The effects of C-phycocyanin (C-pc), a phycobiliprotein, on the expression of pro-fibrotic mediators in hyper-tropic scarring such as connective tissue growth factor (CTGF) and α-smooth muscle actins (α-SMA) were investigated in relation to trans-differentiation of fibroblast to myo-fibroblast, an icon of scar formation. C-pc was isolated from Spirulina Platensis extract using sonication method and C-pc concentration was determined by Bennet and Bogorad equation. α-SMA and CTGF levels in wounded primary human dermal fibroblasts were determined by western blot analysis and immuno-fluorescence confocal microscope was employed. Fibroblast contractility was examined by three-dimensional collagen lattice contraction assay. There was an elevation of α-SMA (121%) and CTGF (143%) levels in wound cells as compared with non-wound cells. The does-response profiles of down regulation demonstrated that the maximum inhibitions of α-SMA by 63% (p<0.05) and CTGF by 50% (p<0.1) were achieved by C-pc (6 nM) treated cells. In confocal assay, non-wound fibroblasts exhibited basal level of α-SMA staining, while wounded cells without C-pc treatment showed strong up-regulation of α-SMA by 147% (p<0.05). C-pc (6 nM) inhibited α-SMA expression by 70% (p<0.05) and reduced collagen contraction by 29% (p<0.05). C-pc seemed to lessen the over expression of CTGF, α-SMA, subsequently alleviating the fibrotic contracture. This study suggests the potential application of C-pc to regulation of the expression of pro-fibrotic mediators in scarring process and its potential usage as an efficient means for anti-fibrosis therapy.
Actins* ; Blotting, Western ; Cicatrix ; Collagen ; Connective Tissue Growth Factor* ; Connective Tissue* ; Contracture ; Down-Regulation ; Fibroblasts ; Humans ; Methods ; Myofibroblasts ; Phycocyanin* ; Sonication ; Spirulina ; Up-Regulation ; Wound Healing ; Wounds and Injuries

PURPOSE: Epidermal growth factor receptor (EGFR) is considered a potential therapeutic target for anti-EGFR therapy in triple-negative breast cancer (TNBC). However, the frequency of EGFR gene mutation in TNBC is low and varies with ethnicity. This study aimed to investigate the incidence of EGFR gene mutation in TNBC. METHODS: EGFR protein expression was evaluated by immunohistochemistry in tissue microarrays of 493 TNBC cases using four different primary antibodies, which included mutation-specific antibodies. For cases with an immunoreactivity level ≥1+, we performed pyrosequencing analysis for EGFR gene mutation. A case was considered mutation-positive when its mutation frequency minus its limit of detection (LOD) was >10%. Cases with mutation frequency higher than LOD were assessed for EGFR gene mutation status using the Cobas assay and by peptide nucleic acid-mediated polymerase chain reaction (PNA-clamping). RESULTS: Among 493 TNBCs, 148 (30.0%) exhibited staining ≥1+ for EGFR, including 78 with 1+, 49 with 2+, and 21 with 3+. Positive EGFR expression (≥2+) was significantly associated with lymphovascular invasion (p=0.010), but not with overall survival (p=0.444) or disease-free survival (p=0.388). None of the 493 TNBCs harbored an EGFR gene mutation. Among 148 cases with an EGFR staining result ≥1+, five (3.4%) showed mutation frequencies (4.4%–10.9%) higher than LOD (2.6%–4.3%) in exons 19 (L747_P753>Q) or 21 (L858R and L861Q) as determined by pyrosequencing. However, Cobas and PNA-clamping failed to detect the presence of EGFR gene mutation in these five cases. CONCLUSION: No activating mutation of EGFR gene of clinical significance was observed in 148 TNBC cases using three commercially available methods. Thus, EGFR gene mutation appears to be an extremely rare event in patients with TNBC.
Antibodies ; Breast Neoplasms ; Disease-Free Survival ; Epidermal Growth Factor* ; Exons ; Genes, erbB-1 ; Humans ; Immunohistochemistry ; Incidence ; Limit of Detection ; Mutation Rate ; Polymerase Chain Reaction ; Receptor, Epidermal Growth Factor* ; Triple Negative Breast Neoplasms*

