Role for Caspase-8 in the Release of IL-1ß and Active Caspase-1 from Viable Human Monocytes during Toxoplasma gondii Infection.

Monocytes are actively recruited to sites of infection and produce the potent proinflammatory cytokine IL-1ß. We previously showed that IL-1ß release during Toxoplasma gondii infection of primary human monocytes requires the NLRP3 inflammasome and caspase-1 but is independent of gasdermin D and pyroptosis. To investigate mechanisms of IL-1ß release, we generated caspase-1, -4, -5, or -8 knockout (KO) THP-1 monocytic cells. Genetic ablation of caspase-1 or -8, but not caspase-4 or -5, decreased IL-1ß release during T. gondii infection without affecting cell death. In contrast, TNF-α and IL-6 secretion were unperturbed in caspase-8 KO cells during T. gondii infection. Dual pharmacological inhibition of caspase-8 and RIPK1 in primary monocytes also decreased IL-1ß release without affecting cell viability or parasite infection. Caspase-8 was also required for the release of active caspase-1 from T. gondii-infected cells and for IL-1ß release during infection with the related apicomplexan parasite Neospora caninum. Surprisingly, caspase-8 deficiency did not impair synthesis or cleavage of pro-IL-1ß, but resulted in the retention of mature IL-1ß within cells. Generation of gasdermin E KO and ATG7 KO THP-1 cells revealed that the release of IL-1ß was not dependent on gasdermin E or ATG7. Collectively, our data indicate that during T. gondii Infection of human monocytes, caspase-8 functions in a novel gasdermin-independent mechanism controlling IL-1ß release from viable cells. This study expands on the molecular pathways that promote IL-1ß in human immune cells and provides evidence of a role for caspase-8 in the mechanism of IL-1ß release during infection.

PTEN/AKT signaling pathway related to hTERT downregulation and telomere shortening induced in Toxoplasma GRA16-expressing colorectal cancer cells.

This study investigated whether the molecular mechanism of granule protein 16 (GRA16), a dense granule protein of Toxoplasma gondii (T. gondii) that induces cancer cell apoptosis, results in telomere shortening in cancer cells. The molecular mechanism of GRA16 responsible for regulating telomerase reverse transcriptase (hTERT) activity and telomere shortening was investigated using GRA16-transferred HCT116 human colorectal cancer cells (GRA16-stable cells). GRA16 directly decreased hTERT expression by downregulating the expression and phosphorylation of hTERT transcriptional factors accompanied by decreased expression of shelterin complex molecules. Moreover, GRA16 resulted in cancer cell death through reduction of telomerase activity which leads to telomere shortening (decreased relative ratio of telomeric repeat-amplified sequence to that of a single-copy gene) (T/S ratio)), and at the same time gamma-H2A histone family member X (γ-H2A.X) stained nucleus was increased in the cells. The molecular mechanism between GRA16 and hTERT inactivation was revealed using inhibitors for phosphatase and tensin homolog (PTEN) and protein phosphatase 2A (PP2A) as well as siRNAs against PTEN and PP2A. hTERT dephosphorylation was induced effectively by the signaling pathway of HAUSP/PTEN/p-AKT(S473) but not by PP2A-B55/p-AKT(T308). Inhibition of the PTEN signaling pathway increased mRNA expressions in hTERT transcriptional factors, cell cycle activating factors, and apoptosis-inhibiting factors. When HCT116 cells were infected with T. gondii, the number of γ-H2A.X-stained nuclei also increased and p-hTERT/hTERT decreased as in GRA16-stable cells. Altogether, our results emphasize that GRA16 is a novel promising telomerase inhibitor that causes telomere shortening through telomerase inactivation by inducing the activation of the tumor suppressor PTEN.

Reduction of Amyloid Burden by Proliferated Homeostatic Microglia in Toxoplasma gondii-Infected Alzheimer's Disease Model Mice.

