Distinct effects of different adjuvants in the mouse model of allergic airway inflammation.

BACKGROUND: Allergic asthma was typically considered as an inflammatory disease mediated by type 2 immunity. However, recent studies revealed that asthma is a complex disease displaying a variety of phenotypes and endotypes. OBJECTIVE: We examined cellular phenotypes in the mouse model of allergic asthma sensitized with different adjuvants. The aim of our study was to determine immunologic cellular characteristics in mouse asthma models induced by ovalbumin (OVA) and a variety of adjuvants. METHODS: Mice were sensitized intraperitoneally with the admixture of OVA and various adjuvants such as Alhydrogel (alum), papain, lipopolysaccharide (LPS), or CpG, and subsequently challenged with OVA intranasally. The cells in bronchoalveolar lavage (BAL) fluid, lung, and mediastinal lymph node (mLN) were examined by flow cytometric analyses. RESULTS: In the lung and BAL fluid, the highest eosinophil levels were observed in the alum group while the highest neutrophil levels were detected in the LPS group. Meanwhile, the LPS group exhibited the most elevated levels of both RORγt+ innate lymphoid cells (ILCs) and IL-17A+ Th cells in the lung and mediastinal lymph node. In the lung, the number of T-bet+ ILCs was highest in the papain group whereas the number of IFN-γ+ Th cells was highest in the CpG group. CONCLUSIONS: Notable variances are found in the composition of immune cells and expression of cytokines at the site of pathogenesis among the different mouse models of allergic asthma created by the sensitization with different adjuvants.

Upregulated microRNA-193a-3p is responsible for cisplatin resistance in CD44(+) gastric cancer cells.

Cisplatin is a well-known anticancer drug used to treat various cancers. However, development of cisplatin resistance has hindered the efficiency of this drug in cancer treatment. Development of chemoresistance is known to involve many signaling pathways. Recent attention has focused on microRNAs (miRNAs) as potentially important upstream regulators in the development of chemoresistance. CD44 is one of the gastric cancer stem cell markers and plays a role in regulating self-renewal, tumor initiation, metastasis and chemoresistance. The purpose of the present study was to examine the mechanism of miRNA-mediated chemoresistance to cisplatin in CD44-positive gastric cancer stem cells. We sorted gastric cancer cells according to level of CD44 expression by FACS and analyzed their miRNA expression profiles by microarray analysis. We found that miR-193a-3p was significantly upregulated in CD44(+) cells compared with CD44(-) cells. Moreover, SRSF2 of miR-193a-3p target gene was downregulated in CD44(+) cells. We studied the modulation of Bcl-X and caspase 9 mRNA splicing by SRSF2 and found that more pro-apoptotic variants of these genes were generated. We also found that downstream anti-apoptotic genes such as Bcl-2 were upregulated, whereas pro-apoptotic genes such as Bax and cytochrome C were downregulated in CD44(+) cells compared to CD44(-) cells. In addition, we found that an elevated level of miR-193a-3p triggered the development of cisplatin resistance in CD44(+) cells. Inhibition of miR-193a-3p in CD44(+) cells increased SRSF2 expression and also altered the levels of multiple apoptotic genes. Furthermore, inhibition of miR-193a-3p reduced cell viability and increased the number of apoptotic cells. Therefore, miR-193a-3p may be implicated in the development of cisplatin resistance through regulation of the mitochondrial apoptosis pathway. miR-193a-3p could be a promising target for cancer therapy in cisplatin-resistant gastric cancer.

Hypoxia-Activated Adipose Mesenchymal Stem Cells Prevents Irradiation-Induced Salivary Hypofunction by Enhanced Paracrine Effect Through Fibroblast Growth Factor 10.

