OBJECTIVE: Plant-derived cytotoxic transgene expression, such as trichosanthin (tcs), regulated by recombinant adeno-associated virus (rAAV) vector is a promising cancer gene therapy. However, the cytotoxic transgene can hamper the vector production in the rAAV producer cell line, human embryonic kidney (HEK293) cells. Here, we explored microRNA-122 (miR122) and its target sequence to limit the expression of the cytotoxic gene in the rAAV producer cells. METHODS: A miR122 target (122T) sequence was incorporated into the 3' untranslated region of the tcs cDNA sequence. The firefly luciferase (fluc) transgene was used as an appropriate control. Cell line HEK293-mir122 was generated by the lentiviral vector-mediated genome integration of the mir122 gene in parental HEK293 cells. The effects of miR122 overexpression on cell growth, transgene expression, and rAAV production were determined. RESULTS: The presence of 122T sequence significantly reduced transgene expression in the miR122-enriched Huh7 cell line (in vitro), fresh human hepatocytes (ex vivo), and mouse liver (in vivo). Also, the normal liver physiology was unaffected by delivery of 122T sequence by rAAV vectors. Compared with the parental cells, the miR122-overexpressing HEK293-mir122 cell line showed similar cell growth rate and expression of transgene without 122T, as well as the ability to produce liver-targeting rAAV vectors. Fascinatingly, the yield of rAAV vectors carrying the tcs-122T gene was increased by 77.7-fold in HEK293-mir122 cells. Moreover, the tcs-122T-containing rAAV vectors significantly reduced the proliferation of hepatocellular carcinoma cells without affecting the normal liver cells. CONCLUSION HEK293-mir122 cells along with the 122T sequence provide a potential tool to attenuate the cytotoxic transgene expression, such as tcs, during rAAV vector production.
Animals ; Dependovirus/genetics* ; Genetic Therapy ; Genetic Vectors/genetics* ; HEK293 Cells ; Humans ; Mice ; MicroRNAs/genetics* ; Trichosanthin

Sequence type (ST) 33 of Shiga toxin-producing Escherichia coli (STEC) strain O91:H14 has been proposed as a potential domestic clone of STEC in Korea because of its high prevalence among human patients with mild diarrhea or asymptomatic carriers. Herein, the clonal diversity of 17 STEC O91:H14 isolates of ST33 during 2003 to 2014 was analyzed by pulsed-field gel electrophoresis, including 14 isolates from human patients and 3 from retail meats. Their virulence characteristics, acid resistance, and antimicrobial susceptibility were also determined. Our results showed that all isolates were clustered mainly into three different pulsotypes and were likely low pathogenic without antimicrobial resistance.
Clone Cells ; Diarrhea ; Electrophoresis, Gel, Pulsed-Field ; Escherichia coli ; Humans ; Korea ; Meat ; Molecular Epidemiology ; Prevalence ; Shiga Toxin ; Shiga-Toxigenic Escherichia coli ; Virulence

PURPOSE: To investigate the efficacy, and identify predictors of success of selective laser trabeculoplasty (SLT) in open-angle glaucoma (OAG) patients after adjusting for intraocular pressure (IOP) changes in the untreated fellow eye. METHODS: This retrospective chart review included 52 eyes of 52 OAG patients who underwent SLT in one eye and were followed-up for at least 1 year after the procedure. The IOP was measured before the treatment, at 1, 2, and 3 months posttreatment, and every 3 months thereafter. To account for the possible influence of IOP fluctuations on laser outcomes, post-laser IOP values of the treated eye of each patient were also analyzed, after adjusting for IOP changes in the untreated fellow eye. Success was defined as an IOP decrease ≥20% of the pretreatment IOP. The success rate was determined based on Kaplan-Meier survival analysis and factors predictive of success were analyzed using the Cox proportional hazard model. RESULTS: The mean pretreatment IOP was 23.17 ± 6.96 mmHg. The mean IOP reduction was 5.59 ± 4.78 mmHg (29.7%) and the success rate was 65.4% at 1 year. The adjusted mean IOP reduction was 4.70 ± 4.67 mmHg (23.9%) and the adjusted success rate was 53.9%. Pretreatment IOP was associated with SLT success; the higher the pretreatment IOP, the greater the post-laser IOP reduction (p = 0.025). Age and mean deviation index did not show a significant association with SLT success (p = 0.066 and p = 0.464, respectively). CONCLUSIONS: SLT is a safe and effective alternative method of IOP reduction in OAG patients. Herein, pretreatment IOP was the only factor significantly associated with SLT success. IOP fluctuations of the untreated eye should be considered for a better understanding of the impact of treatment.
Glaucoma, Open-Angle ; Humans ; Intraocular Pressure ; Methods ; Proportional Hazards Models ; Retrospective Studies ; Shiga Toxin 1 ; Trabeculectomy

