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Induced pluripotent stem cells (iPSCs) are thought to be highly beneficial in the field of regenerative medicine and are believed to overcome immunogenic barriers to cell transplantation. However, issues remain regarding their safety and efficiency for medical use. Furthermore, some recent reports have suggested that iPSCs could be targeted by the autologous immune system. To promote practical applications of iPSCs, in depth research using appropriate animal models is needed and porcine species appear to provide an ideal model. Recent studies have focused on the generation of porcine iPSC cells, but no investigations of their immunological properties have been conducted to date. In the present study, we generated putative iPSCs from porcine somatic cells and measured major histocompatibility complex (MHC) expression on the iPSCs and their derivatives. Compact colonies that expressed pluripotent markers appeared 11 days after viral infection. Embryonic bodies (EB) were produced and differentiated into three germ layers in vitro. Karyotyping and swine leukocyte antigen (SLA) typing showed that the iPSCs were identical to parental somatic cells. Porcine iPSCs expressed only low levels of MHC class I and moderately increased levels on their differentiated derivatives, whereas MHC class II was rarely expressed. In the presence of interferon-gamma (IFN-γ), the expression of MHC class I was elevated on differentiated iPSCs, and gradually decreased after withdrawal of the cytokine. Our data suggest that porcine iPSCs could be useful for preclinical studies of the efficiency and viability of iPSCs, and for devising strategies to rescue transplanted cells from the autologous immune system.

An 8-year-old male mongrel dog that had undergone renal transplantation was presented 25 days later with an acute cough, anorexia and exercise intolerance. During the investigation, neutrophilic leukocytosis was noted, and thoracic radiographs revealed caudal lung lobe infiltration. While being treated with two broad-spectrum antibiotics, clinical signs worsened. Pneumonia due to infection with multidrug-resistant (MDR) Pseudomonas (P.) aeruginosa, sensitive only to imipenem and amikacin, was confirmed by bacteria isolation. After treatment with imipenem-cilastatin without reducing the immunosuppressant dose, clinical signs completely resolved. During the 2-year follow-up period, no recurrence was observed. To the best of authors' knowledge, this is the first report of pneumonia caused by MDR P. aeruginosa in a renal recipient dog and successful management of this disease.

Clostridium acetobutylicum has been considered as an attractive platform host for biorefinery due to its metabolic diversity. Considering its capability to overproduce butanol through butyrate, it was thought that butyric acid can also be efficiently produced by this bacterium through metabolic engineering. The pta-ctfB-deficient C. acetobutylicum CEKW, in which genes encoding phosphotransacetylase and CoA-transferase were knocked out, was assessed for its potential as a butyric acid producer in fermentations with four controlled pH values at 5.0, 5.5, 6.0, and 6.4. Butyric acid could be best produced by fermentation of the CEKW at pH 6.0, resulting in the highest titer of 26.6 g/l, which is 6.4 times higher than that obtained with the wild type. However, due to the remaining solventogenic ability of the CEKW, 3.6 g/l solvents were also produced. Thus, the CEKW was further engineered by knocking out the adhE1-encoding aldehyde/alcohol dehydrogenase to prevent solvent production. Batch fermentation of the resulting C. acetobutylicum HCEKW at pH 6.0 showed increased butyric acid production to 30.8 g/l with a ratio of butyric-to-acetic acid (BA/AA) of 6.6 g/g and a productivity of 0.72 g/l/h from 86.9 g/l glucose, while negligible solvent (0.8 g/l ethanol only) was produced. The butyric acid titer, BA/AA ratio, and productivity obtained in this study were the highest values reported for C. acetobutylicum, and the BA/AA ratio and productivity were also comparable to those of native butyric acid producer Clostridium tyrobutyricum. These results suggested that the simultaneous deletion of the pta-ctfB-adhE1 in C. acetobutylicum resulted in metabolic switch from biphasic to acidogenic fermentation, which enhanced butyric acid production.

Purpose: The aims of this study were to investigate the effects of sterilization with peracetic acid (PAA) and ethanol on the biological activity of porcine liver scaffolds and to develop a new technique for sterilization using slightly acidic electrolyzed water (SAEW)., Methods: Decellularization of liver slices was performed using 0.1% sodium-dodecyl-sulfate, then evaluated by histological and polymerase chain reaction analyses. Decellularized slices were treated with either PAA or ethanol or SAEW, and then DNA content was quantified. We determined sterilization efficiency by culturing scaffolds in culture medium and on blood agar. We next analyzed the glycosaminoglycan and collagen contents of the scaffolds. Finally, we tested the cytotoxicity of the scaffolds as well as the effects of sterilization on host cell attachment and proliferation., Results: Complete cell and antigenic epitopes removal emphasized the decellularization efficiency. PAA and SAEW treatments achieved the highest efficiency of sterilization compared to that of the ethanol treated scaffolds, and were able to remove a considerable fraction of DNA from decellularized livers. The retained glycosaminoglycan content decreased in all treatments in the following order: SAEW, ethanol, and PAA. Ethanol caused a significant loss in collagen content compared to the other groups. A cytotoxicity evaluation revealed that all scaffolds were nontoxic. SAEW-treated scaffolds supported cell attachment and proliferation at a significantly higher rate than other groups., Conclusions: These data suggest that SAEW is highly efficient for sterilizing scaffolds and allowed the scaffolds to retain their bioactivity in addition to its high efficiency for cell remnant removal.

