An Norovirus Outbreak at a Local Festival in Chungnam Korea

Noroviruses (NoV) are the major viral pathogen causing epidemic acute gastroenteritis and outbreaks of foodborne and waterborne illness. During the local festival in Chungnam province, group food poisoning occurred outbreak by NoV infections in Jan 2019. In this study, epidemiological analysis and molecular characterization were conducted such as genotyping, phylogeny. The prevalent genotypes of food poisoning events were NoV GII.3 and GII.17, and NoV GII.3 and GII.17 isolates of this study were completely matched in nucleotide sequence comparison of capsid gene region, respectively. In underground water and stream water, various multiple genotypes of noroviruses were detected including NoV GII.3, GII.8 and GI.4 in aquatic environment of the local festival site. Among 32 worker samples, various NoVs of five genotypes (GI.7, GI.8, GII.3, GII.8, GII.17) were detected in 12 samples and expected to causing NoV contaminated by exposure to groundwater. NoV genotype GII.3, which was detected from groundwater 2, was completely consistent with that of patients and workers. Therefore, groundwater within the local festival site could be main cause of food poisoning event. Because NoV outbreaks are caused by fecal to oral transmission, proper management of sewage purification facilities, groundwater and sanitary toilets is required for many visitors, and efforts are needed to maintain clean environment.

Immunogenic Cell Death Induced by Ginsenoside Rg3: Significance in Dendritic Cell-based Anti-tumor Immunotherapy

Cancer is one of the leading causes of morbidity and mortality worldwide; therefore there is a need to discover new therapeutic modules with improved efficacy and safety. Immune-(cell) therapy is a promising therapeutic strategy for the treatment of intractable cancers. The effectiveness of certain chemotherapeutics in inducing immunogenic tumor cell death thus promoting cancer eradication has been reported. Ginsenoside Rg3 is a ginseng saponin that has antitumor and immunomodulatory activity. In this study, we treated tumor cells with Rg3 to verify the significance of inducing immunogenic tumor cell death in antitumor therapy, especially in DC-based immunotherapy. Rg3 killed the both immunogenic (B16F10 melanoma cells) and non-immunogenic (LLC: Lewis Lung Carcinoma cells) tumor cells by inducing apoptosis. Surface expression of immunogenic death markers including calreticulin and heat shock proteins and the transcription of relevant genes were increased in the Rg3-dying tumor. Increased calreticulin expression was directly related to the uptake of dying tumor cells by dendritic cells (DCs): the proportion of CRT+ CD11c+ cells was increased in the Rg3-treated group. Interestingly, tumor cells dying by immunogenic cell death secreted IFN-gamma, an effector molecule for antitumor activity in T cells. Along with the Rg3-induced suppression of pro-angiogenic (TNF-alpha) and immunosuppressive cytokine (TGF-beta) secretion, IFN-gamma production from the Rg3-treated tumor cells may also indicate Rg3 as an effective anticancer immunotherapeutic strategy. The data clearly suggests that Rg3-induced immunogenic tumor cell death due its cytotoxic effect and its ability to induce DC function. This indicates that Rg3 may be an effective immunotherapeutic strategy.

Human Circadian Rhythms / 수면정신생리

A 'circadian rhythm' is a self-sustained biological rhythm (cycle) that repeats itself approximately every 24 hours. Circadian rhythms are generated by an internal clock, or pacemaker, and persist even in the absence of environmental time cues, collectively termed 'zeitgebers.' Although organisms generate circadian rhythms internally, they are entrained by environmental stimuli, particularly the light-dark cycle. Measurement of the endogenous melatonin rhythm provides relatively reliable surrogate way of assessing the timing of the internal circadian clock. Also, core body temperature and cortisol can be used as markers of circadian rhythms. The sleep-wake cycle, body temperature, and melatonin rhythm have a stable internal phase relationship in humans and other diurnal species. They play an important role in controlling daily behavioral rhythms including task performance, blood pressure, and synthesis and secretion of several hormones. In this review, we address not only the properties, methods of measurement, and markers of circadian rhythms, but also the physiological and psychological importance of human circadian rhythms.

Chemotherapeutic Candidate Inducing Immunological Death of Human Tumor Cell Lines

The immunological death induction by EY-6 on the human tumor cell lines was screened. Human colon carcinoma (HCT15, HCT116), gastric carcinoma (MKN74, SNU668), and myeloma (KMS20, KMS26, KMS34) cells were died by EY-6 treatment with dose-dependent manner. CRT expression, a typical marker for the immunological death, was increased on the EY-6-treated colorectal and gastric cancer cells. Interestingly, the effects on the myeloma cell lines were complicated showing cell line dependent differential modulation. Cytokine secretion from the EY-6 treated tumor cells were dose and cell-dependent. IFN-gamma and IL-12 secretion was increased in the treated cells (200% to over 1000% of non-treated control), except HCT116, SNU668 and KMS26 cells which their secretion was declined by EY-6. Data suggest the potential of EY-6 as a new type of immuno-chemotherapeutics inducing tumor-specific cell death. Further studies are planned to confirm the efficacy of EY-6 including in vivo study.

