Targeting of the osteoclastogenic RANKL-RANK axis prevents osteoporotic bone loss and soft tissue calcification in coxsackievirus B3-infected mice.

Bone mineralization is a normal physiological process, whereas ectopic calcification of soft tissues is a pathological process that leads to irreversible tissue damage. We have established a coxsackievirus B3 (CVB3)-infected mouse model that manifests both osteoporosis and ectopic calcification specifically in heart, pancreas, and lung. The CVB3-infected mice showed increased serum concentrations of both cytokines including IL-1ß, TNF-α, and the receptor activator of NF-κB ligand (RANKL) that stimulate osteoclast formation and of the osteoclast-derived protein tartrate-resistant acid phosphatase 5b. They exhibited more osteoclasts in bone, with no change in the number of osteoblasts, and a decrease in bone formation and the serum concentration of osteoblast-produced osteocalcin. These results indicate that CVB3-induced osteoporosis is likely due to upregulation of osteoclast formation and function, in addition to decreased osteoblast activity. In addition, the serum in the CVB3-infected mice contained a high inorganic phosphate content, which causes ectopic calcification. RANKL treatment induced an increase in the in vitro cardiac fibroblast calcification by inorganic phosphate via the upregulation of osteogenic BMP2, SPARC, Runx2, Fra-1, and NF-κB signaling. We finally observed that i.p. administration of RANK-Fc, a recombinant antagonist of RANKL, prevented bone loss as well as ectopic calcification in CVB3-infected mice. Thus, our results indicate that RANKL may contribute to both abnormal calcium deposition in soft tissues and calcium depletion in bone. In addition, our animal model should provide a tool for the development of new therapeutic agents for calcium disturbance in soft and hard tissues.

Comparison of 12-lipoxygenase expression in vascular smooth muscle cells from old normotensive Wistar-Kyoto rats with spontaneously hypertensive rats.

Vascular aging and essential hypertension cause similar structural and molecular modifications in the vasculature. The 12-lipoxygenase (LO) pathway of arachidonic acid metabolism is linked to cell growth and the pathology of hypertension. Thus, elevated expression of 12-LO has been observed in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR). In the present study, we investigated the differences in 12-LO expression and activity between VSMCs from old normotensive Wistar-Kyoto rats (old WKY, 90-week old) and SHR (13-week old). The protein and mRNA expression of basal or angiotensin II (Ang II)-induced 12-LO in old WKY VSMCs were higher than those in SHR VSMCs. The degradation rate of 12-LO mRNA in old WKY VSMCs was slower than that in SHR VSMCs. However, basal or Ang II-induced 12-LO mRNAs in both old WKY and SHR VSMCs decayed more rapidly than that in young WKY (13-week old) VSMCs. Higher expression of 12-LO in old WKY VSMCs than in SHR VSMCs was correlated with the expression level of Ang II subtype 1 receptor (AT(1)R). The reduced levels of nitric oxide (NO) in old WKY and SHR VSMCs compared with young WKY VSMCs were similar, and there was no significant difference in NO production between old WKY and SHR VSMCs transfected with 12-LO siRNA. In addition, in contrast to the proliferation of SHR VSMCs, the proliferation of old WKY VSMCs was not dependent on 12-LO activation. These results suggest that the potential role of 12-LO in normotensive aging vasculature may be different from that in SHR vasculature.

Sequencing and characterization of Varicella-zoster virus vaccine strain SuduVax.