Non-structural protein 1 (NS1) of the influenza A virus (IAV) inhibits the host's innate immune response by suppressing the induction of interferons (IFNs). Therefore, blocking NS1 activity can be a potential strategy in the development of antiviral agents against IAV infection. In the present study, we selected a single-stranded DNA aptamer specific to the IAV NS1 protein after 15 cycles of systematic evolution of ligands by exponential enrichment (SELEX) procedure and examined the ability of the selected aptamer to inhibit the function of NS1. The selected aptamer binds to NS1 with a Kd of 18.91±3.95nM and RNA binding domain of NS1 is determined to be critical for the aptamer binding. The aptamer has a G-rich sequence in the random sequence region and forms a G-quadruplex structure. The localization of the aptamer bound to NS1 in cells was determined by confocal images, and flow cytometry analysis further demonstrated that the selected aptamer binds specifically to NS1. In addition, luciferase reporter gene assay, quantitative RT-PCR, and enzyme-linked immunosorbent assay (ELISA) experiments demonstrated that the selected aptamer had the ability to induce IFN-β by suppressing the function of NS1. Importantly, we also found that the selected aptamer was able to inhibit the viral replication without affecting cell viability. These results indicate that the selected ssDNA aptamer has strong potential to be further developed as a therapeutic agent against IAV., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Introduction: Adult mesenchymal stem cells (MSCs) have potential to differentiate into various lineages, replacing cells during normal turnover and tissue regeneration to replace damaged or lost adult tissues during osteoporosis and arthritis, or traumatic injuries. We investigated the osteogenic signature in mouse adipose tissue (AD)- and bone marrow (BM)-derived MSCs., Materials and Methods: MSCs from adipose tissue and bone marrow were compared for osteogenic endogenous mRNA markers by reverse-transcription polymerase chain reaction (RT-PCR). Cellular proliferation and immunophenotype analyzed by flow cytometry revealed that mouse AD-MSCs and BM-MSCs shared similar characteristics., Result: Isolated AD-MSC and BM-MSC showed high proliferation rates and fibroblast morphology. Flow cytometry revealed positive markers for mesenchyme, but negative for primitive hematopoietic and endothelial cells. At day 21, Alizarin red S and Von-kossa staining of differentiated cells showed high calcium deposits compared with undifferentiated cells. After 21 days of osteogenic differentiation, AD-MSCs expressed osteocalcin and parathyroid hormone (PTH) compared with undifferentiated cells. Osteogenic-specific transcript of osteocalcin (OC), bone gamma carboxyglutamate protein, and PTH receptor (PTHr) were detected only in differentiated not undifferentiated cells. Undifferentiated BM-MSCs, expressed all markers at low intensity, which amplified during differentiation., Conclusion: Our findings suggest that the OC and PTHr can be used as differentiation markers for osteogenesis of mouse AD-MSC., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: The increasing market in biological pharmaceuticals raises the demand for human test systems. Although 2-dimensional (2D) models are mostly used for these purposes, these models not mimic responses of 3-dimensional (3D) native tissue., Methods: After generation of a rat liver scaffold using 0.1% sodium dodecyl sulfate, we characterized the histology, blood vessel integrity, and residual DNA as well as retained amounts of collagen and glycosaminoglycan (GAG). Then, we examined the susceptibility of extracellular matrix (ECM) to enzymatic remodeling. Finally, a mixed lymphocyte reaction (MLR) was performed to evaluate the in vitro immunogenicity of the ECM against human peripheral blood mononuclear cells (PBMCs)., Results: Histologic examination of decellularized liver revealed the removal of nuclear and cytoplasmic materials with preservation of architecture. The vascular network was intact after decellularization. Biochemical analysis of ECM components revealed that only a negligible amount of DNA was retained compared with the native liver with preservation of large amounts of GAG and collagen. Scaffolds were degraded in response to collagenase treatment. MLR demonstrated that decellularized matrices did not exert any xenostimulatory response against human PBMCs., Conclusion: Our findings suggested that naturally derived rat liver scaffolds show natural biocompatibility besides the ability to preserve the intact 3D structure and components. Because of these characteristics, the whole decellularized rat liver can retain many aspects of native tissue structure and function upon recellularization enabling it to be used for drug screening., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: The genetic heterogeneity of hearing loss makes genetic diagnosis expensive and time consuming using available methods. Whole-exome sequencing has recently been introduced as an alternative approach to identifying causative mutations in Mendelian disorders., Methods: To identify the hidden mutations that cause autosomal recessive nonsyndromic hearing loss (ARNSHL), we performed whole-exome sequencing of 13 unrelated Korean small families with ARNSHL who were negative for GJB2 or SLC26A4 mutations., Results: We found two novel compound heterozygous mutations, IVS11 + 1 and p.R2146Q, of MYO15A in one (SR903 family) of the 13 families with ARNSHL. In addition to these causative mutations, 13 nonsynonymous variants, including variants with uncertain pathogenicity (SR285 family), were identified in the coding exons of MYO15A from Korean exomes., Conclusion: This is the first report of MYO15A mutations in an East Asian population. We suggest that close attention should be paid to this gene when performing genetic testing of patients with hearing loss in East Asia. The present results also indicate that whole-exome sequencing is a valuable method for comprehensive medical diagnosis of a genetically heterogeneous recessive disease, especially in small-sized families.

