Reconstruction of stratum corneum in organotypically cultured canine keratinocyte-derived CPEK cells

The stratum corneum of epidermis is an essential barrier against the external environment and water loss. This study aimed to develop an organotypic culture model that targets the reconstruction of the stratum corneum using canine keratinocyte-derived CPEK cells. The CPEK cells cultured at the air-liquid interface became stratified and formed a stratum corneum-like layer on stratum spinosum- and stratum granulosum-like layers. The CPEK cells in the stratum granulosum-like layer expressed the cornified cell envelope (CCE)-related proteins loricrin and keratinocyte differentiation-associated protein. Organotypically cultured CPEK cells were considered to form a CCE at the stratum granulosum-like layer, allowing the formation of a stratum corneum-like layer. The organotypic culture of CPEK cells could be useful for studying the barrier function of canine stratum corneum.     

Nocturnal isoprene emission from mature trees and diurnal acceleration of isoprene oxidation rates near Quercus serrata Thunb. leaves

Quercus serrata Thunb. ex Murray is a widespread deciduous oak in China, the Korean Peninsula, and Japan, and a strong isoprene emitter. Establishing accurate inventories of this species and estimating net carbon budgets, including biogenic volatile organic compounds (BVOC), necessitates detailed evaluation of BVOC emission and oxidation characteristics. Emissions of isoprene, the most abundant BVOC, presumably contribute to atmospheric chemistry through the formation of photochemical oxidants and secondary organic aerosols. We built an isoprene flux monitoring system to simultaneously reveal characteristics of the flux and fate of isoprene at multiple locations in Q. serrata forests. We used proton transfer reaction mass spectrometry (PTR-MS) and an automated closed chamber to measure isoprene emissions from soil and leaves in a warm-temperate Q. serrata forest. We used a relaxed eddy accumulation system with PTR-MS to simultaneously measure the canopy flux. In continuous foliage chamber measurements, we observed daily variations of isoprene emissions and continuous nocturnal emissions from leaves. Nocturnal emissions exceeded 25 % of total daily leaf emissions and were relatively high at sunset and low at sunrise. These results suggest that nocturnal emissions from mature trees may not be negligible. When leaf emissions were high in the daytime, the canopy isoprene flux tended to plateau at an upper limit. Observations of isoprene concentrations and gradients suggest that the plateau was caused by acceleration of isoprene oxidation, and sequential formation of secondary organic aerosols may occur near the leaf just after emission. Elucidation of these linkages may require continuous field measurements with a simultaneous multi-flux monitoring system.     

Rate of gas phase association of hydroxyl radical and nitrogen dioxide

The reaction of OH and NO(2) to form gaseous nitric acid (HONO(2)) is among the most influential in atmospheric chemistry. Despite its importance, the rate coefficient remains poorly determined under tropospheric conditions because of difficulties in making laboratory rate measurements in air at 760 torr and uncertainties about a secondary channel producing peroxynitrous acid (HOONO). We combined two sensitive laser spectroscopy techniques to measure the overall rate of both channels and the partitioning between them at 25°C and 760 torr. The result is a significantly more precise value of the rate constant for the HONO(2) formation channel, 9.2 (±0.4) × 10(-12) cm(3) molecule(-1) s(-1) (1 SD) at 760 torr of air, which lies toward the lower end of the previously established range. We demonstrate the impact of the revised value on photochemical model predictions of ozone concentrations in the Los Angeles airshed.     