In this study, we confirmed that the number of resident homeostatic microglia increases during chronic Toxoplasma gondii infection. Given that the progression of Alzheimer's disease (AD) worsens with the accumulation of amyloid ß (Aß) plaques, which are eliminated through microglial phagocytosis, we hypothesized that T. gondii-induced microglial proliferation would reduce AD progression. Therefore, we investigated the association between microglial proliferation and Aß plaque burden using brain tissues isolated from 5XFAD AD mice (AD group) and T. gondii-infected AD mice (AD + Toxo group). In the AD + Toxo group, amyloid plaque burden significantly decreased compared with the AD group; conversely, homeostatic microglial proliferation, and number of plaque-associated microglia significantly increased. As most plaque-associated microglia shifted to the disease-associated microglia (DAM) phenotype in both AD and AD + Toxo groups and underwent apoptosis after the lysosomal degradation of phagocytosed Aß plaques, this indicates that a sustained supply of homeostatic microglia is required for alleviating Aß plaque burden. Thus, chronic T. gondii infection can induce microglial proliferation in the brains of mice with progressed AD; a sustained supply of homeostatic microglia is a promising prospect for AD treatment.

Chronic Toxoplasma gondii Infection Alleviates Experimental Autoimmune Encephalomyelitis by the Immune Regulation Inducing Reduction in IL-17A/Th17 Via Upregulation of SOCS3.

Experimental autoimmune encephalomyelitis (EAE) is a mouse model of multiple sclerosis (MS), a demyelinating autoimmune disease caused by the infiltration of a harmful autoreactive Th1 and Th17 cells. To mitigate MS, which is impossible to cure with medication only, immunomodulatory interventions that prevent Th17 cell activation are ideal. The objective of the present study was to analyze the effect of Toxoplasma gondii infection on the onset of EAE. Our results found that Toxoplasma gondii infection in the brain increases SOCS3 expression and decreases the phosphorylation of STAT3, resulting in reducing IL-17A and IL-23, which suppress the differentiation and expansion of pathogenic Th17 cells, an important factor in MS development. These immune responses resulted in a reduction in the clinical scoring of EAE induced by myelin oligodendrocyte glycoprotein 35-55 immunization. In the EAE group with T. gondii infection (Tg + EAE group), Th17-related immune responses that exacerbate the onset of EAE were reduced compared to those in the EAE group. This study suggests that the alleviation of EAE after T. gondii infection is regulated in a SOCS3/STAT3/IL-17A/blood-brain barrier integrity-dependent manner. Although parasite infection would not be permitted for MS treatment, this study using T. gondii infection identified potential targets that contribute to disease attenuation.

Toxoplasma GRA16 Inhibits NF-κB Activation through PP2A-B55 Upregulation in Non-Small-Cell Lung Carcinoma Cells.

Nuclear factor kappa B (NF-κB) activation is a well-known mechanism by which chemoresistance to anticancer agents is reported. It is well-known that irinotecan as a chemotherapeutic drug against non-small-cell lung carcinoma (NSCLC) has limited anticancer effect due to NF-κB activation. In this study, we propose the novel role of GRA16, a dense granule protein of Toxoplasma gondii, as an anticancer agent to increase the effectiveness of chemotherapy via the inhibition of NF-κB activation. To demonstrate this, H1299 cells were stably transfected with GRA16. The anticancer effects of GRA16 were demonstrated as a reduction in tumor size in a mouse xenograft model. GRA16 directly elevated B55 regulatory subunit of protein phosphatase 2A (PP2A-B55) expression in tumor cells, thereby decreasing GWL protein levels and ENSA phosphorylation. This cascade, in turn, induced PP2A-B55 activation and suppressed AKT/ERK phosphorylation and cyclin B1 levels, suggesting reduced cell survival and arrested cell cycle. Moreover, PP2A-B55 activation and AKT phosphorylation inhibition led to NF-κB inactivation via the reduction in inhibitory kappa B kinase beta (IKKß) levels, de-phosphorylation of inhibitor of kappa B alpha (IκBα), and reduction in the nuclear transit of NF-κB p65. Furthermore, this molecular mechanism was examined under irinotecan treatment. The PP2A-B55/AKT/NF-κB p65 pathway-mediated anticancer effects were only induced in the presence of GRA16, but not in the presence of irinotecan. Moreover, GRA16 synergistically promoted the anticancer effects of irinotecan via the induction of the sub-G1 phase and reduction of cell proliferation. Collectively, irinotecan and GRA16 co-treatment promotes the anticancer effects of irinotecan via NF-κB inhibition and cell cycle arrest induced by GRA16, subsequently increasing the chemotherapeutic effect of irinotecan to NSCLC cells via NF-κB inhibition.

