Sa-Hyun Kim

Characterization, in vitro cytotoxicity assessment, and in vivo visualization of multimodal, RITC-labeled, silica-coated magnetic nanoparticles for labeling human cord blood-derived mesenchymal stem cells.

UNLABELLED Live imaging is a powerful technique that can be used to characterize the fate and location of stem cells in animal models. Here we investigated the characteristics and in vitro cytotoxicity of human mesenchymal stem cells (MSCs) labeled with silica-coated magnetic nanoparticles incorporating rhodamine B isothiocyanate, MNPs@SiO2(RITC). We also conducted various in vivo-uptake tests with nanoparticle-labeled human MSCs. MNPs@SiO2(RITC) showed photostability against ultraviolet light exposure and were nontoxic to human MSCs, based on the MTT, apoptosis, and cell cycle arrest assays. In addition, MNPs@SiO2(RITC) did not affect the surface phenotype or morphology of human MSCs. We also demonstrated that MNPs@SiO2(RITC) have stable retention properties in MSCs in vitro. Furthermore, using optical and magnetic resonance imaging, we successfully detected a visible signal from labeled human MSCs that were transplanted into NOD.CB17-Prkdc(SCID) (NOD-SCID) mice. These results demonstrate that MNPs@SiO2(RITC) are biocompatible and useful tools for human MSC labeling and bioimaging. FROM THE CLINICAL EDITOR The characteristics and in vitro cytotoxicity of human mesenchymal stem cells (MSCs) labeled with silica-coated magnetic nanoparticles incorporating rhodamine B isothiocyanate, RITC were investigated in this study. RITC showed photostability against ultraviolet light exposure and was nontoxic to human MSCs. Using both optical and magnetic resonance imaging, successful detection of signal from labeled human MSCs transplanted into mice is demonstrated.

Development of Functional Human Immune System With the Transplantations of Human Fetal Liver/Thymus Tissues and Expanded Hematopoietic Stem Cells in RAG2―/―γc―/― MICE

BACKGROUND There is an increasing need for suitable animal models for the study of the human immune system and disease. The purpose of this study was to develop a practical in vivo model of human immune cell repopulation using ex vivo expanded human fetal liver-derived CD34(+) hematopoietic stem cells and subrenally coimplanted fetal liver/thymus tissues. METHODS Freshly isolated fetal liver-derived CD34(+) hematopoietic stem cells were frozen until injected and ex vivo expanded with various cytokines for 7 days. After fetal liver/thymus tissues were subrenally coimplanted into preirradiated Rag2(-/-)gamma(c)(-/-) mice, frozen and ex vivo expanded CD34(+) cells were injected intravenously. The peripheral blood of the mice was monitored for the detection of human cell engraftment using flow cytometry. Then we confirmed human T-cell function by in vitro function assays. RESULTS After fetal liver/thymus tissues were coimplanted into the irradiated Rag2(-/-)gamma(c)(-/-) mice, with frozen and ex vivo expanded CD34(+) hematopoietic stem cells, human cell engraftments were determined using hCD45 and multilineage markers. The cultured cells with the cytokine combination of stem cell factor, thrombopoietin, Flk2/Flk3 ligand (FL), and interleukin-3 showed stable and long-term engraftment compared to other combinations. The ex vivo expanded human fetal liver-derived CD34(+) hematopoietic stem cells, under our culture conditions, accomplished a large volume of expanded cells that were sustained, demonstrating self-renewal of the evaluated markers, which may have indicated long- term repopulation activity. CONCLUSION The results of this study demonstrated a practical mouse model of expanded human immune cells especially T cells in Rag2(-/-)gamma(c)(-/-) mice.

