Volumetric Reconstruction of a Human Retinal Pigment Epithelial Cell Reveals Specialized Membranes and Polarized Distribution of Organelles.

Purpose: Despite the centrality of the retinal pigment epithelium (RPE) in vision and retinopathy our picture of RPE morphology is incomplete. With a volumetric reconstruction of human RPE ultrastructure, we aim to characterize major membranous features including apical processes and their interactions with photoreceptor outer segments, basolateral infoldings, and the distribution of intracellular organelles. Methods: A parafoveal retinal sample was acquired from a 21-year-old male organ donor. With serial block-face scanning electron microscopy, a tissue volume from the inner-outer segment junction to basal RPE was captured. Surface membranes and complete internal ultrastructure of an individual RPE cell were achieved with a combination of manual and automated segmentation methods. Results: In one RPE cell, apical processes constitute 69% of the total cell surface area, through a dense network of over 3000 terminal branches. Single processes contact several photoreceptors. Basolateral infoldings facing the choriocapillaris resemble elongated filopodia and comprise 22% of the cell surface area. Membranous tubules and sacs of endoplasmic reticulum represent 20% of the cell body volume. A dense basal layer of mitochondria extends apically to partly overlap electron-dense pigment granules. Pores in the nuclear envelope form a distinct pattern of rows aligned with chromatin. Conclusions: Specialized membranes at the apical and basal side of the RPE cell body involved in intercellular uptake and transport represent over 90% of the total surface area. Together with the polarized distribution of organelles within the cell body, these findings are relevant for retinal clinical imaging, therapeutic approaches, and disease pathomechanisms.

Trends of axillary surgery in breast cancer patients with axillary lymph node metastasis: a comprehensive single-center retrospective study.

Purpose: Based on the results of previous trials, de-escalation of axillary surgery after neoadjuvant chemotherapy (NAC) has increased in patients with axillary lymph node (ALN) metastasis at presentation. This study aimed to review the trends of axillary surgery by time period and molecular subtype in patients with ALN metastasis. Methods: We analyzed the rates of sentinel lymph node biopsy (SLNB) and ALN dissection (ALND) based on time period and subtype. The time period was divided into 3 subperiods to determine the rate of axillary surgery type over time (period 1, from 2009 to 2012; period 2, from 2013 to 2016; and period 3, from 2017 to July 2019). Results: From 2009 to July 2019, 2,525 breast cancer patients underwent surgery. Based on subtype, the ALND rate of hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) disease decreased by 13.0% from period 1 to period 3 (period 1, 99.4%; period 2, 97.5%; and period 3, 86.4%; P < 0.001). Conversely, the ALND rate in HR+/HER2+, HR-/HER2+, and triple-negative breast cancer (TNBC) significantly decreased by 43.7%, 48.8%, and 35.2% in period 1, period 2, and period 3, respectively (P < 0.001). In the patient group receiving NAC, HR+/HER2- had a significantly higher ALND rate (84.1%) than HR+/HER2+, HR-/HER2+, and TNBC (60.8%, 62.3%, and 70.7%, respectively; P < 0.001). Conclusion: The SLNB rate in patients with ALN metastasis has increased over time. However, the ALND rate in HR+/HER2- was significantly higher than in other subtypes.

Prognostic Impact of Elevation of Cancer Antigen 15-3 (CA15-3) in Patients With Early Breast Cancer With Normal Serum CA15-3 Level.