The present study investigated caffeinated beverage consumption and screen time in the association with excessive daytime sleepiness (EDS) and sleep duration. We conducted a cross-sectional study including 249 Korean male high school students. These participants responded to a questionnaire inquiring the information on lifestyle factors, consumption of caffeinated beverages, time spent for screen media, and sleep duration as well as to the Epworth Sleepiness Scale (ESS) questionnaire. EDS was defined as ESS scores of 9 or greater. Students with EDS consumed greater amount of chocolate/cocoa drinks and spent longer time for a TV and a mobile phone than those without EDS (p < 0.05). In addition, students with short sleep (≤ 6 hours) consumed greater amount of coffee than others whereas students with long sleep (> 8 hours) consumed greater amount of chocolate/cocoa drinks than others (p < 0.05). Screen time did not differ according to the categories of sleep duration. Although these findings do not support causal relationships, they suggest that screen time is associated with EDS, but not with sleep duration, and that consumption of certain types of caffeinated beverages is associated with EDS and sleep duration. Adolescents may need to reduce screen time and caffeine consumption to improve sleep quality and avoid daytime sleepiness.
Adolescent ; Beverages* ; Caffeine ; Cell Phones ; Coffee ; Cross-Sectional Studies ; Humans ; Life Style ; Male

BACKGROUND/OBJECTIVES: Unregulated inflammatory responses caused by hyperglycemia may induce diabetes complications. Hesperetin, a bioflavonoid, is a glycoside in citrus fruits and is known to have antioxidant and anticarcinogenic properties. However, the effect of inflammation on the diabetic environment has not been reported to date. In this study, we investigated the effect of hesperetin on proinflammatory cytokine secretion and its underlying mechanistic regulation in THP-1 macrophages with co-treatment LPS and hyperglycemic conditions.MATERIALS/METHODS: THP-1 cells differentiated by PMA (1 μM) were cultured for 48 h in the presence or absence of hesperetin under normoglycemic (5.5 mM/L glucose) or hyperglycemic (25 mM/L glucose) conditions and then treated with LPS (100 ng/mL) for 6 h before harvesting. Inflammation-related proteins and mRNA levels were evaluated by enzyme-linked immunosorbent assay, western blot, and quantitative polymerase chain reaction analyses. RESULTS: Hesperetin (0–100 μM, 48 h) treatment did not affect cell viability. The tumor necrosis factor-α and interleukin-6 levels increased in cells co-treated with LPS under hyperglycemic conditions compared to normoglycemic conditions, and these increases were decreased by hesperetin treatment. The TLR2/4 and MyD88 activity levels increased in cells co-treated with LPS under hyperglycemic conditions compared to normoglycemic conditions; however, hesperetin treatment inhibited the TLR2/4 and MyD88 activity increases. In addition, nuclear factor-κB (NF-κB) and Acetyl-NF-κB levels increased in response to treatment with LPS under hyperglycemic conditions compared to normoglycemic conditions, but those levels were decreased when treated with hesperetin. SIRT3 and SIRT6 expressions were increased by hesperetin treatment. CONCLUSIONS Our results suggest that hesperetin may be a potential agent for suppressing inflammation in diabetes.

BACKGROUND/OBJECTIVES: Unregulated inflammatory responses caused by hyperglycemia may induce diabetes complications. Hesperetin, a bioflavonoid, is a glycoside in citrus fruits and is known to have antioxidant and anticarcinogenic properties. However, the effect of inflammation on the diabetic environment has not been reported to date. In this study, we investigated the effect of hesperetin on proinflammatory cytokine secretion and its underlying mechanistic regulation in THP-1 macrophages with co-treatment LPS and hyperglycemic conditions.MATERIALS/METHODS: THP-1 cells differentiated by PMA (1 μM) were cultured for 48 h in the presence or absence of hesperetin under normoglycemic (5.5 mM/L glucose) or hyperglycemic (25 mM/L glucose) conditions and then treated with LPS (100 ng/mL) for 6 h before harvesting. Inflammation-related proteins and mRNA levels were evaluated by enzyme-linked immunosorbent assay, western blot, and quantitative polymerase chain reaction analyses. RESULTS: Hesperetin (0–100 μM, 48 h) treatment did not affect cell viability. The tumor necrosis factor-α and interleukin-6 levels increased in cells co-treated with LPS under hyperglycemic conditions compared to normoglycemic conditions, and these increases were decreased by hesperetin treatment. The TLR2/4 and MyD88 activity levels increased in cells co-treated with LPS under hyperglycemic conditions compared to normoglycemic conditions; however, hesperetin treatment inhibited the TLR2/4 and MyD88 activity increases. In addition, nuclear factor-κB (NF-κB) and Acetyl-NF-κB levels increased in response to treatment with LPS under hyperglycemic conditions compared to normoglycemic conditions, but those levels were decreased when treated with hesperetin. SIRT3 and SIRT6 expressions were increased by hesperetin treatment. CONCLUSIONS Our results suggest that hesperetin may be a potential agent for suppressing inflammation in diabetes.