Immune-triggering effect of the foodborne parasite Kudoa septempunctata through the C-type lectin Mincle in HT29 cells.

Kudoa septempunctata is a myxozoan parasite that causes food poisoning in individuals consuming olive flounder. The present study aimed to investigate the currently insufficiently elucidated early molecular mechanisms of inflammatory responses in the intestine owing to parasite ingestion. After Kudoa spores were isolated from olive flounder, HT29 cells were exposed to spores identified to be alive using SYTO-9 and propidium iodide staining or to antigens of Kudoa spores (KsAg). IL-1ß, IL-8, TNF-α and NFKB1 expression and NF-κB activation were assessed using real-time PCR, cytokine array and western blotting. The immunofluorescence of FITC-conjugated lectins, results of ligand binding assays using Mincle-Fc and IgG-Fc, CLEC4E expressions in response to KsAg stimulation, and Mincle-dependent NF-κB activation were assessed to clarify the early immunetriggering mechanism. Inflammatory cytokines (IL-1α, GM-CSF and TNF-α), chemokines (IL-8, CCL2, CCL5 and CXCL1) and NF-κB activation (pNF-κB/NF-κB) in HT29 cells increased following stimulation by KsAg. The immunofluorescence results of spores and lectins (concanavalin A and wheat germ agglutinin) suggested the importance of Mincle in molecular recognition between Kudoa spores and intestinal cells. Practically, data for Mincle-Fc and KsAg binding affinity, CLEC4E mRNA expression, Mincle immunofluorescence staining and hMincledependent NF-κB activation demonstrated the involvement of Mincle in the early immune-triggering mechanism. The present study newly elucidated that the molecular recognition and immune-triggering mechanism of K. septempunctata are associated with Mincle on human intestinal epithelial cells. [BMB Reports 2020; 53(9): 478-483].

Increase in the nuclear localization of PTEN by the Toxoplasma GRA16 protein and subsequent induction of p53-dependent apoptosis and anticancer effect.

This study investigated the efficacy of Toxoplasma GRA16, which binds to herpes virus-associated ubiquitin-specific protease (HAUSP), in anticancer treatment, and whether the expression of GRA16 in genetically modified hepatocellular carcinoma (HCC) cells (GRA16-p53-wild HepG2 and GRA16-p53-null Hep3B) regulates PTEN because alterations in phosphatase and tensin homologue (PTEN) and p53 are vital in liver carcinogenesis and the abnormal p53 gene appears in HCC. For this purpose, we established the GRA16 cell lines using the pBABE retrovirus system, assessed the detailed mechanism of PTEN regulation in vitro and established the anticancer effect in xenograft mice. Our study showed that cell proliferation, antiapoptotic factors, p-AKT/AKT ratio, cell migration and invasive activity were decreased in GRA16-stable HepG2 cells. Conversely, the apoptotic factors PTEN and p53 and apoptotic cells were elevated in GRA16-stable HepG2 cells but not in Hep3B cells. The change in MDM2 was inconspicuous in both HepG2 and Hep3B; however, the PTEN level was remarkably elevated in HepG2 but not in Hep3B. HAUSP-bound GRA16 preferentially increased p53 stabilization by the nuclear localization of PTEN rather than MDM2-dependent mechanisms. These molecular changes appeared to correlate with the decreased tumour mass in GRA16-stable-HepG2 cell-xenograft nude mice. This study establishes that GRA16 is a HAUSP inhibitor that targets the nuclear localization of PTEN and induces the anticancer effect in a p53-dependent manner. The efficacy of GRA16 could be newly highlighted in HCC treatment in a p53-dependent manner.