Pharmacology of grapiprant,a novel EP4 antagonist: receptor binding,efficacy in a rodent postoperative pain model,and a dose estimation for controlling pain in dogs

Grapiprant is an analgesic and anti‐inflammatory drug in the piprant class that was approved in March 2016 by FDA's Center for Veterinary Medicine for the control of pain and inflammation associated with osteoarthritis (OA) in dogs. Grapiprant functions as a selective antagonist of the EP4 receptor, one of the four prostaglandin E₂ (PGE₂) receptor subtypes. The EP4 receptor mediates PGE₂‐elicited nociception, and grapiprant has been shown to decrease pain in several rat inflammatory pain models. It was also effective in reducing pain associated with OA in humans, providing evidence for its mechanism of action in these species. The estimated canine efficacy dose of between 1.3 and 1.7 mg/kg, p.o. with a methylcellulose suspension, once a day, was predicted based on calculations from comparative affinity of grapiprant to the dog, rat, and human EP4 receptors, serum protein binding, effective doses defined in rat models of pain and inflammation, and human clinical studies. The results of this study guided the doses to be tested in the pilot study and demonstrated the usefulness of the efficacy dose prediction method. The approved commercial tablet dose of grapiprant is 2 mg/kg once a day for the control of pain and inflammation associated with OA in dogs.     

Inhibitory effects of pentosan polysulfate sodium on MAP-kinase pathway and NF-κB nuclear translocation in canine chondrocytes in vitro

Pentosan polysulfate sodium (PPS) has a heparin-like structure and is purificated from the plant of European beech wood. PPS has been used for the treatment of interstitial cystitis for human patients. Recent years, it was newly recognised that PPS reduce pain and inflammation of OA. The molecular biological mechanism of PPS to express its clinical effects is not fully understood. The purpose of the present study is to investigate a mechanism of action of PPS on inflammatory reaction of chondrocytes in vitro. It was evaluated that effects of PPS on interleukin (IL)-1β-induced phosphorylation of mitogen-actiated protein kinases (MAPKs), such as p38, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), nuclear translocation of nuclear factor-kappa B (NF-κB), and matrix metalloproteinase (MMP)-3 production in cultured articular chondrocytes. As a result, in the presence of PPS existence, IL-1β-induced phosphorylation of p38 and ERK were certainly inhibited, while JNK phosphorylation was not affected. Nuclear translocation of NF-κB and MMP-3 production were suppressed by PPS pretreatment prior to IL-1β stimulation. In conclusion, it is strongly suggested that PPS treatment prevents inflammatory intracellular responses induced by IL-1 β through inhibition of phosphorylation of certain MAPKs, p38 and ERK and then nuclear translocation of NF-κB in cultured chondrocytes. These PPS properties may contribute to suppressive consequence of catabolic MMP-3 synthesis. These data might translate the clinical efficacy as PPS treatment could inhibit the cartilage catabolism and related clinical symptoms of OA in dogs.     

Efficacy of enamel matrix proteins on apical periodontal regeneration after experimental apicoectomy in dogs

Adult dogs have a complex apical delta structure in all root apexes of teeth. This complex structure may affect the formation of apical lesions in the teeth such as apical abscesses. The purpose of this study was to evaluate the efficacy of enamel matrix protein (EMP) which was used for periodontal regeneration therapy after an experimental apicoectomy for an assumed apical lesions of the teeth in dogs. The maxillar canine roots and maxillar fourth premolar buccal mesial roots in five beagles were experimentally apicoectomized under general inhalation anesthesia. After the root apex was exposed and excised, EMP was applied on the surface of the exposed dentin. After 12 weeks, dogs were euthanized. and the experimental teeth together with the surrounding soft and hard periodontal tissues were collected for histological evaluation under a light microscope. In the EMP group, the size of the defect where the root apex was removed was smaller than that of the control group. New cementum was dominantly achieved in the EMP group compared to the control group. Furthermore, new collagen fibers that bridged area between the new cementum and new alveolar bone were detected only in the EMP group. The present results demonstrated marked apical periodontal regeneration after apicoectomy in the EMP group. These results, therefore, suggest that the application of EMP can effectively induce the regeneration of periodontal strUctures in apicoectomized dogs.     