Enterotoxigenic Escherichia coli (ETEC) causes diarrhea in pigs, referred to as colibacillosis. The aim of this study was to optimize multiplex polymerase chain reaction (PCR) and immunohistochemistry (IHC) analyses of paraffin-embedded material to detect pathogenic E. coli strains causing colibacillosis in pigs. Multiplex PCR was optimized for fimbriae (F18, F4, F6, F5, and F41) and toxins (types A and B heat-stable toxins [STaP and STb], heat-labile toxin [LT], and type 2 Shiga toxin [ST(x2e)]), and IHC was optimized for an anti-E. coli polyclonal antibody. Samples (132) from pigs received between 2006 and 2014 with clinical and histopathological diagnoses of colibacillosis were analyzed. E. coli was detected by IHC in 78.7%, and at least one virulence factor gene was detected in 71.2%. Pathogenic strains of ETEC with at least one fimbria and one toxin were detected in 40% of the samples in multiplex PCR. The most frequent virulence types were F18-STaP (7.5%), F18-STaP-STb (5.7%), and F4-STaP (3.8%). A statistically significant association was noted between virulence factors F4, F18, STaP, and STb and positive immunostaining results. Colibacillosis diagnosis through multiplex PCR and IHC of paraffin-embedded tissues is a practical approach, as samples can be fixed and stored for long periods before analysis.
Diagnosis ; Diarrhea ; Enteritis ; Enterotoxigenic Escherichia coli ; Immunohistochemistry ; Multiplex Polymerase Chain Reaction ; Shiga Toxin ; Swine ; Swine Diseases ; Virulence ; Virulence Factors

Herein, we report the pathogenic and phylogenetic characteristics of seven Shiga toxin (Stx)-producing Escherichia coli (STEC) isolates from 434 retail meats collected in Korea during 2006 to 2012. The experimental analyses revealed that all isolates (i) were identified as non-O157 STEC, including O91:H14 (3 isolates), O121:H10 (2 isolates), O91:H21 (1 isolate), and O18:H20 (1 isolate), (ii) carried diverse Stx subtype genes (stx₁, stx(2c), stx(2e), or stx₁ + stx(2b)) whose expression levels varied strain by strain, and (iii) lacked the locus of enterocyte effacement (LEE) pathogenicity island, a major virulence factor of STEC, but they possessed one or more alternative virulence genes encoding cytotoxins (Cdt and SubAB) and/or adhesins (Saa, Iha, and EcpA). Notably, a significant heterogeneity in glutamate-induced acid resistance was observed among the STEC isolates (p < 0.05). In addition, phylogenetic analyses demonstrated that all three STEC O91:H14 isolates were categorized into sequence type (ST) 33, of which two beef isolates were identical in their pulsotypes. Similar results were observed with two O121:H10 pork isolates (ST641; 88.2% similarity). Interestingly, 96.0% of the 100 human STEC isolates collected in Korea during 2003 to 2014 were serotyped as O91:H14, and the ST33 lineage was confirmed in approximately 72.2% (13/18 isolates) of human STEC O91:H14 isolates from diarrheal patients.
Cytotoxins ; Enterocytes ; Escherichia coli ; Genomic Islands ; Humans ; Korea ; Meat ; Population Characteristics ; Red Meat ; Shiga Toxin ; Shiga-Toxigenic Escherichia coli ; Virulence ; Virulence Factors

