Sang-Ki Kim

Apoptotic Effect of Quercetin on HT-29 Colon Cancer Cells via the AMPK Signaling Pathway

Activation of AMP-activated protein kinase (AMPK), a physiological cellular energy sensor, strongly suppresses cell proliferation in both nonmalignant and tumor cells. This study demonstrates the mechanism of quercetin-induced apoptosis in HT-29 colon cancer cells. Treatment of cells with quercetin significantly decreased cell viability in a dose-dependent manner. Notably, quercetin increased cell cycle arrest in the G1 phase and up-regulated apoptosis-related proteins, such as AMPK, p53, and p21, within 48 h. Furthermore, in vivo experiments showed that quercetin treatment resulted in a significant reduction in tumor volume over 6 weeks, and apoptosis-related protein induction by quercetin was significantly higher in the 100 mg/kg treated group compared to the control group. All of these results indicate that quercetin induces apoptosis via AMPK activation and p53-dependent apoptotic cell death in HT-29 colon cancer cells and that it may be a potential chemopreventive or therapeutic agent against HT-29 colon cancer.

Molecular epidemiology of bovine noroviruses in South Korea

Abstract Since the prevalence of bovine norovirus (BNoV) and their genetic diversity have only been reported in the USA, England, Germany and The Netherlands, this study examined the prevalence and genetic diversity of BNoVs in diarrheic calves in South Korea using 645 diarrheic fecal specimens from calves by RT-PCR and nested PCR assays. Overall, 9.3% of the diarrheic fecal samples tested positive for BNoVs by either RT-PCR or nested PCR, of which 5.9% samples also tested positive for other enteric pathogens including the bovine coronavirus, bovine viral diarrhea virus, bovine torovirus, bovine groups A, B and C rotaviruses, bovine enteric Nebraska-like calicivirus and Escherichia coli. The genetic diversity was determined by direct sequencing of the partial RdRp region of 12 BNoVs detected from the fecal samples by nested PCR. Among the BNoVs examined, one Korean BNoV strain had the highest nucleotide (86.8%) and amino acid (99.1%) identity with the genotype 1 BNoV (GIII-1) strain, while the remaining 11 Korean BNoVs shared a higher nucleotide (88.0–90.5%) and amino acid (93.5–99.1%) identity with the genotype 2 BNoV (GIII-2) strains. The phylogenetic data for the nucleotide and amino acid sequences also demonstrated that one Korean BNoV strain clustered with GIII-1 but the remaining eleven strains clustered with GIII-2. In conclusion, BNoV infections are endemic and there are two distinct genotypes with GIII-2 being the main genotype circulating in the calf population in South Korea.

Detection and Characterization of Bovine Coronaviruses in Fecal Specimens of Adult Cattle with Diarrhea during the Warmer Seasons

ABSTRACT Bovine coronavirus (BCoV) is an etiological agent associated with winter dysentery (WD), prevalent in adult cattle during the winter. Although we previously detected, isolated, and characterized BCoV strains from adult cattle with WD (WD-BCoV strains) during the winter in South Korea, the precise epidemiology, as well as the causative agent of diarrhea in adult cattle in the warmer seasons, has not been examined. We examined 184 diarrheic fecal specimens collected from 75 herds of adult cattle from seven provinces during the spring (warm), autumn (warm), and summer (hot) seasons. Bovine coronavirus-positive reactions were detected for 107 (58.2%) diarrheic fecal samples (in 47/75 herds). Of these 107 positive samples, 90 fecal samples from 33 herds tested positive for BCoV alone and 17 fecal samples from 14 herds also tested positive for other pathogens. Biological comparisons between the 9 BCoV strains isolated in this study and the 10 previously isolated WD-BCoV strains revealed that there was no receptor-destroying enzyme (RDE) activity against mouse erythrocytes in the 9 BCoV strains but the 10 WD-BCoV strains had high RDE activity. Phylogenetic analysis of the spike (S) and hemagglutinin/esterase (HE) proteins revealed that all the Korean BCoVs clustered together regardless of season and were distinct from the other known BCoVs, suggesting a distinct evolutionary pathway for the Korean BCoVs. These and previous results revealed a high prevalence and widespread geographical distribution of BCoV, suggesting that this virus is endemic in adult cattle with diarrhea in all seasons in South Korea.