Introduction: Graft rejection remains a major cause of morbidity and mortality following renal transplantation. One of the main determinants of success after renal transplantation is histocompatibility between donor and recipient. Most of the research on this topic has addressed human leukocyte antigen (HLA), but the roles played by minor histocompatibility antigens (mHAgs), such as mitochondrially transmitted antigens, are poorly understood. In this study, we evaluated immune responses induced by minor antigens originating from mitochondrial DNA (mtDNA) in a large animal model., Methods: To characterize whole swine leukocyte antigen (SLA) allele in 8 cloned pigs, we performed SLA genotyping for SLA-1, SLA-2, SLA-3, SLA-DQB1, and SLA-DRB1 as well as the hypervariable region 1 (HV1) of mtDNA. Renal transplantation was performed using SLA-matched pigs with different mtDNA as well as SLA-mismatched cloned animals. Cytokine profiling was performed by incubating peripheral leukocytes with cellular components from SLA-matched different mtDNA and SLA-mismatched cells to evaluate mtDNA-mediated immune response., Results: SLA types were confirmed to be identical, but mtDNA sequences of HV1 varied among cloned pigs. Rejection episodes in the SLA-matched group with different mtDNA were similar to those in the SLA-mismatched group; that is, plasma creatinine and BUN levels were increased and mononuclear cell infiltration was observed in perivascular regions in the matched and SLA-mismatched groups. Furthermore, in vitro studies showed interleukin (IL)-1β expression to be elevated in SLA-matched and SLA-mismatched groups., Conclusion: Cloned pigs are a useful preclinical model to evaluate the immunogenicity of mtDNA encoding minor antigens. The mtDNA originating from nongenomic DNA induced cell-mediated immune rejection after kidney transplantation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Mitotically inactivated feeder cells such as mouse embryonic fibroblast (MEFs) cells have been widely applied for physical and physiological support in the pluripotency maintenance of human pluripotent stem cells (hPSCs). However, accurate supporting mechanism or factors of feeder cells are poorly understood. Here, we isolated differentially expressed genes between wild-type MEFs and mitotically inactivated MEFs (miMEFs) by employing annealing control primer-based GeneFishing polymerase chain reaction. We identified a secreted protein acidic cysteine-rich glycoprotein (SPARC) gene that is upregulated in miMEFs. Suppression of SPARC expression in miMEFs using small interference RNA (siRNA) displayed gradual detachment of miMEFs. Furthermore, we found a significant reduction of OCT4- and SSEA3-positive hPS cell population maintained on SPARC siRNA-miMEFs compared to on miMEFs by flow cytometrical analysis. These findings suggest that SPARC plays a critical role in the maintenance of miMEFs without loss of cell number and might be a key component for supporting the culture of hPSCs.

Introduction: Alloantigen recognition in skin transplantation is the bane for surgeons. Several studies have mainly focused on the immunogenicity of major histocompatibility (MHC) antigens and H-Y minor histocompatibility antigens. However, the roles of the mitochondrial DNA (mtDNA) encorded miHA have not been identified. Therefore, we sought to address the antigenicity of the hypervariable region 1 (HV-1) of mtDNA in skin transplantation using cloned pig models., Methods: Swine leukocyte antigen and HV-1 of mtDNA were analyzed using sequencing methods. Skin transplantation was performed between MHC-matched, mtDNA-mismatched cloned miniature pigs. Full-thickness skin was grafted between cloned pigs without any immunosuppressants. The grafted tissues were observed for 3 months and evaluated histologically., Results: The cloned pigs shared identical MHC but mtDNA mismatched at 9 positions. Skin grafts between the cloned pigs were accepted and hair growth maintained, whereas MHC-mismatched grafts showed acute rejection within 7 days after transplantation and were replaced by hairless scar tissue., Conclusions: HV-1 disparate skin grafts were not recognized as alloantigenic by MHC-matched cloned pigs., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Decellularized biologic matrices are plausible biomedical materials for the bioengineering in liver transplantation. However, one of the concerns for safe medical application is the lack of objective assessment of the immunogen within the materials and the in vivo immune responses to the matrices. The purpose of this study was the production of immunogen-reduced and biocompatible matrices from porcine liver. In the present study, 0.1% SDS solution was effective for removing DNA fragments and sequences encoding possible immunogenic and viral antigens within the matrices. The PCR analysis showed that galactose-α-1,3 galactose β-1,4-N-acetylglucosamine (1,3 gal), swine leukocyte antigen (SLA), and porcine endogenous retrovirus (PERV) were completely removed in the matrices. Collagen and glycosaminoglycans (GAGs) were preserved over 63%-71%, respectively, compared to those of native liver. The implanted decellularized tissues showed minimal host responses and naturally degraded within 10 weeks. In this study, we produced immunogen-reduced and biocompatible extracellular matrices from porcine liver. Although future investigations would be required to determine the mechanism of the host reaction, this study could provide useful information of porcine liver-derived biologic matrices for liver researches., (Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The objective of the study was to investigate the disease-causing mutation in an autosomal dominant Charcot-Marie-Tooth disease type 2 family and examine the clinical and histopathological evaluation. We enrolled a family of Korean origin with axonal Charcot-Marie-Tooth disease neuropathy (FC305; 13 males, six females) and applied genome-wide linkage analysis. Whole exome sequencing was performed for two patients. In addition, sural nerve biopsies were obtained from two patients. Through whole exome sequencing, we identified an average of 20,336 coding variants from two patients. We also found evidence of linkage mapped to chromosome 11p11-11q13.3 (LOD score of 3.6). Among these variants in the linkage region, we detected a novel p.S90W mutation in the Berardinelli-Seip congenital lipodystrophy 2 (BSCL2) gene, after filtering 31 Korean control exomes. Our p.S90W patients had frequent sensory disturbances, pyramidal tract signs, and predominant right thenar muscle atrophy in comparison with reported p.S90L patients. The phenotypic spectra were wide and demonstrated intrafamilial variability. Two patients with different clinical features underwent sural nerve biopsies; the myelinated fiber densities were increased slightly in both patients, which differed from two previous case reports of BSCL2 mutations (p.S90L and p.N88S). This report expands the variability of the clinical spectrum associated with the BSCL2 gene and describes the first family with the p.S90W mutation.

Repair of bone defects is a difficult clinical problem for reconstructive surgeons. Bone tissue engineering using an appropriate scaffold with cells is a new therapy for the repair of bone defects. The aim of this study was to evaluate the in vitro osteogenesis of canine adipose tissue-derived mesenchymal stem cells (Ad-MSCs) cultured in a combination of collagen I gel and a porous serum-derived albumin scaffold. A serum-derived albumin scaffold was prepared with canine serum by cross-linking and freeze-drying procedures. Ad-MSCs were seeded into serum-derived albumin scaffolds with or without collagen I gel, and were exposed to osteogenic differentiation conditions in vitro. After 28 days of in vitro culture, the distribution and osteogenic differentiation of Ad-MSCs cultured in the scaffold were evaluated by scanning electron microscopy, histology, immunohistochemistry, alkaline phosphatase (ALP) activity assay, and calcium colorimetric assay. Ad-MSCs showed more homogeneous distribution and osteogenic differentiation in the scaffold with collagen I gel than without collagen I gel. ALP activity and extracellular matrix mineralization in the construct with type I collagen were significantly higher than in the construct without type I collagen (p < 0.05). In conclusion, the combination of collagen I gel and the serum-derived albumin scaffold enhanced osteogenic differentiation and homogenous distribution of Ad-MSCs.

