Isolation and Characterization of Multipotent Mesenchymal Stem Cells Adhering to Adipocytes in Canine Bone Marrow.

The ceiling culture method has been used to isolate mature adipocytes from adipose tissue that can be dedifferentiated into fibroblastic cells, also known as dedifferentiated fat (DFAT) cells that self-renew and are multipotent, with much higher homogeneity and colony-forming efficiency than those of adipose tissue-derived mesenchymal stem cells. We cultured adipocytes from canine bone marrow using this technique, with the expectation of obtaining DFAT cells. However, contrary to our expectations, continuous monitoring of ceiling cultures by time-lapse microscopy revealed many small cells adhering to adipocytes that proliferated rapidly into cells with a fibroblastic morphology and without any dedifferentiation from adipocytes. We named these cells bone marrow peri-adipocyte cells (BM-PACs) and demonstrated the multipotent properties of BM-PACs compared to that of conventionally cultured canine bone marrow mesenchymal stem cells (BMMSCs). BM-PACs showed significantly greater clonogenicity and proliferation ability than BMMSCs. An in vitro trilineage differentiation assay revealed that BM-PACs possess adipogenic, osteogenic, and chondrogenic capacities superior to those of BMMSCs. Flow cytometric analysis revealed that the expression of CD73, which plays an important role in cell growth and differentiation, was significantly higher in BM-PACs than in BMMSCs. These results indicate that canine BM-PACs have stem cell characteristics that are superior to those of BMMSCs, and that these mesenchymal stem cells (MSCs) appear to be a feasible source for cell-based therapies in dogs.

Evaluation of ABCG2 and p63 expression in canine cornea and cultivated corneal epithelial cells.

OBJECTIVE: To examine the expressions of ABCG2 and p63 in canine corneal epithelia and to evaluate their significance in corneal regeneration. PROCEDURES: Canine corneal and limbal epithelial cells were obtained from five healthy beagle dogs. We analyzed the morphological properties of cultivated limbal and corneal epithelial cells. We compared the expressions of ABCG2 and p63 in the limbus and central cornea by immunohistochemistry and real-time quantitative PCR. We analyzed the expression of these markers in cultivated cells by immunocytochemistry and real-time quantitative PCR. RESULTS: The limbal epithelial cells were smaller and proliferated more rapidly than the corneal epithelial cells in primary cultures. The corneal cells failed to be subcultured, whereas the limbal cells could be subcultured with increasing cell size. ABCG2 was localized in the basal layer of the limbal epithelium, and p63 was widely detected in the entire corneal epithelia. ABCG2 expression was significantly higher, and p63 was slightly higher in the limbus compared with the central cornea. ABCG2 was detected only in limbal cells in primary culture, not in corneal cells or passaged limbal cells. p63 was detected in both limbal and corneal cells and decreased gradually in the limbal cells with the cell passages. CONCLUSIONS: ABCG2 was localized in canine limbal epithelial cells, and p63 was widely expressed in canine corneal epithelia. ABCG2 and p63 could prove to be useful markers in dogs for putative corneal epithelial stem cells and for corneal epithelial cell proliferation, respectively.

Comparison of the canine corneal epithelial cell sheets cultivated from limbal stem cells on canine amniotic membrane, atelocollagen gel, and temperature-responsive culture dish.

OBJECTIVE: The current study compared canine corneal epithelial cell sheets cultivated from limbal stem cells on amniotic membrane, atelocollagen gel, and temperature-responsive culture dish. PROCEDURES: We collected limbal epithelial cells from the intact eyes of beagles and cultivated the cells on denuded canine amniotic membranes, temperature-responsive cell culture labware, and collagen gel with 3T3 feeder cells. Immunofluorescence staining for Ki-67 was used to analyze the capacity of cell proliferation in the sheets. Immunofluorescence staining was also performed for the corneal epithelium-specific marker cytokeratin 3 and putative stem cell markers ABCG2 and p63. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to detect ABCG2 and p63. RESULTS: The growth rates of the cultivated cells, or the times it took them to reach confluency, were different for the three scaffolds. The cultivated sheet on the temperature-responsive dish consisted of 2-3 layers, while those on the collagen gel and on the amniotic membrane consisted of 5-8 layers. The basal layer cells grown on all three scaffolds expressed putative stem cell markers. In real-time RT-PCR analysis, the highest level of p63 was observed in the sheets grown on collagen gel. CONCLUSIONS: In this study, the cells cultured on the collagen gel demonstrated a capacity for cell proliferation, and the expressions of stem cells in the sheets suggested that collagen gel is the most suitable carrier for clinical use.

