Hak Sun Yu

Changes in Gene Cassettes of Class 1 Integrons among Escherichia coli Isolates from Urine Specimens Collected in Korea during the Last Two Decades

ABSTRACT Gene cassettes of class 1 integrons in Escherichia coli isolates from urine specimens collected in Korea during the last 2 decades were characterized. intI1 was detected in 54% of the isolates, yet gene cassette regions were amplified in only 43% of the isolates. intI2 was detected in 29 (5%) isolates, and no intI3 was detected in this study. Twenty-one different genes, including genes encoding resistance to antibiotics, an alcohol dehydrogenase gene (adhE), and unknown genes, were detected. The genes most commonly found in class 1 integrons were those for aminoglycoside and trimethoprim resistance. The occurrence of aminoglycoside resistance genes in class 1 integrons decreased, and the presence of dfr genes increased rapidly, during the last 2 decades. Single-gene cassettes were predominant during the 1980s, while multigene cassettes predominated from the 1990s on. The aadA1, aadA2, and blaP1-aadA2 gene cassettes were frequently found in isolates from the 1980s but were not detected in isolates recovered since 2000. dfrA12-aadA2 and dfrA17-aadA5 were the most prevalent gene cassettes among isolates recovered from the 1990s on. In conclusion, class 1 integrons would appear to be responsible for resistance to antibiotics commonly used to treat urinary tract infections, and selection of a specific gene cassette was found to occur over the course of time.

Macrophage Migration Inhibitory Factor Homologs of Anisakis simplex Suppress Th2 Response in Allergic Airway Inflammation Model via CD4+CD25+Foxp3+ T Cell Recruitment

We have cloned the macrophage migration inhibitory factor (MIF)-like protein (Anisakis simplex (As)-MIF) from larvae of the whale worm (Anisakis simplex third-stage larvae). Asthma was induced in the mice using OVA/alum, with or without various concentrations of rAs-MIF treatment before OVA/alum challenge. Treatment with rAs-MIF coupled with OVA/alum during the challenge period induced a complete inhibition of eosinophilia and goblet cell hyperplasia within the lung and profoundly ameliorated the development of lung hyperreactivity. Also, rAs-MIF was shown to reduce profoundly the quantity of Th2-related cytokines (IL-4, IL-5, and IL-13) in the bronchial alveolar lavage fluid and allergen-specific IgG2a in sera. IL-10 and TGF-β levels in the bronchoalveolar lavage fluid of the rAs-MIF-treated group were significantly higher than in the other groups. Additionally, CD4+CD25+Foxp3+ T cells (regulatory T) were recruited to the spleen and lungs of the rAs-MIF-treated mice, but this recruitment was inhibited by anti-rAs-MIF Ab.

Changes in Patterns of Antimicrobial Susceptibility and Integron Carriage among Shigella sonnei Isolates from Southwestern Korea during Epidemic Periods

ABSTRACT Shigella sonnei isolates from southwestern Korea during the epidemic periods of 1998 to 2000 were genetically related. The antimicrobial susceptibilities of the outbreak-related isolates changed annually. All isolates carried class 2 integrons, and the outbreak-related isolates from 1999 also carried class 1 integrons. The antimicrobial susceptibilities of S. sonnei isolates are readily changed by antibiotic selective pressures, and integrons are responsible for resistance to antimicrobial agents commonly used to treat shigellosis.

Trichinella spiralis: Infection reduces airway allergic inflammation in mice

In an effort to define the mechanism underlying the host immune downregulation inherent to Trichinella spiralis infection, we compared the levels of Th1, Th2, and regulatory cytokines and CD4(+)CD25(+) forkhead box P3 (FoxP3)(+) T (T(reg)) cell recruitment, as well as cellular pathology in the airway between T. spiralis infected and uninfected asthma-induced mice. After the induction of allergic airway inflammation, we noted influxes of inflammatory cells into the peribronchial tree. However, in the T. spiralis infection groups, cellular infiltration was minimal around the bronchial tree, with only a smattering of inflammatory cells. In the OVA-challenged group after T. spiralis infection, the numbers of macrophages and eosinophils in the bronchial alveolar lavage fluid were reduced by 23% and 52%, respectively, as compared to those of the OVA-challenged group. Airway hyperresponsiveness of OVA-challenged mice after T. spiralis infection was significantly suppressed as compared to the OVA-only challenged mice. The T. spiralis-infected mice exhibited a significant reduction in IL-5 concentrations relative to that noted in the OVA-challenged group (p<0.01). Nevertheless, the regulatory cytokines IL-10 and TGF-β levels were increased significantly as the result of T. spiralis infection, and we verified the recruitment of T(reg) cells in lung draining lymph nodes via T. spiralis infection. Therefore, T(reg) cells, which were recruited by T. spiralis infection, might ameliorate lung function and reduce allergic airway inflammation.