PURPOSE: To report a case of hyphema after selective laser trabeculoplasty (SLT) in a patient with pseudoexfoliative glaucoma. CASE SUMMARY: A 77-year-old female was referred for elevation of intraocular pressure (IOP). Previously, she had been diagnosed with pseudoexfoliative glaucoma in the right eye and was using topical IOP-lowering agents. The best corrected visual acuity was 20/100 in the right eye and 20/40 in the left eye. IOP, measured with Goldmann applanation tonometer, was 32 mm Hg in the right eye and 20 mm Hg in the left eye. Gonioscopy revealed open-angle glaucoma with +2 trabecular meshwork pigmentation but without peripheral anterior synechiae or neovascularization. SLT was performed in the right eye. Two days later, the patient had sudden onset of blurred vision and pain in the right eye. Visual acuity was limited to light perception, and IOP was 34 mm Hg in the right eye. Slit-lamp examination revealed 1.1 mm hyphema with 4+ red blood cell count in the anterior chamber. Three weeks after the SLT, hyphema in the right eye disappeared, but IOP was measured to be 42 mm Hg. The patient underwent trabeculectomy in the right eye. CONCLUSIONS: SLT is an effective means of lowering IOP with low risk of complications. However, hyphema can rarely occur after SLT and can affect the outcome of the treatment.
Aged ; Anterior Chamber ; Erythrocyte Count ; Female ; Glaucoma ; Glaucoma, Open-Angle ; Gonioscopy ; Humans ; Hyphema* ; Intraocular Pressure ; Pigmentation ; Shiga Toxin 1 ; Trabecular Meshwork ; Trabeculectomy* ; Visual Acuity

PURPOSE: Reduced brain glucose metabolism and basal forebrain cholinergic neuron degeneration are common features of Alzheimer's disease and have been correlated with memory function. Although regions representing glucose hypometabolism in patients with Alzheimer's disease are targets of cholinergic basal forebrain neurons, the interaction between cholinergic denervation and glucose hypometabolism is still unclear. The aim of the present study was to evaluate glucose metabolism changes caused by cholinergic deficits. MATERIALS AND METHODS: We lesioned basal forebrain cholinergic neurons in rats using 192 immunoglobulin G-saporin. After 3 weeks, lesioned animals underwent water maze testing or were analyzed by 18F-2-fluoro-2-deoxyglucose positron emission tomography. RESULTS: During water maze probe testing, performance of the lesioned group decreased with respect to time spent in the target quadrant and platform zone. Cingulate cortex glucose metabolism in the lesioned group decreased, compared with the normal group. Additionally, acetylcholinesterase activity and glutamate decarboxylase 65/67 expression declined in the cingulate cortex. CONCLUSION: Our results reveal that spatial memory impairment in animals with selective basal forebrain cholinergic neuron damage is associated with a functional decline in the GABAergic and cholinergic system associated with cingulate cortex glucose hypometabolism.
Acetylcholine/metabolism ; Alzheimer Disease ; Animals ; Antibodies, Monoclonal/*pharmacology ; Basal Forebrain/*drug effects/metabolism ; Cholinergic Agents/administration & dosage/*pharmacology ; Cholinergic Neurons/*drug effects/metabolism ; Fluorodeoxyglucose F18 ; GABAergic Neurons/*drug effects/metabolism ; Glucose/*metabolism ; Gyrus Cinguli/*drug effects/metabolism ; Humans ; Injections ; Maze Learning ; Motor Activity/physiology ; Positron-Emission Tomography ; Rats ; Ribosome Inactivating Proteins, Type 1/*pharmacology

Ribosome-inactivating proteins (RIPs) belong to a family of enzymes that attack eukaryotic ribosomes and potently inhibit cellular protein synthesis. RIPs possess several biomedical properties, including anti-viral and anti-tumor activities. Multiple RIPs are known to inhibit tumor cell proliferation through inducing apoptosis in a variety of cancers, such as breast cancer, leukemia/lymphoma, and hepatoma. This review focuses on the anti-tumor activities of RIPs and their apoptotic effects through three closely related pathways: mitochondrial, death receptor, and endoplasmic reticulum pathways.
Animals ; Antineoplastic Agents ; Apoptosis ; Endoplasmic Reticulum ; Humans ; Mitochondria ; Plant Proteins ; Receptors, Death Domain ; Ribosome Inactivating Proteins ; Ribosomes

OBJECTIVELittle effort has been made to study the protein-encoding genes isolated from traditional Chinese medicine (TCM) drugs, and the delivery of these genes into malignant cells through recombinant adeno-associated virus (rAAV) vectors has not been attempted.