Dendritic Cell (DC) Vaccine in Mouse Lung Cancer Minimal Residual Model; Comparison of Monocyte-derived DC vs. Hematopoietic Stem Cell Derived-DC

The anti-tumor effect of monocyte-derived DC (MoDC) vaccine was studied in lung cancer model with feasible but weak Ag-specific immune response and incomplete blocking of tumor growth. To overcome this limitation, the hematopoietic stem cell-derived DC (SDC) was cultured and the anti-tumor effect of MoDC & SDC was compared in mouse lung cancer minimal residual model (MRD). Therapeutic DCs were cultured from either CD34+ hematopoietic stem cells with GM-CSF, SCF and IL-4 for 14 days (SDC) or monocytes with GM-CSF and IL-4 for 7 days (MoDC). DCs were injected twice by one week interval into the peritoneum of mice that are inoculated with Lewis Lung Carcinoma cells (LLC) one day before the DC injection. Anti-tumor responses and the immune modulation were observed 3 weeks after the final DC injection. CD11c expression, IL-12 and TGF-beta secretion were higher in SDC but CCR7 expression, IFN-gamma and IL-10 secretion were higher in MoDC. The proportion of CD11c+CD8a+ cells was similar in both DC cultures. Although both DC reduced the tumor burden, histological anti-tumor effect and the frequencies of IFN-gamma secreting CD8+ T cells were higher in SDC treated group than in MoDC. Conclusively, although both MoDC and SDC can induce the anti-tumor immunity, SDC may be better module as anti-tumor vaccine than MoDC in mouse lung cancer.

Cellular Mechanism of Newly Synthesized Indoledione Derivative-induced Immunological Death of Tumor Cell

BACKGROUND: EY-6 is one of the newly synthesized indoledione derivatives to induce tumor cell-specific cell death. In this study, we investigated the mechanism of immunological death induced by EY-6 at mouse colon cancer cell as well as at the normal immune cell represented by dendritic cell. METHODS: C57BL/6 mouse syngeneic colon cancer cell MC38 was treated with EY-6, and analyzed by MTT for viability test, flow cytometry for confirming surface expressing molecules and ELISA for detection of cytokine secretion. Normal myeloid-dendritic cell (DC) was ex vivo cultured from bone marrow hematopoietic stem cells of C57BL/6 mice with GM-CSF and IL-4 to analyze the DC uptake of dead tumor cells and to observe the effect of EY-6 on the normal DC. RESULTS: EY-6 killed the MC38 tumor cells in a dose dependent manner (25, 50 and 100 microM) with carleticulin induction. And EY-6 induced the secretion of IFN-gamma but not of TNF-alpha from the MC38 tumor cells. EY-6 did not kill the ex-vivo cultured DCs at the dose killing tumor cells and did slightly but not significantly induced the DC maturation. The OVA-specific cross-presentation ability of DC was not induced by chemical treatment (both MHC II and MHC I-restricted antigen presentation). CONCLUSION: Data indicate that the EY-6 induced tumor cell specific and immunological cell death by modulation of tumor cell phenotype and cytokine secretion favoring induction of specific immunity eliminating tumor cells.

Immunotherapy of Malignant Melanoma with Tumor Lysate-Pulsed Autologous Monocyte-Derived Dendritic Cells

PURPOSE: Dendritic cell (DC) vaccination for melanoma was introduced because melanoma carries distinct tumor-associated antigens. The purpose of this study was to investigate the efficacy and safety of DC vaccination for melanoma in Korea. MATERIALS AND METHODS: Five patients with stage IV and one with stage II were enrolled. Autologous monocyte-derived DCs (MoDCs) were cultured and pulsed with tumor-lysate, keyhole limpet hemocyanin, and cytokine cocktail for mature antigen-loaded DC. DC vaccination was repeated four times at 2-week intervals and 2-4x107 DC were injected each time. RESULTS: Reduced tumor volume was observed by PET-CT in three patients after DC vaccination. Delayed type hypersensitivity responses against tumor antigen were induced in five patients. Tumor antigen-specific IFN-gamma-producing peripheral blood mononuclear cells were detected with enzyme-linked immunosorbent spot in two patients. However, the overall clinical outcome showed disease progression in all patients. CONCLUSION: In this study, DC vaccination using tumor antigen-loaded, mature MoDCs led to tumor regression in individual melanoma patients. Further standardization of DC vaccination protocol is required to determine which parameters lead to better anti-tumor responses and clinical outcomes.