Cysticidal effect on acanthamoeba and toxicity on human keratocytes by polyhexamethylene biguanide and chlorhexidine.

Purpose: To evaluate the cysticidal effect of polyhexamethylene biguanide (PHMB) and chlorhexidine on Acanthamoeba and its toxic effect on cultured human keratocytes. Methods: Each well of a twofold-diluted Acanthamoeba cyst-containing suspension of 5 × 104 cysts/mL was treated with PHMB and chlorhexidine for 8, 24, and 48 hours to determine the minimal cysticidal concentration (MCC) of each disinfectant. Human corneal keratocytes (5 × 104cells/mL) were exposed to PHMB and chlorhexidine for the same time to determine the survival rate of keratocytes. Inverted phase-contrast and electron microscopy were used to observe the morphologic changes. Results: The mean MCC of PHMB for 8, 24, and 48 hours was 9.42, 5.62, and 2.37 μg/mL, respectively. The mean MCC of chlorhexidine for 8, 24, and 48 hours was 24.32, 10.02, and 7.02 μg/mL, respectively. The respective survival rate of keratocytes at the MCC was 91.7%, 64.6%, and 49.7% for PHMB and 95.7%, 90.6%, and 78.1% for chlorhexidine, respectively. The cysts and keratocytes showed more damaged appearances after treatment with PHMB than chlorhexidine. Conclusions: PHMB and chlorhexidine showed a similar amoebicidal efficacy. However, PHMB seemed to be more toxic to keratocytes than chlorhexidine.

Inhibition of dextran sulfate sodium (DSS)-induced intestinal inflammation via enhanced IL-10 and TGF-β production by galectin-9 homologues isolated from intestinal parasites

We isolated a galectin-9 (Gal-9) homologue gene (Tl-gal) from an adult worm of the canine gastrointestinal nematode parasite, Toxascaris leonina, via random cDNA library sequencing. The deduced amino acid sequence of the Tl-gal genes evidenced an identity of 89% with the galectin of Dirofilaria immitis, 87% identity with the galectin of Brugia malayi, and 35% identity with the human GAL-9 gene. To evaluate immune modulate function of Tl-GAL in host inflammatory response, we constructed recombinant Tl-GAL (rTl-GAL) using an Escherichia coli expression vector system and treated to intestinal inflammation mice. Although the carbohydrate-binding ability of rTl-GAL was less than that of rat galectin, we confirmed that recombinant rTl-GAL has carbohydrate-binding activity. The clinical symptoms of dextran sulfate sodium (DSS)-treated mice after rTl-GAL pre-treatment were found to be minimized, or less profound, as compared to those of the rTl-GAL untreated group. Additionally, the DSS-treated mice exhibited a significant shortening of the colon, but the large intestines of the rTl-GAL pre-treated mice were longer than those of the control group (P<0.05). Additionally, the rTl-GAL treated group exhibited significantly increased the levels of TGF-beta and IL-10 (P<0.05). The production of these regulatory cytokines may ameliorate intestinal inflammation. These findings demonstrate that rTl-GAL could inhibit inflammation reactions via the inhibition of Th1 and Th2 cytokine production by increasing the production of TGF-beta and IL-10 cytokines. The rTl-GAL may induce TGF-beta expression, primarily via the activation of the p38 pathway. In conclusion, rTl-GAL may function like a host galectin, thus functioning as a regulatory molecule in the host immune system; rTl-GAL may prove useful in the design of novel therapeutic intervention strategies for the treatment of allergic and immune diseases.

Parasitic Helminth Cystatin Inhibits DSS-Induced Intestinal Inflammation Via IL-

Many immune down-regulatory molecules have been isolated from parasites, including cystatin (cystain protease inhibitor). In a previous study, we isolated and characterized Type I cystatin (CsStefin-1) of the liver fluke, Clonorchis sinensis. To investigate whether the CsStefin-1 might be a new host immune modulator, we induced intestinal inflammation in mice by dextran sodium sulfate (DSS) and treated them with recombinant CsStefin-1 (rCsStefin-1). The disease activity index (DAI) increased in DSS only-treated mice. In contrast, the DAI value was significantly reduced in rCsStefin-1-treated mice than DSS only-treated mice. In addition, the colon length of DSS only-treated mice was shorter than that of rCsStefin-1 treated mice. The secretion levels of IFN-γ and TNF-α in the spleen and mesenteric lymph nodes (MLNs) were significantly increased by DSS treatment, but the level of TNF-α in MLNs was significantly decreased by rCsStefin-1 treatment. IL-10 production in both spleen and MLNs was significantly increased, and IL-10+F4/80+ macrophage cells were significantly increased in the spleen and MLNs of rCsStefin-1 treated mice after DSS treatment. In conclusion, rCsStefin-1 could reduce the intestinal inflammation occurring after DSS treatment, these effects might be related with recruitment of IL-10 secreting macrophages.