All cellular phenomena and developmental events, including inner ear development, are modulated through harmonized signaling networks. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor, is a major signaling component involved in cross talk with key regulators of development; i.e., Wnt, Notch, and bone morphogenetic proteins. Although Pten function has been studied in various systems, its role in inner ear development is poorly understood. Here, we used inner ear-specific Pten conditional knockout mice and examined the characteristics of the inner ear. In a detailed analysis of the phenotype, reduced cochlear turning and widened epithelia were observed. Phalloidin staining of sensory epithelium revealed that hair cell patterns were disturbed; i.e., additional rows of hair cells were discovered. The neural abnormality revealed a reduction in and disorganization of nerve fibers, including apoptosis at the neural precursor stage. Pten deficiency induced increased phosphorylation of Akt at Ser473. The elevation of inhibitory glycogen synthase kinase 3β Ser9 phosphorylation (pGSK3β) was sustained until the neuronal differentiation stage at embryonic day 14.5, instead of pGSK3β downregulation. This is the first report on the influence of Pten/Akt/GSK3β signaling on the development of spiral ganglia. These results suggest that Pten is required for the maintenance of neuroblast number, neural precursors, and differentiation in the inner ear.

Previous animal studies have shown that transplantation of mesenchymal stem cells (MSCs) into spinal cord lesions enhances axonal regeneration and promotes functional recovery. We isolated the MSCs derived from fat, bone marrow, Wharton's jelly and umbilical cord blood (UCB) positive for MSC markers and negative for hematopoietic cell markers. Their effects on the regeneration of injured canine spinal cords were compared. Spinal cord injury was induced by balloon catheter compression. Dogs with injured spinal cords were treated with only matrigel or matrigel mixed with each type of MSCs. Olby and modified Tarlov scores, immunohistochemistry, ELISA and Western blot analysis were used to evaluate the therapeutic effects. The different MSC groups showed significant improvements in locomotion at 8 weeks after transplantation (P<0.05). This recovery was accompanied by increased numbers of surviving neuron and neurofilament-positive fibers in the lesion site. Compared to the control, the lesion sizes were smaller, and fewer microglia and reactive astrocytes were found in the spinal cord epicenter of all MSC groups. Although there were no significant differences in functional recovery among the MSCs groups, UCB-derived MSCs (UCSCs) induced more nerve regeneration and anti-inflammation activity (P<0.05). Transplanted MSCs survived for 8 weeks and reduced IL-6 and COX-2 levels, which may have promoted neuronal regeneration in the spinal cord. Our data suggest that transplantation of MSCs promotes functional recovery after SCI. Furthermore, application of UCSCs led to more nerve regeneration, neuroprotection and less inflammation compared to other MSCs.

Charcot-Marie-Tooth disease (CMT) is one of the most common inherited neuropathies and is a genetically and clinically heterogeneous disorder with variable inheritance modes. As several molecules have been reported to have therapeutic effects on CMT, depending on the underlying genetic causes, exact genetic diagnostics have become very important for executing personalized therapy. Whole-exome sequencing has recently been introduced as an available method to identify rare or novel genetic defects from genetic disorders. Particularly, CMT is a model disease to apply exome sequencing because more than 50 genes (loci) are involved in its development with weak genotype-phenotype correlation. This study performed the exome sequencing in 25 unrelated CMT patients who revealed neither 17p12 duplication/deletion nor several major CMT genes. This study identified eight causative heterozygous mutations (32%). This detection rate seems rather high because each sample was tested before the study for major genetic causes. Therefore, this study suggests that the exome sequencing can be a highly exact, rapid, and economical molecular diagnostic tool for CMT patients who are tested for major genetic causes., (© 2012 Wiley Periodicals, Inc.)

Alternative sources of mesenchymal stem cells (MSCs) for replacing bone marrow (BM) have been extensively investigated in the field of bone tissue engineering. The purpose of this study was to compare the osteogenic potential of canine MSCs derived from adipose tissue (AT), BM, umbilical cord blood (UCB), and Wharton's jelly (WJ) using in vitro culture techniques and in vivo orthotopic implantation assays. After canine MSCs were isolated from various tissues, the proliferation and osteogenic potential along with vascular endothelial growth factor (VEGF) production were measured and compared in vitro. For the in vivo assay, MSCs derived from each type of tissue were mixed with β-tricalcium phosphate and implanted into segmental bone defects in dogs. Among the different types of MSCs, AT-MSCs had a higher proliferation potential and BM-MSCs produced the most VEGF. AT-MSCs and UCB-MSCs showed greater in vitro osteogenic potential compared to the other cells. Radiographic and histological analyses showed that all tested MSCs had similar osteogenic capacities, and the level of new bone formation was much higher with implants containing MSCs than cell-free implants. These results indicate that AT-MSCs, UCB-MSCs, and WJ-MSCs can potentially be used in place of BM-MSCs for clinical bone engineering procedures.