To explore the effects and mechanisms of paracrine factors secreted from human adipose mesenchymal stem cell (hAdMSCs) that are activated by hypoxia on radioprotection against irradiation-induced salivary hypofunction in subjects undergoing radiotherapy for head and neck cancers. An organotypic spheroid coculture model to mimic irradiation (IR)-induced salivary hypofunction was set up for in vitro experiments. Human parotid gland epithelial cells were organized to form three-dimensional (3D) acinus-like spheroids on growth factor reduced -Matrigel. Cellular, structural, and functional damage following IR were examined after cells were cocultured with hAdMSCs preconditioned with either normoxia (hAdMSCNMX ) or hypoxia (hAdMSCHPX ). A key paracrine factor secreted by hAdMSCsHPX was identified by high-throughput microarray-based enzyme-linked immunosorbent assay. Molecular mechanisms and signaling pathways on radioprotection were explored. Therapeutic effects of hAdMSCsHPX were evaluated after in vivo transplant into mice with IR-induced salivary hypofunction. In our 3D coculture experiment, hAdMSCsHPX significantly enhanced radioresistance of spheroidal human parotid epithelial cells, and led to greater preservation of salivary epithelial integrity and acinar secretory function relative to hAdMSCsNMX . Coculture with hAdMSCsHPX promoted FGFR expression and suppressed FGFR diminished antiapoptotic activity of hAdMSCsHPX . Among FGFR-binding secreted factors, we found that fibroblast growth factor 10 (FGF10) contributed to therapeutic effects of hAdMSCsHPX by enhancing antiapoptotic effect, which was dependent on FGFR-PI3K signaling. An in vivo transplant of hAdMSCsHPX into irradiated salivary glands of mice reversed IR-induced salivary hypofunction where hAdMSC-released FGF10 contributed to tissue remodeling. Our results suggest that hAdMSCsHPX protect salivary glands from IR-induced apoptosis and preserve acinar structure and functions by activation of FGFR-PI3K signaling via actions of hAdMSC-secreted factors, including FGF10. Stem Cells 2018;36:1020-1032.

Chest wall recurrence in pT1-2N0-1 breast cancer patients after mastectomy without radiotherapy.

PURPOSE: In correlation with the nodal status in the era of modern radiotherapy, the chest wall recurrence (CWR) rate was investigated in pT1-2N0-1 breast cancer patients after a mastectomy without post-mastectomy radiotherapy (PMRT). METHODS: The data from the patients participating in two South Korean multi-institutional studies (KROG 14-22; N = 1842 and KROG 14-23; N = 1382) were analyzed. In total, 3224 pT1-2N0-1 breast cancer patients who underwent mastectomy without PMRT were analyzed. RESULTS: The median follow-up time was 72.2 months (range 0.8-125.2 months). The overall CWRs during the follow-up period were 1.68% in N0 patients and 2.82% in N1 patients. There was no statistically significant difference in 5-year and 10-year CWR-free survival (CWRFS) between the N0 and N1 patients. Of the 70 patients with CWR, 33 (1% of all the patients) had isolated CWR, and the 10-year overall survival rate in this group was 96.9%. After the propensity score matching of the N0 and N1 groups, there was still no difference in CWRFS by nodal status. CONCLUSIONS: The incidence of CWR in pT1-2N0-1 breast cancer patients is very low, especially with isolated recurrence. Also, the obtained data showed that the nodal status had no impact on CWRFS.

Clinical efficacy of combination therapy with an alpha blocker and low-dose sildenafil on post-therapy lower urinary tract symptoms after low-dose-rate brachytherapy for prostate cancer.

PURPOSE: To investigate the efficacy of tamsulosin monotherapy and tamsulosin with low-dose sildenafil combination therapy on lower urinary tract symptoms (LUTS) following low-dose-rate (LDR) brachytherapy in early prostate cancer patients. METHODS: From March 2008 to June 2014, of the 212 prostate cancer patients with a Gleason score ≤7 who received LDR brachytherapy, 80 patients with a prostate volume ≤35 g and progressed LUTS following implantation were selected. All 80 patients took tamsulosin 0.4-mg monotherapy until 1 month after implantation. Then, the patients were divided into two groups; 45 patients received tamsulosin 0.4-mg monotherapy, and 35 patients received tamsulosin 0.4-mg plus sildenafil 25-mg combination therapy due to erectile dysfunction. LUTS were compared between the two groups using the International Prostate Symptom Score (IPSS), the mean maximum flow rate (Q max) and the pre-implantation post-voiding residual (PVR) volume at 1 and 3 months after implantation. RESULTS: The pre-implantation total IPSS, Q max and PVR for the monotherapy and combination therapy groups were 14.0 ± 6.7, 14.3 ± 3.2 ml/s and 36.3 ± 16.7 ml and 15.3 ± 5.6, 13.7 ± 4.5 ml/s and 39.0 ± 23.4 ml, respectively. At 1 month post-implantation, both groups showed increases in total IPSS and PVR, but no statistically significant differences were observed (P = 0.078, P = 0.23). At 3 months post-implantation, the combination therapy group showed a greater decrease in total IPSS compared with the monotherapy group (P = 0.035), but there were no statistically significant differences in the Q max and PVR between the two groups. CONCLUSION: Tamsulosin plus low-dose sildenafil combination therapy is a beneficial treatment for post-implantation progression of LUTS.