Serum keratan sulphate as a cartilage metabolic marker in horses: the effect of exercise

Keratan sulphate (KS) concentration in sera from resting horses and horses training daily on a racetrack was measured by an inhibition enzyme-linked immunosorbent assay (ELISA) using anti-equine KS antibody 1/14/16H9. For the in-training horses, serum KS concentrations in 2-year-old-horses was significantly higher than 3- or 4-year-old-horses. A higher concentration of serum KS was found in the in-training group than in the long-term resting group in 2-year-old-horses. Serum KS concentration increased remarkably immediately after training in healthy horses, and at 1, 5, 9 and 24 h after training remained at similar levels to the pre-training concentration. The results suggest that serum KS concentration could represent the situation of joint loading, induced by daily racetrack training, affecting the metabolic activities in joint cartilage.     

Inhibitory effects of 22-oxa-calcitriol and all- trans retinoic acid on the growth of a canine osteosarcoma derived cell-line in vivo and its pulmonary metastasis in vivo

Pulmonary metastasis is a major cause of death and a major obstacle to the successful treatment of canine osteosarcoma. However, the residual capacity of the neoplasia for differentiation and its susceptibility to undergo apoptosis may be used to suppress its growth and metastatic properties. The highly metastasizing POS (HMPOS) canine osteosarcoma cell line which preferentially metastasize to the lungs was used to test the possible efficacy of 22-oxa-calcitriol (OCT) and all- trans retinoic acid (ATRA) to inhibit growth and pulmonary metastasis of the subcutaneously grown osteosarcoma in nude mice. Treatments in vitro, morphologically elongated and increased alkaline phosphatase activity and staining of cells. Tumour growth in vivo was inhibited significantly and the combination treatment of OCT and ATRA (OCT + ATRA) exerted a synergistic and stronger suppression at concentration of 1.0 microg kg(-1)body weight when given subcutaneously three times a week for 5 weeks. The subcutaneous tumours of the control mice consisted of osteoblast-like cells and isolated chondroblast-like cells, but formed several areas of osteoid and increased amount of collagen tissue in all treated mice. Pinpoint macrometastatic nodules developed only in all control mice. Micrometastatic nodule developed only in two of six mice treated with ATRA. However, nodule size and number, and lung wet weight were all reduced significantly. Metastasis were not seen in the mice treated with OCT or OCT + ATRA. This study demonstrated that inhibition of growth and pulmonary metastasis was induced by subcutaneous treatment with these drugs and suggest that both its differentiating and apoptotic inducing activities may be responsible for the antitumour effects. These drugs may be useful in the clinic as an adjunct for the treatment of canine osteosarcoma.     

Consideration of the role of antigenic keratan sulphate reacting to a 1/14/16H9 antibody as a molecular marker to monitor cartilage metabolism in horses

The role of keratan sulphate (KS) as a marker of cartilage metabolism was evaluated by using an in vitro model of equine articular cartilage. Articular cartilage was harvested from clinically healthy 6-month-old foals (n=3). Chondrocytes were centrifuged and cultured as pellets. Chondrocyte pellets were stimulated by insulin-like growth factor (IGF)-Ialpha or interleukin (IL)-1alpha for 2 weeks. The sulfated glycosaminoglycans (GAG) and antigenic KS concentrations in the culture media were measured by a 1,9-dimethyl-methylene blue (DMMB) colorimetric assay and an inhibition ELISA using a 1/14/16H9 antibody, respectively. Concentration of GAG was significantly increased in the media of pellets stimulated by both IGF-Ialpha and IL-1alpha. Antigenic KS concentration was significantly increased in those stimulated by IL-1alpha, while no significant change was found in those stimulated by IGF-Ialpha. A high correlation between GAG and antigenic KS concentrations was found in the media of pellets stimulated by IL-1alpha (r=0.87), but not in those stimulated by IGF-Ialpha (r=0.43). The results suggest that the concentration of antigenic KS reacting to 1/14/16H9 mirrors the GAG concentration during the stage of cartilage catabolism, but not during the cartilage anabolic stage. The concentration of antigenic KS reacting to 1/14/16H9 antibody in biological fluids could therefore be a useful marker to further understand principally the catabolic and slightly the anabolic process of articular cartilage metabolism.