Blackcurrant extract promotes differentiation of MC3T3­E1 pre­osteoblasts.

Osteoporosis risk increases in menopausal individuals owing to the decrease in estrogen secretion. Blackcurrant extract (BCE) ameliorates osteoporosis; however, the underlying mechanisms are unclear. Furthermore, although BCE has phytoestrogenic activity, its effects on osteoblasts are unknown. In the present study, we investigated BCE-mediated attenuation of osteoporosis using mouse MC3T3-E1 pre-osteoblasts, with a focus on osteogenesis. After treating MC3T3-E1 cells with BCE for 48 h, cell proliferation was assessed using Cell Counting Kit-8. Levels of osteoblast differentiation markers, namely alkaline phosphatase activity and total collagen content in the cells, were evaluated after 3 and 14 days of BCE treatment, respectively. The expression of genes encoding osteoblast differentiation markers, including collagen type I (Col-I), alkaline phosphatase (Alp), bone γ-carboxyglutamate protein (Bglap), and runt-related transcription factor 2 (Runx2), was evaluated using reverse transcription-quantitative polymerase chain reaction. Mineralization of the cells was evaluated using Alizarin Red staining. Femoral tissues of ovariectomized (OVX) rats with or without 3% BCE were stained using ALP to evaluate osteogenic differentiation in femoral tissue. After treating MC3T3-E1 cells with BCE, cell proliferation had increased. BCE treatment increased Alp activity and total collagen content. Moreover, the expression of Col-I, Alp, Bglap, and Runx2 increased in BCE-treated cells. Furthermore, when MC3T3-E1 cells were treated with BCE for 21 days, the levels of calcified nodules increased. Alp staining intensity was stronger in the epiphyses on femoral tissue of OVX rats treated with 3% BCE than in those of untreated OVX rats. The results suggest that BCE may promote osteogenesis by inducing osteoblast differentiation.

Morphometrical Differences among Endometrial Endometrioid Carcinoma Grade 1, Grade 3, and Serous Carcinoma in Endometrial Liquid-Based Cytology Preparations.

INTRODUCTION: In Japan, the direct smearing preparation (conventional preparation) has been widely used for cytological examination of the endometrium. Problems with the conventional preparation can be dissolved by liquid-based cytology (LBC) preparation. The Yokohama System is a method for reporting endometrial cytology, but the system lumps cancers together and does not distinguish between histological types. The objective of this study was to clarify morphometrical differences among endometrial endometrioid carcinoma grade 1 (G1), grade 3 (G3), and serous carcinoma (Serous) by image analysis of endometrial LBC. METHODS: Using Papanicolaou smears prepared by LBC after sampling with a brush from 32 G1, 16 G3, and 16 Serous patients, image analysis was performed concerning the following 11 items: (1) number of layers of cluster, (2) area of cluster, (3) perimeter of cluster, (4) roundness of cluster, (5) complexity of cluster, (6) area of nucleus, (7) perimeter of nucleus, (8) roundness of nucleus, (9) complexity of nucleus, (10) area of nucleolus, and (11) nucleolus/nucleus (N/N) ratio. The data were statistically compared among G1, G3, and Serous. RESULTS: Significant differences were observed in the number of layers of cluster (G1G3G3, G1>Serous), complexity of cluster (G1Serous), and N/N ratio (G1>G3, G3

Effects of high-frequency near infrared laser irradiation on experimental tooth movement-induced pain in rats.