Endoplasmic reticulum stress as a novel target to ameliorate epithelial-to-mesenchymal transition and apoptosis of human peritoneal mesothelial cells.

Epithelial-to-mesenchymal transition (EMT) and apoptosis of peritoneal mesothelial cells are known to be the earliest mechanisms of peritoneal fibrosis in peritoneal dialysis (PD). Endoplasmic reticulum (ER) stress with an unfolded protein response is regarded to have a role in the development of organ fibrosis. To investigate the potential role of ER stress as a target to prevent and/or delay the development of peritoneal fibrosis, we examined the effect of ER stress on EMT or apoptosis of human peritoneal mesothelial cells (HPMCs) and elucidated the mechanisms underlying the protective effect of ER stress preconditioning on TGF-ß1-induced EMT. ER stress inducers, tunicamycin (TM) and thapsigargin (TG), induced EMT with Smad2/3 phosphorylation, an increased nuclear translocation of ß-catenin and Snail expression. Low concentrations of TM and TG did not induce apoptosis within 48 h; however, high concentrations of TM- (>1 ng/ml) and TG- (>1 nM) induced apoptosis at 12 h with a persistent increase in C/EBP homologous protein. TGF-ß1 induced EMT and apoptosis in HPMCs, which was ameliorated by taurine-conjugated ursodeoxycholic acid, an ER stress blocker. Interestingly, pre-treatment with TM or TG for 4 h also protected the cells from TGF-ß1-induced EMT and apoptosis, demonstrating the role of ER stress as an adaptive response to protect HPMCs from EMT and apoptosis. Peritoneal mesothelial cells isolated from PD patients displayed an increase in GRP78/94, which was correlated with the degree of EMT. These findings suggest that the modulation of ER stress in HPMCs could serve as a novel approach to ameliorate peritoneal damage in PD patients.

Renoprotective effect of red ginseng in gentamicin-induced acute kidney injury.

Aminoglycoside-induced nephrotoxicity is one of the prevalent causes of acute kidney injury (AKI). Oxidative stress-mediated apoptosis of renal tubular cells is known to be a major mechanism of renal injury. Red ginseng extract (RGE) has been reported to possess antioxidant and immune-modulatory activities. We investigated the effect of RGE on gentamicin (GM)-induced apoptosis and oxidative stress in cultured renal tubular cells and animal model of GM-induced AKI. GM induced the generation of reactive oxygen species (ROS) with an increase in NADPH oxidase (NOX) activity and mitochondrial oxidation in NRK-52E cells that were ameliorated with RGE. GM-induced apoptosis of NRK-52E cells, which was associated with an increased expression of mitochondrial Bax, cytosolic cytochrome c, and cleaved caspase-9 and -3, along with a decrease in bcl-2 expression, was also blocked by RGE. In an animal model of GM-induced AKI, RGE treatment significantly attenuated renal dysfunction, cell apoptosis, and tubular damage. RGE ameliorated ROS production in rats with GM-induced AKI, as demonstrated by an increase in the reduced form of glutathione in renal cortex and a decrease in urinary excretion of 8-hydroxy-2'-deoxyguanosine. Our results suggest that RGE protects the kidney from GM-induced AKI via the mechanism of modulation of oxidative stress.

miR-106b modulates cancer stem cell characteristics through TGF-ß/Smad signaling in CD44-positive gastric cancer cells.

