes associated with bleomycin-injured small airways in a sheep model of pulmonary fibrosis. MATERIALS AND METHODS: Two separate lung segments in ten sheep received two challenges of either 3 IU bleomycin, or saline (control), two weeks apart. The animals were euthanized seven weeks after the final bleomycin injury. Airflow resistance in the infused segments was measured with a wedged-bronchoscope procedure. This parameter was measured at baseline before bleomycin/saline-infusion, and at 2-, 4-, and 7-weeks after the final bleomycin-infusion. Inflammation and fibrosis in the airways were assessed by semi-quantitative morphological parameters. The density of blood vessels in the small airway walls was assessed in lung tissue sections immuno-stained with antibodies against collagen type IV. RESULTS: There were a number of changes in the distal airways of bleomycin-infused lung segments. Bleomycin exposure significantly elevated airway resistance in these lung segments when compared to saline-infused control lung segments. In the peribronchial and peribronchiolar regions of the small airways, there were significantly increased levels of inflammation, fibrosis, airway wall area, and collagen deposition in bleomycin-infused airways when compared to saline-infused airways. Bronchial blood vessel density was not significantly different between bleomycin-and saline-infused lung segments. CONCLUSIONS: In summary, our results indicate that the distal airways are involved in the pathology induced by bleomycin in this sheep model. This suggests that the sheep model may be useful for studying small airway remodeling in pulmonary fibrosis.

KCa3.1 channel blockade attenuates microvascular remodelling in a large animal model of bleomycin-induced pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease with limited therapeutic options and poor prognosis. IPF has been associated with aberrant vascular remodelling, however the role of vascular remodelling in pulmonary fibrosis is poorly understood. Here, we used a novel segmental challenge model of bleomycin-induced pulmonary fibrosis in sheep to evaluate the remodelling of the pulmonary vasculature, and to investigate the changes to this remodelling after the administration of the KCa3.1 channel inhibitor, senicapoc, compared to the FDA-approved drug pirfenidone. We demonstrate that in vehicle-treated sheep, bleomycin-infused lung segments had significantly higher blood vessel density when compared to saline-infused control segments in the same sheep. These microvascular density changes were significantly attenuated by senicapoc treatment. The increases in vascular endothelial growth factor (VEGF) expression and endothelial cell proliferation in bleomycin-infused lung segments were significantly reduced in sheep treated with the senicapoc, when compared to vehicle-treated controls. These parameters were not significantly suppressed with pirfenidone treatment. Senicapoc treatment attenuated vascular remodelling through inhibition of capillary endothelial cell proliferation and VEGF expression. These findings suggest a potential new mode of action for the novel drug senicapoc which may contribute to its efficacy in combatting pulmonary fibrosis.

Normal inflammation and regeneration of muscle following injury require osteopontin from both muscle and non-muscle cells.

BACKGROUND: Osteopontin is secreted by skeletal muscle myoblasts and macrophages, and its expression is upregulated in muscle following injury. Osteopontin is present in many different structural forms, which vary in their expression patterns and effects on cells. Using a whole muscle autograft model of muscle injury in mice, we have previously shown that inflammation and regeneration of muscle following injury are delayed by the absence of osteopontin. The current study was undertaken to determine whether muscle or non-muscle cells provide the source of osteopontin required for its role in muscle regeneration. METHODS: The extensor digitorum longus muscle of wild-type and osteopontin-null mice was removed and returned to its bed in the same animal (autograft) or placed in the corresponding location in an animal of the opposite genotype (allograft). Grafts were harvested at various times after surgery and analysed by histology, flow cytometry and quantitative polymerase chain reaction. Data were analysed using one- or two-way ANOVA or Kruskal-Wallis test. RESULTS: Immunohistochemistry confirmed that osteopontin was expressed by macrophages in osteopontin-null muscle allografts in wild-type hosts and by myoblasts in wild-type allografts in osteopontin-null hosts. The decrease in muscle fibre number observed in wild-type autografts following grafting and the subsequent appearance of regenerating fibres were delayed in both types of allografts to a similar extent as in osteopontin-null autografts. Infiltration of neutrophils, macrophages and M1 and M2 macrophage subtypes were also delayed by lack of osteopontin in the muscle and/or host. While the proportion of macrophages showing the M1 phenotype was not affected, the proportion showing the M2 phenotype was decreased by osteopontin deficiency. Expression of tumour necrosis factor-α and interleukin-4 was lower in osteopontin-null than in wild-type autografts, and there was no difference between the osteopontin-null graft types. CONCLUSIONS: Osteopontins from muscle and non-muscle sources are equally important in the acute response of muscle to injury, and cannot substitute for each other, suggesting that they play distinct roles in regulation of cell behaviour. Future studies of mechanisms of osteopontin's roles in acute muscle inflammation and regeneration will need to investigate responses to osteopontins derived from both myoblasts and macrophages.

