Geriatric oral and maxillofacial dysfunctions in the context of geriatric syndrome.

OBJECTIVES: To propose the application of the concept of geriatric syndrome for common geriatric oral and maxillofacial dysfunctions and to suggest the necessity of developing effective evaluation methods for oral and maxillofacial frailty. DESIGN: The concepts of frailty and geriatric syndrome based on multi-morbidity and polypharmacy were applied to five common geriatric oral medicinal dysfunctional problems: salivary gland hypofunction (dry mouth), chronic oral mucosal pain disorders (burning mouth symptoms), taste disorders (taste disturbances), swallowing disorders (dysphagia), and oral and maxillofacial movement disorders (oromandibular dyskinesia and dystonia). RESULTS: Each of the dysfunctions is caused by various kinds of diseases and/or conditions and medications, thus the concept of geriatric syndrome could be applied. These dysfunctions, suggested as components of oral and maxillofacial geriatric syndrome, are associated and interacted with each other in a complexity of vicious cycle. The resulting functional impairments caused by this syndrome can cause oral and maxillofacial frailty. CONCLUSIONS: Geriatric oral and maxillofacial dysfunctions could be better appreciated in the context of geriatric syndrome. The development of effective methods for evaluating the severity of these dysfunctions and the resulting frailty is essential.

Interferon-inducible T-cell alpha chemoattractant (ITAC) induces the melanocytic migration and hypopigmentation through destabilizing p53 via histone deacetylase 5: a possible role of ITAC in pigment-related disorders.

BACKGROUND: Cell migration plays a major role in the immune response and in tumorigenesis. Interferon-inducible T-cell alpha chemoattractant (ITAC) elicits a strong chemotactic response from immune cells. OBJECTIVES: To examine the effect of ITAC on melanocyte migration and pigmentation and its involvement in related disorders, and to investigate potential key players in these processes. METHODS: Human melanocytes or melanoma cells were treated with ITAC and a migration assay was carried out. Global gene expression analysis was performed to find genes regulated by ITAC treatment. The function of key players involved in ITAC-induced cellular processes was addressed using knockdown or overexpression experiments in combination with ITAC treatment. ITAC expression in the inflammation-associated hypopigmentary disorder, vitiligo, was examined. RESULTS: Among CXCR3 ligands, only ITAC induced melanocyte migration. ITAC treatment upregulated the expression of histone deacetylase 5 (HDAC5) and downregulated that of p53, a known target of HDAC5. Through knockdown or overexpression of HDAC5 and p53, we confirmed that HDAC5 mediates ITAC-induced migration by decreasing levels of p53 via deacetylation. In addition, ITAC treatment could decrease pigmentation in a p53- and HDAC5-dependent manner. Finally, the increased migration of human melanoma cells by ITAC treatment and the increased ITAC expression in the epidermis of vitiligo skin were verified. CONCLUSIONS: This study provides in vitro evidence for the migratory and hypopigmentation effects of ITAC on melanocytic cells, gives translational insights into the roles of ITAC in pathological conditions, and suggests that HDAC5 and its substrate p53 are potent targets for regulating ITAC-induced cellular processes.

Complete mitochondrial genome of the Amur hedgehog Erinaceus amurensis (Erinaceidae) and higher phylogeny of the family Erinaceidae.

We sequenced and characterized the complete mitogenome (KX964606) of the Amur hedgehog Erinaceus amurensis to provide more data for comparative mitogenomics of the genus Erinaceus (Erinaceidae). The mitogenome of E. amurensis is a circular molecule 16,941 bp long, consisting of a control region and a conserved set of 37 genes containing 13 protein-coding genes, 22 tRNA genes, and two rRNA genes (12S rRNA and 16S rRNA). The mitogenome of E. amurensis is AT-biased, with a nucleotide composition of 33.9% A, 21.1% C, 32.6% T, and 12.4% G. The mitogenomes of E. amurensis and the closely related hedgehog species E. europaeus, excluding the control region (66.7%), share over 90% sequence similarity. According to the inter-generic relationship based on six mitogenomes described from five genera of Erinaceidae, the subfamilies Erinaceinae and Galericinae are strongly supported as monophyletic groups, with each genus well placed within its own subfamily. Within the subfamily Erinaceinae, E. amurensis is a sister species to E. europaeus, and the relationship between Hemiechinus and Erinaceus is strongly supported. Within the subfamily Galericinae, the clade of Hylomys + Neotetracus was sister to that of Echinosorex, with clades supported by high values. Our findings will help to understand the codon usage pattern and molecular evolution of E. amurensis, and provide insight into inter-generic relationships within the family Erinaceidae. In future studies, the inclusion of mitogenomes from other genera would greatly enhance our understanding of higher phylogeny within the Erinaceidae.