The purpose of this study is to evaluate the effect of acellular dermal matrix (ADM) as a delivery carrier of adipose-derived mesenchymal stem cells (ASCs) on bone regeneration in athymic murine calvarial bone defect. Paired-critical size defects in nude rat skull were made. The right-side defects received ASCs/ADM or only ADM, whereas the left-side defect was not treated. In 3D images, new bone formation in the ASCs/ADM group was apparent at 4 wk, but in the ADM group at 8 wk. At 4 and 8 wk, bone mineral density and tissue volume in rats that received ASCs/ADM were significantly greater than rats that received ADM and control groups. Histological examination revealed that the defect was repaired by bone in the ASCs/ADM group, whereas only minimal bone island with fibrous connection was observed in the control group. In histomorphometric analysis, the total healing score in the ASCs/ADM group at 4 wk was significantly higher than the ADM and negative control group, whereas the score of 8 wk was similar between the ASCs/ADM and ADM group. ASCs/ADM implants promote new bone formation more rapidly than ADM only or no treatment. ADM seeded with ASCs may be potentially useful as a future biomaterial option in bone implants., (Copyright © 2012 Wiley Periodicals, Inc.)

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) are a promising source of cells for bone tissue engineering. Matrigel is a basement membrane extract containing multiple extracellular components. This mixture may promote the osteogenic differentiation of MSCs and provide a more appropriate microenvironment for transplanted cells. Here, we investigated the effect of Matrigel on the osteogenic potential of Ad-MSCs. Canine Ad-MSCs were cultured in 2D and 3D matrices and implanted into subcutaneous pouches of dogs either with or without Matrigel. Culture mineralization, cell adhesion efficiency, cell proliferation, osteoid matrix production and alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase activities were quantified and compared. Ad-MSCs grown in 2D cultures with Matrigel showed higher levels of calcium deposition and ALP activity than those grown in the absence of Matrigel under osteogenic conditions. In 3D cultures, the cells cultivated with Matrigel showed greater attachment, proliferation and osteogenic differentiation than those grown without Matrigel. In vivo, Ad-MSCs implanted with Matrigel showed higher osteogenic potential than those without Matrigel. In conclusion, these data suggest that the use of Matrigel can increase the osteogenic potential of canine Ad-MSCs.

Porcine organs are attractive for xenotransplantation, if severe immunologic concerns can be overcome. Recently, reengineered organs, with heterologous cellular materials removed but preserved organ architecture and vasculature have been created using small rodents in an effort to produce customized bioengineered organs. However, few studies have been performed to generate bioengineered organs from porcine sources. The aim of this work was to produce 3-D bioengineered scaffolds from major porcine organs, preserving the native morphology and vascular structures with complete removal of cellular and nuclear materials. We decellularized porcine heart, liver, and kidney using a peristaltic pump system with 1% sodium dodecyl sulfate. The preservation of major architecture and vasculature was confirmed by gross findings, ultrasonography, and angiography. Hematoxylin and eosin staining revealed no evidence of nuclear or cytoplasmic residues. Quantitative DNA analysis demonstrated a substantial reduction (0%-8%) of porcine DNA in the scaffolds. These results suggested that 3-D bioengineered scaffolds of porcine organs may have tremendous potential to produce non-immunogenic transplantable organs as well as beneficial tools for biomedical studies on organ re-engineering and repair., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Introduction: Bioscaffolds derived from animal organs are promising materials for xenotransplantation and regenerative medicine. For effective generation of biological scaffolds from diverse organs, there have been many technical challenges. In this study, we introduced a novel approach to create multiorgan bioscaffolds through systemic decellularization., Methods and Materials: To obtain acellular bioscaffolds, the healthy adult rats were systemically perfused with ionic detergent through the carotid artery. Additional liver perfusion was set up to prevent potential obstruction from the influx of the decellularized debris via the portal vein. The perfusion system was controlled to maintain a constant physiological cardiac output of approximately 50 mL/min and was designed to minimize air entrapment. After decellularization, every organ designated for bioscaffold was harvested for evaluation of vascular structure and histology., Results: The perfusion times were different for each organ. In our histological analysis, the decellularized bioscaffolds harvested from most organs including major solid organs (ie, heart, liver, and kidney) as well as the others (such as stomach, intestines, spleen, etc) represented no evidence of residual cellular materials. Furthermore, the well-preserved collagen materials and intact vascular structures were also confirmed., Conclusion: The results from this study suggested that this systemic decellularization has the advantages to obtain a variety of bioscaffolds from single donor, and we can even decellularize organs with complex influx vascular structures. This method may also be used to study organ bioengineering for patients who need simultaneous combined organ transplantation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The outbreak of severe acute respiratory syndrome (SARS) in 2002 affected thousands of people and an efficient diagnostic system is needed for accurate detection of SARS coronavirus (SARS CoV) to prevent or limit future outbreaks. Of the several SARS CoV structural proteins, the nucleocapsid protein has been shown to be a good diagnostic marker. In this study, an ssDNA aptamer that specifically binds to SARS CoV nucleocapsid protein was isolated from a DNA library containing 45-nuceotide random sequences in the middle of an 88mer single-stranded DNA. After twelve cycles of systematic evolution of ligands by exponential enrichment (SELEX) procedure, 15 ssDNA aptamers were identified. Enzyme-linked immunosorbent assay (ELISA) analysis was then used to identify the aptamer with the highest binding affinity to the SARS CoV nucleocapsid protein. Using this approach, an ssDNA aptamer that binds to the nucleocapsid protein with a K(d) of 4.93±0.30nM was identified. Western blot analysis further demonstrated that this ssDNA aptamer could be used to efficiently detect the SARS CoV nucleocapsid protein when compared with a nucleocapsid antibody. Therefore, we believe that the selected ssDNA aptamer may be a good alternative detection probe for the rapid and sensitive detection of SARS., (Copyright © 2011 Elsevier B.V. All rights reserved.)