Generation and Qualification of Functionally Active Leukemia-derived DCs from Malignant Blasts in Acute Leukemia

BACKGROUND: Dendritic cells (DCs) are increasingly being utilized for anti-cancer immunotherapy. Acute myeloid leukemia (AML) blasts are able to generate leukemia-derived DC. Advances in culture techniques and AML-DC characterization justify possible clinical applications. We investigated the ability of AML, acute lymphoblastic leukemia (ALL) and biphenotypic acute leukemia (BAL) blasts to differentiate into DCs in vitro and the qualified function of the leukemia-derived DCs. METHODS: Leukemia cells from 11 patients with AML, 3 patients with ALL and 2 patients with BAL were cultured with GM-CSF, IL-4 and with or without SCF. Cultured leukemia cells were evaluated by phenotype, mixed lymphocyte reaction (MLR), cytokine production and cytotoxic T cell (CTL) inducing activity. RESULTS: DCs were generated with GM-CSF and IL-4 from the leukemic blasts in 72% of the AML patient cells. MHC class I/II, CD11c and ICAM-1 were highly expressed in the AML-derived DCs. MLR and enzyme linked immunospot (ELISPOT) assays demonstrated that AML-DCs were able to induce T cell proliferation and activation into IFN-gamma secreting effector cells. The ALL blasts from two out of three patients differentiated into DCs with MHC class I/II+, CD11c+ only in the presence of GM-CSF, SCF and IL-4 for 14 days. CONCLUSION: These results suggest that functionallyactive DCs can be differentiated from AML blasts using GM-GSF and IL-4 and ALL, BAL blasts were differentiated into DCs only under stem cell-DC culture conditions.

Mechanism of Differential Ag-specific Immune Induction by Different Tumor Cell Lysate Pulsed DC

BACKGROUND: Tumor cell lysate has been considered as a preferential antigen source for the therapeutic dendritic cell pulsing. Our experiences with in vivo study with animal tumor model indicate the tumor cell lysate dependent differential effect of DC therapy. Our previous data show that MC38 lysate pulsed-DC induced stronger ag-specific immunity than CT26 lysate pulsed-DC in vitro. In this study we tried to reveal the mechanism for differential induction of ag-specific immunity of different colon cancer cell lysate pulsed-DCs. METHODS: MC38 and CT26 cell lines were prepared as lysate by freezing-thawing procedure. Tumor cell antigenicity was confirmed by detecting the surface expression of MHC I/II & B7.1/2 molecules. IL-10, IL-12 and TGF-beta in the tumor cell lysate were detected by ELISA and the presence of heat shock proteins were analysed by western blotting. RESULTS: The secretion of IL-10, a immune-inhibitory cytokine was about 470% higher in CT26 lysate than in MC38. Hsp 70 was detected only in the MC38 lysate but not in the CT26. On the other hand, Hsp 60 and 90 expression were not different in two colon cancer cell lysates. CONCLUSION: In two different colon caner cell lysate, immune inhibitory IL-10 (higher in CT26) and Hsp70 (MC38 superiority) were differentially expressed. These data indicate that higher ag- specific immunity induction by MC38 lysate pulsed-DC may due to the expression of hsp70 and lower secretion of IL-10, a immune-inhibitory cytokine than CT26 lysate. The significance of other cytokine and the surface marker expression will be discussed.

Anti-cancer Effect of Hematopoietic Stem Cell-derived Allogeneic-DC Vaccine in Melanoma Metastasis Model