BACKGROUND: Varicella-zoster virus (VZV) causes chickenpox in children and shingles in older people. Currently, live attenuated vaccines based on the Oka strain are available worldwide. In Korea, an attenuated VZV vaccine has been developed from a Korean isolate and has been commercially available since 1994. Despite this long history of use, the mechanism for the attenuation of the vaccine strain is still elusive. We attempted to understand the molecular basis of attenuation mechanism by full genome sequencing and comparative genomic analyses of the Korean vaccine strain SuduVax. RESULTS: SuduVax was found to contain a genome that was 124,759 bp and possessed 74 open reading frames (ORFs). SuduVax was genetically most close to Oka strains and these Korean-Japanese strains formed a strong clade in phylogenetic trees. SuduVax, similar to the Oka vaccine strains, underwent T- > C substitution at the stop codon of ORF0, resulting in a read-through mutation to code for an extended form of ORF0 protein. SuduVax also shared certain deletion and insertion mutations in ORFs 17, 29, 56 and 60 with Oka vaccine strains and some clinical strains. CONCLUSIONS: The Korean VZV vaccine strain SuduVax is genetically similar to the Oka vaccine strains. Further comparative genomic and bioinformatics analyses will help to elucidate the molecular basis of the attenuation of the VZV vaccine strains.

Seroepidemiologic survey of varicella-zoster virus in korean adults using glycoprotein enzyme immuno assay and fluorescent antibody to membrane antigen test.

BACKGROUND: Herpes zoster (HZ) occurs mainly in the elderly and Korea is rapidly becoming an aging society. Therefore, it is important to know the immune status against varicella-zoster virus (VZV) in Korean adults to prevent the disease. OBJECTIVE: The aim of this study was to survey the immune status of Korean adults over 40 years of age against VZV. METHODS: Antibody titer was measured using a VaccZyme™ VZV glycoprotein enzyme immunoassay (gpEIA) (Binding Site, UK). Fluorescent antibody to membrane antigen (FAMA) test was performed to measure the seropositive rate. RESULTS: HZ incidence in the 214 adults enrolled in this study was 10.3%. The gpEIA geometric mean titer (GMT) was 490 mIU/ml and 90.2% of the subjects had a protective level of gpEIA antibody titer against varicella. The average gpEIA GMT of adults who previously had HZ was 1,122 mIU/ml, which was higher than the average gpEIA GMT of 457 mIU/ml in adults who had not had HZ. The FAMA positive rate was 98.6%. CONCLUSION: Most (90.2%) Korean adults ≥40-years-of-age have a protective level of gpEIA antibody against varicella and 98.6% were FAMA seropositive. The GMT of gpEIA antibody was significantly increased with age, and was higher in adults with a history of HZ.

Endothelial colony-forming cell coating of pig islets prevents xenogeneic instant blood-mediated inflammatory reaction.

Instant blood-mediated inflammatory reaction (IBMIR) causes rapid islet loss in islet transplantation. Endothelial colony-forming cells (ECFCs) display unique abilities to promote angiogenesis and repair vascular injury compared to those of endothelial cells (ECs), which inhibits the allogeneic and xenogeneic IBMIR. We investigated the coating of pig islets with ex vivo-expanded ECFCs as a strategy to overcome xenogeneic IBMIR. Porcine islets were cocultured with human ECFCs in a specially modified culture medium for 2 days to obtain 70-90% coverage. The coating of pig islets with human ECFCs did not affect the glucose-stimulated insulin secretion capacity or diabetes reversal rate after the transplantation of a marginal islet mass under the kidney capsules of diabetic nude mice compared to that of untreated islets. Uncoated islets, PBS control without islets, and the ECFC-coated islets were examined with an in vitro tubing loop assay using human blood. After 60 min of incubation in human blood, the ECFC-coated islets showed platelet consumption inhibition and low C3a and TAT assay results compared to those of the uncoated islets. Furthermore, there was very little macroscopic or microscopic clotting in the human ECFC-coated pig islets. The protective effect was more prominent compared to that of human EC coating of pig islets in our previous study. We investigated the changes in human-specific MCP-1, IL-8, and tissue factor (TF) levels after the coating of pig islets with human ECFCs or human ECs. The IL-8 levels after coating pig islets with ECFCs were significantly lower than those after coating pig islets with ECs, but there were no significant differences in the MCP-1 or TF levels between the ECFCs and ECs. In conclusion, the coating of pig islets with ECFCs completely prevented all components of xenogeneic IBMIR. ECFCs may be a better source of protection against xenogeneic IBMIR than are mature ECs.