Keratinocyte-specific ablation of protease-activated receptor 2 prevents gingival inflammation and bone loss in a mouse model of periodontal disease.

Chronic periodontitis is characterised by gingival inflammation and alveolar bone loss. A major aetiological agent is Porphyromonas gingivalis, which secretes proteases that activate protease-activated receptor 2 (PAR2 ). PAR2 expressed on oral keratinocytes is activated by proteases released by P. gingivalis, inducing secretion of interleukin 6 (IL-6), and global knockout of PAR2 prevents bone loss and inflammation in a periodontal disease model in mice. To test the hypothesis that PAR2 expressed on gingival keratinocytes is required for periodontal disease pathology, keratinocyte-specific PAR2 -null mice were generated using K14-Cre targeted deletion of the PAR2 gene (F2rl1). These mice were subjected to a model of periodontitis involving placement of a ligature around a tooth, combined with P. gingivalis infection ("Lig + Inf"). The intervention caused a significant 44% decrease in alveolar bone volume (assessed by microcomputed tomography) in wildtype (K14-Cre:F2rl1wt/wt ), but not littermate keratinocyte-specific PAR2 -null (K14-Cre:F2rl1fl/fl ) mice. Keratinocyte-specific ablation of PAR2 prevented the significant Lig + Inf-induced increase (2.8-fold) in the number of osteoclasts in alveolar bone and the significant up-regulation (2.4-4-fold) of the inflammatory markers IL-6, IL-1ß, interferon-γ, myeloperoxidase, and CD11b in gingival tissue. These data suggest that PAR2 expressed on oral epithelial cells is a critical regulator of periodontitis-induced bone loss and will help in designing novel therapies with which to treat the disease.

The vacuolar H+ ATPase V0 subunit d2 is associated with chondrocyte hypertrophy and supports chondrocyte differentiation.

Chondrocyte hypertrophy makes important contributions to bone development and growth. We have investigated a number of novel cartilage genes identified in a recent transcriptomic study to determine whether they are differentially expressed between different zones of equine foetal growth cartilage. Twelve genes (ATP6V0D2, BAK1, DDX5, GNB1, PIP4K2A, RAP1B, RPS7, SRSF3, SUB1, TMSB4, TPI1 and WSB2) were found to be more highly expressed in the zone of hypertrophic chondrocytes than in the reserve or proliferative zones, whereas FOXA3 and SERPINA1 were expressed at lower levels in the hypertrophic zone than in the reserve zone. ATP6V0D2, which encodes vacuolar H+ ATPase (V-ATPase) V0 subunit d2 (ATP6V0D2), was selected for further study. Immunohistochemical analysis of ATP6V0D2 in growth cartilage showed stronger staining in hypertrophic than in reserve zone or proliferative chondrocytes. Expression of ATP6V0D2 mRNA and protein was up-regulated in the mouse chondrocytic ATDC5 cell line by conditions inducing expression of hypertrophy-associated genes including Col10a1 and Mmp13 (differentiation medium). In ATDC5 cells cultured in control medium, knockdown of Atp6v0d2 or inhibition of V-ATPase activity using bafilomycin A1 caused a decrease in Col2a1 expression, and in cells cultured in differentiation medium the two treatments caused a decrease in nuclear area. Inhibition of V-ATPase, but not Atp6v0d2 knockdown, prevented the upregulation of Col10a1, Mmp13 and Vegf by differentiation medium, while Atp6v0d2 knockdown, but not inhibition of V-ATPase, caused an increase in the number of ATDC5 cells cultured in differentiation medium. These observations identify ATP6V0D2 as a novel chondrocyte hypertrophy-associated gene. The results are consistent with roles for V-ATPase, both ATP6V0D2-dependent and -independent, in supporting chondrocyte differentiation and hypertrophy.