Development of Molecular Diagnosis Using Multiplex Real-Time PCR and T4 Phage Internal Control to Simultaneously Detect Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis from Human Stool Samples.

This study aimed to develop a new multiplex real-time PCR detection method for 3 species of waterborne protozoan parasites (Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis) identified as major causes of traveler's diarrhea. Three target genes were specifically and simultaneously detected by the TaqMan probe method for multiple parasitic infection cases, including Cryptosporidium oocyst wall protein for C. parvum, glutamate dehydrogenase for G. lamblia, and internal transcribed spacer 1 for C. cayetanensis. Gene product 21 for bacteriophage T4 was used as an internal control DNA target for monitoring human stool DNA amplification. TaqMan probes were prepared using 4 fluorescent dyes, FAMTM, HEXTM, Cy5TM, and CAL Fluor Red® 610 on C. parvum, G. lamblia, C. cayetanensis, and bacteriophage T4, respectively. We developed a novel primer-probe set for each parasite, a primer-probe cocktail (a mixture of primers and probes for the parasites and the internal control) for multiplex real-time PCR analysis, and a protocol for this detection method. Multiplex real-time PCR with the primer-probe cocktail successfully and specifically detected the target genes of C. parvum, G. lamblia, and C. cayetanensis in the mixed spiked human stool sample. The limit of detection for our assay was 2×10 copies for C. parvum and for C. cayetanensis, while it was 2×103 copies for G. lamblia. We propose that the multiplex real-time PCR detection method developed here is a useful method for simultaneously diagnosing the most common causative protozoa in traveler's diarrhea.

Effects of Antioxidants in Reducing Accumulation of Fat in Hepatocyte.

The progress of the hepatic steatosis (HS), a clinicopathological status, is influenced by cellular oxidative stress, lipogenesis, fatty acid (FA) oxidation, and inflammatory responses. Because antioxidants are gaining attention as potent preventive agents for HS, we aimed to investigate anti-lipogenic effects of the antioxidants vitamin C (VC), N-acetylcysteine (NAC), and astaxanthin (ATX) using hepatocytes. For this, we established an in vitro model using 1 mM oleic acid (OA) and human liver hepatocellular carcinoma (HepG2) cells; 10 µM antioxidants were evaluated for their ability to reduce fat accumulation in hepatocytes. Our results showed that all three antioxidants were effective to reduce fat accumulation for the molecular targets such as reduction in lipid droplets, triglyceride (TG) concentration, reactive oxygen species (ROS) production, and cell apoptosis, as well as in gene expressions of endoplasmic reticulum (ER) stress-related effectors, lipogenesis, and inflammatory cytokines. There were simultaneous increases in diphenyl-1-picrylhydrazyl (DPPH) radical scavenging effect, cell survival, AMPK phosphorylation, NRF2-related gene expression for cellular defense, and FA ß-oxidation. However, among these, ATX more effectively inhibited ER stress and lipogenesis at the intracellular level than VC or NAC. Consequently, ATX was also more effective in inhibiting cell death, lipotoxicity, and inflammation. Our result emphasizes that ATX achieved greater lipotoxicity reduction than VC and NAC.

Characteristics of Infection Immunity Regulated by Toxoplasma gondii to Maintain Chronic Infection in the Brain.