Cancer stem cells have the capacity to form new tumors and are thus considered to be a cause of metastasis and tumor recurrence. However, many of the mechanisms determining cancer stem cell characteristics are still unknown. MicroRNAs (miRNAs) are possible modulators of cancer stem cell generation and may be involved in the retention of cancer stem cell characteristics. The aim of this study was to examine the miRNA expression profiles regulating the cancer stem-like cell characteristics in gastric cancer. We sorted gastric cancer stem-like cells using the stem cell marker CD44 by fluorescence-activated cell sorting. CD44(+) cells formed more and larger spheres compared with CD44(-) cells. Cancer stem cell markers were overexpressed in CD44(+) cells. CD44(+) cells showed increased expression of mesenchymal cell markers, whereas epithelial markers were downregulated. In miRNA microarray, the miR-106b family comprising miR-106b, miR-93, and miR-25 was significantly upregulated in CD44(+) cells than in CD44(-) cells. Smad7, which inhibits transforming growth factor-ß (TGF-ß)/Smad signaling as a target of the miR-106b family, was downregulated in CD44(+) cells. Furthermore, expression of TGF-ß/Smad signal molecules was activated in CD44(+) cells, in accordance with the action of the miR-106b family. Inhibition of miR-106b showed suppression of the TGF-ß/Smad signaling pathway and decreased self-renewal capacity and cell invasiveness. Our study suggests that CD44(+) gastric cancer cells show cancer stem cell properties with epithelial-mesenchymal transition (EMT). Increased miR-106b family expression regulated cancer stem-like cell properties, particularly EMT characteristics, through the TGF-ß/Smad signaling pathway in CD44(+) stem-like cells. Taken together, these results indicate that targeting miR-106b may be an effective form of cancer therapy in gastric cancer through the modulation of cancer stem cell characteristics.

Uric acid induces fat accumulation via generation of endoplasmic reticulum stress and SREBP-1c activation in hepatocytes.

Non-alcoholic fatty liver disease (NAFLD) is currently one of the most common types of chronic liver injury. Elevated serum uric acid is a strong predictor of the development of fatty liver as well as metabolic syndrome. Here we demonstrate that uric acid induces triglyceride accumulation by SREBP-1c activation via induction of endoplasmic reticulum (ER) stress in hepatocytes. Uric acid-induced ER stress resulted in an increase of glucose-regulated protein (GRP78/94), splicing of the X-box-binding protein-1 (XBP-1), the phosphorylation of protein kinase RNA-like ER kinase (PERK), and eukaryotic translation initiation factor-2α (eIF-2α) in cultured hepatocytes. Uric acid promoted hepatic lipogenesis through overexpression of the lipogenic enzyme, acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FAS), and stearoyl-CoA desaturase 1 (SCD1) via activation of SREBP-1c, which was blocked by probenecid, an organic anion transport blocker in HepG2 cells and primary hepatocytes. A blocker of ER stress, tauroursodeoxycholic acid (TUDCA), and an inhibitor of SREBP-1c, metformin, blocked hepatic fat accumulation, suggesting that uric acid promoted fat synthesis in hepatocytes via ER stress-induced activation of SREBP-1c. Uric acid-induced activation of NADPH oxidase preceded ER stress, which further induced mitochondrial ROS production in hepatocytes. These studies provide new insights into the mechanisms by which uric acid stimulates fat accumulation in the liver.

Proteomic analysis of CD44(+) and CD44(-) gastric cancer cells.

CD44 is a cell surface protein and it is widely used as a cancer stem cell marker in various cancer types including gastric cancer. We conducted proteomic analysis in CD44(+) and CD44(-) gastric cancer cells to understand characteristics of CD44(+) and CD44(-) cells. In the present study, we sorted cells from the gastric cancer cell line MKN45 according to CD44 expression to separate out CD44(+) and CD44(-) cells. And we conducted RT-PCR to identify mRNA expression of cancer stem cell markers in CD44(+) and CD44(-) cells. Cancer stem cell markers showed upregulated expression in CD44(+) cells. Next, we performed two-dimensional electrophoresis analysis to determine the differential expression pattern of proteins in each group; control, CD44(+), and CD44(-) MKN45 cells. We found a total of 113 spots that varied in expression between CD44(+) and CD44(-) cells, and subjected 20 of those protein spots to MALDI-MS. We selected the three proteins (HSPA8; heat shock cognate 71 kDa protein isoform 1, ezrin, α-enolase) upregulated in CD44(+) cells than CD44(-) cells and one protein (prohibitin) showed increased expression in CD44(-) cells. We validated the protein expression levels of four selected proteins by Western blot. We suggest that our study could be a helpful background to study CD44(+) cancer stem-like cells and differences between CD44(+) and CD44(-) cells in gastric cancer.

Risk Factors for Distant Metastasis in Extrahepatic Bile Duct Cancer after Curative Resection (KROG 1814).

