Kyoung-Ju Song

Rapid and safe learning of robotic gastrectomy for gastric cancer: Multidimensional analysis in a comparison with laparoscopic gastrectomy

BACKGROUND The learning curve of robotic gastrectomy has not yet been evaluated in comparison with the laparoscopic approach. We compared the learning curves of robotic gastrectomy and laparoscopic gastrectomy based on operation time and surgical success. METHODS We analyzed 172 robotic and 481 laparoscopic distal gastrectomies performed by single surgeon from May 2003 to April 2009. The operation time was analyzed using a moving average and non-linear regression analysis. Surgical success was evaluated by a cumulative sum plot with a target failure rate of 10%. Surgical failure was defined as laparoscopic or open conversion, insufficient lymph node harvest for staging, resection margin involvement, postoperative morbidity, and mortality. RESULTS Moving average and non-linear regression analyses indicated stable state for operation time at 95 and 121 cases in robotic gastrectomy, and 270 and 262 cases in laparoscopic gastrectomy, respectively. The cumulative sum plot identified no cut-off point for surgical success in robotic gastrectomy and 80 cases in laparoscopic gastrectomy. Excluding the initial 148 laparoscopic gastrectomies that were performed before the first robotic gastrectomy, the two groups showed similar number of cases to reach steady state in operation time, and showed no cut-off point in analysis of surgical success. CONCLUSIONS The experience of laparoscopic surgery could affect the learning process of robotic gastrectomy. An experienced laparoscopic surgeon requires fewer cases of robotic gastrectomy to reach steady state. Moreover, the surgical outcomes of robotic gastrectomy were satisfactory.

Effect of Therapeutic Chemical Agents In Vitro and on Experimental Meningoencephalitis Due to Naegleria fowleri

ABSTRACT Naegleria fowleri is a ubiquitous, pathogenic free-living amoeba; it is the most virulent Naegleria species and causes primary amoebic meningoencephalitis (PAME) in laboratory animals and humans. Although amphotericin B is currently the only agent available for the treatment of PAME, it is a very toxic antibiotic and may cause many adverse effects on other organs. In order to find other potentially therapeutic agents for N. fowleri infection, the present study was undertaken to evaluate the in vitro and in vivo efficacies of miltefosine and chlorpromazine against pathogenic N. fowleri. The result showed that the growth of the amoeba was effectively inhibited by treatment with amphotericin B, miltefosine, and chlorpromazine. When N. fowleri trophozoites were treated with amphotericin B, miltefosine, and chlorpromazine, the MICs of the drug were 0.78, 25, and 12.5 μg/ml, respectively, on day 2. In experimental meningoencephalitis of mice that is caused by N. fowleri, the survival rates of mice treated with amphotericin B, miltefosine, and chlorpromazine were 40, 55, and 75%, respectively, during 1 month. The average mean time to death for the amphotericin B, miltefosine, and chlorpromazine treatments was 17.9 days. In this study, the effect of drugs was found to be optimal when 20 mg/kg was administered three times on days 3, 7, and 11. Finally, chlorpromazine had the best therapeutic activity against N. fowleri in vitro and in vivo. Therefore, it may be a more useful therapeutic agent for the treatment of PAME than amphotericin B.

Cytopathic changes and pro-inflammatory cytokines induced by Naegleria fowleri trophozoites in rat microglial cells and protective effects of an anti-Nfa1 antibody

Naegleria fowleri, a free‐living amoeba, causes fatal primary amoebic meningoencephalitis in experimental animals and humans. The nfa1 gene (360 bp) was previously cloned from a cDNA library of pathogenic N. fowleri by immunoscreening, and produced a 13·1‐kDa recombinant protein that showed pseudopodia‐specific localization by immunocytochemistry. On the basis of an idea that the pseudopodia‐specific Nfa1 protein seems to be involved in the pathogenicity of N. fowleri, the cytopathic activity of N. fowleri trophozoites co‐cultured with rat microglial cells was observed, and the effects of an anti‐Nfa1 antibody in a co‐culture system were elucidated. Using light, scanning and transmission electron microscopy, it was seen that N. fowleri trophozoites in contact with microglial cells produced vigorous pseudopodia and a food‐cup structure. Microglial cells were destroyed by N. fowleri trophozoites as seen from necrotic cell death in a time‐dependent manner. In a51Cr release assay, N. fowleri showed 17·8%, 24·9%, 54·6% and 98% cytotoxicity against microglial cells at 3, 6, 12 and 24 h post‐incubation, respectively. However, when anti‐Nfa1 antibody was added in a coculture system, N. fowleri cytotoxicity was reduced to 15·5%, 20·3%, 46·7% and 66·9%, respectively. Moreover, microglial cells co‐cultured with N. fowleri trophozoites secreted the pro‐inflammatory cytokines, TNF‐α, IL‐1β and IL‐6. In the presence of anti‐Nfa1 antibody, the secretion of TNF‐α was slightly, but not significantly, decreased.