To examine the immune environment of chronic Toxoplasma gondii infection in the brain, the characteristics of infection-immunity (premunition) in infection with T. gondii strain ME49 were investigated for 12 weeks postinfection (PI). The results showed that neuronal cell death, microglia infiltration and activation, inflammatory and anti-inflammatory cytokine expression, Stat1 phosphorylation, and microglia activation and inflammatory gene transcripts related to M1 polarization in the brain were increased during the acute infection (AI) stage (within 6 weeks PI), suggesting that innate and cellular inflammatory response activation and neurodegeneration contributed to excessive inflammatory responses. However, these immune responses decreased during the chronic infection (CI) stage (over 6 weeks PI) with reductions in phosphorylated STAT1 (pSTAT1) and eosinophilic neurons. Notably, increases were observed in transcripts of T-cell exhaustion markers (TIM3, LAG3, KLRG1, etc.), suppressor of cytokines signaling 1 protein (SOCS1), inhibitory checkpoint molecules (PD-1 and PD-L1), and Arg1 from the AI stage (3 weeks PI), implying active immune intervention under the immune environment of M1 polarization of microglia and increases in inflammatory cytokine levels. However, when BV-2 microglia were stimulated with T. gondii lysate antigens (strain RH or ME49) in vitro, nitrite production increased and urea production decreased. Furthermore, when BV-2 cells were infected by T. gondii tachyzoites (strain RH or ME49) in vitro, nitric oxide synthase and COX-2 levels decreased, whereas Arg1 levels significantly increased. Moreover, Arg1 expression was higher in ME49 infection than in RH infection, whereas nitrite production was lower in ME49 infection than in RH infection. Accordingly, these results strongly suggest that immune triggering of T. gondii antigens induces M1 polarization and activation of microglia as well as increase NO production, whereas T. gondii infection induces the inhibition of harmful inflammatory responses, even with M1 polarization and activation of microglia and Th1 inflammatory responses, suggesting a host-parasite relationship through immune regulation during CI. This is a characteristic of infection immunity in infection with T. gondii in the central nervous system, and SOCS1, a negative regulator of toxoplasmic encephalitis, may play a role in the increase in Arg1 levels to suppress NO production.

Preventive Effects of Resveratrol-enriched Extract of Peanut Sprout on Bacteria- and Estradiol-induced Prostatitis in Mice.

The present study investigated the effect of peanut sprout extract (PSE) as a natural resveratrol supplement on chronic bacterial prostatitis (CBP) and estradiol- induced benign prostatic hyperplasia (BPH). PSE contained a high level of resveratrol (148.51 ± 3.05 µg/g), and was tested on the mouse models of CBP . (induced by Escherichia coli 292 infection) and BPH (induced by treatment with ß-estradiol and dihydrotestosterdne). PSE toxicity was assessed on the basis of changes in body weight, alanine aminotransferase activity (an indicator of hepatotoxicity), and expression of the kidney injury marker KIM-1. The effects of PSE on the histopathology of prostate tissue, the proportion of neutrophils, and immune cell profiles in the blood and spleen were examined. PSE administration did not result in any toxicity but reduced the bacterial burden and histopathological changes in the prostate. In addition, lymphocytes (CD4⁺, CD8⁺, and CD 19⁺) in the spleen were significantly increased after PSE administration in CBP mice, suggesting immune enhancement. PSE treatment of bone Snarrow-derived macrophages increased the expression of CD40, which is related to the pro-inflammatory function and host defense against pathogens. It is concluded that PSE would be a good supplement for the mitigation of prostate hyperplasia and prostatitis.

Development of a Multiplex PCR System for the Simultaneous Detection of the Shrimp Species Fenneropenaeus chinensis, Litopenaeus vannamei, and Penaeus monodon.

Due to mixed use of shrimp in foodstuffs, it is important to distinguish Fenneropenaeus chinensis from other shrimp species, such as Litopenaeus vannamei and Penaeus monodon. For this purpose, we have developed a multiplex PCR method to detect the simultaneous presence of all three species. Universal primers specific to the 16S ribosomal RNA subunit were used to sequence and ascertain the species in the samples. In this study, specific primers were designed to result in a unique PCR product size (143, 260, and 348 bp for L. vannamei, F. chinensis and P. monodon, respectively). In addition, duplex and triplex PCR protocols were developed to concurrently and rapidly detect these three shrimp species without nonspecific gene amplification. This multiplex PCR system is expected to be widely used to distinguish shrimp species that should be monitored to prevent their mixed use in food.

Multiplex-Touchdown PCR to Simultaneously Detect Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis, the Major Causes of Traveler's Diarrhea.