Activities of daily living and oral hygiene status of older Korean patients in a long-term care facility.

OBJECTIVES: This study investigated the relationship between oral hygiene conditions, activities of daily living (ADL) and cognitive ability in older Korean patients in long-term care facilities. METHODS: Ninety older persons (65+) were randomly sampled from a possible 112 residents in a single facility. They participated in a 2-month-long survey. The Korean Modified Barthel Index was used to measure the ADL, and cognitive ability was measured using the Mini-Mental State Examination, Korean version. Oral hygiene status was measured using the Simplified Oral Hygiene Index and the Tongue Coating Index (TCI). RESULTS: Older participants with complete dependence manifested significantly poorer oral hygiene (P < 0.05). Scores on the TCI were significantly higher in participants who were dentulous with partial dependence (P < 0.05). A multiple regression analysis showed that dependence and being dentulous significantly predicted poorer oral hygiene (P < 0.05). CONCLUSIONS: This study suggests that older participants with complete dependence had poor oral hygiene on tooth surfaces, while participants with partial dependence had poor tongue hygiene. In addition, dentulous older participants had poorer tongue hygiene than edentulous ones. This indicates the need to assess tooth status and provide oral care services via ADL in long-term care facilities.

Inactivation of Bacillus cereus spores in a tsuyu sauce using continuous ohmic heating with five sequential elbow-type electrodes.

AIMS: The effect of ohmic heating (OH) in a pilot plant system which had a zig-zag shaped (elbow-type) ohmic heater with five sequential voltage electrodes was investigated on Bacillus cereus spores in a commercial tsuyu sauce. METHODS AND RESULTS: The electrical field was fixed at 26·7 V cm(-1) with an alternating current frequency of 25 kHz. Raw tsuyu sauce (50 l) inoculated with B. cereus spores was submitted in a 4 × 3 factorial design to the OH system and heated at 95, 105, 115, and 125°C each for 30, 60, and 90 s. Survival of B. cereus spores and colour change in the commercial tsuyu sauce were both measured before and after treatment. As the treatment temperature and time increased, the number of surviving B. cereus spores decreased. The OH treatment in a bath-type process at 105°C for more than 30 s resulted in the total inactivation of the inoculated B. cereus spores (average 5·4 log reductions to undetectable levels after treatment). The OH protocol of heating at 105°C for 60 s which ensure complete eradication of the inoculated spores without compromising product quality was chosen and investigated for its suitability for commercial application on bulk quantities of samples (500 l). Reliable and reproducible reductions in B. cereus spore counts of 4·7-5·5 log CFU ml(-1) (mean ± standard deviation = 5·1 ± 0·3 CFU ml(-1) ) were achieved by the selected protocol of the continuous OH treatment (105°C for 60 s). CONCLUSION: This study suggests that OH treatment with five sequential elbow-type electrodes has great potential as an industrial sterilizing method for liquid food contaminated with B. cereus spores. SIGNIFICANCE AND IMPACT OF THE STUDY: This procedure will enhance the microbiological quality of liquid foods while minimizing quality deterioration.

Destruction of Bacillus cereus spores in a thick soy bean paste (doenjang) by continuous ohmic heating with five sequential electrodes.