Increased unbound retinol-binding protein 4 concentration induces apoptosis through receptor-mediated signaling.

The increase of apo-/holo-retinol-binding protein 4 (RBP4) concentrations has been found in subjects with renal dysfunction and even in diabetic patients with microalbuminuria. Holo-RBP4 is recognized to possess cytoprotective function. Therefore, we supposed that the relative increase in apo-RBP4 might induce cell damage. In this study, we investigated the signal transduction that activated apoptosis in response to the increase of apo-/holo-RBP4 concentration. We found that increase of apo-/holo-RBP4 concentration ratio delayed the displacement of RBP4 with "stimulated by retinoic acid 6" (STRA6), enhanced Janus kinase 2 (JAK2)/STAT5 cascade, up-regulated adenylate cyclase 6 (AC6), increased cAMP, enhanced JNK1/p38 cascade, suppressed CRBP-I/RARα (cellular retinol-binding protein/retinoic acid receptor α) expression, and led to apoptosis in HK-2 and human umbilical vein endothelial cells. Furthermore, STRA6, JAK2, STAT5, JNK1, or p38 siRNA and cAMP-PKA inhibitor reversed the repression of CRBP-I/RARα and apoptosis in apo-RBP4 stimulation. In conclusion, this study indicates that the increase of apo-/holo-RBP4 concentration may influence STRA6 signaling, finally causing apoptosis.

The PNPLA3 I148M polymorphism is associated with insulin resistance and nonalcoholic fatty liver disease in a normoglycaemic population.

BACKGROUND AND AIMS: The adiponutrin/patatin-like phospholipase-3 (PNPLA3) I148M polymorphism has recently been found to contribute to differences in hepatic lipid content. Nonalcoholic fatty liver disease (NAFLD) has recently been considered a hepatic component of insulin resistance and a risk factor in the emergence of type 2 diabetes. However, whether there is an association between PNPLA3 I148M and insulin resistance and NAFLD in a normoglycaemic population is still unknown. METHODS: This study enrolled 156 normoglycaemic individuals with NAFLD and 723 controls. All participants received complete biochemical and clinical workups including liver ultrasonography. They were then genotyped for the PNPLA3 I148M polymorphism. RESULTS: We found significant differences in the genotype and the dominant model of the PNPLA3 I148M polymorphism between the NAFLD groups and the controls (P=0.018 and P=0.01 respectively). Furthermore, there was a dose effect of the PNPLA3 I148M genotype, in that CG heterozygotes had a risk of NAFLD between CC and GG homozygotes [adjusted odds ratio (OR)=2.03, 95% confidence interval (CI)=1.23-3.375 for the GG genotype and adjusted OR=1.55, 95% CI=1.02-2.35 for the CG genotype]. The dominant model of the PNPLA3 I148M polymorphism showed higher waist circumference, fasting insulin, Homeostasis Model Assessment (HOMA-IR), alanine aminotransferase concentrations and ferritin level. Multivariate analysis showed the PNPLA3 I148M polymorphism to be independently and significantly associated with NAFLD in our normoglycaemic participants. CONCLUSION: This study reports an association between the PNPLA3-I148M polymorphism and insulin resistance and NAFLD in a normoglycaemic population.

Association of polymorphisms of heat shock protein 70 with susceptibility to noise-induced hearing loss in the Taiwanese population.

Noise-induced hearing loss (NIHL) is the major cause of adult sensorineural hearing loss. It is a complex disease caused by the interaction of environmental and genetic factors. Previous studies found that heat shock proteins (HSPs) were associated with the development of NIHL. Specifically, polymorphisms in the heat shock protein 70 (HSP70) gene family are associated with a susceptibility to NIHL. In this study, three single nucleotide polymorphisms (SNPs) of the HSP70 family (SNP1: rs2075800; SNP2: rs1043618; SNP3: rs2763979) were genotyped in 349 noise-exposed Taiwanese workers. The subjects were categorized into noise-susceptible (NS; n = 27) and general susceptibility (GS; n = 322) groups by the change of a 4K-weighted audiometric average in an interval of 5 years. The G/C genotype of SNP2 was found to be associated with NIHL susceptibility (adjusted OR = 2.634; 95% CI = 1.096-6.328). No significant association was found for SNP1 and SNP3 with NIHL susceptibility. Analysis of haplotypes composed of these three SNPs revealed a significant association between NIHL susceptibility and haplotype CCC (OR = 2.197; 95% CI = 1.110-4.370). In conclusion, the genetic polymorphisms in the HSP70 genes seem to be associated with the individual's susceptibility to NIHL in the Taiwanese population. These findings could be used as a reference in the understanding and prevention of NIHL.