This study aimed to develop a multiplex-touchdown PCR method to simultaneously detect 3 species of protozoan parasites, i.e., Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis, the major causes of traveler's diarrhea and are resistant to standard antimicrobial treatments. The target genes included the Cryptosporidium oocyst wall protein for C. parvum, Glutamate dehydrogenase for G. lamblia, and 18S ribosomal RNA (18S rRNA) for C. cayetanensis. The sizes of the amplified fragments were 555, 188, and 400 bps, respectively. The multiplex-touchdown PCR protocol using a primer mixture simultaneously detected protozoa in human stools, and the amplified gene was detected in >1×103 oocysts for C. parvum, >1×104 cysts for G. lamblia, and >1 copy of the 18S rRNA gene for C. cayetanensis. Taken together, our protocol convincingly demonstrated the ability to simultaneously detect C. parvum, G. lamblia, and C. cayetanenesis in stool samples.

Improvement in the Electrical Performance of Ge-Doped InZnO Thin-Film Transistor.

We fabricated amorphous oxide semiconductor thin-film transistors (TFTs) using Ge-doped InZnO (Ge-IZO) thin films as active-channel layers. The Ge-IZO thin films were deposited at room temperature by radio-frequency (RF) magnetron co-sputtering system, and then annealed in air for 1 h at 300 °C. Some processing parameters such as sputtering oxygen partial pressure [O2/(Ar + O2)] and sputtering power for GeO2 target were changed to investigate what was the optimal amount of Ge in the Ge-IZO active layer. A small concentration of Ge added to IZO by co-sputtering enhanced the carrier concentration, mobility, and conductivity; but further increase in Ge concentration degraded the device performance. In order to optimize the electrical properties of Ge-IZO TFTs, we tried to adjust the processing parameters and the best Ge-IZO TFT was obtained at a co-sputtering oxygen partial pressure of 2% and GeO2 target power of 10 W. The fabricated Ge-IZO TFT exhibited an on/off ratio of 3.0 x 10(7), a saturation mobility of 13.05 cm2/V·s, a subthreshold swing of 0.95 V/dec, and a threshold voltage of 0 V. XPS and XRD analyses of Ge-IZO films were performed to investigate the binding energies of atoms in Ge-IZO films and the crystallinity of the films. 90% transmittance of visible light was achieved, which makes the technology useful for transparent devices.

Effects of Double Active Layer and Acetic Acid Stabilizer on the Electrical Properties of a Solution-Processed Zinc Tin Oxide Thin-Film Transistor.

We investigated the effects of a double active layer (DAL) and acetic acid stabilizer on zinc tin oxide (ZTO) thin-film transistors (TFTs) fabricated using a solution process. The DAL was composed of two layers created by a ZTO solution doped with the same or different percentiles of an atomic Sn concentration (30 at.%, 60 at.%). The electrical performance of the ZTO TFTs significantly was improved after we added acetic acid (AA) instead of monoethanolamine (MEA). This was accomplished by applying a type 2 DAL (bottom layer: Sn 60 at.%, top layer: Sn 30 at.%, 60/30) instead of other types (30/30 or 60/60). It was demonstrated that AA plays a role in lowering the decomposition temperature, enhancing the metal-oxygen bridge, and decreasing hydroxyl groups in the film. In addition, the type 2 DAL structure (60/30) lowered the Ioff of the ZTO TFT and controlled the carrier concentration in the channel. The best performances were obtained at a Sn concentration of 60 at.% in the bottom ZTO layer and 30 at.% in the top ZTO layer, with AA added as a stabilizer. The ZTO TFT exhibited an on/off ratio of 1.1 x 10(9), a saturation mobility of 5.04 cm2/V·s, a subthreshold slope of 0.11 V/decade, and a threshold voltage of 1.6 V.

Sequential magnetic resonance spectroscopic changes in a patient with nonketotic hyperglycinemia.