To obtain selective and potent inhibitor for T-type calcium channel by ligand based drug design, 4-piperidinecarboxylate and 4-piperidinecyanide derivatives were prepared and evaluated for in vitro and in vivo activity against α(1G) calcium channel. Among them, several compounds showed good T-type calcium channel inhibitory activity and minimal off-target activity over hERG channel (% inhibition at 10 μM=61.85-71.99, hERG channel IC(50)=1.57 ± 0.14-4.98 ± 0.36 μM). Selected compound 31a was evaluated on SNL model of neuropathic pain and showed inhibitory effect on mechanical allodynia., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The linkage search for susceptibility loci using SNP markers in hereditary hearing loss has proven challenging due to genetic heterogeneity. We conducted a genome-wide linkage analysis using high-density SNP markers in two Korean families (families coded SD-J and SR-167) with autosomal dominant non-syndromic hearing loss (ADNSHL). Evidence was found of linkage at 8q24.13~q24.3 and 10p11.21~q22.2 (LOD 3.01) in the SD-J family. In the case of family SR-167, which had the most affected members, the parametric LOD score was low owing to the lack of power for linkage analysis. However, using non-parametric linkage analysis, it was possible to obtain significant evidence for linkage at 10q22.1~q23.31 (LOD 1.79; NPL 6.47, P<0.00001). There is an overlapping region with a significant LOD score between the SD-J and SR-167 families, which encompasses 4 cM at 10q22.1~22.2. Interestingly, the characteristics of hearing loss in both families were similar, and the haplotype within overlapping region was shared in the affected individuals of the two families. We performed direct sequencing of the candidate genes that are thought to be causing the condition, but no disease-causing mutations were identified.

In this study, we analyzed the influence of phosphate (P(i)) limitation on the metabolism of Corynebacterium glutamicum. Metabolite analysis by gas chromatography-time-of-flight (GC-TOF) mass spectrometry of cells cultivated in glucose minimal medium revealed a greatly increased maltose level under P(i) limitation. As maltose formation could be linked to glycogen metabolism, the cellular glycogen content was determined. Unlike in cells grown under P(i) excess, the glycogen level in P(i)-limited cells remained high in the stationary phase. Surprisingly, even acetate-grown cells, which do not form glycogen under P(i) excess, did so under P(i) limitation and also retained it in stationary phase. Expression of pgm and glgC, encoding the first two enzymes of glycogen synthesis, phosphoglucomutase and ADP-glucose pyrophosphorylase, was found to be increased 6- and 3-fold under P(i) limitation, respectively. Increased glycogen synthesis together with a decreased glycogen degradation might be responsible for the altered glycogen metabolism. Independent from these experimental results, flux balance analysis suggested that an increased carbon flux to glycogen is a solution for C. glutamicum to adapt carbon metabolism to limited P(i) concentrations.

The principal objective of this study was to investigate the prevalence of toxoplasmosis in household and stray cats in Seoul, Republic of Korea. We collected blood samples from 72 stray and 80 household cats, and all samples were examined by ELISA and nested PCR. The overall positive rates of Toxoplasma gondii in stray cats were 38.9% (28/72), with 15.3% (11/72) in ELISA and 30.6% (22/72) in PCR. The positive rate in male stray cats was slightly higher than that of female stray cats. The highest positive rate of T. gondii infection was noted in Gangnam and Songpa populations in ELISA and in Gwangjin population in PCR. In household cats, however, we could not detect any specific antibodies or DNA for T. gondii. In conclusion, we recognized that the infection rate of toxoplasmosis in stray cats in Seoul was considerably high but household cats were free from infection.

The identification of genes to be deleted or amplified is an essential step in metabolic engineering for strain improvement toward the enhanced production of desired bioproducts. In the past, several methods based on flux analysis of genome-scale metabolic models have been developed for identifying gene targets for deletion. Genome-wide identification of gene targets for amplification, on the other hand, has been rather difficult. Here, we report a strategy called flux scanning based on enforced objective flux (FSEOF) to identify gene amplification targets. FSEOF scans all the metabolic fluxes in the metabolic model and selects fluxes that increase when the flux toward product formation is enforced as an additional constraint during flux analysis. This strategy was successfully employed for the identification of gene amplification targets for the enhanced production of the red-colored antioxidant lycopene. Additional metabolic engineering based on gene knockout simulation resulted in further synergistic enhancement of lycopene production. Thus, FSEOF can be used as a general strategy for selecting genome-wide gene amplification targets in silico.

Background: The metabolomic approaches for mining biomarkers of women's cancers based on gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry combined with partial least squares-discriminant analysis are described., Methods: To identify urinary potential biomarkers, the qualitative and quantitative analyses were introduced with 10 breast, 9 ovarian and 12 cervical cancer patients as well as 22 normal controls, which were considered with their ages and menopausal state., Results: For comprehensive metabolomic approaches, the non-targeted qualitative profiling was first achieved to get metabolic patterns of collected samples and the targeted quantitative analysis focused on hormonal metabolism was also conducted. Two known biomarkers, i.e., 5-hydroxymethyl-2-deoxyuridine and 8-hydroxy-2-deoxyguanosine, in breast cancer were also confirmed using the present methods. In addition, 3 potential biomarkers for ovarian cancer i.e. 1-methyladenosine, 3-methyluridine, and 4-androstene-3,17-dione, which were categorized in significantly increased level using one way of variance analysis (p<0.05), were identified as quantitatively targeted metabolites with pattern analysis. The cancer markers identified in this study are highly related to metabolites which are responsible for oxidative DNA damage and DNA methylation process., Conclusion: The present metabolomic approaches are not only useful for diagnostic tools and patient stratification, but may be mapped on metabolic network to reflect disease states.