Induction of multiple myeloma-specific cytotoxic T lymphocyte stimulation by dendritic cell pulsing with purified and optimized myeloma cell lysates

We investigated the possibility of immunotherapy for multiple myeloma (MM) using myeloma-specific cytotoxic T lymphocytes (CTLs) that were stimulated in vitro by dendritic cells (DCs) pulsing with purified and optimized myeloma lysates. CD14+ cells were cultured in the presence of GM-CSF and IL-4. On day 6, the immature DCs were pulsed with the purified myeloma cell lysates, and then maturation of the DCs was induced by the addition of a cytokine cocktail. There were no differences in the phenotypic expressions of mature DCs that were generated by pulsing with CD138+ cell lysates or total cell lysates. In optimization of the concentration of myeloma lysates, DCs pulsed with 10 µg/mL of myeloma lysate had greater allogeneic T-cell stimulatory capacities than those pulsed with higher concentrations of myeloma lysates. The CTL lines generated by DCs pulsed with myeloma lysates demonstrated potent cytotoxic activities against autologous target cells, but not against HLA-A2− cell lines or K562 cell lines. The DCs pulsed with myeloma lysates demonstrated a higher stimulatory capacity for autologous CTL compared with mature nonpulsed DCs. These results suggested that the DCs pulsed with purified and optimized myeloma cell lysates could generate potent myeloma-specific CTLs for approaches in MM.

Dual enteric and respiratory tropisms of winter dysentery bovine coronavirus in calves

SummaryAlthough winter dysentery (WD), which is caused by the bovine coronavirus (BCoV) is characterized by the sudden onset of diarrhea in many adult cattle in a herd, the pathogenesis of the WD-BCoV is not completely understood. In this study, colostrum-deprived calves were experimentally infected with a Korean WD-BCoV strain and examined for viremia, enteric and nasal virus shedding as well as for viral antigen expression and virus-associated lesions in the small and large intestines and the upper and lower respiratory tract from 1 to 8 days after an oral infection. The WD-BCoV-inoculated calves showed gradual villous atrophy in the small intestine and a gradual increase in the crypt depth of the large intestine. The WD-BCoV-infected animals showed epithelial damage in nasal turbinates, trachea and lungs, and interstitial pneumonia. The WD-BCoV antigen was detected in the epithelium of the small and large intestines, nasal turbinates, trachea and lungs. WD-BCoV RNA was detected in the serum from post-inoculation day 3. These results show that the WD-BCoV has dual tropism and induces pathological changes in both the digestive and respiratory tracts of calves. To our knowledge, this is the first detailed report of dual enteric and respiratory tropisms of WD-BCoV in calves. Comprehensive studies of the dual tissue pathogenesis of the BCoV might contribute to an increased understanding of similar pneumoenteric CoV infections in humans.

Genetic diversity of porcine sapoviruses.

Abstract Sapoviruses (SaVs) within the Caliciviridae family are an important cause of gastroenteritis in both humans and animals. Although the widespread occurrence of divergent human SaV strains has been reported, there have only been a few studies of porcine SaVs examining their genetic diversity. The aim of this study was to assess the genetic diversity of porcine SaVs in piglets with diarrhea in South Korea. Two hundred and thirty-seven fecal specimens from piglets with diarrhea were examined from 78 farms over a 2-year period from six provinces in South Korea. Overall, 69 (29.1%) of the samples from five provinces tested positive for porcine SaVs by either RT-PCR or nested PCR with the primer pairs specific to porcine SaVs. An analysis of the partial capsid gene (757bp) of 12 porcine SaVs detected from fecal samples showed genetic divergence, not only among the Korean porcine SaVs (67.7–99.1%), but also between Korean and American porcine SaVs (69.1–90.2%). Interestingly, one strain (Po/SaV/JN-MA113/05/Korea), formed a second porcine SaV/GIII genotype, and is tentatively called GIII/2. This strain had a 0.236–0.405 inter-cluster distance with the other strains in the same genogroup, which is comparable to the distances between the established GI and GII SaVs. Furthermore, two potential recombinant porcine SaVs were identified. In conclusion, porcine SaV infections are common in diarrheic piglets in South Korea. The infecting strains are genetically diverse, and include a newly recognized genotype and recombinant viruses within genogroup III.

Ex vivo expansion of canine cytotoxic large granular lymphocytes exhibiting characteristics of natural killer cells.