Discomfort and dull pain are known side effects of orthodontic treatment. Pain is expected to be reduced by near-infrared (NIR) lasers; however, the mechanism underlying effects of short-pulse NIR lasers in the oral and maxillofacial area remains unclear. This study aimed to examine the effects of high-frequency NIR diode laser irradiation on pain during experimental tooth movement (ETM) on 120 J. NIR laser with 910 nm wavelength, 45 W maximum output power, 300 mW average output power, and 200 ns pulse width (Lumix 2; (Lumix 2; Fisioline, Verduno CN, Italy) was used for the experiment. A nickel-titanium-closed coil was used to apply a 50-gf force between the maxillary left-side first molar and incisor in 7-week-old Sprague-Dawley rats (280-300 g) to induce ETM. We measured facial-grooming frequency and vacuous chewing movement (VCM) period between laser-irradiation and ETM groups. We performed immunofluorescent histochemistry analysis to quantify levels of Iba-1, astrocytes, and c-fos protein-like immunoreactivity (Fos-IR) in the trigeminal spinal nucleus caudalis (Vc). Compared with the ETM group, the laser irradiation group had significantly decreased facial-grooming frequency (P = 0.0036), VCM period (P = 0.043), Fos-IR (P = 0.0028), Iba-1 levels (P = 0.0069), and glial fibrillary acidic protein (GFAP) levels (P = 0.0071). High-frequency NIR diode laser irradiation appears to have significant analgesic effects on ETM-induced pain, which involve inhibiting neuronal activity, microglia, and astrocytes, and it inhibits c-fos, Iba-1, and GFAP expression, reducing ETM-induced pain in rats. High-frequency NIR diode laser application could be applied to reduce pain during orthodontic tooth movement.

Exosomal MicroRNA as Biomarkers for Diagnosing or Monitoring the Progression of Ovarian Clear Cell Carcinoma: A Pilot Study.

Ovarian cancer is the most common cause of gynecological malignancy-related mortality since early-stage disease is difficult to diagnose. Advanced clear cell carcinoma of the ovary (CCCO) has dismal prognosis, and its incidence has been increasing in Japan, emphasizing the need for highly sensitive diagnostic and prognostic CCCO biomarkers. Exosomal microRNAs (miRNAs) secreted by tumor cells are known to play a role in carcinogenesis; however, their involvement in ovarian cancer is unclear. In this study, we performed expression profiling of miRNAs from exosomes released by five cell lines representing different histological types of ovarian cancer. Exosomes isolated from culture media of cancer and normal cells were compared for miRNA composition using human miRNA microarray. We detected 143 exosomal miRNAs, whose expression was ≥1.5-fold higher in ovarian cancer cells than in the control. Among them, 28 miRNAs were upregulated in cells of all histological ovarian cancer types compared to control, and three were upregulated in CCCO cells compared to other types. Functional analyses indicated that miR-21 overexpressed in CCCO cells targeted tumor suppressor genes PTEN, TPM1, PDCD4, and MASP1. The identified miRNAs could represent novel candidate biomarkers to diagnose or monitor progression of ovarian cancer, particularly CCCO.

Usefulness of Fractal Analysis of Kirsch Edge Images for the Tissue Fragment Inner Structure in Breast FNAB.

OBJECTIVE: Recent advances in high-precision mammography and ultrasound screening have led to an increase in the detection of early lesions (ductal carcinoma in situ and small cancers) appearing as microcalcified lesions or microcystic images, and there needs to be an improvement in the accuracy of breast fine-needle aspiration biopsy (FNAB) assessing these lesions. The objective of this study was to investigate whether fractal analysis of Kirsch edge images for the tissue fragment inner structure (FKT) is useful in breast FNAB. FKT measures tissue fragment chromasia of hyperchromatic crowded tissue fragments (HCG), tissue fragment shape unevenness, and tissue fragment inner structure complexity. Study Design Materials: Nineteen epithelial tissue fragments of fibroadenoma (FA) from 7 patients and 52 tissue fragments of invasive breast carcinoma of no special type (IBC-NST) (grade 1-2) from 11 patients were assessed. First, tissue fragments were classified into small (smaller than 60 × 102 µm2), medium, and large (100 × 102 µm2 or larger), and the appearance rate of each size was determined. Second, for FKT, the luminance value of tissue fragment chromasia, the unevenness and fractal value, and the tissue fragment inner structure complexity were determined. In statistical analysis, the Steel-Dwass test, nonlinear discriminant analysis, and receiver operating characteristic analysis were performed, setting the significance level at p < 0.05. RESULTS: "Unevenness of the tissue fragment shape," "fractal value of the tissue fragment shape," and "fractal value of the tissue fragment inner structure" were significantly higher in small and large tissue fragments in IBC-NST compared with those in FA. The specificity and sensitivity were the highest (100%) in small tissue fragments in multivariate analysis using 4 variables ("luminance value of tissue fragment chromasia," "unevenness of tissue fragment shape," "fractal value of the tissue fragment shape," and "fractal value of the tissue fragment inner structure"). CONCLUSION: FKT, which evaluates "tissue fragment darkness," "tissue fragment shape unevenness," and "tissue fragment inner structure complexity" focusing on small tissue fragments of HCG in breast FNAB, is useful as a system that assists cytopathological assessment of breast FNAB.