PURPOSE: Cancer antigen 15-3 (CA15-3) is a serum tumor marker for breast cancer (BC) extensively used in clinical practice. CA15-3 is non-invasive, easily available, and a cost-effective tumor marker for immediate diagnosis, monitoring and prediction of BC recurrence. We hypothesized that an elevation of CA15-3 may have prognostic impact in patients with early BC with normal serum CA15-3 level. METHODS: This was a retrospective cohort study, which included patients with BC who received curative surgery at a comprehensive single institution between 2000 and 2016. CA15-3 levels from 0 to 30 U/mL were considered normal, and patients who had CA15-3 > 30 U/mL, were excluded from the study. RESULTS: The mean age of study participants (n = 11,452) was 49.3 years. The proportion of participants with elevated CA15-3 ≥ 1 standard deviation (SD) compared with the previous examination during follow-up was 23.3% (n = 2,666). During the follow-up (median follow-up 5.8 years), 790 patients experienced recurrence. The fully-adjusted hazard ratio (HR) for recurrence comparing participants with stable CA15-3 level to subjects with elevated CA15-3 level was 1.76 (95% confidence interval [CI], 1.52-2.03). In addition, if the CA15-3 was elevated ≥ 1 SD, the risk was much higher (HR, 6.87; 95% CI, 5.81-8.11) than in patients without elevated CA15-3 ≥ 1 SD. In sensitivity analysis, the recurrence risk was consistently higher in participants with elevated CA15-3 levels than in participants without elevated CA15-3 levels. The association between elevated CA15-3 levels and incidence of recurrence was observed in all subtypes and the association was stronger in patients with N+ than in patients with N0 stage (p-value for interaction < 0.01). CONCLUSION: The results of the present study demonstrated that elevation of CA15-3 in patients with early BC and initial normal serum CA15-3 levels has a prognostic impact.

Primary Angiosarcoma of the Breast: A Single-Center Retrospective Study in Korea.

Due to the rarity of primary angiosarcoma of the breast, optimal management is based on expert opinion. The aim of this study was to review all primary angiosarcomas of the breast obtained from a single center in terms of clinicopathologic characteristics, treatment, and survival outcomes. From 1997 to 2020, 15 patients with primary angiosarcoma of the breast underwent either mastectomy or wide excision. We analyzed the clinicopathologic data to assess disease-free survival and overall survival. Fifteen women with primary angiosarcoma of the breast were identified. The mean age at diagnosis was 33 years (range: 14-63 years). The overall mean tumor size was 7.7 cm (range 3.5-20 cm). Upon histological grading, there were three cases of low grade, five intermediate grade, six high grade, and one unidentified grade. The five-year disease-free survival rate was 24.4%, and the five-year survival rate was 37.2%. The survival rate of the low-grade patient group was statistically higher than that of the intermediate- or high-grade patient groups (p = 0.024). Primary angiosarcoma of the breast is a rare aggressive tumor characterized by high grade and poor outcome. Histologic grade appears to be a reliable predictor of survival. There are no standard treatment guidelines; thus, optimal R0 surgical resection remains the best approach. The roles of neoadjuvant, adjuvant chemotherapy, and radiotherapy remain unclear.

A randomized controlled trial of the effect of ramosetron on postoperative restoration of bowel motility after gynecologic laparoscopic surgery.

OBJECTIVE: To investigate the effect of ramosetron after gynecological laparoscopic surgery on the recovery of bowel function. METHODS: A prospective randomized controlled trial conducted at Kyung Hee University hospital, South Korea, from August 2016 to September 2017. Patients were randomized to receive either 10 mg dexamethasone before induction of anesthesia (control group C), followed by intravenous administration of patient-controlled analgesia (IV-PCA) or 2 ml normal saline before induction of anesthesia and 0.6 mg ramosetron (study group R) administered with IV-PCA. RESULTS: A total of 88 patients were enrolled. Times to first flatus (group C 23.98 ± 6.31 vs. group R 27.14 ± 9.56 h; P = 0.148) and first defecation (group C 36.16 ± 16.04 vs. group R 43.41 ± 20.01 h; P = 0.138) showed no statistically significant differences. No significant differences were observed in the frequency of postoperative nausea and vomiting (PONV) and demand for additional analgesics. Multiple linear regression for analysis of factors affecting time to first flatus revealed no significant results. CONCLUSION: Ramosetron did not delay bowel movement recovery after gynecologic laparoscopic surgery and was as effective as dexamethasone in regulating PONV. Ramosetron can be used with IV-PCA without concerns about delay in recovery of bowel function. CLINICALTRIALS: gov registration number: NCT02849483.

The Relationship of Lifestyle Factors with the Prevalence of Major Depressive Disorder by Ecological Factors.

