To determine the effect of the tumor necrosis factor-α (TNF-α) in odontoclast formation, we administrated a TNF-α inhibitor in rats with diabetes rats with periodontitis. The rats included in the study were divided into three groups: control rats without diabetes or periodontitis (the C group), rats with periodontitis and diabetes (the PD group), and rats with periodontitis and diabetes treated by infliximab, the TNF inhibitor (the PD+infliximab group). The PD and PD+ infliximab groups received intravenous administrations of streptozotocin (STZ, 50 mg/kg) to induce diabetes. After 7 days of STZ injections, the mandibular first molars were ligatured to induce periodontitis. The PD+infliximab group was intrapenitoneally administrated by infliximab (5 mg/kg). On days 3 and 20 after the ligature administration, odontoclast formation along root surfaces was evaluated by tartrate resistant acid phosphatase (TRAP) staining and cathepsin K immunohistochemistry. On day 3, the number of TRAP- and cathepsin K-positive cells increased more so in the PD group than in the C group. The PD+infliximab group showed a lower number of positive cells than the PD group. There was no difference in all the groups on day 20. On day 3, the cathepsin-K positive multinucleated and mononucleated cells were higher in the PD group than in the C group. The number of cathepsin-K positive multinucleated cells was lower in the PD+infliximab group than in the PD group. The PD group showed more cathepsin K-positive cells in the furcation and distal surfaces than the c group. The Cathepsin K-positive cells of the PD+infliximab group were lower than that of the PD group in furcation. These results suggest that TNF-α stimulates odontoclast formation in diabetes with periodontitis.

Transplantation is considered to be a very useful approach to improve human welfare and to prolong life-span. Heterologous organ transplantation using pig organs which are similar to human beings and easy to make mass-production has known as one of the alternatives. To ensure potential usage of the pig organ for transplantation application, it is essentially required to generate transgenic pig modifying immuno-related genes. Previously, we reported production of heterozygous α 1,3-galactosyltransferase (GalT) knock-out and human membrane cofactor protein (MCP) expressing pig (GalT-MCP/+), which is enforced for suppression of hyperacute and acute immunological rejection. In this study, we reported generation of homozygous pig (GalT-MCP/-MCP) by crossbreeding GalT-MCP/+ pigs. Two female founders gave birth to six of GalT-MCP/-MCP, and seven GalT-MCP/+ pigs. We performed quantitative real-time PCR, western blot, and flow cytometry analyses to confirm GalT and MCP expression. We showed that fibroblasts of the GalT-MCP/-MCP pig do not express GalT and its product Gal antigen, while efficiently express MCP. We also showed no expression of GalT, otherwise expression of MCP at heart, kidney, liver and pancreas of transgenic pig. Taken together, we suggest that the GalT-MCP/-MCP pig is a useful candidate to apply xenotransplantation study.

This study was conducted to evaluate effect of α-linolenic acid (ALA) and bovine serum albumin (BSA) on viability, acrosome reaction and mitochondrial intact in frozen-thawed boar sperm. The boar semen was collected by gloved-hand method and cryopreserved using freezing extender containing 3 ng/mL ALA and/or 20 μg/mL BSA. Cryopreserved boar sperms were thawed in 37°C water-bath for 45 sec to analysis. Viability, acrosome reaction, and mitochondrial intact were analyzed using flow cytometry. In results, viability of frozen-thawed boar sperm was significantly higher in only ALA+BSA supplement group than control group (p<0.05), whereas there was no difference either in ALA or BSA supplement. However, acrosome reacted sperm in both of live and all sperm population were significantly decreased in all treatment groups than control (p<0.05). Interestingly, mitochondrial intact of boar sperm was enhanced in ALA and ALA+BSA groups compared with control (p<0.05). In this study, we showed that supplementation of ALA and BSA in freezing extender enhanced the sperm viability, mitochondrial intact and decrease acrosomal membrane damage. In conclusion, our findings suggest that quality of frozen-thawed sperm in mammalians could improve by using of ALA and BSA.

The aim of this study was to evaluate effect of α-linolenic acid (ALA) on viability, acrosome reaction and mitochondrial intact in frozen-thawed boar sperm. The boar semen was collected by gloved-hand method and cryopreserved in 20% egg yolk freezing extender containing ALA (0, 3, 5, and 10 ng/mL) with 0.05% ethanol. The frozen-boar spermatozoa were thawed at 37.5°C for 45 sec in water-bath. The spermatozoa samples were evaluated the plasma membrane integrity, acrosome reaction, and mitochondrial integrity using flow cytometry. In results, population of live sperm with intact plasma membrane was significantly higher in control and 3 ng/mL ALA treatment group than ethanol group (p<0.05). In contract, dying sperms were higher in ethanol group than 3 ng/mL ALA treatment (p<0.05). Acrosomal membrane damage in all sperm population was reduced in 3 ng/mL ALA groups compared with ethanol treatment (p<0.05). However, acrosome damage in live sperm population was no significant difference among the all treatment groups. Mitochondrial integrity was not influenced by ALA treatments in both of live and all sperm population. In conclusion, this results show that supplement of ALA during the cryopreservation process could reduce the membrane damages including plasma and acrosomal membrane, whereas ALA did not influence to mitochondria in boar spermatozoa. Therefore, these results suggest that ALA can protect against the membrane damage derived cryo-stress, and cryopreservation efficiency of boar semen would be improved by use of ALA.