Potential Vasculoprotective Effects of Blackcurrant (Ribes nigrum) Extract in Diabetic KK-Ay Mice.

Polyphenols are bioactive compounds found naturally in fruits and vegetables; they are widely used in disease prevention and health maintenance. Polyphenol-rich blackcurrant extract (BCE) exerts beneficial effects on vascular health in menopausal model animals. However, the vasculoprotective effects in diabetes mellitus (DM) and atherosclerotic vascular disease secondary to DM are unknown. Therefore, we investigated whether BCE is effective in preventing atherosclerosis using KK-Ay mice as a diabetes model. The mice were divided into three groups and fed a high-fat diet supplemented with 1% BCE (BCE1), 3% BCE (BCE2), or Control for 9 weeks. The mice in the BCE2 group showed a considerable reduction in the disturbance of elastic lamina, foam cell formation, and vascular remodeling compared to those in the BCE1 and Control groups. Immunohistochemical staining indicated that the score of endothelial nitric oxide synthase staining intensity was significantly higher in both BCE2 (2.9) and BCE1 (1.9) compared to that in the Control (1.1). Furthermore, the score for the percentage of alpha-smooth muscle actin was significantly lower in the BCE2 (2.9%) than in the Control (2.1%). Our results suggest that the intake of anthocyanin-rich BCE could have beneficial effects on the blood vessels of diabetic patients.

Hypocholesterolemic Effect of Blackcurrant (Ribes nigrum) Extract in Healthy Female Subjects: A Pilot Study.

Blackcurrant extract (BCE) ameliorates dyslipidemia in menopausal model animals and in elderly women at a risk of dyslipidemia. However, it is unknown whether the daily intake of BCE can prevent lipid abnormalities in healthy individuals. Lipids are essential for the body, but they also cause arteriosclerosis. In this noncomparative pilot study, we examined the effects of BCE administered for 29 days on serum lipids in young healthy women. Blood samples were collected before and on days 4 and 29 after BCE intake, and 20 lipoprotein fractions in the serum were separated using a gel-permeation high-performance liquid chromatography method to measure the triacylglycerol and cholesterol levels in lipoproteins. There were no effects on lipids on day 4 of BCE intake, but the total cholesterol level decreased on day 29. Furthermore, the levels of total very-low-density lipoprotein (VLDL) cholesterol, small VLDL cholesterol, and large low-density lipoprotein cholesterol were significantly decreased. These results suggest that the daily intake of BCE has a hypocholesterolemic effect in healthy women, and that it is effective in preventing atherosclerosis.

Morphological Differences between Liquid-Based Cytology and Conventional Preparation in Endometrial Endometrioid Carcinoma Grade 1 and Grade 3, and the Differentiation of Grades in Each Method.

INTRODUCTION: Direct smearing preparation (conventional preparation [CP]) has been widely used for endometrial cytology in Japan. In CP, sampling and screening errors are problematic. In liquid-based cytology preparation (LBC), the problems of CP can be solved. But there is a problem that cytological findings of LBC are different from those of CP. The purpose of this study was to evaluate the differences of morphological findings of endometrial cytology between LBC and CP, and the usefulness of the endometrial LBC to differentiate endometrioid carcinoma grade 1 (G1) from grade 3 (G3). METHODS: Thirteen cases of endometrioid carcinoma G1, and 5 cases of G3 collected by the Softcyte device and prepared by LBC and CP (split specimen) were used. We focused on the following items: (1) the number of clusters per cm2, (2) the number of layers of clusters, (3) area of clusters, (4) perimeter of clusters, (5) roundness of clusters, (6) complexity of clusters, (7) area of nucleus, (8) perimeter of nucleus, (9) roundness of nucleus, (10) complexity of nucleus, (11) area of nucleolus, and (12) nucleolus-nucleus ratio (N/N). RESULTS: Compared with CP, the number of clusters and layers of the clusters in LBC were significantly larger in G1. The area and perimeters of the clusters and the nucleus were significant smaller, and the N/N ratio was greater in LBC than that in CP in both G1 and G3. Regarding morphological differences between G1 and G3 in LBC and CP, the number of layers was significantly larger in G1 than in G3 in LBC and CP. The area of the clusters in LBC was significantly larger in G1 than in G3. The area and perimeters of the nucleus in CP and the area of the nucleolus and N/N ratio in LBC and CP were significantly smaller in G1 than in G3. CONCLUSION: In the endometrial cytology, it became clear that the cell image was different between LBC and CP and between G1 and G3. By microscopic examination understanding the characteristics of the cell image in LBC, endometrial LBC could be useful to diagnose endometrial carcinoma.