Mouse mammary tumor virus-like nucleotide sequences in canine and feline mammary tumors.

Mouse mammary tumor virus (MMTV) has been speculated to be involved in human breast cancer. Companion animals, dogs, and cats with intimate human contacts may contribute to the transmission of MMTV between mouse and human. The aim of this study was to detect MMTV-like nucleotide sequences in canine and feline mammary tumors by nested PCR. Results showed that the presence of MMTV-like env and LTR sequences in canine malignant mammary tumors was 3.49% (3/86) and 18.60% (16/86), respectively. For feline malignant mammary tumors, the presence of both env and LTR sequences was found to be 22.22% (2/9). Nevertheless, the MMTV-like LTR and env sequences also were detected in normal mammary glands of dogs and cats. In comparisons of the MMTV-like DNA sequences of our findings to those of NIH 3T3 (MMTV-positive murine cell line) and human breast cancer cells, the sequence similarities ranged from 94 to 98%. Phylogenetic analysis revealed that intermixing among sequences identified from tissues of different hosts, i.e., mouse, dog, cat, and human, indicated the MMTV-like DNA existing in these hosts. Moreover, the env transcript was detected in 1 of the 19 MMTV-positive samples by reverse transcription-PCR. Taken together, our study provides evidence for the existence and expression of MMTV-like sequences in neoplastic and normal mammary glands of dogs and cats.

The 8-oxoguanine glycosylase I (hOGG1) Ser326Cys variant affects the susceptibility to multi-vessel disease in Taiwan coronary artery disease patients.

8-hydroxydeoxyguanosine, the key lesion of oxidative DNA damage, contributes to the development of coronary artery disease (CAD). In humans, 8-hydroxydeoxyguanosine is repaired by the enzyme 8-oxoguanine glycosylase I (hOGG1). We investigated the association between the hOGG1 Ser(326)Cys polymorphism and the presence and the severity of CAD in a Taiwan population. Genotypes of the hOGG1 Ser(326)Cys polymorphism were determined from 1397 participants enrolled in this study (378 CAD patients and 1019 controls). CAD severity was indicated both by number of vessels affected (single-vessel disease, SVD vs. multi-vessel disease, MVD), and by individual diffuse score. Real-time polymerase chain reaction was used to determine genotype, using allele-specific TaqMan probes. We found that presence of the hOGG1 Ser(326)Cys polymorphism was associated with a significantly increased risk of CAD and multi-vessel disease when assuming a dominant model of inheritance (OR: 1.52 [95%:1.082~2.133], p=0.015; OR: 2.26 [95%:1.232~4.156], p=0.007). This result was confirmed by multivariate analysis, after adjustment for age, gender, body-mass index, diabetes hypertension, hypercholesterolemia and smoking (OR: 1.78 [95%:1.127~2.806], p<0.005; OR: 2.44 [95%:1.276~4.651], p<0.001). In the present study, hOGG1 Ser(326)Cys polymorphism is a novel genetic marker to be independently associated with the development and severity of CAD in Taiwanese population.

Stability of miniplates and miniscrews used for orthodontic anchorage: experience with 492 temporary anchorage devices.

OBJECTIVES: The aim of this retrospective study was to evaluate systematically the potential factors that influence failure rates of temporary anchorage devices (TADs) used for orthodontic anchorage. MATERIALS AND METHODS: Data on 492 TADs (miniplates, pre-drilling miniscrews, and self-drilling miniscrews) in 194 patients were collected. The factors related to TAD failure were evaluated using univariate analysis and multivariate forward stepwise logistic regression analysis. RESULTS: There were no significant differences in failure rates among the TADs for the following variables: gender, type of malocclusion, facial divergency, implantation site (buccal, lingual, or crestal/midpalatal), location (anterior or posterior), method of force application (power chain or Ni-Ti coil spring), arch (upper or lower), type of soft tissue (attached gingiva or removable mucosa), and most of the cephalometric measurements that reflect dento-cranio-facial characteristics. An increased failure rate was noted for the self-drilling miniscrew type of TAD, TADs used for tooth uprighting, those inserted on bone with lower density, those associated with local inflammation of the surrounding soft tissue, those loaded within 3 weeks after insertion, and those placed in patients with greater mandibular retrusion. Failure rates of the self-drilling miniscrews installed by an oral surgeon and by an orthodontist did not differ significantly. CONCLUSIONS: Inflammation of soft tissue surrounding a TAD and early loading within 3 weeks after insertion were the most significant factors predicting TAD failure. Both orthodontists and oral surgeons who install orthodontic TADs must undergo sufficient training to achieve clinical excellence.