UNLABELLED: This study selected spores from Bacillus cereus FSP-2 strain (the isolate from a commercial doenjang processing line) as the test strain which showed significantly higher thermal resistance (P < 0·05) than B. cereus reference strain (ATCC 27348). The spores in doenjang were subjected to ohmic heating (OH) at 95, 105, 115 and 125°C for 30, 60 or 90 s using a five sequential electrode system (electrical field: 26·7 V cm(-1) ; alternating current frequency: 25 kHz). OH at 105°C for 30-90 s reduced the B. cereus spore count in doenjang samples to <4 log CFU g(-1) . Since OH treatment at 115 and 125°C caused a perceivable colour change in the product (>1·5 National Bureau of Standards units), treatment at 105°C for 60 s was selected and applied on a large scale (500 kg of product). Reliable and reproducible destruction of B. cereus spores occurred; the reductions achieved (to < 4 log CFU g(-1) ) met the Korean national standards. Scanning electron microscopy revealed microstructural alterations in the spores (shrinkage and a distorted outer spore coat). OH is an effective method for destroying B. cereus spores to ensure the microbiological quality and safety of a thick, highly viscous sauce. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that an ohmic heating (OH) using a five sequential electrode system can effectively destroy highly heat-resistant Bacillus cereus spores which have been frequently found in a commercial doenjang processing line without perceivable quality change in the product. In addition, it may demonstrate high potential of the unique OH system used in this study that will further contribute to ensure microbiological quality and safety of crude sauces containing high levels of electrolyte other than doenjang as well.

Effects of low-fat milk consumption on metabolic and atherogenic biomarkers in Korean adults with the metabolic syndrome: a randomised controlled trial.

BACKGROUND: Previous studies of the health effects of low-fat milk or dairy consumption on the metabolic syndrome have yielded inconsistent results. The present study aimed to investigate the effects of low-fat milk consumption on traits associated with the metabolic syndrome, as well as inflammatory and atherogenic biomarkers, in Korean adults with the metabolic syndrome. METHODS: Overweight Koreans with the metabolic syndrome (n = 58) were recruited and randomly assigned to either the low-fat milk or control group. The low-fat milk group was instructed to consume two packs of low-fat milk per day (200 mL twice daily) for 6 weeks, and the control group was instructed to maintain their habitual diet. Clinical investigations were conducted during the screening visit, on study day 0, and after 6 weeks. RESULTS: No significant differences in changes in body mass index, blood pressure, lipid profile and adiponectin levels, as well as levels of inflammatory markers, oxidative stress markers and atherogenic markers, were found between the low-fat milk and control groups. However, compared to the controls, significant favourable decreases in serum soluble vascular adhesion molecule-1 and endothelin-1 levels were found in the 12 subjects with high blood pressure and in the 18 subjects with hypertriglyceridaemia in the low-fat milk group. CONCLUSIONS: The present study did not demonstrate an overall beneficial effect of low-fat milk consumption in subjects with the metabolic syndrome. However, low-fat milk consumption may have a favourable effect on atherogenic markers in subjects with high blood pressure or hypertriglyceridaemia.

Optimization of low-temperature blanching combined with calcium treatment to inactivate Escherichia coli O157:H7 on fresh-cut spinach.

AIMS: To develop a mild blanching method with calcium salts to ensure microbiological safety and quality of fresh-cut spinach. METHODS AND RESULTS: The antimicrobial efficacy of eight calcium salts was evaluated on Escherichia coli O157:H7 at 45-65°C and calcium hydroxide (Ca(OH)2 ) showed the greatest synergistic antimicrobial effect with heat. Combinations of low temperature treatments (45, 55, 65°C), time (20, 40, 60 s) and Ca(OH)2 (0·25, 0·50, 0·75%) were applied for E. coli O157:H7 disinfection on fresh-cut spinach to develop a predictive model using a Box-Behnken experimental design. A suitable quadratic model was produced (R(2) = 0·98, P < 0·001) and the optimum condition (64·9°C with 0·52% Ca(OH)2 for 42·4 s) was drawn by reducing 6·6 log CFU g(-1) of E. coli O157:H7 on fresh-cut spinach. Blanching at 61·9°C with 0·52% Ca(OH)2 for 41·7 s can inactivate E. coli O157:H7 on spinach by 5·4 log CFU g(-1) . The new method was comparable to the CDC recommendation for safe spinach cooking (71·1°C, 15 s; 5·0 log CFU g(-1) reduction) with lower levels of weight loss of the spinach (P < 0·05). CONCLUSIONS: This study suggests an efficient spinach blanching method for E. coli O157:H7 disinfection. SIGNIFICANCE AND IMPACT OF THE STUDY: This blanching method will enhance microbiological safety of fresh-cut produce while minimizing the use of energy and chemicals.