Blackcurrant (Ribes nigrum L.) Extract Exerts Potential Vasculoprotective Effects in Ovariectomized Rats, Including Prevention of Elastin Degradation and Pathological Vascular Remodeling.

Estrogen exerts cardioprotective effects in menopausal women. Phytoestrogens are plant-derived substances exhibiting estrogenic activity that could beneficially affect vascular health. We previously demonstrated that blackcurrant (Ribes nigrum L.) extract (BCE) treatment exerted beneficial effects on vascular health via phytoestrogenic activity in ovariectomized (OVX) rats, which are widely used as menopausal animal models. Here, we examined whether BCE treatment reduced elastin degradation and prevented pathological vascular remodeling in OVX rats fed a regular diet (OVX Control) or a 3% BCE-supplemented diet (OVX BCE), compared with sham surgery rats fed a regular diet (Sham) for 3 months. The results indicated a lower staining intensity of elastic fibers, greater elastin fragmentation, and higher α-smooth muscle actin protein expression in OVX Control rats than in OVX BCE and Sham rats. Pathological vascular remodeling was only observed in OVX Control rats. Additionally, we investigated matrix metalloproteinase (MMP)-12 mRNA expression levels to elucidate the mechanism underlying elastin degradation, revealing significantly upregulated MMP-12 mRNA expression in OVX Control rats compared with that in Sham and OVX BCE rats. Together, we identify BCE as exerting a vascular protective effect through reduced MMP-12 expression and vascular smooth muscle cell proliferation. To our knowledge, this is the first report indicating that BCE might protect against elastin degradation and pathological vascular remodeling during menopause.

Fractal Analysis Method for the Complexity of Cell Cluster Staining on Breast FNAB.

OBJECTIVE: Because of the increased precision of ultrasound breast cancer screening, early cancer cases with no clear mass or extraction of microcysts on imaging have recently increased, and improvement of the accuracy of breast fine-needle aspiration biopsy (FNAB) cytology is needed. The objective of this study was to investigate the usefulness of cluster gray image-fractal analysis evaluating the darkness of clusters, cluster unevenness, and complexity of hyperchromicity (cluster density) of deep-stained cell clusters, known as hyperchromatic crowded cell groups (HCG), on FNAB as a cytology assistance system for breast FNAB. STUDY DESIGN: One hundred clusters collected from 10 patients with fibroadenoma (FA), 90 clusters from 9 patients with ductal carcinoma in situ (DCIS), and 122 clusters from 11 patients with invasive breast carcinoma of no special type (IBC-NST) were used. (1) Cluster size classification: clusters were classified into small, middle, and large clusters (small cluster: smaller than 40 × 102 µm2; large cluster: 100 × 102 µm2 or larger; middle cluster: intermediate), and their frequency was calculated. (2) Cluster gray image-fractal analysis: (a) the darkness of clusters (luminance), (b) cluster unevenness (complexity), and (c) complexity of cluster density (roundness-corrected fractal value) were assessed. For statistical analysis, the multiple comparison Steel-Dwass test was used, with a significance level of p < 0.05. RESULTS: (1) Cluster size classification: in FA, small, middle, and large clusters appeared at a similar frequency, and the frequency (30%) of large clusters was significantly higher than that in other diseases. In IBC-NST, many small clusters (61%) appeared and their frequency was significantly higher than that in other diseases, whereas the frequency of large clusters was significantly lower. (2) Cluster gray image-fractal analysis: in IBC-NST, the luminance of small clusters was low (dark), the cluster unevenness was high, and the complexity of cluster density was high, whereas the luminance of large clusters was high (bright), the cluster unevenness was high, and complexity of cluster density was high compared with those in FA. CONCLUSION: Cluster gray image-fractal analysis evaluating the darkness of clusters, cluster unevenness, and complexity of cluster density in breast FNAB HCG is a useful cytology assistance system for breast FNA.