Nonketotic hyperglycinemia (NKH) is a rare inborn error of amino acid metabolism. A defect in the glycine cleavage enzyme system results in highly elevated concentrations of glycine in the plasma, urine, cerebrospinal fluid, and brain, resulting in glycine-induced encephalopathy and neuropathy. The prevalence of NKH in Korea is very low, and no reports of surviving patients are available, given the scarcity and poor prognosis of this disease. In the current study, we present a patient with NKH diagnosed on the basis of clinical features, biochemical profiles, and genetic analysis. Magnetic resonance spectroscopy (MRS) allowed the measurement of absolute glycine concentrations in different parts of the brain that showed a significantly increased glycine peak, consolidating the diagnosis of NKH. In additional, serial MRS follow-up showed changes in the glycine/creatinine ratios in different parts of the brain. In conclusion, MRS is an effective, noninvasive diagnostic tool for NKH that can be used to distinguish this disease from other glycine metabolism disorders. It may also be useful for monitoring NKH treatment.

International travel of Korean children and Dengue fever: A single institutional analysis.

PURPOSE: Dengue fever occurs in many popular tourist destinations and is increasingly imported by returning travelers in Korea. Since Korea is not an endemic country for dengue fever, pediatricians do not usually suspect dengue fever in febrile children even with typical presentation and exposure history. This study was performed to describe the international travel experiences and dengue fever in Korean children. METHODS: Travel histories were collected based on questionnaires completed by all patients' guardians who visited the pediatric infectious diseases clinic at Samsung Medical Center from January 2008 to December 2008. For patients who were suspected of dengue fever, a serological test was performed. RESULTS: Five hundred and seventeen children visited the pediatric infectious diseases clinic for the first time during this period. About 30% of patients who responded to the questionnaire (101/339) had experienced international travel within the last 2 years. Four patients were diagnosed with dengue fever by serological test. CONCLUSION: Increasing numbers of Korean children visit dengue endemic areas and they may return home with dengue fever. Dengue fever should be suspected in patients who have a travel history to endemic areas.

Development of enrichment semi-nested PCR for Clostridium botulinum types A, B, E, and F and its application to Korean environmental samples.

An enrichment semi-nested PCR procedure was developed for detection of Clostridium botulinum types A, B, E, and F. It was applied to sediment samples to examine the prevalence of C. botulinum in the Korean environment. The first pair of primers for the semi-nested PCR was designed using a region shared by the types A, B, E, and F neurotoxin gene sequences, and the second round employed four nested primers complementary to the BoNT/A, /B, /E, and /F encoding genes for simultaneous detection of the four serotypes. Positive results were obtained from the PCR analysis of five of 44 sediments (11%) collected from Yeong-am Lake in Korea; all were identified as deriving from type B neurotoxin (bontb) genes. Two of the C. botulinum type B organisms were isolated, and their bontb genes sequenced. The deduced amino acid sequences of BoNT/B showed 99.5 and 99.8% identity with the amino acid sequence of accession no. AB084152. Our data suggest that semi-nested PCR is a useful tool for detecting C. botulinum in sediments, and renders it practicable to conduct environmental surveys.

Determination of neurotoxin gene expression in Clostridium botulinum type A by quantitative RT-PCR.

Real time reverse transcription (RT)-PCR was used to quantify the expression of the botulinum neurotoxin type A (BoNT/A) gene (cntA) by normalization with the expression of 16S rRNA. The method were confirmed by monitoring the mRNA levels of cntA during growth in five type A strains. In all but one of the strains the expression of cntA mRNA was maximal in the late exponential phase, and approximately 35-fold greater than in the early exponential phase. The concentration of the extracellular BoNT/A complex detected by ELISA was highest in stationary phase. Sodium nitrite and sorbic acid completely inhibited growth at 20 ppm and 4 mg ml-1, respectively. CntA expression became lower in proportion to the concentration of sorbic acid, and this reduction was confirmed by mouse bioassay. Our results show that real time RT-PCR can be used to quantify levels of C. botulinum type A neurotoxin transcripts and to assess the effects of food additives on botulinal risk.