CD44+/CD24- Cancer Stem Cells Are Associated With Higher Grade of Canine Mammary Carcinomas.

The CD44+/CD24- phenotype identifies cancer stem cell (CSC) properties in canine mammary carcinoma (MC); however, the histopathological features associated with this phenotype remain to be elucidated. Here, we determined whether the CD44+/CD24- phenotype was associated with hormonal receptor (HR; estrogen receptor [ER] and/or progesterone receptor [PR]) status and/or triple (ER, PR, and human epithelial growth factor receptor 2)-negative (TN) subtype; conventional histological evaluation was also performed. We found that, as single markers, both CD44+ and CD24+ were associated with less aggressive histological types, low grade, and a non-TN subtype; both markers were associated with HR positivity. On the other hand, a CD44+/CD24- phenotype was associated with higher grade of carcinoma. Therefore, our results suggest that immunohistochemical phenotyping for CD44/CD24 is useful for the evaluation of tumor behavior as well as CSC-like properties in canine MCs.

Effects of Obesity and Obesity-Related Molecules on Canine Mammary Gland Tumors.

Obesity can affect the clinical course of a number of diseases, including breast cancer in women and mammary gland tumors in female dogs, via the secretion of various cytokines and hormones. The objective of this study was to examine the expression patterns of obesity-related molecules such as aromatase, leptin, and insulin-like growth factor 1 receptor (IGF-1 R) in canine mammary carcinomas (CMCs) on the basis of the body condition score (BCS). Comparative analyses of the expression of these molecules, together with prognostic factors for CMCs, including hormone receptors (HRs; estrogen and progesterone receptors), lymphatic invasion, central necrosis of the tumor, and histologic grade, were performed on 56 CMCs. The mean age of CMC onset was lower in the overweight or obese group (8.7 ± 1.9 years) than in the lean or ideal body weight group (10.4 ± 2.7 years). The proportion of poorly differentiated (grade III) tumors was significantly higher in the overweight or obese female dogs. Aromatase expression was significantly higher in the overweight or obese group and was correlated with the expression of HRs (P = .025). These findings suggest that overweight or obese status might affect the development and behavior of CMCs by tumor-adipocyte interactions and increased HR-related tumor growth.

Community periodontal treatment needs in South Korea.

OBJECTIVES: This study aimed to assess the relationship between socio-economic factors and community periodontal treatment needs in Korea. METHODS: Data were obtained from the year 2009 Korean National Health and Nutrition Examination Survey. Our analysis included 7510 survey participants over the age of 19 years. To assess the relationship between socio-economic factors and the need for periodontal scaling, we performed multivariate logistic regression analyses for data with a complex sampling structure. PASW statistics 19.0 (SPSS Inc., Chicago, IL, USA) was used to perform the statistical analyses, and the results were expressed as odds ratios (OR) with corresponding 95% confidence intervals (CIs). RESULTS: A very high percentage of Korean adults required periodontal scaling (71.5%). After adjusting for sex, age, and socio-economic factors, the need for periodontal scaling was associated with low levels of education (OR: 1.41, 95% CI: 1.03-1.93), low incomes (OR: 1.27, 95% CI: 1.01-1.60), employment as a service and sales worker (OR: 1.39, 95% CI: 1.10-1.77), and employment as a manual worker (OR: 1.31, 95% CI: 1.02-1.69). CONCLUSIONS: In South Korea, the need for periodontal scaling was associated with socio-economic factors, such as low levels of education, low incomes, employment as a service and sales worker and employment as a manual worker. Consequently, clinical and community dental hygienists should consider adults with these risk factors as belonging to high-priority subgroups to whom they should respond first.

Gender differences in chewing discomfort in older South Koreans.