Outer Cutoff Value for the Box-Counting Method for Fractal Analysis of the Nucleus Using Kirsch Edge Detection.

OBJECTIVE: The complexity of chromatin (i.e., irregular geometry and distribution) is one of the important factors considered in the cytological diagnosis of cancer. Fractal analysis with Kirsch edge detection is a known technique to detect irregular geometry and distribution in an image. We examined the outer cutoff value for the box-counting (BC) method for fractal analysis of the complexity of chromatin using Kirsch edge detection. MATERIALS: The following images were used for the analysis: (1) image of the nucleus for Kirsch edge detection measuring 97 × 122 pix (10.7 × 13.4 µm) with a Feret diameter of chromatin mesh (n = 50) measuring 17.3 ± 1.8 pix (1.9 ± 0.5 µm) and chromatin network distance (n = 50) measuring 4.4 ± 1.6 pix (0.49 ± 0.18 µm), and (2) sample images for Kirsch edge detection with varying diameters (10.4, 15.9, and 18.1 µm) and network width of 0.4 µm. METHODS: Three types of bias that can affect the outcomes of fractal analysis in cytological diagnosis were defined. (1) Nuclear position bias: images of 9 different positions generated by shifting the original position of the nucleus in the middle of a 256 × 256 pix (28.1 µm) square frame in 8 compass directions. (2) Nuclear rotation bias: images of 8 different rotations obtained by rotating the original position of the nucleus in 45° increments (0°, 45°, 90°, 135°, 180°, 225°, 270°, and 315°). (3) Nuclear size bias: images of varying size (diameter: 190 pix [10.4 µm], 290 pix [15.9 µm], and 330 pix [18.1 µm]) with the same mesh pattern (network width: 8 pix [0.4 µm]) within a 512 × 512 pix square. Different outer cutoff values for the BC method (256, 128, 64, 32, 16, and 8 pix) were applied for each bias to assess the fractal dimension and to compare the coefficient of variation (CV). RESULTS: The BC method with the outer cutoff value of 32 pix resulted in the least variation of fractal dimension. Specifically, with the cutoff value of 32 pix, the CV of nuclear position bias, nuclear rotation bias, and nuclear size bias were <1% (0.1, 0.4, and 0.3%, respectively), with no significant difference between the position and rotation bias (p = 0.19). Our study suggests that the BC method with the outer cutoff value of 32 pix is suitable for the analysis of the complexity of chromatin with chromatin mesh.

Blackcurrant (Ribes nigrum) Extract Prevents Dyslipidemia and Hepatic Steatosis in Ovariectomized Rats.

Estrogen is involved in lipid metabolism. Menopausal women with low estrogen secretion usually gain weight and develop steatosis associated with abnormal lipid metabolism. A previous study showed that blackcurrant (Ribes nigrum L.) extract (BCE) had phytoestrogen activity. In this study, we examined whether BCE improved lipid metabolism abnormalities and reduced liver steatosis in ovariectomized rats, as a menopausal animal model. Twelve-week-old ovariectomized (OVX) rats were fed a regular diet (Ctrl) or a 3% BCE supplemented diet while sham rats were fed a regular diet for three months. Body weight, visceral fat weight, levels of serum triglycerides, total cholesterol, and LDL cholesterol decreased in the BCE-treated OVX and sham rats, but not in OVX Ctrl rats. The results of hematoxylin and eosin staining revealed that BCE decreased the diameters of adipocytes and the nonalcoholic fatty liver disease activity score. Furthermore, quantitative RTPCR indicated a decreased expression of hepatitis-related genes, such as tumor necrosis factor-α, IL-6, and IL-1ß in OVX rats after BCE treatment. This is the first study that reported improvement of lipid metabolism abnormalities in OVX rats by BCE administration. These results suggest that the intake of BCE alleviated dyslipidemia and prevented nonalcoholic steatohepatitis during menopause in this animal model.

