Extracellular Vesicles Derived from Three-Dimensional-Cultured Human Umbilical Cord Blood Mesenchymal Stem Cells Prevent Inflammation and Dedifferentiation in Pancreatic Islets.

It is unclear whether extracellular vesicles (EVs) from mesenchymal stem cells (MSCs) have a direct protective effect on pancreatic islets. In addition, whether culturing MSCs in three dimensions (3D) instead of a monolayer (2D) can induce changes in the cargo of EVs that facilitate the polarization of macrophages into an M2 phenotype has not been investigated. We sought to determine whether EVs from MSCs cultured in 3D can prevent inflammation and dedifferentiation in pancreatic islets and, if so, whether the protective effect is superior to that of EVs from 2D MSCs. Human umbilical cord blood- (hUCB-) MSCs cultured in 3D were optimized according to cell density, exposure to hypoxia, and cytokine treatment based on the ability of the hUCB-MSC-derived EVs to induce the M2 polarization of macrophages. Islets isolated from human islet amyloid polypeptide (hIAPP) heterozygote transgenic mice were cultured in serum-deprived conditions with hUCB-MSC-derived EVs. EVs derived from 3D hUCB-MSCs had more abundant microRNAs involved in M2 polarization of macrophages and had an enhanced M2 polarization ability on macrophages, which was optimized when the 3D culture condition was 2.5 × 104 cells per spheroid without preconditioning with hypoxia and cytokine exposure. When islets isolated from hIAPP heterozygote transgenic mice were cultured in serum-deprived conditions with hUCB-MSC-derived EVs, the EVs derived from 3D hUCB-MSCs suppressed the expression of proinflammatory cytokines and caspase-1 in pancreatic islets and increased the proportion of M2-polarized islet-resident macrophages. They improved glucose-stimulated insulin secretion, reduced the expression of Oct4 and NGN3, and induced the expression of Pdx1 and FoxO1. The greater suppression of IL-1ß, NLRP3 inflammasome, caspase-1, and Oct4 and induction of Pdx1 and FoxO1 were found in islets cultured with the EVs derived from 3D hUCB-MSCs. In conclusion, EVs derived from 3D hUCB-MSCs optimized for M2 polarization attenuated nonspecific inflammation and preserved ß-cell identity of pancreatic islets.

Protective effect of a novel clinical-grade small molecule necrosis inhibitor against oxidative stress and inflammation during islet transplantation.

Inhibition of mitochondrial reactive oxygen species (ROS) and subsequent damage-associated molecular patterns (DAMPs)-induced inflammatory responses could be a novel target in clinical islet transplantation. We investigated the protective effects of NecroX-7, a novel clinical-grade necrosis inhibitor that specifically targets mitochondrial ROS, against primary islet graft failure. Islets from heterozygote human islet amyloid polypeptide transgenic (hIAPP+/- ) mice and nonhuman primates (NHPs) were isolated or cultured with or without NecroX-7 in serum-deprived medium. Supplementation with NecroX-7 during hIAPP+/- mouse islet isolation markedly increased islet viability and adenosine triphosphate content, and attenuated ROS, transcription of c-Jun N-terminal kinases, high mobility group box 1, interleukin-1beta (IL-1 ß ), IL-6, and tumor necrosis factor-alpha. Supplementation of NecroX-7 during serum-deprived culture also protected hIAPP+/- mouse and NHP islets against impaired viability, serum deprivation-induced ROS, proinflammatory response, and accumulation of toxic IAPP oligomer. Supplementation with NecroX-7 during isolation or serum-deprived culture of hIAPP+/- mouse and NHP islets also improved posttransplant glycemia in the recipient streptozotocin-induced diabetic hIAPP-/- mice and BALB/c-nu/nu mice, respectively. In conclusion, pretransplant administration of NecroX-7 during islet isolation and serum-deprived culture suppressed mitochondrial ROS injury, generation of DAMPs-induced proinflammatory responses, and accumulation of toxic IAPP oligomers ex vivo, and improved posttransplant glycemia in vivo.

Mir214-3p and Hnf4a/Hnf4α reciprocally regulate Ulk1 expression and autophagy in nonalcoholic hepatic steatosis.