AIMS: The purpose of this study was to identify gender differences in chewing discomfort among elderly Koreans. METHODS: This study used data from 56 616 (weighted sample: 5 638 394) subjects aged over 65 years who participated in the 2011 Community Health Survey in Korea. Of them, 23 059 (weighted sample: 2 368 200, 42.0%) were men and 33 357 (weighted sample: 3 270 194, 58.0%) were women. Data were analysed using chi-square tests and hierarchical logistic regression analyses, with SPSS 20.0. Chewing discomfort was set as the dependent variable, and independent variables were divided into socio-economic factors (place of residence, age, education, monthly household income, basic living security stipend, private insurance, economic activity, living arrangements), general health factors (hypertension, diabetes) and oral health factors (tooth defects, denture use, subjective periodontal health status). RESULTS: A greater proportion of women (50.2%) than men (42.6%) exhibited chewing discomfort (P < 0.001). In men, place of residence, monthly household income, private health insurance, tooth defects and periodontal health were associated with chewing discomfort (P < 0.05). In women, age, education level, basic living security stipend and denture use were associated with chewing discomfort (P < 0.05). CONCLUSIONS: Elderly Korean women experience more severe chewing discomfort than their male counterparts. The factors associated with chewing also differ by gender.

Enhanced bactericidal action of acidified sodium chlorite caused by the saturation of reactants.

AIMS: Factors affecting the antibacterial action of acidified sodium chlorite (ASC), a widely used disinfectant, have not been determined. This study investigated the significant factors suggesting efficient production method to maximize bactericidal action of ASC. METHODS AND RESULTS: The effects of (i) preparation procedures (total three methods); (ii) initial concentrations of reactants: sodium chlorite (SC) and citric acid (CTA) (up to maximum solubility of each reactant) and (iii) final pH values (3·0 and 2·5) to the bactericidal action of ASC were investigated with a fixed final concentration of SC (10 ppm) using various foodborne pathogens (Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium and Staphylococcus aureus). The antimicrobial compounds produced and the bactericidal effects depended on the preparation procedure and the initial concentrations of the reactants. The ASC prepared by premixing highly concentrated reactants (in particular > 40%) followed by dilution (dilution after reaction, DAR) was more effective in inactivating foodborne pathogens, and it produced higher antimicrobial compound (Cl(2) and ClO(2)) yields than the other procedures. A 5-min treatment with ASC, produced using the other procedures, resulted in a reduction of < 3·5 log CFU ml(-1) (Gram positive = 0·18-0·78; Gram negative = 0·03-3·49 log CFU ml(-1)), whereas ASC produced with the DAR procedure using the saturated reactants completely inactivated all of the test pathogens within 5 min without recovery (initial concentration = 6·94-7·08 log CFU ml(-1)). CONCLUSION: The ASC production with the DAR procedure using the saturated reactants maximizes both the antimicrobial compound yields and bactericidal effects of the ASC solutions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study will contribute to increase the efficiency of ASC treatments for disinfections reducing the effective SC concentrations for industrial use.

Analysis of a new histological and molecular-based classification of canine mammary neoplasia.

Canine mammary tumors (CMTs) are morphologically and biologically heterogeneous, prompting several attempts to classify such tumors on the basis of their histopathological characteristics. Recently, molecular-based analysis methods borrowed from human breast cancer research have also been applied to the classification of CMTs. In this study, canine mammary neoplasms (n = 648) occurring in Korea from 2008 to 2011 were analyzed according to the histological classification and grading system proposed by Goldschmidt et al. Furthermore, randomly selected mammary carcinomas (n = 159) were classified according to the molecular subtype using immunohistochemical characteristics. Canine mammary neoplasia accounted for 52.6% (648/1250) of the tumors in female dogs, and 51.7% (340/648) of these were malignant. All of the carcinoma-anaplastic subtypes were grade III tumors (5/5, 100%), while most of the carcinoma-tubular subtypes (15/18, 83.3%) and carcinoma arising in a complex adenoma/mixed-tumor subtype (115/135, 85.2%) were grade I tumors. Tumor cell invasion into lymphatic vessels was most common in the comedocarcinoma, carcinoma-anaplastic, and inflammatory carcinoma subtypes. The most frequently occurring molecular subtype (70/159, 44%) was luminal A. However, the basal-like subtype was the most malignant and was frequently associated with grade III tumors and lymphatic invasion. The carcinoma-solid subtypes were also often of the basal-like subtype. Reclassification of CMTs using the newly proposed histopathological classification system and molecular subtyping could aid in determining the prognosis and the most suitable anticancer treatment for each case.