PURPOSE: Risk factors predicting distant metastasis (DM) in extrahepatic bile duct cancer (EHBDC) patients treated with curative resection were investigated. MATERIALS AND METHODS: Medical records of 1,418 EHBDC patients undergoing curative resection between Jan 2000 and Dec 2015 from 14 institutions were reviewed. After resection, 924 patients (67.6%) were surveilled without adjuvant therapy, 297 (21.7%) were treated with concurrent chemoradiotherapy (CCRT) and 148 (10.8%) with CCRT followed by chemotherapy. To exclude the treatment effect from innate confounders, patients not treated with adjuvant therapy were evaluated. RESULTS: After a median follow-up of 36.7 months (range, 2.7 to 213.2 months), the 5-year distant metastasis-free survival (DMFS) rate was 57.7%. On multivariate analysis, perihilar or diffuse tumor (hazard ratio [HR], 1.391; p=0.004), poorly differentiated histology (HR, 2.014; p < 0.001), presence of perineural invasion (HR, 1.768; p < 0.001), positive nodal metastasis (HR, 2.670; p < 0.001) and preoperative carbohydrate antigen (CA) 19-9 ≥ 37 U/mL (HR, 1.353; p < 0.001) were significantly associated with inferior DMFS. The DMFS rates significantly differed according to the number of these risk factors. For validation, patients who underwent adjuvant therapy were evaluated. In patients with ≥ 3 factors, additional chemotherapy after CCRT resulted in a superior DMFS compared with CCRT alone (5-year rate, 47.6% vs. 27.7%; p=0.001), but the benefit of additional chemotherapy was not observed in patients with 0-2 risk factors. CONCLUSION: Tumor location, histologic differentiation, perineural invasion, lymph node metastasis, and preoperative CA 19-9 level predicted DM risk in resected EHBDC. These risk factors might help identifying a subset of patients who could benefit from additional chemotherapy after resection.

Uric acid-induced phenotypic transition of renal tubular cells as a novel mechanism of chronic kidney disease.

Recent experimental and clinical studies suggest a causal role of uric acid in the development of chronic kidney disease. Most studies have focused on uric acid-induced endothelial dysfunction, oxidative stress, and inflammation in the kidney. The direct effects of uric acid on tubular cells have not been studied in detail, and whether uric acid can mediate phenotypic transition of renal tubular cells such as epithelial-to-mesenchymal transition (EMT) is not known. We therefore investigated whether uric acid could alter E-cadherin expression and EMT in the kidney of hyperuricemic rats and in cultured renal tubular cells (NRK cells). Experimental hyperuricemia was associated with evidence of EMT before the development of significant tubulointerstitial fibrosis at 4 wk, as shown by decreased E-cadherin expression and an increased α-smooth muscle actin (α-SMA). Allopurinol significantly inhibited uric acid-induced changes in E-cadherin and α-SMA with an amelioration of renal fibrosis at 6 wk. In cultured NRK cells, uric acid induced EMT, which was blocked by the organic anion transport inhibitor probenecid. Uric acid increased expression of transcriptional factors associated with decreased synthesis of E-cadherin (Snail and Slug). Uric acid also increased the degradation of E-cadherin via ubiquitination, which is of importance since downregulation of E-cadherin is considered to be a triggering mechanism for EMT. In conclusion, uric acid induces EMT of renal tubular cells decreasing E-cadherin synthesis via an activation of Snail and Slug as well as increasing the degradation of E-cadherin.

Effects of dexamethasone on the TGF-ß1-induced epithelial-to-mesenchymal transition in human peritoneal mesothelial cells.

The epithelial-to-mesenchymal transition (EMT) is known to have a role in appropriate embryonic development, the physiological response to injury and pathological events such as organ fibrosis and cancer progression. Glucocorticoid (GC), one of the most commonly used anti-inflammatory drugs, inhibits the deposition of extracellular matrix independent of its anti-inflammatory effect. The EMT of human peritoneal mesothelial cells (HPMCs) is a key mechanism of peritoneal fibrosis; however, it has not yet been investigated whether GC imposes any effect on the EMT of HPMCs. To investigate the therapeutic potential of GC on preserving peritoneal membrane function, we studied the effect of dexamethasone (DEXA), a synthetic GC, on the transforming growth factor-ß1 (TGF-ß1)-induced EMT in HPMCs. As assessed by changes in cell morphology, the expression of epithelial and mesenchymal cell markers (such as E-cadherin, ZO-1 and α-SMA, α-smooth muscle actin) and cell migration, DEXA inhibited the TGF-ß1-induced EMT. RU486, a glucocorticoid receptor (GR) antagonist, blocked the effect of DEXA on the TGF-ß1-induced EMT. Importantly, DEXA also induced the mesenchymal-to-epithelial transition of TGF-ß1-stimulated HPMCs. The beneficial effect of DEXA on the TGF-ß1-induced EMT was mediated through the amelioration of ERK and p38 mitogen-activated protein kinase (MAPK) phosphorylation; however, this effect was not related to the TGF-ß1-induced activation of Smad2/3 signaling. DEXA inhibited glycogen synthase kinase-3ß (GSK-3ß) phosphorylation and the Snail upregulation induced by TGF-ß1, which were also ameliorated by inhibitors of MAPK. In conclusion, this is the first study demonstrating the protective effect of DEXA on the EMT in TGF-ß1-stimulated HPMCs by inhibiting MAPK activation, GSK-3ß phosphorylation and Snail upregulation.