Baicalin inhibits root resorption during tooth movement in a rodent model.

OBJECTIVE: Baicalin mediates bone metabolism and has shown protective activity against periodontal tissue damage in a rat model of periodontitis. Therefore, we hypothesized that baicalin may inhibit the root resorption that occurs during orthodontic tooth movement and examined its effect on the histological changes in periodontal tissue that occur during tooth movement. METHODS: First molars of rats were subjected to traction using excessive orthodontic force to produce a root resorption model. Rats in the baicalin group received baicalin for 3 weeks during tooth movement, and the amount of first molar movement on day 21 after the initiation of traction was measured by three-dimensional micro-computed tomography analysis. After tooth movement, tissue samples from the mesial and tension sides were collected, and successive horizontal sections were prepared and examined using hematoxylin-eosin and tartrate-resistant acid phosphatase (TRAP) staining and immunohistochemical staining for the receptor activator of NF-kB ligand (RANKL) and osteoprotegerin (OPG). The severity of root resorption was also determined by histological analysis. RESULTS: There was no significant intergroup difference in tooth movement during the experimental exaggerated tooth movement. In comparison with the control group, the baicalin-treated group showed increased OPG expression, suppressed RANKL expression, and significantly fewer TRAP-positive cells in the first molars. The root resorption area was significantly smaller in the baicalin group. CONCLUSIONS: Treatment with baicalin prevented root resorption without preventing tooth movement. Baicalin may be useful for the management of root resorption during orthodontic treatment.

Expression of CA2 and CA9 carbonic anhydrases in ulcerative colitis and ulcerative colitis-associated colorectal cancer.

Ulcerative colitis (UC) is characterized by chronic inflammation in the colonic mucosa and submucosa with repeating relapse and remission, but the pathogenesis is unknown. Patients with long-standing UC are at high risk of neoplasm development. The aim of the present study was to identify molecules whose expression is associated with UC and UC-associated colorectal cancer (UCCA). Biopsy specimens from UC and normal colonic mucosae were analyzed using a proteomics approach, in which carbonic anhydrase 2 (CA2) was identified as a molecule downregulated in UC mucosae. Immunohistochemically, CA2 expression was detected in normal and diverticulitis mucosal epithelia, and its expression decreased as UC activity increased. CA2 expression was almost undetectable in UCCA. We also analyzed the expression of another carbonic anhydrase, CA9, and its upstream molecule, hypoxia-inducible factor-1α (HIF-1α), both of which are induced under hypoxic conditions. It was revealed that CA9 expression was relatively low in normal, diverticulitis and UC mucosae, and was upregulated in UCCA. HIF-1α expression was consistently low in all tissue types examined. In conclusion, these results suggest that CA2 and CA9 may be possible indicators of UC activity and UCCA development, respectively.

Bmi-1 Immunohistochemical Expression in Endometrial Carcinoma is Correlated with Prognostic Activity.

Background and objectives: B-lymphoma Mo-MLV insertion region 1 (Bmi-1) is a stem cell factor that is overexpressed in various human cancer tissues. It has been implicated in cancer cell proliferation, cell invasion, distant metastasis, and chemosensitivity, and is associated with patient survival. Several reports have also identified Bmi-1 protein overexpression in endometrial carcinoma; however, the relationship between Bmi-1 expression and its significance as a clinicopathological parameter is still insufficiently understood. Accordingly, the present study aimed to clarify whether immunohistochemical staining for Bmi-1 in human endometrial carcinoma and normal endometrial tissues can be used as a prognostic and cell proliferation marker. Materials and Methods: Bmi-1 expression was assessed in endometrioid carcinoma (grade 1-3) and normal endometrial tissues (in the proliferative and secretory phases) by immunohistochemistry; protein expression was evaluated using the nuclear labeling index (%) in the hot spot. Furthermore, we examined other independent prognostic and proliferation markers, including the protein levels of Ki-67, p53, and cyclin A utilizing semi-serial sections of endometrial carcinoma tissues. Results: The expression of the Bmi-1 protein was significantly higher in all grades of endometrial carcinoma than in the secretory phase of normal tissues. Moreover, Bmi-1 levels tended to be higher in G2 and G3 tissues than in G1 tissue, without reaching significance. Bmi-1 expression showed no notable differences among International Federation of Gynecology and Obstetrics (FIGO) stages in endometrial carcinoma. Furthermore, we observed a significant positive relationship between Bmi-1 and Ki-67, cyclin A, or p53 by Spearman's rank correlation test, implying that high Bmi-1 expression can be an independent prognostic marker in endometrial carcinoma. Conclusions: Our study suggests that Bmi-1 levels in endometrial carcinoma tissues may be useful as a reliable proliferation and prognostic biomarker. Recently, the promise of anti-Bmi-1 strategies for the treatment of endometrial carcinoma has been detected. Our results provide fundamental data regarding this anti-Bmi-1 strategy.