Obesity is consistently increasing in prevalence and can trigger insulin resistance and type 2 diabetes. Many lines of evidence have shown that macrophages play a major role in inflammation associated with obesity. This study was conducted to determine metformin, a widely prescribed drug for type 2 diabetes, would regulate inflammation through down-regulation of scavenger receptors in macrophages from obesity-induced type 2 diabetes. RAW 264.7 cells and peritoneal macrophages were stimulated with LPS to induce inflammation, and C57BL/6N mice were fed a high-fat diet to generate obesity-induced type 2 diabetes mice. Metformin reduced the production of NO, PGE2 and pro-inflammatory cytokines (IL-1beta, IL-6 and TNF-alpha) through down-regulation of NF-kappaB translocation in macrophages in a dose-dependent manner. On the other hand, the protein expressions of anti-inflammatory cytokines, IL-4 and IL-10, were enhanced or maintained by metformin. Also, metformin suppressed secretion of TNF-alpha and reduced the protein and mRNA expression of TNF-alpha in obese mice as well as in macrophages. The expression of scavenger receptors, CD36 and SR-A, were attenuated by metformin in macrophages and obese mice. These results suggest that metformin may attenuate inflammatory responses by suppressing the production of TNF-alpha and the expressions of scavenger receptors.
Animals ; Cytokines ; Diet, High-Fat ; Dinoprostone ; Down-Regulation ; Hand ; Inflammation ; Insulin Resistance ; Interleukin-10 ; Interleukin-4 ; Interleukin-6 ; Macrophages ; Macrophages, Peritoneal ; Metformin ; Mice ; Mice, Obese ; NF-kappa B ; Obesity ; Prevalence ; Receptors, Scavenger ; RNA, Messenger ; Tumor Necrosis Factor-alpha

Dioscoreae Rhizome (DR) has been used in traditional medicine to treat numerous diseases and is reported to have anti-diabetes and anti-tumor activities. To identify a bioactive traditional medicine with anti-inflammatory activity of a water extract of DR (EDR), we determined the mRNA and protein levels of proinflammatory cytokines in macrophages through RT-PCR and western blot analysis and performed a FACS analysis for measuring surface molecules. EDR dose-dependently decreased the production of NO and pro-inflammatory cytokines such as IL-1beta, IL-6, TNF-alpha, and PGE2, as well as mRNA levels of iNOS, COX-2, and pro-inflammatory cytokines, as determined by western blot and RT-PCR analysis, respectively. The expression of co-stimulatory molecules such as B7-1 and B7-2 was also reduced by EDR. Furthermore, activation of the nuclear transcription factor, NF-kappaB, but not that of IL-4 and IL-10, in macrophages was inhibited by EDR. These results show that EDR decreased pro-inflammatory cytokines via inhibition of NF-kappaB-dependent inflammatory protein level, suggesting that EDR could be a useful immunomodulatory agent for treating immunological diseases.
Blotting, Western ; Cytokines ; Dinoprostone ; Dioscorea ; Immune System Diseases ; Inflammation ; Interleukin-10 ; Interleukin-4 ; Interleukin-6 ; Macrophages ; Medicine, Traditional ; NF-kappa B ; Rhizome ; RNA, Messenger ; Transcription Factors ; Tumor Necrosis Factor-alpha ; Water

Epithelial-myoepithelial carcinoma is a very rare malignant tumor accounting for less than one percent of sa1ivary gland neoplasms. Histologically, the tumor consists of a mixture of trabecular structure with double-layered arrangement of inner ductal cells and outer myoepithelial cells. Immunohistochemistry and electron microscopy confirm dual differentiation toward myoepithelial and ductal cells. We report a 22-year-old man with epithelial-myoepithelial carcinoma arising from nasal cavity, one of the most unusual locations.
Humans ; Immunohistochemistry ; Microscopy, Electron ; Nasal Cavity ; Young Adult