Cyanidin 3-O-Glucoside Reduces Helicobacter pylori VacA-Induced Cell Death of Gastric KATO III Cells through Inhibition of the SecA Pathway

Two key virulence factors of Helicobacter pylori are the secreted virulent proteins of vacuolating toxin A (VacA) and cytotoxin associated protein A (CagA) which lead to damages of gastric epithelial cells. We previously identified that the cyanidin 3-O-glucoside (C3G) inhibits the secretion of both VacA and CagA. In the current report, we show that C3G inhibits VacA secretion in a dose-dependent manner by inhibiting secretion system subunit protein A (SecA) synthesis. As SecA is involved in translocation of bacterial proteins, we predicted that inhibition of the SecA pathway by C3G should decrease H. pylori-induced cell death. To test this hypothesis, the human gastric cell line KATO III cells were co-cultured with H. pylori 60190 (VacA+/CagA+) and C3G. We found that C3G treatment caused a decrease in activation of the pro-apoptotic proteins caspase-3/-8 in H. pylori-infected cells leading to a decrease in cell death. Our data suggest that consumption of foods containing anthocyanin may be beneficial in reducing cell damage due to H. pylori infection.

Inhibitory effect of piperine on Helicobacter pylori growth and adhesion to gastric adenocarcinoma cells

BackgroundPiperine is a compound comprising 5-9% of black pepper (Piper nigrum), which has a variety of biological roles related to anticancer activities. Helicobacter pylori has been classified as a gastric carcinogen, because it causes gastritis and gastric cancer by injecting the virulent toxin CagA and translocating VacA. The present study investigated the inhibitory action of piperine on H. pylori growth and adhesion.MethodsInhibition of H. pylori growth was determined by the broth macrodilution method, and adhesion to gastric adenocarcinoma cells validated by urease assay. Motility test was performed by motility agar and the expression of adhesion gene and flagellar gene in response to the piperine treatment was assessed by RT-PCR and immunoblotting.ResultsAdministrated piperine suppressed the level of H. pylori adhesion to gastric adenocarcinoma cells in a dose dependent manner and the inhibition was statistically significant as determined by Student’s t-test. In addition, piperine treatment effects on the flagellar hook gene flgE and integral membrane component of the export apparatus gene flhA expression to be suppressed and piperine diminished the H. pylori motility.ConclusionsflhA, encodes an integral membrane component of the export apparatus, which is also one of the regulatory protein in the class 2 genes expression and flgE is one of them that encodes hook part of the flagella. Suppression of both genes, leads to less motility results in the organism attracted less towards to the gastric epithelial cells might be the possible reason in the adhesion inhibition. To our knowledge, this is the first report published on the inhibitory effects of piperine against the adhesion of H. pylori to gastric adenocarcinoma cells.

The Effect of Human Fetal Liver-Derived Mesenchymal Stem Cells on CD34+ Hematopoietic Stem Cell Repopulation in NOD/Shi-scid/IL-2Rãnull Mice

Mesenchymal stem cells (MSCs) are progenitors that are capable of differentiating into mesenchymal tissues. They are known to support allogeneic hematopoietic stem cell transplantation by facilitating engraftment without increasing the risk of graft-versus-host disease. We optimized culture conditions for human fetal liver-derived MSCs (hFL-MSCs) to investigate the role of hFL-MSCs on repopulation of hematopoietic stem cells in NOD/Shi-scid/IL-2Rγ(null) (NOG) mice using CD34(+) hematopoietic stem cells (HSCs) derived from umbilical cord blood (UCB). FL-MSCs and CD34(+) HSCs were prepared from fetal liver and UCB, respectively. Twenty-four hours after irradiation, CD34(+) HSCs and hFL-MSCs were injected intravenously and intratibially into NOG mice. During 24 weeks posttransplantation, engraftment levels of human cells were analyzed in bone marrow, peripheral blood, and spleen of transplanted mice by flow cytometry. hFL-MSCs showed a fibroblast-like morphology and immunophenotypic characteristics appropriate for MSCs. hFL-MSCs prolonged the survival of NOG mice that had been cotransplanted with UCB CD34(+) cells. Fluorescence-activated cell-sorting analysis showed that engraftment of human cells was increased by cotransplantation of hFL-MSCs. However, significant enhancement of human cell engraftment was not detected in NOG mice regardless of the number of cotransplanted MSCs. Although survival of repopulating NOG mice and engraftment of human cells were prolonged by cotransplantation of hFL-MSCs, 8.0 × 10(6) MSCs were not sufficient to increase HSC engraftment in irradiated NOG mice in vivo.

Cartilage oligomeric matrix protein-angiopoientin-1 enhances angiogenesis of isolated islet and maintains normoglycemia following transplantation.