OBJECTIVE: The association between ecological/lifestyle factors and major depressive disorder (MDD) have been provided but was inconsistent as characteristics of population including race, gender, etc. METHODS: Data were extracted from the Korean National Health and Nutrition Examination Survey and consisted of 35,839 adults including 1,537 with MDD. Ecological factors included age, sex, married status, education, family income, residence, occupation, BMI, self-recognition stress, and history of non-communicable disease. Smoking, drinking, regular exercise, total energy intake, and sleep was consisted for lifestyle factors. The relationship between MDD and ecological/lifestyle factors, was evaluated using the multiple logistic regression model after adjustment for covariates. RESULTS: The increased prevalence of MDD in men was related aged, unmarried, low educated, unoccupied, high BMI, and high self-recognition stress. To women, MDD prevalence was increased as aged, low educated and family income, resided in urban, unoccupied, high self-recognition stress and history of non-communicable disease. Current smoking/drinking and lack of sleep was positively related with prevalence of MDD in women. The relationship between lifestyle factors and MDD prevalence was influenced by ecological status, predominantly in women. CONCLUSION: The relationship of lifestyle factors with MDD prevalence were observed and could be attenuated by various ecological factors, in women.

Clinical outcomes of pancreaticoduodenectomy for pancreatic ductal adenocarcinoma depending on preservation or resection of pylorus.

BACKGROUNDS/AIMS: The comparative effectiveness of pylorus-resecting pancreaticoduodenectomy (PRPD) and pylorus- preserving pancreaticoduodenectomy (PPPD) in pancreatic head cancer is still disputed. The aim of this study was to analyze the data obtained from a large, single center with PPPD compared with PRPD in terms of postoperative outcomes, including blood glucose levels and survival in patients with pancreatic head cancer. METHODS: Between January 2007 and December 2016, a total of 556 patients with pancreatic head cancer underwent either PPPD or PRPD. We analyzed the clinicopathologic data to assess short- and long-term outcomes retrospectively. RESULTS: For underlying disease, patients with DM in PPPD were fewer than in PRPD (33.0% vs. 46.2%, p=0.002). The median value of CA19-9 was significantly higher in PRPD than in PPPD (129.36 vs. 86.47, p=0.037). The incidence of Clavien-Dindo grade III to V major complications in PPPD was significantly higher than in PRPD (20.4% vs. 13.4%, p=0.032). Resection of pylorus was shown to reduce complications in univariate and multivariate analyses (p=0.032 and = 0.021, respectively). The 5-year survival rates were 27.6% in the PPPD group and 22.4% in the PRPD group (p=0.015). CONCLUSIONS: The results of PPPD and PRPD showed no significant differences from those reported conventionally in previous studies. Although further well-designed studies are needed, it is more important to select the range of surgical resection for the patient's disease regardless of resection of pylorus.

Comparison of DRY and WET vaginal swabs with cervical specimens in Roche Cobas 4800 HPV and Abbott RealTime High Risk HPV tests.

BACKGROUND: Self-collected vaginal swab samples have been proposed as an alternative specimen collection method for human papillomavirus (HPV) DNA detection. OBJECTIVES: Two vaginal swabs (a cone-shaped flocked swab (DRY) and a L-shape FLOQSwab with 2mL eNAT transport medium (WET)) were compared to standard cervical samples for HPV DNA testing. Additionally, they were also compared by using Roche Cobas 4800 HPV (Roche_HPV) and Abbott Real-time High Risk HPV (Abbott_HPV) tests. STUDY DESIGN: Ninety-six women were prospectively enrolled from the National Cancer Center in Korea between June and August 2015. WET and DRY vaginal swabs and cervical specimens were collected. Roche_HPV and Abbott_HPV tests were performed. The Roche_HPV test on cervical specimens was used as reference. RESULTS: The observed agreements (kappa) of Roche_HPV and Abbott_HPV between WET and DRY swabs were 89.6% (0.790, 95% confidence interval (95% CI): 0.667-0.913) and 91.7% (0.833, 95%CI: 0.723-0.943), respectively. No statistical difference was observed between WET and DRY swabs (p>0.05 for all comparisons). For HPV16/18, the sensitivity/specificity of Roche_HPV on the DRY and WET samples presented 93.8%/96.3% and 87.5%/97.5%, respectively. For other High Risk HPV (hrHPV), the sensitivity/specificity of Roche_HPV on the DRY and WET swabs presented 91.9%/91.5% and 97.3%/98.3, respectively. The sensitivity/specificity of the Abbott_HPV on the DRY and WET swabs were 93.8%/98.8%, 87.5%/98.8% for HPV16/18, and 91.9%/93.2%, 100.0%/93.2% for other hrHPV, respectively. CONCLUSIONS: HPV tests performed similarly when using vaginal DRY and WET swab samples. Using DRY and WET swabs to collect vaginal specimens could be an alternative to collecting cervical samples for HPV DNA testing.