Macroautophagy/autophagy, a self-degradative process, regulates metabolic homeostasis in response to various stress conditions and is a therapeutic target for nonalcoholic fatty liver disease. We found that autophagic activity was inhibited as a result of a significant reduction in the expression of autophagy-related genes such as Ulk1 in a mouse model and patients with fatty liver. This downregulation was caused by increased Mir214-3p levels and decreased Hnf4a/Hnf4α mRNA levels in hepatocytes. Mir214-3p suppressed Ulk1 expression through direct binding at a 3' untranslated region sequence. Hnf4a directly activated transcription of Ulk1. We investigated lipid accumulation and the expression of autophagy-related genes in the livers of mice treated with anti-Mir214-3p. Hepatic steatosis was alleviated, and Ulk1 mRNA levels were significantly increased by locked nucleic acid-mediated Mir214-3p silencing. Additionally, autophagosome formation and MAP1LC3/LC3-II protein levels were increased, indicating an increase in autophagic activity. Interestingly, suppression of Mir214-3p did not ameliorate fatty liver under Ulk1 suppression, suggesting that reduced Mir214-3p levels mitigate hepatic steatosis through upregulation of Ulk1. These results demonstrate that inhibition of Mir214-3p expression ameliorated fatty liver disease through increased autophagic activity by increasing the expression of Ulk1. Thus, Mir214-3p is a potential therapeutic target for nonalcoholic fatty disease.Abbreviations: AMPK: adenosine monophosphate-activated protein kinase; ATG: autophagy-related; ChIP: chromatin immunoprecipitation; CTSB: cathepsin B; CTSL: cathepsin L; CQ: chloroquine; HFD: high-fat diet; HNF4A: hepatocyte nuclear factor 4, alpha; IF: immunofluorescence; IHC: immunohistochemistry; LDs: lipid droplets; Leup: leupeptin; LFD: low-fat diet; LNA: locked nucleic acid; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; miRNA: microRNA; MTOR: mechanistic target of rapamycin kinase; NAFLD: non-alcoholic fatty liver disease; NASH: non-alcoholic steatohepatitis; PCR: polymerase chain reaction; TEM: transmission electron microscopy; TF: transcription factor; TLDA: TaqMan low-density array; ULK1: unc-51 like kinase 1; UTR: untranslated region.

Clinical significance of Ki-67 and p53 expression in curatively resected non-small cell lung cancer.

The aim of this study is to explore the association of Ki-67 and p53 expression with prognosis in non-small cell lung cancer (NSCLC) patients who underwent curative resection. We retrospectively identified 116 consecutive patients with stages I-III NSCLC who underwent curative resection at a single center from January 2007 to December 2012. Ki-67 and p53 expression was assessed by immunohistochemistry. Data on clinicopathologic features and survival were collected retrospectively. Ki-67 expression in 109 samples and p53 expression in 115 patients were analyzed. According to the results, 108 patients (99 %) showed at least some expression of Ki-67. The median Ki-67 expression level was 30 %. Positive p53 expression was observed in 91 (79 %) patients. Higher Ki-67 expression (>40 %) was significantly more frequent in male (26 vs. 4 % in female, p=0.002), ever-smoker (31 vs. 10 % in never-smoker, p=0.024), and non-adenocarcinoma (30 vs. 11 % of adenocarcinoma, p=0.012) patients. In univariable analysis, median disease-free survival (DFS) was shorter with higher Ki-67 expression (16.1 vs. 61.9 months in those with lower Ki-67 expression, p=0.005), and p53 expression did not show an association with DFS. Among 42 patients with stage I NSCLC who did not receive adjuvant chemotherapy, DFS was significantly worse in patients with higher Ki-67 expression (2-year DFS rate 57 vs. 88 %, p=0.018). In a Cox regression model, higher Ki-67 expression (>40 %) was a significant independent prognostic factor associated with poorer DFS (HR 2.9, 95 % CI 1.3-6.2) along with TNM stage and age. Higher Ki-67 expression (>40 %) showed an independent association with shorter DFS in NSCLC patients who underwent curative resection.

Retained placenta accreta after a first-trimester abortion manifesting as an uterine mass.

Placenta accreta during the first trimester of pregnancy is rare. Only a few cases of placenta accreta manifesting as a uterine mass have been published. Most patients with placenta accreta present with vaginal bleeding during or after pregnancy. This report describes a patient with placenta accreta that caused vaginal bleeding three years after a first trimester abortion. The patient had regular menstruation for three years after the abortion. Initially endometrial cancer or a uterine myoma with degeneration was suspected. This is the first report of a placenta accreta detected as a uterine mass long after a first trimester abortion with delayed vaginal bleeding.

Intramyometrial uterine cysts with special reference to ultrastructural findings: report of two cases.

An intramyometrial cyst is an extremely rare condition that is characterized by a benign, endometrial, epithelium-lined cyst within the thickened myometrium. Few cases of intramyometrial cysts have currently been reported in the literature, with or without microscopic description. We have experienced two cases of intramyometrial cysts. One was a 6.5 cm-sized cyst and the other was a 3.0 cm-sized cyst accompanied by adenomyosis. Case 1 was a 41-year-old female and case 2 was a 51-year-old female who had been suffering from menorrhagia for several days. A total hysterectomy was performed for both women. Histological examination showed that the huge cysts were composed of single-layered, ciliated, cuboidal epithelia surrounded by diffusely thickened myometrium. Ultrastructural examination of case 1 showed the lining cells of the cyst consisted of the basalis-type endometrial epithelial cells that have surface microvilli. The cells were surrounded by a duplicated basal lamina and joined by well-formed desmosomes. We report here on two cases of intramyometrial cyst with special reference to the ultrastructural examination, and we discuss the pathogenesis of this rare lesion.

Recurrent and metastatic trichilemmal carcinoma of the skin over the thigh: a case report.

Trichilemmal carcinoma (TC) is an uncommon cutaneous neoplasm that develops from the external root sheath of the hair follicle. It is considered to be a low-grade carcinoma with low metastatic potential. Local recurrence and metastasis are rare after surgical excision. We report here on a case of metastatic TC in the skin over the thigh, and this tumor was treated with cisplatin and cyclophosphamide combination chemotherapy.