Radical prostatectomy versus high dose permanent prostate brachytherapy using iodine-125 seeds for patients with high risk prostate cancer: a matched cohort analysis.

OBJECTIVE: To compare the biochemical outcomes reported after radical prostatectomy (RP) versus high dose permanent prostate brachytherapy (HDPPB) using iodine-125 seeds in the treatment of matched high risk prostate cancer (HiPCa). METHODS: In this retrospective review, 55 HiPCa patients treated between March 2006 and August 2011, who underwent HDPPB using iodine-125 seeds combined with external beam radiation therapy (EBRT) or androgen deprivation therapy (ADT), were compared with 55 HiPCa patients who underwent RP. Patients were matched for age, prostate-specific antigen (PSA), clinical stage, and Gleason scores. The biochemical outcomes after HDPPB and RP were compared via Kaplan-Meier analysis. RESULTS: Of the 110 patients analyzed, the mean ages, PSA, and Gleason biopsy scores were similar between the two cohorts. Among patients who underwent HDPPB, 20 patients (36.4%) had received adjuvant EBRT. Of this subsample, most patients (98.2%) had received adjuvant ADT for 3 months. Among patients with RP, 20 patients (36.4%) had received adjuvant EBRT, whereas 28 patients had received adjuvant ADT. The mean implanted seed numbers were 92.8, the mean D90 was 218.7 Gy, and the mean V100 was 96.1% after HDPPB. With regard to oncological outcomes, biochemical disease-free survival rates were similar between the two cohorts (82.6 vs. 81.1%, p = 0.982). Urethrorectal fistula developed in one patient with HDPPB. CONCLUSION: RP and HDPPB, using iodine-125 seeds with combined treatment modalities, exhibited similar biochemical recurrence-free survival rates among HiPCa patients. Further prospective studies with greater sample sizes and longer follow-up periods are needed to validate these results.

Placenta-derived mesenchymal stem cells have an immunomodulatory effect that can control acute graft-versus-host disease in mice.

BACKGROUND AND AIMS: Immunomodulatory properties of mesenchymal stem cells (MSCs) have been applied to reduce the incidence of graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation (HSCT). Among the various sources of MSCs that have immunomodulatory effects in vitro, only placenta-derived MSCs (PD-MSCs) have not been evaluated in an in vivo model of GVHD. In this study, we investigated the immunomodulatory properties of PD-MSCs in vitro and evaluated their clinical potential for controlling GVHD in an animal model. METHODS: A GVHD animal model was established by transplanting C57BL/6 donor bone marrow cells and spleen cells into lethally irradiated BALB/c recipient mice. To control GVHD, human PD-MSCs were transplanted into recipient mice (5 × 10(5) or 1 × 10(6) cells). RESULTS: PD-MSCs suppressed mitogen-stimulated T cell proliferation in vitro in a dose-dependent manner. Moreover, PD-MSCs inhibited cytokine secretion (interleukin-12, tumor necrosis factor-α and interferon-γ) of activated T cells. In vivo, the survival rate in the PD-MSC group (transplanted with 1 × 10(6) cells) was higher than that in the control group and histological scores were low in the PD-MSC group. CONCLUSION: We present the first evidence that human PD-MSCs can efficiently control GVHD in an HSCT in vivo model.

Pancreatoduodenectomy with uncut-Roux-en-Y reconstruction in patients with previous radical gastrectomy.