METHODSWe synthesized the cDNAs of five known cytotoxic proteins isolated from TCM drugs and the FLAG epitope-tagged cDNAs were subcloned into a rAAV plasmid vector. The protein expression was confirmed by Western blot assay. Various cancer cell lines were transfected with the above plasmids and cell growth was monitored both in vitro and in vivo. The best cytotoxic gene was further packaged into rAAV vectors, under the control of a liver cancer-specific promoter. The liver tumor growth was then monitored following intratumor administration of the rAAV vectors.

RESULTSThe expression plasmids, encoding individual potential cytotoxic genes tagged with FLAG epitope, were successfully generated and sequenced. Among these genes, trichosanthin (TCS) gene yielded the most promising results for the inhibition of cancer cell growth in vitro. The over-expressed TCS functioned as a type I ribosome-inactivating protein, followed by inducing apoptosis that is associated with the Bcl-PARP signaling pathway. Furthermore, intratumor injection of rAAV vectors containing the TCS gene significantly inhibited the growth of human hepatocellular carcinoma tumors in a murine xenograft model.

CONCLUSIONOur studies suggest that the use of TCM cytotoxic genes is a useful therapeutic strategy for treating human cancers in general, and liver tumors in particular.

Animals ; Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; physiology ; Cell Line, Tumor ; DNA, Complementary ; Dependovirus ; Genetic Vectors ; Humans ; Liver Neoplasms ; physiopathology ; Medicine, Chinese Traditional ; methods ; Mice ; Neoplasms ; physiopathology ; Trichosanthin ; genetics ; pharmacology

A total of 156 Shiga-like toxin producing Escherichia coli (STEC) were isolated from fecal samples of Korean native (100/568, 18%) and Holstein dairy cattle (56/524, 11%) in Korea between September 2010 and July 2011. Fifty-two STEC isolates (33%) harbored both of shiga toxin1 (stx1) and shiga toxin2 (stx2) genes encoding enterohemolysin (EhxA) and autoagglutinating adhesion (Saa) were detected by PCR in 83 (53%) and 65 (42%) isolates, respectively. By serotyping, six STEC from native cattle and four STEC from dairy cattle were identified as O-serotypes (O26, O111, O104, and O157) that can cause human disease. Multilocus sequence typing and pulsed-field gel electrophoresis patterns highlighted the genetic diversity of the STEC strains and difference between strains collected during different years. Antimicrobial susceptibility tests showed that the multidrug resistance rate increased from 12% in 2010 to 42% in 2011. Differences between isolates collected in 2010 and 2011 may have resulted from seasonal variations or large-scale slaughtering in Korea performed to control a foot and mouth disease outbreak that occurred in early 2011. However, continuous epidemiologic studies will be needed to understand mechanisms. More public health efforts are required to minimize STEC infection transmitted via dairy products and the prevalence of these bacteria in dairy cattle.
Animals ; Anti-Bacterial Agents/pharmacology ; Cattle/microbiology ; Drug Resistance, Multiple, Bacterial ; Electrophoresis, Gel, Pulsed-Field/veterinary ; Escherichia coli Infections/epidemiology/microbiology/*veterinary ; Female ; Genes, Bacterial/genetics ; Latex Fixation Tests/veterinary ; Microbial Sensitivity Tests/veterinary ; Multilocus Sequence Typing/veterinary ; Prevalence ; Republic of Korea/epidemiology ; Shiga Toxin 1/genetics ; Shiga Toxin 2/genetics ; *Shiga-Toxigenic Escherichia coli/drug effects/genetics