Diagnostic Value of Elevated D-Dimer Level in Venous Thromboembolism in Patients With Acute or Subacute Brain Lesions.

OBJECTIVE: To define the risk factors that influence the occurrence of venous thromboembolism (VTE) in patients with acute or subacute brain lesions and to determine the usefulness of D-dimer levels for VTE screening of these patients. METHODS: Medical data from January 2012 to December 2013 were retrospectively reviewed. Mean D-dimer levels in those with VTE versus those without VTE were compared. Factors associated with VTE were analyzed and the odds ratios (ORs) were calculated. The D-dimer cutoff value for patients with hemiplegia was defined using a receiver operating characteristic (ROC) curve. RESULTS: Of 117 patients with acute or subacute brain lesions, 65 patients with elevated D-dimer levels (mean, 5.1±5.8 mg/L; positive result >0.55 mg/L) were identified. Logistic regression analysis showed that the risk of VTE was 3.9 times higher in those with urinary tract infections (UTIs) (p=0.0255). The risk of VTE was 4.5 times higher in those who had recently undergone surgery (p=0.0151). Analysis of the ROC showed 3.95 mg/L to be the appropriate D-dimer cutoff value for screening for VTE (area under the curve [AUC], 0.63; 95% confidence interval [CI], 0.5-0.8) in patients with acute or subacute brain lesions. This differs greatly from the conventional D-dimer cutoff value of 0.55 mg/L. D-dimer levels less than 3.95 mg/L in the absence of surgery showed a negative predictive value of 95.8% (95% CI, 78.8-99.8). CONCLUSION: Elevated D-dimer levels alone have some value in VTE diagnosis. However, the concomitant presence of UTI or a history of recent surgery significantly increased the risk of VTE in patients with acute or subacute brain lesions. Therefore, a different D-dimer cutoff value should be applied in these cases.

Akt enhances Runx2 protein stability by regulating Smurf2 function during osteoblast differentiation.

Runx2 plays essential roles in bone formation and chondrocyte maturation. Akt promotes osteoblast differentiation induced by the bone morphogenetic proteins BMP2 and enhances the function and transcriptional activity of Runx2. However, the precise molecular mechanism underlying the relationship between Runx2 and Akt is not well understood. In this study, we examined the role of Akt in regulating Runx2 function. We found that Akt increases the stability of Runx2 protein. However, the level of Runx2 mRNA was not affected by Akt, and we did not find any evidence for direct modification of Runx2 by Akt. Instead, we found evidence that Akt induces the phosphorylation of the Smad ubiquitination regulatory factor Smurf2 and decreases the level of Smurf2 protein through ubiquitin/proteasome-mediated degradation of Smurf2. Akt also alleviates Smurf2-mediated suppression of Runx2 transcriptional activity. Taken together, our results suggest that Akt regulates osteoblast differentiation, at least in part, by enhancing the protein stability and transcriptional activity of Runx2 through regulation of ubiquitin/proteasome-mediated degradation of Smurf2.

Prolyl isomerase Pin1 enhances osteoblast differentiation through Runx2 regulation.

Peptidyl-prolyl isomerase 1 (Pin1) is the only enzyme known to catalyze isomerization of the pSer/Thr-Pro peptide bond. Pin1 induces conformational change of substrates and subsequently regulates diverse cellular processes. However, its role in osteoblast differentiation is not well understood. Here we show that Pin1 enhances osteoblast differentiation. Pin1 interacts and affects the protein stability and transcriptional activity of an important osteogenic transcriptional factor Runx2. Our results indicate that this regulation is likely due to suppression of poly-ubiquitination-mediated proteasomal degradation of Runx2. Our current finding suggests that Pin1 is a novel regulator of osteoblast differentiation that acts through the regulation of Runx2 function.