Objective: To investigate the development of gingival hyperplasia in dogs after renal transplantation and administration of microemulsified cyclosporine A (MCsA)., Study Design: Experimental study., Animals: Healthy adult mongrel dogs (n=5)., Methods: As part of study on renal transplantation, dogs administered MCsA (20 mg/kg/day), azathioprine, and prednisolone to prevent graft rejection were monitored for development of gingival changes. Prednisolone was discontinued after 3 months. MCsA dose was adjusted to maintain whole blood trough concentration of 400-700 ng/mL. Gingival change was evaluated by weekly examination and photodocumentation, and gingival biopsy for histopathology was performed at 28 weeks., Results: One dog was lost because of acute graft rejection. Gingival hyperplasia developed in 3 of 4 dogs. The earliest gingival changes occurred in the interdental papillae at 20 weeks after transplantation. On histopathology, the underlying connective tissue was thickened and contained increase numbers of fibroblasts and inflammatory infiltrates., Conclusions: Long-term immunosuppression with an MCsA-based treatment likely induces substantial gingival hyperplasia when therapeutic, immunosuppressive blood levels of MCsA were maintained for 32 weeks., Clinical Relevance: MCsA is used for immune-mediated diseases and preventing rejection after transplant in dogs. MCsA blood levels, and gingival hyperplasia should be monitored by routine examination of the interdental papilla in dogs administered MCsA for long periods.

An 8-year-old Yorkshire terrier was presented with tracheal collapse. Two intraluminal nitinol stents were implanted. The implanted stents were found to be fractured 4 weeks after implantation. The fractured stents were removed. To restore the collapsed trachea, ring prostheses were applied. However, the dog was euthanized because of a bad outcome following surgery.

Metabolomics has focused on toxicological applications to (1) understand the mechanisms of toxicity, (2) identify novel biomarkers of toxicity, and (3) provide in vivo assessment in animal models through simple and fast methods to date. The toxicological effects of nonylphenol (NP) were evaluated after intraperitoneal injection of rats with 0, 50, and 250 mg kg(-1) day(-1) NP for four consecutive days. In the nontargeted approach, different extraction conditions were introduced to investigate the effects of NP on rats through gas chromatography/mass spectrometry (GC/MS). The GC/MS data obtained were further analyzed with partial least-squares discriminant analysis to compare toxicological effects between control and treated groups. The targeted approach was also used in combination with GC/MS to quantify endocrine hormones and to identify possible biomarkers in rat urine under optimal extraction conditions. In addition, we considered the metabolic trajectory to examine the metabolite profiles and patterns related to steroid metabolism in rats that were treated with NP, considering both treatment amount and time. The data suggest that tetrahydrocorticosterone and 5alpha-tetrahydrocorticosterone are possible urinary biomarkers of NP-induced toxicity. This metabolomic approach is a promising tool to assist with screening in toxicological studies.

Inflammation plays a key role in obesity-related pathologies such as cardiovascular disease, type II diabetes, and several types of cancer. Obesity-induced inflammation entails the enhancement of the recruitment of macrophages into adipose tissue and the release of various proinflammatory proteins from fat tissue. Therefore, the modulation of inflammatory responses in obesity may be useful for preventing or ameliorating obesity-related pathologies. Some spice-derived components, which are naturally occurring phytochemicals, elicit antiobesity and antiinflammatory properties. In this study, we investigated whether active spice-derived components can be applied to the suppression of obesity-induced inflammatory responses. Mesenteric adipose tissue was isolated from obese mice fed a high-fat diet and cultured to prepare an adipose tissue-conditioned medium. Raw 264.7 macrophages were treated with the adipose tissue-conditioned medium with or without active spice-derived components (i.e., diallyl disulfide, allyl isothiocyanate, piperine, zingerone and curcumin). Chemotaxis assay was performed to measure the degree of macrophage migration. Macrophage activation was estimated by measuring tumor necrosis factor-alpha (TNF-alpha), nitric oxide, and monocyte chemoattractant protein-1 (MCP-1) concentrations. The active spice-derived components markedly suppressed the migration of macrophages induced by the mesenteric adipose tissue-conditioned medium in a dose-dependent manner. Among the active spice-derived components studied, allyl isothiocyanate, zingerone, and curcumin significantly inhibited the cellular production of proinflammatory mediators such as TNF-alpha and nitric oxide, and significantly inhibited the release of MCP-1 from 3T3-L1 adipocytes. Our findings suggest that the spice-derived components can suppress obesity-induced inflammatory responses by suppressing adipose tissue macrophage accumulation or activation and inhibiting MCP-1 release from adipocytes. These spice-derived components may have a potential to improve chronic inflammatory conditions in obesity.

Nerve growth factor in the tear film and corneal epithelium is hypothesized to play an important role in ocular surface maintenance and corneal wound healing. The purpose of this study was to determine the expression of nerve growth factor and its high affinity (trkA) receptor in tears, cornea, and lacrimal glands of normal dogs, the modulation of nerve growth factor and its trkA receptor during corneal wound healing, and the effect of topical nerve growth factor application on canine corneal epithelial wound healing. In the first of three experiments, the nerve growth factor content of tears, corneal epithelium, lacrimal gland, and 3rd eyelid gland was determined in normal dogs by enzyme-linked immunosorbent assay and the expression of nerve growth factor and its trkA receptor were evaluated in the cornea and lacrimal glands by immunohistochemistry. In a second experiment, unilateral corneal epithelial defects were created, and tissues were evaluated for changes in nerve growth factor or trkA expression for 1 week. In a third experiment, bilateral corneal epithelial defects were created and the right eyes in each animal were treated 4 times daily with either recombinant human nerve growth factor, murine nerve growth factor, or nerve growth factor-blocking antibody. The results of this study showed that nerve growth factor levels in normal dog tears, corneal epithelium, third eyelid gland and lacrimal gland were 15.4+/-4.6 ng ml(-1), 33.5+/-12.3, 52.4+/-17.4 and 48.8+/-9.4 ng g(-1), respectively. NGF and trkA receptors were identified by immunohistochemistry in all tissues examined. After unilateral corneal wounding, nerve growth factor concentration increased in the tears bilaterally for 3 days, especially in the wounded eye, and then returned to pre-wounding values. Nerve growth factor content, and immunohistochemical staining for nerve growth factor and trkA, increased significantly in the ipsilateral cornea epithelium following unilateral wounding. Nerve growth factor concentrations in lacrimal and third eyelid glands also increased bilaterally (p<0.01) after unilateral wounding. Time to wound closure and rate of epithelial migration did not differ significantly between nerve growth factor-treated, nerve growth factor antibody-treated, and control eyes. In conclusion, nerve growth factor is present under resting physiologic conditions in normal canine tears, and nerve growth factor and its trkA receptor are present under resting conditions in normal canine corneal epithelium, lacrimal gland and third eyelid gland. Nerve growth factor is elevated in the tears, cornea, and lacrimal glands after corneal epithelial wounding; however, topical application of nerve growth factor, or its blocking antibody does not modulate corneal wound healing in the normal dog eye.