10^+F4/80^+

The limited treatment option for recurrent prostate cancer and the eventual resistance to conventional chemotherapy drugs has fueled continued interest in finding new anti‐neoplastic agents of natural product origin. We previously reported anti‐proliferative activity of deoxypodophyllotoxin (DPT) on human prostate cancer cells. Using the PC‐3 cell model of human prostate cancer, the present study reveals that DPT induced apoptosis via a caspase‐3‐dependent pathway that is activated due to dysregulated mitochondrial function. DPT‐treated cells showed accumulation of the reactive oxygen species (ROS), intracellular Ca  i2+ surge, increased mitochondrial membrane potential (MMP, ΔΨm), Bax protein translocation to mitochondria and cytochrome c release to the cytoplasm. This resulted in caspase‐3 activation, which in turn induced apoptosis. The antioxidant N‐acetylcysteine (NAC) reduced ROS accumulation, MMP and Ca  i2+ surge, on the other hand the Ca2+ chelator BAPTA inhibited the Ca  i2+ overload and MMP without affecting the increase of ROS, indicating that the generation of ROS occurred prior to Ca2+ flux. This suggested that both ROS and Ca  i2+ signaling play roles in the increased MMP via Ca  i2+ ‐dependent and/or ‐independent mechanisms, since ΔΨm elevation was reversed by NAC and BAPTA. This study provides the first evidence for the involvement of both ROS‐ and Ca  i2+ ‐activated signals in the disruption of mitochondrial homeostasis and the precedence of ROS production over the failure of Ca2+ flux homeostasis. J. Cell. Biochem. 114: 1124–1134, 2013.

Macrophage Recruitment

Infection by Trichinella spiralis takes place in two distinct phases: one is the intestinal phase and the other is the muscle phase. To evaluate alterations in cytokine production during a T. spiralis infection, we periodically assessed the cytokine production of splenocytes in mice after infection (AI). The levels of Th2-related cytokines immediately increased after the initiation of T. spiralis larval intestinal invasion (1 week AI). These early elevations in the Th2 response might be associated with the innate immune responses of intestine epithelial cells against T. spiralis larval invasion. IL-4 and IL-13 levels reached a peak prior to the initiation of nurse cell formation (2 weeks AI). Additionally, all Th17-related cytokines, except for IL-17, increased slightly until 2 weeks AI. However, expression levels for all of the Th2 and Th17-related cytokines began to decrease after the initiation of nurse cell formation and reached basal levels at 4 weeks AI, except for IL-5. At the same time, the CD4(+)CD25(+)Foxp3(+) T (regulatory T, T(reg)) cell population increased significantly in the spleen. Additionally, the number of cells in the peripheral lymph nodes increased. In conclusion, T. spiralis larva intestinal invasion induced the production of Th2 and Th17 cell-related cytokines, and the cytokines decreased with T(reg) cell-related cytokine.

Interplay of reactive oxygen species, intracellular Ca2+ and mitochondrial homeostasis in the apoptosis of prostate cancer cells by deoxypodophyllotoxin

ABSTRACT The resistance to ampicillin and nalidixic acid in Shigella sonnei isolates obtained in Korea during the period 1998 to 2000 was characterized. Recently (J. Y. Oh, H. S. Yu, S. K. Kim, S. Y. Seol, D. T. Cho, and J. C. Lee, J. Clin. Microbiol. 41:421-423, 2003) ampicillin and nalidixic acid resistance was found in 49 and 70%, respectively, of the 67 S. sonnei isolates obtained during this period. We analyzed 138 S. sonnei isolates collected during the same period. Ampicillin and nalidixic acid resistance was found in 30 and 86% of the isolates, respectively. The ampicillin resistance was mediated by a TEM-1β -lactamase, and TEM-52 extended-spectrum β-lactamase was identified in one sporadic S. sonnei isolate from 1999. blaTEM-1 and blaTEM-52 were located in conjugative R-plasmids. Tn3 was detected in 41% of the ampicillin-resistant isolates. The R-plasmids from the transconjugants that transferred resistance to ampicillin exhibited different restriction fragment length polymorphism patterns, and a blaTEM-1 probe was hybridized with the different fragments. The nalidixic acid resistance was exclusively associated with an amino acid substitution, Ser83→Leu (TCG→TTG), in gyrA. These findings indicate that the genetically related S. sonnei strains readily acquire resistance to ampicillin, streptomycin, trimethoprim, and sulfamethoxazole but not nalidixic acid through conjugative R-plasmids from difference sources when confronted by antibiotic selective pressures.