Changes in hepatic gene expression upon oral administration of taurine-conjugated ursodeoxycholic acid in ob/ob mice.

Nonalcoholic fatty liver disease (NAFLD) is highly prevalent and associated with considerable morbidities. Unfortunately, there is no currently available drug established to treat NAFLD. It was recently reported that intraperitoneal administration of taurine-conjugated ursodeoxycholic acid (TUDCA) improved hepatic steatosis in ob/ob mice. We hereby examined the effect of oral TUDCA treatment on hepatic steatosis and associated changes in hepatic gene expression in ob/ob mice. We administered TUDCA to ob/ob mice at a dose of 500 mg/kg twice a day by gastric gavage for 3 weeks. Body weight, glucose homeostasis, endoplasmic reticulum (ER) stress, and hepatic gene expression were examined in comparison with control ob/ob mice and normal littermate C57BL/6J mice. Compared to the control ob/ob mice, TUDCA treated ob/ob mice revealed markedly reduced liver fat stained by oil red O (44.2±5.8% vs. 21.1±10.4%, P<0.05), whereas there was no difference in body weight, oral glucose tolerance, insulin sensitivity, and ER stress. Microarray analysis of hepatic gene expression demonstrated that oral TUDCA treatment mainly decreased the expression of genes involved in de novo lipogenesis among the components of lipid homeostasis. At pathway levels, oral TUDCA altered the genes regulating amino acid, carbohydrate, and drug metabolism in addition to lipid metabolism. In summary, oral TUDCA treatment decreased hepatic steatosis in ob/ob mice by cooperative regulation of multiple metabolic pathways, particularly by reducing the expression of genes known to regulate de novo lipogenesis.

ATP measurement predicts porcine islet transplantation outcome in nude mice.

Current nude mice islet transplantation studies cannot be used prospectively. Therefore, to predict transplantation outcomes, reliable and rapid assays for islet quality assessment are warranted. This study evaluated the predictive power of the porcine islet ATP content on the outcomes of islet transplantation in nude mice. Here, we report that the ATP measurement using a small number of handpicked islets with a diameter of 100 to 150 mum is a good predictor of islet graft efficacy in nude mice. Using receiver-operator characteristic analysis, the area under the curve of the ATP content using a small number of handpicked islets was 0.867 (95% confidence interval 0.744-0.989, P<0.001). The sensitivity and the specificity measured were 83.3% and 73.3%, respectively. In conclusion, a simple and a rapid measurement of intraislet ATP content could be a promising substitute for current nude mice islet transplantation studies.

Interferon-stimulated genes response in endothelial cells following Hantaan virus infection.

The regulation mechanism of interferon (IFN) and IFN-stimulated genes is a very complex procedure and is dependent on cell types and virus species. We observed molecular changes related to anti-viral responses in endothelial cells during Hantaan virus (HTNV) infection. We found that there are two patterns of gene expression, the first pattern of gene expression being characterized by early induction and short action, as in that of type I IFNs,' and the other being characterized by delayed induction and long duration, as those of IRF-7, MxA, and TAP-1/2. Even though there are significant differences in their induction folds, we found that all of IFN-alpha/beta, IRF- 3/7, MxA, and TAP-1/2 mRNA expressions reached the peak when the viral replication was most active, which took place 3 days of post infection (d.p.i.). In addition, an interesting phenomenon was observed; only one gene was highly expressed in paired genes such as IFN-alpha/beta' (3/277-folds), IRF-3/7 (2.2/29.4-folds), and TAP- 1/2 (26.2/6.1-folds). Therefore, IFN-beta, IRF-7, and TAP-1 seem to be more important for the anti-viral response in HTNV infection. MxA was increased to 296-folds at 3 d.p.i. and kept continuing 207-folds until 7 d.p.i.. The above results indicate that IFN-beta works for an early anti-viral response, while IRF7, MxA, and TAP-1 work for prolonged anti-viral response in HTNV infection.

Influence of strain and age differences on the yields of porcine islet isolation: extremely high islet yields from SPF CMS miniature pigs.