Expression of cysteine proteinase of Clonorchis sinensis and its use in serodiagnosis of clonorchiasis

A gene encoding cysteine proteinase from Clonorchis sinensis has been cloned and expressed in Escherichia coli. The cysteine proteinase cDNA fragment was amplified by reverse transcription–polymerase chain reaction using degenerate oligonucleotide primers derived from conserved active site of cysteine proteinases. The 5′ and 3′ regions of the gene were amplified using rapid amplification of cDNA ends. The cloned gene has an open reading frame of 696 bp and deduced amino acid sequence of 232. Sequence analysis and alignment showed significant homologies with the eukaryotic cysteine proteinases and conservation of the Cys, His, and Asp residues that form the catalytic triad. Analysis of the expressed protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the molecular weight of the protein was approximately 28.5 kDa. Proteolytic activity of the expressed protein was inhibited by cysteine proteinase inhibitors such as l-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane, iodoacetic acid, and leupeptin. The expressed protein showed biochemical properties similar to those of cysteine proteinases of other parasites. The expressed protein strongly reacted with the sera from patients with clonorchiasis but not with the sera from patients with paragonimiasis, fascioliasis, cysticercosis, and sparganosis, or with sera from normal human controls. These results suggest that the expressed protein may be valuable as a specific diagnostic material for the immunodiagnosis of clonorchiasis.

Role of the Nfa1 Protein in Pathogenic Naegleria fowleri Cocultured with CHO Target Cells

ABSTRACT Naegleria fowleri, a free-living amoeba, exists as a virulent pathogen which causes fatal primary amoebic meningoencephalitis in experimental animals and humans. Using infected and immune mouse sera, we previously cloned an nfa1 gene from a cDNA library of N. fowleri by immunoscreening. The nfa1 gene (360 bp) produced a recombinant 13.1-kDa protein, and the Nfa1 protein showed pseudopodium-specific immunolocalization on a trophozoite of N. fowleri. In this study, the role of the Nfa1 protein as a cell contact mechanism of N. fowleri cocultured with target cells was observed by an immunofluorescence assay with an anti-Nfa1 polyclonal antibody. Using confocal microscopic findings, the Nfa1 protein was located on the pseudopodia of N. fowleri trophozoites. The Nfa1 protein in N. fowleri trophozoites cocultured with CHO target cells was also located on pseudopodia, as well as in a food cup formed as a phagocytic structure in close contact with target cells. The amount of nfa1 mRNA of N. fowleri was strongly increased 6 h after coculture.

Expression of the nfa1 Gene Cloned from Pathogenic Naegleria fowleri in Nonpathogenic N. gruberi Enhances Cytotoxicity against CHO Target Cells In Vitro

ABSTRACT The pathogenic amoeba Naegleria fowleri has a 360-bp nfa1 gene that encodes the Nfa1 protein (13.1 kDa), which is located in the pseudopodia of the amoeba, and an anti-Nfa1 antibody reduces N. fowleri-induced mammalian-cell cytotoxicity in vitro. In contrast, an anti-Nfa1 antibody cannot detect Nfa1 protein expression in the nonpathogenic amoeba Naegleria gruberi, which also possesses the nfa1 gene. In the present study, the nfa1 gene cloned from pathogenic N. fowleri was transfected into nonpathogenic N. gruberi to determine whether it was related to pathogenicity. The nfa1 gene was initially inserted into a eukaryotic transfection vector, pEGFP-C2, containing a cytomegalovirus promoter and the green fluorescent protein (GFP) gene, and was designed as pEGFP-C2/nfa1UTR (nfa1UTR contains 5′ upstream regions, the nfa1 open reading frame, and 3′ downstream regions). After transfection, the green fluorescence was observed in the cytoplasm of N. gruberi trophozoites. These transfectants were preserved for more than 9 months after selection. The transfected nfa1 gene was observed by PCR using nfa1- and vector-specific primers in the genomic DNA of N. gruberi transfected with the pEGFP-C2/nfa1UTR vector. In addition, the nfa1 and GFP genes were identified by reverse transcription-PCR in transgenic N. gruberi. The Nfa1 protein expressed in transgenic N. gruberi was identified as a 13.1-kDa band by Western blotting using an anti-Nfa1 antibody. Finally, N. gruberi transfected with the pEGFP-C2/nfa1UTR vector was found to have enhanced cytotoxicity against CHO cells compared with naïve N. gruberi.

Reactive oxygen species‐dependent necroptosis in Jurkat T cells induced by pathogenic free‐living Naegleria fowleri

Naegleria fowleri, a free‐living amoeba, is the causative pathogen of primary amoebic meningoencephalitis in humans and experimental mice. N. fowleri is capable of destroying tissues and host cells through lytic necrosis. However, the mechanism by which N. fowleri induces host cell death is unknown. Electron microscopy indicated that incubation of Jurkat T cells with N. fowleri trophozoites induced necrotic morphology of the Jurkat T cells. N. fowleri also induced cytoskeletal protein cleavage, extensive poly (ADP‐ribose) polymerase hydrolysis and lactate dehydrogenase (LDH) release. Although no activation of caspase‐3 was observed in Jurkat T cells co‐incubated with amoebae, intracellular reactive oxygen species (ROS) were strongly generated by NADPH oxidase (NOX). Pretreating cells with necroptosis inhibitor necrostatin‐1 or NOX inhibitor diphenyleneiodonium chloride (DPI) strongly inhibited amoeba‐induced ROS generation and Jurkat cell death, whereas pan‐caspase inhibitor z‐VAD‐fmk did not. N. fowleri‐derived secretory products (NfSP) strongly induced intracellular ROS generation and cell death. Necroptotic effects of NfSP were effectively inhibited by pretreating NfSP with proteinase K. Moreover, NfSP‐induced LDH release and intracellular ROS accumulation were inhibited by pretreating Jurkat T cells with DPI or necrostatin‐1. These results suggest that N. fowleri induces ROS‐dependent necroptosis in Jurkat T cells.