Gastric cancer is very common. Many patients have undergone radical gastric cancer surgery in Korea. Recently, the number of cases with secondary cancer occurring in other organs such as periampullary cancers is increasing as survival rate of gastric cancer patients increases. There are some clinical issues in managing patients with periampullary cancer who have undergone radical gastrectomy previously. Considering that pancreatoduodectomy (PD) has two phases (i.e., resection and reconstruction), it can be very complicated and controversial to perform safe and effective reconstruction following PD in patients with a previous radical gastrectomy. In this report, we present our experiences of uncut-Roux-en-Y fashioned reconstruction in PD for patients with a previous radical gastrectomy and discuss its technical characteristics and potential advantages.

Identification of dendritic cell precursor from the CD11c+ cells expressing high levels of MHC class II molecules in the culture of bone marrow with FLT3 ligand.

Dendritic cells (DCs) are readily generated from the culture of mouse bone marrow (BM) treated with either granulocyte macrophage-colony stimulating factor (GM-CSF) or FMS-like tyrosine kinase 3 ligand (FLT3L). CD11c+MHCII+ or CD11c+MHCIIhi cells are routinely isolated from those BM cultures and generally used as in vitro-generated DCs for a variety of experiments and therapies. Here, we examined CD11c+ cells in the BM culture with GM-CSF or FLT3L by staining with a monoclonal antibody 2A1 that is known to recognize mature or activated DCs. Most of the cells within the CD11c+MHCIIhi DC gate were 2A1+ in the BM culture with GM-CSF (GM-BM culture). In the BM culture with FLT3L (FL-BM culture), almost of all the CD11c+MHCIIhi cells were within the classical DC2 (cDC2) gate. The analysis of FL-BM culture revealed that a majority of cDC2-gated CD11c+MHCIIhi cells exhibited a 2A1-CD83-CD115+CX3CR1+ phenotype, and the others consisted of 2A1+CD83+CD115-CX3CR1- and 2A1-CD83-CD115-CX3CR1- cells. According to the antigen uptake and presentation, morphologies, and gene expression profiles, 2A1-CD83-CD115-CX3CR1- cells were immature cDC2s and 2A1+CD83+CD115-CX3CR1- cells were mature cDC2s. Unexpectedly, however, 2A1-CD83-CD115+CX3CR1+ cells, the most abundant cDC2-gated MHCIIhi cell subset in FL-BM culture, were non-DCs. Adoptive cell transfer experiments in the FL-BM culture confirmed that the cDC2-gated MHCIIhi non-DCs were precursors to cDC2s, i.e., MHCIIhi pre-cDC2s. MHCIIhi pre-cDC2s also expressed the higher level of DC-specific transcription factor Zbtb46 as similarly as immature cDC2s. Besides, MHCIIhi pre-cDC2s were generated only from pre-cDCs and common DC progenitor (CDP) cells but not from monocytes and common monocyte progenitor (cMoP) cells, verifying that MHCIIhi pre-cDC2s are close lineage to cDCs. All in all, our study identified and characterized a new cDC precursor, exhibiting a CD11c+MHCIIhiCD115+CX3CR1+ phenotype, in FL-BM culture.

Use of lysates from pooled human mononuclear cells to activate CD3 T cells in humanized mice with low human cell engraftment efficiency.

In regenerative medicine, humanized mice (hu-mice) are extremely valuable for verifying the cross talk between immune cells and therapeutic cells. Given the highly dynamic nature of the activities of immune cells, the in vitro platform does not allow for screening of their exact interactions with different therapeutic cells. By contrast, hu-mice have been widely applied for in vivo studies, especially those on immune rejection. However, the full reconstitution of lymphoid lineage cells in hu-mice remains to be realized. In this study, we investigated whether lysates from healthy donor-derived pooled mononuclear cells (MNCs) can promote the increase of lymphoid lineage cells in hu-mice. The pooled MNC lysate treatment of hu-mice possessing a low proportion of CD45 cells resulted in significant increases in CD3 cells and CD45 cells with the RO phenotype. The diverse epitopes from the pooled MNC lysates significantly induced the proportion of lymphoid lineage cells in the thymus and spleen after therapeutic cells with mismatched HLAs were co-injected into the hu-mice. These findings demonstrate the technical benefits of using pooled MNC lysates for reconstituting lymphoid lineage cells in hu-mice, providing a valuable in vivo platform for investigating the cross talk between lymphoid immune cells and therapeutic cells.