SUMO4 M55V variant is associated with diabetic nephropathy in type 2 diabetes.

OBJECTIVE-SUMO4 mRNA was recently found to be mainly expressed in the kidney, and the methionine-to-valine substitution at codon 55 (M55V) variant of SUMO4 may induce higher nuclear factor-kappaB (NF-kappaB) activity. Because NF-kappaB is known to mediate the development of diabetic nephropathy, we examined the association between the SUMO4 M55V variant and the severity of diabetic nephropathy. RESEARCH DESIGN AND METHODS-We recruited a total of 430 patients with type 2 diabetes. The M55V (rs237025, 163A-->G) polymorphism of SUMO4 was genotyped by real-time PCR, and urine albumin concentration was measured by radioimmunoassay. RESULTS-The frequencies of SUMO4 AA, GA, and GG were 52.6, 40.7, and 6.7%, respectively, in the normoalbuminuric group; 45.5, 47.3, and 7.1% in the microalbuminuric group; and 36.9, 46.2, and 16.9% in the macroalbuminuric group. We detected a significant linear trend for SUMO4 genotype between the macroalbuminuric and normoalbuminuric groups. The mean urine albumin-to-creatinine ratio (42.3 +/- 108.82 mg/mmol) in the GG group was significantly higher than in the AA (14.9 +/- 51.49 mg/mmol) and GA (17.0 +/- 43.74 mg/mmol) groups. Multivariate logistic regression analysis showed the SUMO4 M55V variant to be independently associated with the severity of diabetic nephropathy. CONCLUSIONS-This study indicates that the SUMO4 gene M55V variant is associated with severity of diabetic nephropathy in patients with type 2 diabetes.

The hOGG1 Ser326Cys gene polymorphism is associated with decreased insulin sensitivity in subjects with normal glucose tolerance.

Increased oxidative stress has been observed to contribute the development of insulin resistance. Oxidative stress is known to increase the conversion of deoxyguanosine (dG) to 8-hydroxy-2'-deoxyguanosine (8-OHdG). Human 8-oxoguanine glycosylase (hOGG1) is the key component responsible for the removal of 8-OHdG from oxidatively damaged DNA. The repair activity of the hOGG1 Ser326Cys gene variant has been demonstrated to be lower than that of the hOGG1 Ser/Ser genotype. Therefore, the possible association of the hOGG1 Ser326Cys gene variant with insulin sensitivity was investigated in 279 normal glucose-tolerant subjects without history of cancer. Allele frequency was 21.5% for the Ser/Ser genotype (n = 60), 45.9% for the Ser/Cys genotype (n = 128), and 32.6% for the Cys/Cys genotype (n = 91). Subjects carrying the Cys/Cys genotype had significantly lower insulin sensitivity levels, assessed by homeostasis model assessment-insulin resistance (HOMA-IR), compared with the Ser/Ser and Ser/Cys genotypes (P < 0.001 and P < 0.001, respectively). In a multiple linear regression analysis, the Cys/Cys genotype was a significant determinant of HOMA-IR, independent of age, sex, body mass index, fasting plasma cholesterol, triglyceride, HDL cholesterol, LDL cholesterol, or hypertension. The present study indicates that the hOGG1 gene Cys/Cys variant is associated with a significant decrease in insulin sensitivity in subjects with normal glucose tolerance.

Emphysematous cystitis, a rare complication of urinary tract infection in a male diabetic patient: a case report.

Emphysematous cystitis is a rare complication of urinary tract infection, characterized by spontaneous gas formation in the urinary bladder due to bacterial fermentation. Approximately 50 to 80% of patients with this disease are diabetic, and there is a higher incidence in females. We report a case of emphysematous cystitis in a diabetic male who was admitted under the impressions of hypoglycemia, acute bronchitis, and chronic renal failure. Treatment of the emphysematous cystitis consisted of adequate urinary drainage, empirical antibiotic therapy, and strict blood sugar control. The patient recovered satisfactorily after 9 days of hospitalization.