Effects of Blackcurrant Anthocyanin on Endothelial Function and Peripheral Temperature in Young Smokers.

BACKGROUND: Blackcurrant anthocyanin (BCA) is expected to repair endothelial dysfunction, but it remains unclear whether beneficial effects are present in young healthy persons. This study examines whether supplements containing blackcurrant anthocyanin improve endothelial function and peripheral temperature in young smokers. METHODS: Young, healthy male nonsmokers (N group: n = 11; mean age 22 ± 2 years) and smokers (S group: n = 13; mean age 21 ± 1 years) were enrolled. A randomized and double-blind trial was designed to compare the effects of no supplement, a supplement containing 50 mg of blackcurrant anthocyanin (supplement A), and a supplement containing 50 mg of blackcurrant anthocyanin plus vitamin E (supplement B) on flow-mediated dilatation (FMD) and skin temperature. RESULTS: Under no supplement, FMD was unchanged during the 2 h period after smoking in the N group, whereas it was decreased during the 2 h period after smoking in the S group. Under the A supplement, FMD was decreased 1 h after smoking and returned to the baseline level 2 h after smoking in the S group. The skin temperature in the area of the foot dorsum was decreased in the S group after smoking compared with that in the N group, who did not smoke, whereas under A and B supplements, it was higher in the S group compared with that in the N group. CONCLUSIONS: BCA could attenuate the smoking-induced acute endothelial dysfunction and improve peripheral temperature in young smokers.

Phytoestrogenic Effects of Blackcurrant Anthocyanins Increased Endothelial Nitric Oxide Synthase (eNOS) Expression in Human Endothelial Cells and Ovariectomized Rats.

Phytoestrogens are plant-derived chemicals that are found in many foods and have estrogenic activity. We previously showed that blackcurrant extract (BCE) and anthocyanins have phytoestrogenic activity mediated via estrogen receptors (ERs), and anthocyanins may improve vascular function. BCE contains high levels of anthocyanins, but their health-promoting effects are unclear. This study examined the effects of BCE on the regulation of endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) synthesis in human endothelial cells as key regulators in cardiovascular disease. The results showed that eNOS mRNA levels were significantly upregulated in BCE- or anthocyanin-treated human vascular endothelial cells but decreased in cells treated with fulvestrant, an ER antagonist. These results corresponded with NO levels, suggesting that BCE and anthocyanin may regulate NO synthesis via eNOS expression. Thus, the phytoestrogenic effects exerted by BCE via ERs influenced eNOS mRNA expression and NO synthesis. In vivo, we investigated whether anthocyanin-rich BCE upregulated eNOS protein expression in ovariectomized (OVX) rats, a widely used animal model of menopause. Our results showed that anthocyanin-rich BCE significantly upregulated eNOS mRNA levels and NO synthesis through phytoestrogenic activity and therefore promoted blood vessel health in OVX rats as a postmenopausal model.

Blackcurrant Extract with Phytoestrogen Activity Alleviates Hair Loss in Ovariectomized Rats.