Effect of Elective Internal Mammary Node Irradiation on Disease-Free Survival in Women With Node-Positive Breast Cancer: A Randomized Phase 3 Clinical Trial.

IMPORTANCE: The benefit of internal mammary node irradiation (IMNI) for treatment outcomes in node-positive breast cancer is unknown. OBJECTIVE: To investigate whether the inclusion of IMNI in regional nodal irradiation improves disease-free survival (DFS) in women with node-positive breast cancer. DESIGN, SETTING, AND PARTICIPANTS: This multicenter, phase 3 randomized clinical trial was conducted from June 1, 2008, to February 29, 2020, at 13 hospitals in South Korea. Women with pathologically confirmed, node-positive breast cancer after breast-conservation surgery or mastectomy with axillary lymph node dissection were eligible and enrolled between November 19, 2008, and January 14, 2013. Patients with distant metastasis and those who had received neoadjuvant treatment were excluded. Data analyses were performed according to the intention-to-treat principle. INTERVENTIONS: All patients underwent regional nodal irradiation along with breast or chest wall irradiation. They were randomized 1:1 to receive radiotherapy either with IMNI or without IMNI. MAIN OUTCOMES AND MEASURES: The primary end point was the 7-year DFS. Secondary end points included the rates of overall survival, breast cancer-specific survival, and toxic effects. RESULTS: A total of 735 women (mean [SD] age, 49.0 [9.1] years) were included in the analyses, of whom 373 received regional nodal irradiation without IMNI and 362 received regional nodal irradiation with IMNI. Nearly all patients underwent taxane-based adjuvant systemic treatment. The median (IQR) follow-up was 100.4 (89.7-112.1) months. The 7-year DFS rates did not significantly differ between the groups treated without IMNI and with IMNI (81.9% vs 85.3%; hazard ratio [HR], 0.80; 95% CI, 0.57-1.14; log-rank P = .22). However, an ad hoc subgroup analysis showed significantly higher DFS rates with IMNI among patients with mediocentrally located tumors. In this subgroup, the 7-year DFS rates were 81.6% without IMNI vs 91.8% with IMNI (HR, 0.42; 95% CI, 0.22-0.82; log-rank P = .008), and the 7-year breast cancer mortality rates were 10.2% without IMNI vs 4.9% with IMNI (HR, 0.41; 95% CI, 0.17-0.99; log-rank P = .04). No differences were found between the 2 groups in the incidence of adverse effects, including cardiac toxic effects and radiation pneumonitis. CONCLUSIONS AND RELEVANCE: This randomized clinical trial found that including IMNI in regional nodal irradiation did not significantly improve the DFS in patients with node-positive breast cancer. However, patients with medially or centrally located tumors may benefit from the use of IMNI. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04803266.

Dummy run quality assurance study in the Korean Radiation Oncology Group 19 - 09 multi-institutional prospective cohort study of breast cancer.

BACKGROUND: The Korean Radiation Oncology Group (KROG) 19 - 09 prospective cohort study aims to determine the effect of regional nodal irradiation on regional recurrence rates in ypN0 breast cancer patients. Dosimetric variations between radiotherapy (RT) plans of participating institutions may affect the clinical outcome of the study. We performed this study to assess inter-institutional dosimetric variations by dummy run. METHODS: Twelve participating institutions created RT plans for four clinical scenarios using computed tomography images of two dummy cases. Based on a reference structure set, we analyzed dose-volume histograms after collecting the RT plans. RESULTS: We found variations in dose distribution between institutions, especially in the regional nodal areas. Whole breast and regional nodal irradiation (WBI + RNI) plans had lower inter-institutional agreement and similarity for 95% isodose lines than WBI plans. Fleiss's kappa values, which were used to measure inter-institutional agreement for the 95% isodose lines, were 0.830 and 0.767 for the large and medium breast WBI plans, respectively, and 0.731 and 0.679 for the large and medium breast WBI + RNI plans, respectively. There were outliers in minimum dose delivered to 95% of the structure (D95%) of axillary level 1 among WBI plans and in D95% of the interpectoral region and axillary level 4 among WBI + RNI plans. CONCLUSION: We found inter-institutional and inter-case variations in radiation dose delivered to target volumes and organs at risk. As KROG 19 - 09 is a prospective cohort study, we accepted the dosimetric variation among the different institutions. Actual patient RT plan data should be collected to achieve reliable KROG 19 - 09 study results.