PURPOSE: Intraoperative frozen sectioning (FS) of sentinel lymph nodes (SLNs) is widely used to determine whether total axillary lymph node dissection should be performed in patients with breast cancer. We evaluated the usefulness and limitations of the FS protocol, which has been used in our institution for the intraoperative SLN examination. METHODS: We analyzed the FS results of SLNs in 807 invasive breast carcinoma patients who underwent intraoperative SLN biopsy between January 2005 and December 2007. Lymph nodes larger than 5 mm were sliced at 2 mm intervals and all the slices were submitted for FS. The remaining tissue of the SLN was formalin-fixed and paraffin-embedded for permanent sectioning (PS). If the FS result was negative for tumor cells, then immunohistochemical stain for pancytokeratin was performed. The metastatic SLNs were graded according to the AJCC cancer staging manual (6th edition). The results of FS and PS were compared with regard to the pathologic diagnosis. RESULTS: The average number of SLNs was 2.9 per patients. A total of 204 (25.3%) patients were reported to have a metastatic SLN(s) on the FS. Among the 603 patients with negative FS results, 34 (5.6%) patients showed metastasis on the PS. Another 10 (1.7%) patients who had negative results on FS showed isolated tumor cells on the PS or on the cytokeratin immunohistochemistry. Twenty-nine of the 34 (85.3%) false negative cases showed micrometastasis on the PS. Ten (29.4%) false negative results were caused by interpretation errors and 24 (70.6%) were caused by technical problems. CONCLUSION: The false negative rate of our protocol for FS of a SLN was low. The failure of FS was largely caused by the failure to detect micrometastasis. FS is a reliable method for an intraoperative SLN examination if a very stringent protocol is used.
Biopsy ; Breast ; Breast Neoplasms ; Frozen Sections ; Humans ; Immunohistochemistry ; Keratins ; Lymph Node Excision ; Lymph Nodes ; Neoplasm Metastasis ; Neoplasm Micrometastasis ; Neoplasm Staging ; Nitriles ; Pyrethrins ; Sentinel Lymph Node Biopsy

To evaluate whether Palmitoyl-pentapeptide (Pal-KTTKS), a lipidated subfragment of type 1 pro-collagen (residues 212–216), plays a role in fibroblast contractility, the effect of Pal-KTTKS on the expression of pro-fibrotic mediators in hypertropic scarring were investigated in relation with trans-differentiation of fibroblast to myofibroblast, an icon of scar formation. α-SMA was visualized by immunofluorescence confocal microscopy with a Cy-3-conjugated monoclonal antibody. The extent of α-SMA-positive fibroblasts was determined in collagen lattices and in cell culture study. Pal-KTTKS (0–0.5 µM) induced CTGF and α-SMA protein levels were determined by western blot analysis and fibroblast contractility was assessed in three-dimensional collagen lattice contraction assay. In confocal analysis, fibroblasts were observed as elongated and spindle shapes while myofibroblast observed as squamous, enlarged cells with pronounced stress fibers. Without Pal-KTTKS treatment, three quarters of the fibroblasts differentiates into the myofibroblast; α-SMA-positive stress fibers per field decreased twofold with 0.1 µM Pal-KTTKS treatment (75 ± 7.1 vs 38.6 ± 16.1%, n = 3, p<0.05). The inhibitory effect was not significant in 0.5 µM Pal-KTTKS treatment. Stress fiber level and collagen contractility correlates with α-SMA expression level. In conclusion, Pal-KTTKS (0.1 µM) reduces α-SMA expression and trans-differentiation of fibroblasts to myofibroblast. The degree of reduction is dose-dependent. An abundance of myofibroblast and fibrotic scarring is correlated with excessive levels of α-SMA and collagen contractility. Delicate balance between the wound healing properties and pro-fibrotic abilities of pentapeptide KTTKS should be considered for selecting therapeutic dose for scar prevention.
Actins* ; Blotting, Western ; Cell Culture Techniques ; Cicatrix ; Collagen ; Connective Tissue Growth Factor* ; Connective Tissue* ; Fibroblasts ; Fluorescent Antibody Technique ; Microscopy, Confocal ; Myofibroblasts ; Stress Fibers ; Wound Healing ; Wounds and Injuries*