BACKGROUND: Porcine pancreas is a potential source of material for islet xenotransplantation. However, the difficulty in isolating islets, because of their fragility and the variability of isolation outcome in donor age and breed, represents a major obstacle to porcine islet xenotransplantation. In this study, we compared the islet isolation yield of specific pathogen-free (SPF) Chicago Medical School (CMS) miniature pigs with that of another miniature pig breed and market pigs from a local slaughterhouse. METHODS: Nine adult CMS miniature (ACM) pigs (>12 months), six young CMS miniature (YCM) pigs (6-7 months), four adult Prestige World Genetics (PWG) miniature (APM) pigs (>12 months), and 13 adult market (AM) pigs from a local slaughterhouse were used for islet isolation. RESULTS: The islet yield per gram of pancreas from ACM pigs (9589 +/- 2823 IEQ/g) was significantly higher than that from APM pigs (1752 +/- 874 IEQ/g, P < 0.05), AM pigs (1931 +/- 947 IEQ/g, P < 0.05), or YCM pigs (3460 +/- 1985 IEQ/g, P < 0.05). Isolated islets from ACM pigs were significantly larger than those from AM pigs or YCM pigs. The in vitro and in vivo function of isolated islets showed no difference among experimental groups. The pancreases of ACM pigs contained higher mean islet volume density percentages and larger size of islets than those of AM or APM pigs. CONCLUSIONS: We isolated extremely high yields of well-functioning islets from ACM pigs bred under SPF conditions. SPF CMS miniature pigs should be one of the best porcine islet donors for clinical porcine islet xenotransplantation.

Colocalization of interferon regulatory factor 7 (IRF7) with latent membrane protein 1 (LMP1) of Epstein-Barr virus.

Interferon regulatory factor 7 (IRF7) is one of the transcriptional factors for the activation of type I Interferon (IFN) genes. It is known that IRF7 and the latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) are highly expressed in EBV type III latency cells, and LMP1 induces mRNA expression of IRF7. In this study, the expression pattern of endogenous IRF7 was observed in several B cell lines with or without EBV infection by immunofluorescence staining. IRF7 was localized in the cytoplasm of EBV-negative B cells and EBV type I latency B cell lines. However, IRF7 was located both in the cytoplasm and nucleus of EBV type III latency cell lines. In the Jijoye cell (type III latency cell), IRF7 was colocalized with LMP1 in the cytoplasm in a capping configuration, and their interaction was confirmed by co-immunoprecipitation of LMP1 and IRF7. This colocalization was confirmed by co-transfection of IRF7 and LMP1 plasmids in EBV-negative B cells. These results suggest that the IRF7 and LMP1 interact with each other, and this may relate to the mechanism whereby LMP1 exerts functional effects in B-lymphocytes.

Pirfenidone suppressed the development of glomerulosclerosis in the FGS/Kist mouse.

Pirfenidone (PFD) is a newly developed anti-fibrotic agent. We evaluated the effect of PFD for the prevention of renal fibrosis using a spontaneous progressive glomerulosclerosis animal model, FGS/Kist mice. Male and female FGS/Kist mice were fed a diet containing 0.5% PFD or the same control diet (CD) without PFD, for 1, 2, or 3-month periods. Body weight was monitored for the general effect of PFD on the mice. Proteinuria and glomerular filtration rate (GFR) were evaluated for renal function. The sclerosis index was examined for the morphological changes. There were no significant changes in body weight between the PFD and control groups in both sexes. Proteinuria levels were low in all the PFD groups compared to the corresponding CD groups. The sclerosis scores were also reduced in both sexes of the 3-month PFD groups (p<0.05), and glomerular filtration rates were increased in both sexes of the 3-month PFD groups compared to the CD groups. The treatment of PFD for 1 or 2-month periods did not have statistic significances but the treatment for 3 months had statistic significances in sclerosis and GFR compared to CD groups. These results suggested that long-term administration of PFD suppressed the progression of glomerulosclerosis and improved renal function of the FGS/Kist mice.