Canine NK cells still are not well-characterized due to the lack of information concerning specific NK cell markers and the fact that NK cells are not an abundant cell population. In this study, we selectively expanded the canine cytotoxic large granular lymphocytes (CLGLs) that exhibit morphologic, genetic, and functional characteristics of NK cells from normal donor PBMCs. The cultured CLGLs were characterized by a high proportion of CD5(dim) expressing cells, of which the majority of cells co-expressed CD3 and CD8, but did not express TCRαβ and TCRγδ. The phenotype of the majority of the CLGLs was CD5(dim)CD3(+)CD8(+) TCRαβ(-)TCRγδ(-)CD4(-)CD21(-)CD11c(+/-)CD11d(+/-)CD44(+). The expression of mRNAs for NK cell-associated receptors (NKG2D, NKp30, NKp44, Ly49, perforin, and granzyme B) were highly upregulated in cultured CLGLs. Specifically, NKp46 was remarkably upregulated in the cultured CLGLs compared to PBMCs. The mRNAs for the NKT-associated iTCRα gene in CLGLs was present at a basal level. The cytotoxic activity of the CLGLs against canine NK cell-sensitive CTAC cells was remarkably elevated in a dose-dependent manner, and the CLGLs produced large amounts of IFN-γ. The antitumor activity of CLGLs extended to different types of canine tumor cells (CF41.Mg and K9TCC-pu-AXC) without specific antigen recognition. These results are consistent with prior reports, and strongly suggest that the selectively expanded CLGLs represent a population of canine NK cells. The results of this study will contribute to future research on canine NK cells as well as NK cell-based immunotherapy.

Monocyte-derived dendritic cells from HLA-matched allogeneic donors showed a greater ability to induce leukemic cell-specific T cells in comparison to leukemic cell-derived dendritic cells or monocyte-derived dendritic cells from AML patients

We investigated the usefulness of allogeneic monocyte-derived dendritic cells (allogeneic mDCs) pulsed with leukemic lysates in acute myeloid leukemia (AML). Allogeneic mDCs showed higher expressions of several molecules (HLA-DR, CD80, CD83 or CD86), higher production of IL-12 and higher capacity to stimulate allogeneic T cells compared to both leukemic DCs and autologous mDCs. Autologous T cells primed by allogeneic mDCs displayed a larger number of interferon-gamma-secreting cells against leukemic cells than those primed by either leukemic DCs or autologous mDCs. These results suggest that monocyte-derived DCs from HLA-matched allogeneic donors can be used as an alternative to generate leukemia-specific cytotoxic T cells and to overcome the limitation of leukemic DCs or autologous mDCs.

Down-regulation of cellular vascular endothelial growth factor levels induces differentiation of leukemic cells to functional leukemic-dendritic cells in acute myeloid leukemia

We examined the effect of cellular vascular endothelial growth factor (VEGF) levels on the generation of leukemic dendritic cells (DCs). Leukemic DCs were successfully generated in vitro from bone marrow cells of 16 of 21 acute myeloid leukemia (AML) patients, and the cellular VEGF concentrations in the leukemic cells and the neutralization of VEGF with anti-VEGF antibody were determined. AML cells that failed to generate leukemic DCs showed significantly higher cellular VEGF levels compared with generated leukemic DCs, and down-regulation of cellular VEGF levels induced the generation of leukemic DCs from AML cells. Inhibition of cellular VEGF levels increased interleukin (IL)-12 production and the allostimulatory capacity of leukemic DCs. These results suggest that the generation of leukemic DCs from AML cells is inversely related to the VEGF production of the cells and that the down-regulation of cellular VEGF levels can induce potential differentiation of leukemic cells to functional leukemic DCs in patients with AML.

Molecular characterization of HE, M, and E genes of winter dysentery bovine coronavirus circulated in Korea during 2002-2003.

The different bovine coronavirus (BCoV) strains or isolates exhibited various degrees of substitutions, resulting in altered antigenicity and pathogenicity of the virus. In the previous our study, we demonstrated that the spike glycoprotein gene of Korean winter dysentery (WD) BCoV had a genetic property of both enteric (EBCV) and respiratory BCoV (RBCV) and were significantly distinct from the ancestral enteric strains. In the present study, therefore, we analyzed the other structure genes, the hemagglutinin/esterase (HE) protein, the transmembrane (M) protein and the small membrane (E) protein to characterize 10 WD BCoV circulated in Korea during 2002–2003 and compared the nucleotide and deduced amino acid sequences with the other known BCoV. Phylogenetic analysis indicated that the HE gene among BCoV could be divided into three groups. The first group included only RBCV, while the second group contained calf diarrhea BCoV, RBCV, WD and EBCV, respectively. The third group possessed only all Korean WD strains which were more homologous to each other and were sharply distinct from the other known BCoV, suggesting Korean WD strains had evolutionary distinct pathway. In contrast, the relative conservation of the M and E proteins of BCoV including Korean WD strains and the other coronaviruses suggested that structural constraints on these proteins are rigid, resulting in more limited evolution of these proteins. In addition, BCoV and human coronavirus HCV-OC43 contained four potential O-glycosylation sites in the M gene. However, the M gene sequence of both BCoV and HCV-OC43 might not contain a signal peptide, suggesting the M protein might be unlikely to be exposed to the O-glycosylation machinery in vivo.