Islet transplantation (ITx) has potential as a therapy for patients with type 1 diabetes. For successful engraftment and insulin independence, the transplanted islets must establish an adequate, stable blood supply. Angiopoientin-1 (Ang1) is a specific growth factor that induces vascularization via the Tie2 or Tie1 receptor. In this study, we used an in vitro angiogenesis assay to evaluate islet function following transplantation and the effect of the Ang1 variant cartilage oligomeric matrix protein (COMP) Ang1 on isolated islets. The enhanced function of islets transduced with COMP-Ang1 was also confirmed in a streptozotocin (STZ)-induced diabetic mice model. In a three-dimensional collagen-based culture system, the transduction of COMP-Ang1 into islets significantly increased angiogenesis compared with the bacterial-β-galactosidase (LacZ)-transduced controls and an intact, nontransduced islet negative control group. COMP-Ang1 transduced islets also attenuated hyperglycemia in syngeneic diabetic C57BL/6 mice and enhanced glucose tolerance by areas under the curves of intraperitoneal glucose tolerance tests. These findings demonstrated the capacity of COMP-Ang1 to promote revascularization in cultured islets, which may contribute to successful transplantation in vivo.

Regulatory Effects of Black Rice Extract on Helicobacter pylori Infection-Induced Apoptosis

SCOPE Black rice extract (BRE) contains cyanidin 3-O-glucoside (C3G), an anthocyanin, as the major component. In this study, we found that BRE inhibits the mRNA and protein expression of genes encoding cytotoxin-associated protein A (cagA) and vacuolating protein A (vacA) in Helicobacter pylori 60190 strain. METHODS AND RESULTS We performed RT-PCR and western blotting to show that BRE inhibits the mRNA and protein expression of SecA. Because SecA is involved in VacA export in bacteria, our result suggests a positive correlation between BRE-induced inhibition of secA expression and VacA secretion. Further, we perform MTT assay and flow cytometry to show that BRE decreases the apoptosis of H. pylori-infected KATO III cells. Finally, we perform western blotting to show that the cell-protective effect of BRE is associated with decreased levels of active proapoptotic proteins caspases and PARP and increased levels of antiapoptotic proteins survivin and XIAP in H. pylori-infected cells. CONCLUSION Thus, our results indicate that BRE acts as a potent inhibitor of the biogenesis of H. pylori virulence proteins and decreases the apoptosis of H. pylori-infected cells. Moreover, our results suggest that BRE can be used to exert beneficial effects in patients with gastroduodenal diseases caused by H. pylori.

The first report of NDM-5–producing uropathogenic Escherichia coli isolates in South Korea

•We have described the first report of NDM-5–producing uropathogenic Escherichia coli clinical isolates in South Korea.•Three uropathogenic Escherichia coli (UPEC) strains out of 561 isolates showed resistance to imipenem.•The 3 carbapenem-resistant UPEC isolates belonged to phylogenetic group B1 and ST 101.•The 3 isolates carried a blaNDM-5, blaCTX-M, and blaTEM; however, rmtB gene was detected only in the last 2 isolates.

Anti-inflammatory effects of Kaempferol on Helicobacter pylori-induced inflammation

ABSTRACT Inflammation induced by Helicobacter pylori infection related to gastric carcinogenesis. In this study, we have investigated the anti-inflammatory effect and its mechanism of kaempferol in the inflammatory response caused by H. pylori infection in vitro. We found that kaempferol reduced the expression of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-8) and production of IL-8 in AGS cells. In addition, kaempferol suppressed translocation of cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA) of H. pylori to AGS cells. It was due to decreased transcription of type IV secretion system (T4SS) components involved in CagA injection and secretion system subunit protein A (SecA) of type V secretion system (T5SS) involved in VacA secretion by kaempferol. In conclusion, kaempferol shows the anti-inflammatory effect by suppressing the translocation of CagA and VacA proteins and leading to the down-regulation of pro-inflammatory cytokines. Abbreviations: CagA: cytotoxin-associated gene A; VacA: vacuolating cytotoxin A; T4SS: type IV secretion systems; SecA: secretion system subunit protein A; T5SS: type V secretion system; Graphical Abstract Kaempferol has an anti-inflammatory effect on H. pylori infection by suppressing the translocation of CagA and VacA proteins.