Effects of growth differentiation factor 9 and bone morphogenetic protein 15 on the in vitro maturation of porcine oocytes.

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are members of the transforming growth factor-ß (TGF-ß) family, and their roles in oocyte maturation and cumulus expansion are well known in the mouse and human, but not in the pig. We investigated GDF9 and BMP15 expressions in porcine oocytes during in vitro maturation. A significant increase in the mRNA levels of GDF9 and BMP15 was observed at germinal vesicle breakdown, with expression levels peaking at metaphase I (MI), but decreasing at metaphase II (MII). GDF9 and BMP15 protein localized to the oocyte cytoplasm. While treatment with GDF9 and BMP15 increased the expression of genes involved in both oocyte maturation (c-mos, cyclinb1 and cdc2) and cumulus expansion (has2, ptgs2, ptx3 and tnfaip6), SB431542 (a TGFß-GDF9 inhibitor) decreased meiotic maturation at MII. Following parthenogenetic activation, the percentage of blastocysts in SB431542 treatment was lower than in the control (41.3% and 74.4%, respectively). Treatment with GDF9 and BMP15 also increased the mRNA levels of maternal genes such as c-mos [a regulatory subunit of mitogen-activated protein kinase (MAPK)], and cyclinb1 and cdc2 [regulatory subunits of maturation/M-phase-promoting factor (MPF)]; however, SB431542 significantly decreased their mRNA levels. These data were supported by poly (A)-test PCR and protein activity analyses. Our results show that GDF9 and BMP15 participate in cumulus expansion and that they stimulate MPF and MAPK activities in porcine oocytes during in vitro maturation.

Reduction in dental plaque in patients with mental disorders through the dental hygiene care programme.

OBJECTIVE: To develop a dental hygiene care programme based on the specific needs of patients with mental disorders and to suggest practical guidelines to improve the oral health care of these patients. METHODS: A total of 73 patients with mental illness participated in the study. The patients were randomly classified into three groups and followed over 12 weeks at 4-week intervals. A newly designed dental hygiene care programme using flash-based video, brochures and a toothpick method was implemented by five dental hygienists. Plaque index, stimulated saliva, subjective oral dryness and dental caries activity were analysed as outcome variables. RESULTS: Results showed that the dental plaque index significantly decreased after each session (P < 0.0001) in all three groups, and significant differences were found between groups (P = 0.036). Patients' oral dryness decreased significantly, but stimulated saliva and dental caries activity did not improve. CONCLUSION: The results of this study suggest that the dental hygiene care programme, which made use of a short, 10-min flash-based video and brochures every 4 weeks, was effective in reducing the dental plaque index of patients with mental disorders.

Short communication: retinoic acid plus prolactin to synergistically increase specific casein gene expression in MAC-T cells.

Mammary alveolar (MAC-T) cells, an established bovine mammary epithelial cell line, are frequently used to investigate differentiation. A lactogenic phenotype in these cells is induced by treatment with a combination of hydrocortisone, insulin, and prolactin (PRL). The effect of the vitamin A derivative retinoic acid (RA), which induces differentiation in many cells, has not been studied in MAC-T cells. The objective of this study was to evaluate the differentiation potential of RA (1 µM) in MAC-T cells and to examine the effect of combined treatment with RA (1 µM) and PRL (5 µg/mL). Although RA treatment alone inhibited MAC-T cell proliferation, co-treatment of RA with PRL increased cell growth compared with the control group (treated with 1 µg/mL hydrocortisone and 5 µg/mL insulin). The ratio of Bcl to Bax mRNA was decreased in the RA treatment compared with RA+PRL or control. Retinoic acid-induced differentiation of MAC-T cells was associated with an increase in the mRNA expression of αS1-casein (3.9-fold), αS2-casein (4.5-fold), and ß-casein (4.4-fold) compared with the control group. Expression of αS1-casein, αS2-casein, and ß-casein was increased 12.9-fold, 11.9-fold, and 19.3-fold, respectively, following treatment with RA and PRL combined compared with the control group. These results demonstrate that RA induces differentiation of MAC-T cells and acts synergistically with PRL to increase specific casein gene expression.