Sophoricoside isolated from Sophora japonica ameliorates contact dermatitis by inhibiting NF-κB signaling in B cells.

Sophoricoside (SOPH) is an isoflavone isolated from Sophora japonica (Leguminosae). In this study, the inhibitory effect of SOPH on contact dermatitis was investigated. At dosages of 3 and 10 mg/kg, SOPH ameliorated 2,4-dinitrochlorobenzene-induced acute and chronic contact dermatitis by 50-70%. As cellular targets, SOPH mainly affected the functions of B cells rather than T cells, macrophages and dendritic cells. As signaling targets, SOPH inhibited the phosphorylation and degradation of IκBα/ß and the nuclear translocation of NF-κB p65 in B cells, but not in dendritic cells and macrophages. SOPH did not affect the phosphorylation of ERK, p38, and JNK MAPKs, in B cells, dendritic cells, and macrophages. Taken together, these results suggest that SOPH ameliorates contact dermatitis by inhibiting mainly NF-κB signaling in B cells.

Promoting effect of polysaccharide isolated from Mori fructus on dendritic cell maturation.

Maturation of dendritic cells (DCs) is usually attenuated in the tumor microenvironment, which is an important immunological problem in DC-based immunotherapy of cancer. In this study, we report the effect of a Mori fructus polysaccharide (MFP) on DC maturation. MFP was treated to DCs generated from mouse BM cells. MFP induced phenotypic maturation of DCs, as proven by the increased expression of CD40, CD80/86, and MHC-I/II molecules. MFP induced functional maturation of DCs, in that MFP increased the expression of IL-12, IL-1ß, TNF-α, and IFN-ß, decreased antigen capture capacity, and enhanced allogenic T cell stimulation. MFP efficiently induced maturation of DCs from C3H/HeN mice having normal toll-like receptor4 (TLR4), but not DCs from C3H/HeJ mice having mutated TLR4, suggesting that TLR4 might be one of the membrane receptors of MFP. As a mechanism of action, MFP increased phosphorylation of mitogen-activated protein kinase (MAPKs), and nuclear translocation of NF-κB p65 subunit, which were important signal molecules downstream from TLR4. These data suggest that MFP induces DC maturation through TLR4 and MFP can be used as an adjuvant in DC-based cancer immunotherapy.

A polysaccharide isolated from Pueraria lobata enhances maturation of murine dendritic cells.

Maturation of dendritic cells (DCs) is a critical factor for initiating the immune response. However, DC maturation is usually attenuated in the tumor microenvironment, which is an important immunological problem in DC-based immunotherapy against cancer. Here, we report the effect of a polysaccharide (PLP) isolated from Pueraria lobata on phenotypic and functional maturation of DCs. Phenotypic maturation was demonstrated by increased expression of CD40, CD86, and major histocompatibility complex I/II. PLP induced functional maturation of DCs, as shown by increased production of interleukin (IL)-12, IL-1ß, and tumor necrosis factor-α, decreased antigen capture capacity, and enhanced allogenic T cell stimulation. In addition, PLP activated DCs generated from C3H/HeN mice with normal TLR4, but not DCs from C3H/HeJ mice with mutated TLR4, suggesting that the TLR4 is a membrane receptor of PLP. We showed that PLP increased ERK, JNK, and p38 mitogen-activated protein kinase phosphorylation, and nuclear translocation of the nuclear factor-kappaB p65 subunit, which are signaling molecules downstream of TLR4. These results indicate that PLP induced DC maturation through TLR4 signaling.

Effusanin C inhibits inflammatory responses via blocking NF-κB and MAPK signaling in monocytes.

Effusanin C, a constituent of Isodon japonicus, has been used in oriental countries as a traditional folk medicine to treat inflammatory diseases, but its mechanism of action remains unknown. Here, we investigate the inhibitory activity of effusanin C in inflammatory monocytes. Effusanin C markedly inhibited the production of inflammatory mediators including nitric oxide, IL-1ß, and TNF-α in macrophages and dendritic cells. Furthermore, molecular studies showed that effusanin C inhibited phosphorylation of p38, JNK, and ERK, degradation of IκBß, and nuclear translocation of NF-κB p50/p65 in these cells. Taken together, these data show that effusanin C inhibits inflammatory responses by blocking NF-κB and MAPK signalings in monocytes.