Canine kidney preservation models have historically used autotransplants to avoid the complications of rejection, although clinically all transplants are allografts. This study investigated the effects of preservation time and method on early kidney function in a canine allograft vs. autograft model. Kidneys were harvested from beagles, preserved by cold storage (CS) in UW solution for 0, 24 or 72 h, or by machine perfusion (MP) with Belzer MPS for 72 h. In some experiments 45 min of warm ischemia (WI) was performed in situ before harvest. Allograft recipients received steroid immunosuppression. Kidney function was assessed by serum creatinine and survival for 7 days. Allografts preserved for 0 and 24 h performed as well as autografts. Allografts preserved for 72 h by either CS or MP had a higher incidence of primary nonfunction (PNF) compared with autografts, as determined by survival (50% vs. 100%, p < 0.003). Primary nonfunction kidneys had thrombotic microangiopathy, vascular and peritubular capillary binding of IgM and complement C4d, and evidence of circulating donor-specific antibodies; all consistent with humoral rejection. These responses were dependent on hypothermia time and were not attributable to ischemia, immunosuppression, preservation solution, or cellular rejection. In conclusion, prolonged hypothermia can cause PNF in allografts owing to acute humoral rejection.

Purpose: To determine the effect of chronic corneal epithelial debridement on epithelial and stromal morphology and extracellular matrix components, and to compare those changes to those in spontaneous chronic corneal epithelial defects (SCCED) in dogs., Methods: Axial corneal epithelial wounds, 10 mm in diameter, were created weekly for 8 weeks in five normal adult laboratory beagles. Slit lamp biomicroscopy and corneal pachymetry were performed weekly before wounding. Three days after the last debridement the dogs were killed humanely, and corneas were processed for light and electron microscopy and immunohistochemistry for collagen IV, collagen VII, fibronectin, and laminin., Results: No significant changes in corneal thickness were found. All samples demonstrated epithelial dysmaturation adjacent to the wound edge, and, in four of five, a narrow zone of nonadherent epithelium formed adjacent to the exposed stroma. All samples had a stromal acellular zone in the area of the defect and continuing for a short distance under the adjacent attached epithelium. Experimentally wounded dogs did not form the superficial hyaline acellular lamina found in 92% of dogs with SCCED. Laminin, collagen IV, and fibronectin were present on the stromal surface in all samples, and collagen VII was present in four of five samples. Transmission electron microscopy (TEM) demonstrated the presence of basement membrane on the surface of the exposed stroma., Conclusions: Epithelial changes are similar between experimentally wounded dogs and dogs with SCCED. The stromal acellular zone that forms in experimentally wounded dogs is distinct from the hyaline lamina observed in dogs with SCCED. The difference in the acellular stromal layers between chronically wounded dogs and dogs with SCCED may be of relevance to our understanding of the pathophysiology of persistent epithelial defects.

Aims: To investigate if the amniotic membrane (AM) promotes epithelial migration while inhibiting stromal remodelling associated with corneal haze after excimer laser keratectomy., Methods: A wound 150 microm in depth and 6.0 mm in diameter was produced in 40 rabbits using an excimer laser. One eye was randomly chosen to be covered by the AM while the other eye served as a control. Epithelial wound healing was evaluated, together with any morphological changes of the anterior stroma connected with corneal haze. These morphological changes were histopathologically analysed using dichlortriazinyl aminofluorescein (DTAF), Masson trichrome staining, and an image analyser., Results: The AM group had a short latent phase followed by fast epithelial healing (p<0.001) during the early wound healing period and a significant decrease in the inflammatory response, together with a smaller change in the number of keratocytes than the control group. The mean thickness of the regenerated stroma was significantly thinner in the AM group than in the control group at 8 weeks (p<0.0001). The AM group had a more regular architecture of regenerated stromal lamella at 8 weeks and significantly less haze after 4 weeks than the control group (p<0.05)., Conclusion: Use of the AM as a dressing on a corneal wound created by excimer laser surgery, in which severe haze is expected, may induce rapid epithelial healing with less inflammatory response. The AM may inhibit the irregular synthesis of stromal collagen that is associated with corneal haze.

Purpose: To evaluate the use of multifocal phototherapeutic keratectomy (PTK) for the treatment of indolent and persistent epithelial defect (PED)., Setting: Department of Ophthalmology, Kangnam St. Mary's Hospital, The Catholic University of Korea, College of Medicine, Seoul, Korea., Methods: Fifteen eyes diagnosed with PED were treated with Summit excimer laser PTK. In all patients (N = 15), the epithelium had previously failed to cover with at least 1 method of medical or surgical treatment. Multifocal ablations were delivered to the elevated margin surrounding the epithelial defect area using PTK with a diameter of 1.0 mm and depth of 150 to 200 pulse ablations. The mean follow-up was 17.7 months (range 9 to 37 months)., Results: Complete reepithelialization occurred within 7 days in 13 eyes and within 11 days and 12 days in 1 eye each. All corneas except 1 remained healed for at least 9 months of follow-up. One eye had a recurrence 3 months after PTK and was retreated. Postoperative visual acuity improved in 8 eyes and was unaltered in 7., Conclusion: Multifocal PTK with the 193 nm excimer laser is a safe, effective treatment for PED that is unresponsive to conventional therapy.