Infiltrating Foxp3+ regulatory T cells and histopathological features in canine classical and spermatocytic seminomas.

In humans, regulatory T (T reg) cells are known to play a critical role in both the regulation of immune homoeostasis and the progression of cancer. However, there is little information about the identification, characterization and the function of T reg cells in canine tumours. We identified T reg cells in 28 canine seminoma samples using a Forkhead box P3 (Foxp3) antibody and investigated the relationship between T reg cell infiltration and histopathological features of classical and spermatocytic seminomas (SE and SS, respectively). The Foxp3 protein showed nuclear immunostaining in infiltrating lymphocytes, and Foxp3+ cells were diffused or focally distributed in seminoma tissues. Foxp3+ cells were frequently present in the SS histotype, in seminomas that showed no evidence of tumour cell invasion into the vessels and in seminomas showing a diffuse growth pattern with three cell types. Neither the SE/SS histotype nor the histopathological features of the tumour correlated with Foxp3+ cell counts. These results indicate that Foxp3+ T reg cells may be associated with a less malignant histological phenotype or may not play a critical role in the immune response of canine seminomas. Moreover, Foxp3+ T reg cells may be associated with SS seminoma, but further studies, involving a larger number of samples, are required to better understand whether these cells play a critical role in the immune response in canine seminomas. This is the first report to demonstrate the characteristics of T reg cell infiltration in canine seminoma.

Identification of the DDAH2 protein in pig reproductive tract mucus: a putative oestrus detection marker.

Precisely detecting oestrus is important for artificial insemination. The aims of this study were to identify oestrus-specific sow mucus proteins to determine the optimal time for artificial insemination. The proestrous- and oestrous-stage mucus proteins were purified and analysed with proteomic tools such as two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight analyses. Among the differentially expressed proteins, the dimethylarginine dimethylaminohydrolase 2 (DDAH2) protein showed a 3.6-fold increase during the proestrous stage compared to that during the oestrous stage. A western immunoblot study revealed that two of three sow mucus samples clearly showed negative anti-DDAH2 antibody activity during the oestrous stage. This study demonstrated that the pig DDAH2 mucus protein exists during the proestrous stage, but not during the oestrous stage, suggesting that mucus DDAH2 could be useful as an oestrus detection marker.

Characterization of GFRα-1-positive and GFRα-1-negative spermatogonia in neonatal pig testis.

Type A spermatogonia, including spermatogonial stem cells, are primary cells that maintain spermatogenesis and produce spermatozoa. Many spermatogonial markers have been reported in rodents. However, few markers have been identified in pig spermatogonia. Despite the lack of information, it is necessary to separate pure spermatogonial cells from whole testicular cells to understand the mechanism of spermatogenic meiosis and to establish spermatogonial stem cells for further biotechnological studies. The purpose of this study was to identify glial cell-derived neurotrophic factor receptor alpha-1 (GFRα-1) as a surface marker for early spermatogonia in neonatal pig testes. Histological analysis of 3-day-old pig testes revealed that type A spermatogonia, which lack heterochromatin, could be distinguished in neonatal pig testes. Immunohistochemistry of neonatal pig testes with GFRα-1 antibody identified that some of the spermatogonial cells expressed GFRα-1 on the cell membrane. Co-immunostaining with both GFRα-1 and protein gene product 9.5 (PGP 9.5) detected PGP 9.5 in all spermatogonia of neonatal pig testes, whereas GFRα-1 was not detected on the surface of some PGP 9.5-positive cells, indicating that some of the spermatogonial cells were PGP 9.5 positive and GFRα-1 negative. After immunomagnetic cell sorting using a GFRα-1 antibody, both GFRα-1-positive and GFRα-1-negative cells expressed PGP 9.5. Identifying the differential mRNA expression of both GFRα-1-positive and GFRα-1-negative cells using reverse transcription-polymerase chain reaction analysis revealed the expression of promyelocytic leukaemia zinc finger, octamer-binding protein 4 and homeobox transcription factor in both cell types. These results suggest that GFRα-1-positive and GFRα-1-negative spermatogonia exist in PGP 9.5-positive spermatogonia during the early stage of pig testes spermatogenesis, and that GFRα-1 can be used for sorting PGP 9.5-expressing spermatogonia.