A T cell-specific knockout reveals an important role for protease-activated receptor 2 in lymphocyte development.

Activation of protease-activated receptor-2 (PAR2) expressed by T cells has been linked to the bone loss associated with periodontitis. We generated PAR2 conditional-null mice and crossed these with mice expressing Cre recombinase under control of the Lck proximal promoter, to produce T cell-specific PAR2-null mice in order to further study the cellular mechanism involved in periodontitis. Here we report that efficient deletion of PAR2 in thymocytes isolated from T cell-specific PAR2-null mice resulted in thymic and splenic hypoplasia and a reduction in the cells of the cortex and a loss of distinction between the cortex and the medulla of the thymus. FACS analysis confirmed significant reductions in CD4 and CD8 double negative (DN3 and DN4) sub-populations, as well as double positive and single positive T cells, in T cell-specific PAR2-null mice compared to Cre expressing PAR2 wild-type mice. The proportion of annexin V positive and propidium iodide negative cells was increased in CD4 and CD8 double negative, double positive and single positive T cells from T cell-specific PAR2-null mice. No change in the proportion of Ki67 positive cells was observed in sections of thymus from T cell-specific PAR2-null mice, suggesting that the depletion of T cell sub-populations in T cell-specific PAR2-null mice resulted from increased apoptosis rather than reduced proliferation. Together, these results demonstrate that PAR2 plays an important and previously unrecognised anti-apoptotic role in T cell development and suggest that the PAR2 conditional-null mouse will be an important resource for determining tissue and cell specific effects of PAR2.

The antiepileptic medications carbamazepine and phenytoin inhibit native sodium currents in murine osteoblasts.

OBJECTIVE: Fracture risk is a serious comorbidity in epilepsy and may relate to the use of antiepileptic drugs (AEDs). Many AEDs inhibit ion channel function, and the expression of these channels in osteoblasts raises the question of whether altered bone signaling increases bone fragility. We aimed to confirm the expression of voltage-gated sodium (NaV ) channels in mouse osteoblasts, and to investigate the action of carbamazepine and phenytoin on NaV channels. METHODS: Immunocytochemistry was performed on primary calvarial osteoblasts extracted from neonatal C57BL/6J mice and additional RNA sequencing (RNASeq) was included to confirm expression of NaV . Whole-cell patch-clamp recordings were made to identify the native currents expressed and to assess the actions of carbamazepine (50 µm) or phenytoin (50 µm). RESULTS: NaV expression was demonstrated with immunocytochemistry, RNA sequencing, and functionally, with demonstration of robust tetrodotoxin-sensitive and voltage-activated inward currents. Application of carbamazepine or phenytoin resulted in significant inhibition of current amplitude for carbamazepine (31.6 ± 5.9%, n = 9; p < 0.001), and for phenytoin (35.5 ± 6.9%, n = 7; p < 0.001). SIGNIFICANCE: Mouse osteoblasts express NaV , and native NaV currents are blocked by carbamazepine and phenytoin, supporting our hypothesis that AEDs can directly influence osteoblast function and potentially affect bone strength.

Identification of novel osteochondrosis--Associated genes.

During the early stages of articular osteochondrosis, cartilage is retained in subchondral bone, but the pathophysiology of this condition of growing humans and domestic animals is poorly understood. A subtractive hybridization study was undertaken to compare gene expression between the cartilage of early experimentally induced equine osteochondrosis lesions and control cartilage. Of the many putative differentially expressed genes identified, eight were confirmed by quantitative PCR analysis as differentially expressed, in addition to those already known to be associated with early lesions. Genes encoding vacuolar H(+)-ATPase V0 subunit d2 (ATP6V0D2), cathepsin K, integrin-binding sialoprotein, integrin αV, low density lipoprotein receptor-related protein 4, lumican, osteopontin, and thymosin ß4 (TMSB4) were expressed at higher levels in lesions than in control cartilage. These genes included 34 genes not previously identified in cartilage. Some genes identified as associated with early lesions are known chondrocyte hypertrophy-associated genes, and in transmission electron microscopy studies normal hypertrophic chondrocytes were observed in lesions. Differential expression of ATP6V0D2 and TMSB4 in the cartilage of early naturally occurring osteochondrosis lesions was confirmed by immunohistochemistry. These results identify novel osteochondrosis-associated genes and provide evidence that articular osteochondrosis does not necessarily result from failure of chondrocytes to undergo hypertrophy.