Ancocyanin-rich blackcurrant extract (BCE) has phytoestrogen activity; however, its effect on hair follicles is unknown. Additionally, hair loss is known to occur during menopause in women owing to decreased estrogen secretion. This study examined whether BCE alleviated female pattern hair loss using a rat model. RNA was extracted and analyzed using a microarray and ingenuity pathway analysis. A quantitative polymerase chain reaction revealed that 1 µg/mL BCE altered many genes downstream of beta-estradiol in human hair dermal papilla cells. Additionally, the expression of the hair follicle stem cell marker keratin 19 was greatly enhanced. In a menopause model, ovariectomized rats were fed a diet containing 3% BCE for three months. An analysis of the number of hair shafts revealed that BCE increased the number of hairs by 0.5 hairs/follicular unit. Moreover, immunostaining revealed that the expression of Ki67 also increased by 19%. Furthermore, fluorescent immunostaining showed that the expression of other stem cell markers, including keratin 15, CD34, and keratin 19, was induced in rat hair follicular cells. In conclusion, these findings suggest that BCE has phytoestrogen activity in hair follicles and contributes to the alleviation of hair loss in a menopausal model in rats.

Cyclin A is a reliable proliferation marker in endometrial cancer cell lines.

Cyclin A, a cell cycle regulatory protein, promotes cell proliferation and has been observed to be highly expressed in cancer and to promote tumor growth; however, its value as a marker for endometrial carcinoma has not yet been established. Accordingly, the aim of the present study was to clarify whether cyclin A can be used as a cell proliferation marker using the endometrial carcinoma cell lines Ishikawa and HEC-50B, derived from patients with low-grade and high-grade cancer, respectively. The expression of cyclin A was determined by flow cytometry using double staining with FITC and 7-AAD, and immunocytochemical staining. The results were compared to those of Ki-67, the widely used cell proliferation marker that is considered to be a prognostic marker in endometrial cancer. The flow cytometry results revealed that cyclin A expression was significantly higher in HEC-50B than in Ishikawa cells during the logarithmic growth phase. In addition, cyclin A expression was consistently higher than Ki-67 expression in the examined cell lines. Immunocytochemical staining confirmed cyclin A expression in HEC-50B and Ishikawa cells, demonstrating significantly higher expression during the logarithmic growth phase than during the stationary phase. By contrast, Ki-67 was expressed in almost 90% of the cells, irrespective of their growth state. These results indicate that cyclin A expression is significantly increased in cells with higher proliferative ability and is specifically expressed in cells that have passed the G1-S checkpoint. Therefore, cyclin A may be a reliable proliferation biomarker for endometrioid carcinoma.

Usefulness of Cytological Scoring Method by Breast Fine Needle Aspiration Cytology on Breast Duct Dilatation and Cystic Lesions.

AIM: The objective of this study was to apply a scoring method to fine needle aspiration cytology on breast duct dilatation and cystic lesions, to set an optimum cut-off value to differentiate between benign and malignant cases, and to identify features useful for cell judgment. MATERIALS AND METHODS: Samples were 23 preparations of specimens (12 benign and 11 malignant cases) suspected with intraductal lesions or cystic change by ultrasonography or mammography and cytology. The scoring system comprised the following 10 items, and each item was scored 1-3, with a total score of 10-30. Three items were concerning structural atypia: 1, scattered epithelial cells; 2, uneven irregular cluster edge; and 3, overlapping nuclei of epithelial cells, and seven items were concerning cellular atypia: 4, irregular nuclear size; 5, irregular nuclear morphology; 6, deep dyeing chromatin; 7, chromatin granularity; 8, chromatin distribution; 9, nucleolus; and 10, absence of myoepithelial cells. RESULTS: (1) Scoring cut-off value: malignancy is to be suspected when the score is 20.75 or higher (diagnostic accuracy: 95.7%). (2) Findings useful for cancer judgment: the sensitivity of the following four findings was high: uneven irregular cluster edge, irregular nuclear overlapping, chromatin granularity, and absence of myoepithelial cells. (3) Correlation among the findings: the findings correlated with malignancy were as follows: scattered epithelial cells versus uneven irregular cluster edge (rs = 0.8). CONCLUSION: Cytological evaluation by scoring lesions accompanied by intraductal dilatation and cystic change was a useful method capable of differentiating between benign and malignant cases at a high accuracy.