NOX1 participates in ROS-dependent cell death of colon epithelial Caco2 cells induced by Entamoeba histolytica

Entamoeba histolytica, which causes amebic colitis and occasional liver abscesses in humans, can induce host cell death through apoptosis and necrosis. Recently, we have demonstrated that E. histolytica can induce cell death in neutrophils via diphenyleneiodonium-sensitive NADPH oxidase (NOX)-derived reactive oxygen species (ROS). Although there are enzyme systems similar to the phagocyte NADPH oxidase system in many non-phagocytic cell types, the signaling role of NOX-derived ROS in cell death of human colon epithelial cells induced by E. histolytica remains obscure. Incubation of colon epithelial Caco2 tumor cell lines with amebic trophozoites resulted in intracellular ROS generation and cell death in a caspase-independent manner. Pretreatment with DPI, an inhibitor of NOX, strongly decreased E. histolytica-induced cell death in Caco2 cells. As identified by RT-PCR, NOX1 transcripts were highly expressed in Caco2 cells. siRNA-mediated suppression of NOX1 protein significantly inhibited E. histolytica-induced cell death and ROS response in Caco2 cells. These results suggest that NOX1 participates in the ROS-dependent cell death of colon epithelial cells induced by amebic adhesion during the early phase of intestinal amebiasis.

The Nf-actin gene is an important factor for food-cup formation and cytotoxicity of pathogenic Naegleria fowleri

Naegleria fowleri destroys target cells by trogocytosis, a phagocytosis mechanism, and a process of piecemeal ingestion of target cells by food-cups. Phagocytosis is an actin-dependent process that involves polymerization of monomeric G-actin into filamentous F-actin. However, despite the numerous studies concerning phagocytosis, its role in the N. fowleri food-cup formation related with trogocytosis has been poorly reported. In this study, we cloned and characterized an Nf-actin gene to elucidate the role of Nf-actin gene in N. fowleri pathogenesis. The Nf-actin gene is composed of 1,128-bp and produced a 54.1-kDa recombinant protein (Nf-actin). The sequence identity was 82% with nonpathogenic Naegleria gruberi but has no sequence identity with other mammals or human actin gene. Anti-Nf-actin polyclonal antibody was produced in BALB/c mice immunized with recombinant Nf-actin. The Nf-actin was localized on the cytoplasm, pseudopodia, and especially, food-cup structure (amoebastome) in N. fowleri trophozoites using immunofluorescence assay. When N. fowleri co-cultured with Chinese hamster ovary cells, Nf-actin was observed to localize around on phagocytic food-cups. We also observed that N. fowleri treated with cytochalasin D as actin polymerization inhibitor or transfected with antisense oligomer of Nf-actin gene had shown the reduced ability of food-cup formation and in vitro cytotoxicity. Finally, it suggests that Nf-actin plays an important role in phagocytic activity of pathogenic N. fowleri.

Leukotriene B4 receptor BLT-mediated phosphorylation of NF-κB and CREB is involved in IL-8 production in human mast cells induced by Trichomonas vaginalis-derived secretory products

Trichomonas vaginalis is a protozoan parasite that causes acute tissue inflammation in vaginal trichomoniasis. In this study, we investigated the signaling mechanisms through which T. vaginalis-derived secretory products (TvSP) induce chemokine IL-8 production in human mast cells. Stimulation with TvSP induced up-regulation of IL-8 protein secretion in HMC-1 cells. In addition, TvSP induced phosphorylation of transcription factors NF-κB and CREB in HMC-1 cells. Pretreatment of TvSP with lipase, but not heat or proteinase K strongly abolished the stimulatory effect on IL-8 production. Moreover, TvSP-induced IL-8 production and phosphorylation of NF-κB or CREB were inhibited when HMC-1 cells were stimulated with modified TvSP collected from 5-lipooxygenase inhibitor-treated trichomonads. Indeed, T. vaginalis-secreted lipid mediator LTB(4) (700pg/ml) from 1×10(7) trichomonads. Furthermore, pretreatment of HMC-1 cells with antagonists for LTB(4) receptors BLT1 or BLT2 abolished the stimulatory effects of TvSP. Finally, TvSP-induced IL-8 production was inhibited by pretreatment with IκB or CREB inhibitors. These results suggest that T. vaginalis-derived secretory lipid mediator LTB(4) induces IL-8 production in mast cells via BLT-dependent activation of NF-κB and CREB.