Evidence of apoptosis induced by viral protein 2 of chicken anaemia virus.

Although viral protein 3 (VP3) of chicken anaemia virus (CAV) has been well recognised as an inducer of apoptosis, viral protein 2 (VP2) of the virus has only been speculated to have apoptotic activity. This has not been verified because the open reading frame (ORF) encoding VP2 completely encompasses that encoding VP3, and thus the possibility of expression of VP3 cannot be excluded. The aim of this study was to elucidate the potential role of VP2 as an inducer of apoptosis. Site-directed mutagenesis was used to generate a point mutation that knocked out VP3 by early termination of its translation with a stop codon without imposing any change in the amino acid sequence of VP2. The mutated sequence was inserted into the pCAT plasmid preceded by a favorable Kozak's consensus sequence to create pCAT-VP2(+)VP3(-). The absence of VP3 expression in MSB1 cells transfected with this plasmid was confirmed using Western blotting, and DNA strand breaks and nuclear morphological changes were assessed to detect apoptosis. There was an increased level of apoptotic death in cells transfected with pCAT-VP2(+)VP3(-) compared to those transfected with the vector alone. This provides evidence that CAV VP2 can induce apoptosis.

Tumour progression and cancer-induced pain: a role for protease-activated receptor-2?

The role of proteases in modifying the microenvironment of tumour cells has long been recognised. With the discovery of the protease-activated receptor family of G protein-coupled receptors a mechanism for cells to sense and respond directly to proteases in their microenvironment was revealed. Many early studies described the roles of protease-activated receptors in the cellular events that occur during blood coagulation and inflammation. More recently, studies have begun to focus on the roles of protease-activated receptors in the establishment, progression and metastasis of a variety of tumours. This review will focus on the expression of protease-activated receptor-2 and its activators by normal and neoplastic tissues, and describe current evidence that activation of protease-activated receptor-2 is an important event at multiple stages of tumour progression and in pain associated with cancer.

Omega-1 knockdown in Schistosoma mansoni eggs by lentivirus transduction reduces granuloma size in vivo.

Schistosomiasis, one of the most important neglected tropical diseases worldwide, is caused by flatworms (blood flukes or schistosomes) that live in the bloodstream of humans. The hepatointestinal form of this debilitating disease results from a chronic infection with Schistosoma mansoni or Schistosoma japonicum. No vaccine is available to prevent schistosomiasis, and treatment relies predominantly on the use of a single drug, praziquantel. In spite of considerable research effort over the years, very little is known about the complex in vivo events that lead to granuloma formation and other pathological changes during infection. Here we use, for the first time, a lentivirus-based transduction system to deliver microRNA-adapted short hairpin RNAs (shRNAmirs) into the parasite to silence and explore selected protein-encoding genes of S. mansoni implicated in the disease process. This gene-silencing system has potential to be used for functional genomic-phenomic studies of a range of socioeconomically important pathogens.

Contractile properties of slow and fast skeletal muscles from protease activated receptor-1 null mice.

INTRODUCTION: Protease-activated receptors (PARs) may play a role in skeletal muscle development. We compared the contractile properties of slow-twitch soleus muscles and fast-twitch extensor digitorum longus (EDL) muscles from PAR-1 null and littermate control mice. METHODS: Contractile function was measured using a force transducer system. Fiber type proportions were determined using immunohistochemistry. RESULTS: Soleus muscles from PAR-1 null mice exhibited longer contraction times, a leftward shift in the force-stimulation frequency relationship, and decreased fatiguability compared with controls. PAR-1 null soleus muscles also had increased type 1 and decreased type IIb/x fiber numbers compared with controls. In PAR-1 null EDL muscles, no differences were found, except for a slower rate of fatigue compared with controls. CONCLUSIONS: The absence of PAR-1 results in a slower skeletal muscle contractile phenotype, likely due to an increase in type I and a decrease in type IIb/x fiber numbers. Muscle Nerve 50: 991-998, 2014.