Inhibition of TAK1 by kamebakaurin in dendritic cells.

Kamebakaurin (KA) has anti-cancer and anti-inflammatory activities through direct inhibition of DNA-binding activity of nuclear factor-kappa B (NF-κB) p50. We suggest here another molecular target of KA by the use of lipopolysaccharide-treated dendritic cells. In cell- and enzyme-based assays, KA directly inhibited autophosphorylation and kinase activity of TAK1, followed by the inhibition of TAK1-downstream signaling cascades, such as IKK phosphorylation-IκBα degradation-nuclear translocation of NF-κB, phosphorylation of MEK3/6-p38 mitogen activated protein kinase (MAPK), and MKK4/7-c-Jun N-terminal kinase MAPK. These results demonstrated that TAK1 might be the direct molecular target of KA.

Halofuginone and other febrifugine derivatives inhibit prolyl-tRNA synthetase.

Febrifugine, the bioactive constituent of one of the 50 fundamental herbs of traditional Chinese medicine, has been characterized for its therapeutic activity, though its molecular target has remained unknown. Febrifugine derivatives have been used to treat malaria, cancer, fibrosis and inflammatory disease. We recently demonstrated that halofuginone (HF), a widely studied derivative of febrifugine, inhibits the development of T(H)17-driven autoimmunity in a mouse model of multiple sclerosis by activating the amino acid response (AAR) pathway. Here we show that HF binds glutamyl-prolyl-tRNA synthetase (EPRS), inhibiting prolyl-tRNA synthetase activity; this inhibition is reversed by the addition of exogenous proline or EPRS. We further show that inhibition of EPRS underlies the broad bioactivities of this family of natural product derivatives. This work both explains the molecular mechanism of a promising family of therapeutics and highlights the AAR pathway as an important drug target for promoting inflammatory resolution.

Inhibition of human pancreatic tumor growth by cytokine-induced killer cells in nude mouse xenograft model.

Pancreatic cancer is the fourth commonest cause of cancer-related deaths in the world. However, no adequate therapy for pancreatic cancer has yet been found. In this study, the antitumor activity of cytokine-induced killer (CIK) cells against the human pancreatic cancer was evaluated in vitro and in vivo. Human peripheral blood mononuclear cells were cultured with IL-2-containing medium in anti-CD3 for 14 days. The resulting populations of CIK cells comprised 94% CD3(+), 4% CD3(-)CD56(+), 41% CD3(+)CD56(+), 11% CD4(+), and 73% CD8(+). This heterogeneous cell population was called cytokine-induced killer (CIK) cells. At an effector-target cell ratio of 100:1, CIK cells destroyed 51% of AsPC-1 human pancreatic cancer cells, as measured by the (51)Cr-release assay. In addition, CIK cells at doses of 3 and 10 million cells per mouse inhibited 42% and 70% of AsPC-1 tumor growth in nude mouse xenograft assays, respectively. This study suggests that CIK cells may be used as an adoptive immunotherapy for pancreatic cancer patients.

Adjuvant effect of a natural TLR4 ligand on dendritic cell-based cancer immunotherapy.

The clinical efficacy of dendritic cell (DC) vaccine in cancer patients has been unsatisfactory due, at least in part, to the deficiency of maturation and impaired migration of ex vivo generated DCs to the draining lymph nodes. To solve this problem, we used angelan, a natural TLR4 ligand, to enhance the maturation and migration of DCs. Angelan increased the expression of MHC-I/II, CD80, and CD86, DC maturation markers, through the NF-κB pathway. This compound also increased CCR7 expression in DCs through NF-κB and p38 pathway and enhanced their migration against CCL19, which is a key chemokine that guides DCs into lymph nodes. We also showed that angelan enhanced in vivo DC homing from tissues to draining lymph nodes. When treated to DCs in vitro and vivo, angelan increased antitumor activity of DCs in B16F10 syngeneic tumor model. Taken together, the present data suggest that a natural TLR4 ligand might be helpful for overcoming the disadvantages of DC-based cancer therapy, such as impaired maturation and poor migration in cancer patients.