BACKGROUND: Dendritic cell (DC)-based cancer immunotherapy is studied for several years. However, it is mainly derived from autologous PBMC or leukapheresis from patient, which has limitations about yield and ability of DC production according to individual status. In order to solve these problems, inquiries about allogeneic DCs are performed but there are no preclinical trial answers for effect or toxicity of allogeneic DC to use for clinical trial. In this study, we compared the anti-tumor effect of allogeneic and autologous DCs from mouse bone marrow stem cells in mouse metastatic melanoma model. METHODS: B16F10 melanoma cells (5 x 10(4)/mouse) were injected intravenously into the C57BL/6 mouse. Therapeutic DCs were differentiated from autologous (C57BL/6: CDC) or allogeneic (B6C3F1: BDC) bone marrow stem cells with GM-CSF, SCF and IL-4 for 13days and pulsed with B16F10 tumor cell lysate (Blys) for 18hrs. DC intra-peritoneal injections began on the 8th day after the tumor cell injection by twice with one week interval. RESULTS: Anti-tumor response was observed by DC treatment without any toxicity especially in allogeneic DC treated mice (tumor burden score: 2.667+/-0.184, 2.500+/-0.463, 2.000+/-0.286, 1.500+/-0.286, 1.667+/-0.297 for saline, CDC/unpulsed-DC: U-DC, CDC/Blys-DC, BDC/U-DC and BDC/Blys-DC, respectively). IFN-gamma secretion was significantly increased in allogeneic DC group stimulated with B16F10 cell lysate (2,643.3+/-5,89.7, 8,561.5+/-2,204.9. 6,901.2+/-141.1 pg/1 x 10(6) cells for saline, BDC/U-DC and BDC/Blys-DC, respectively) with increased NK cell activity. CONCLUSION: Conclusively, promising data was obtained that allogeneic DC can be used for DC-based cancer immunotherapy.

Effect of Dendritic Cell Based Cancer Vaccine Using Allogeneic Tumor Cell Lysate in Melanoma Pulmonary Metastasis Model

BACKGROUND: To perform the successful dendritic cell-based cancer immunotherapy one of the main issues to be solved is the source of antigen for DC pulsing. Limitations occur by using auto-tumor lysate due to the difficulties obtaining enough tumor tissue(s) quantitatively as well as qualitatively. In this study the possibility of allogeneic tumor cell lysate as a DC pulsing antigen has been tested in mouse melanoma pulmonary metastasis model. METHODS: B16F10 melanoma cells (1x10(5)/mouse) were inoculated intravenously into the C57BL/6 mouse. Therapeutic DCs were cultured from the bone marrow myeloid lineage cells with GM-CSF and IL-4 (1,000 U/ml each) for 7 days and pulsed with lysate of either autologous B16F10 (B-DC), allogeneic K1735 (C3H/He origin; K-DC) or CloneM3 (DBA2 origin; C-DC) melanoma cells for 18 hrs. Pulsed-DCs (1x10(6)/mouse)[CGP1] were injected i.p. twice with one week interval starting from the day 1 after tumor cell inoculation. RESULTS: Without observable toxicity, allogeneic tumor cell lysate pulsed-DC induced the significantly better anti-tumor response (tumor scale: 2.7+/-0.3, 0.7+/-0.3 and 0.3+/-0.2 for saline, B-DC and C-DC treated group, respectively). Along with increased tumor specific lymphocyte proliferations, induction of IFN-gamma secretion against both auto- and allo-tumor cell lysates was observed from the DC treated mice. (w/B16F10-lysate: 44.97+/-10.31, 1787.94+/-131.18, 1257.15+/-48.27, w/CloneM3 lysate: 0, 1591.13+/-1.83, 1460.47+/-86.05 pg/ml for saline, B-DC and C-DC treated group, respectively) Natural killer cell activity was also increased in the mice treated with tumor cell lysate pulsed-DC (8.9+/-[CGP2]0.1, 11.6+/-0.8 and 12.6+/-0.7% specific NK activity for saline, B-DC and C-DC treated group, respectively). CONCLUSION: Conclusively, promising data were obtained that allogeneic-tumor cell lysate can be used as a tumor antigen for DC-based cancer immunotherapy.

Immunocell Therapy for Lung Cancer: Dendritic Cell Based Adjuvant Therapy in Mouse Lung Cancer Model

BACKGROUND: The anti-tumor therapeutic effect of autologous tumor cell lysate pulsed-dendritic cells (DCs) was studied for non-immunogenic and immune suppressive lung cancer model. To test the possibility as an adjuvant therapy, minimal residual disease model was considered in mouse in vivo experiments. METHODS: Syngeneic 3LL lung cancer cells were inoculated intravenously into the C57BL/6 mouse. Autologous tumor cell (3LL) or allogeneic leukemia cell (WEHI-3) lysate pulsed-DCs were injected twice in two weeks. Intraperitoneal DC injection was started one day (MRD model) after tumor cell inoculation. Two weeks after the final DC injection, tumor formation in the lung and the tumor-specific systemic immunity were observed. Tumor-specific lymphocyte proliferation and the IFN-r secretion were analyzed for the immune monitoring. Therapeutic DCs were cultured from the bone marrow myeloid lineage cells with GM-CSF and IL-4 for 7 days and pulsed with tumor cell lysate for 18 hrs. RESULTS: Compared to the saline treated group, tumor formation was suppressed in 3LL tumor cell lysate pulsed-DC treated group, while 3LL-specific immune stimulation was minimum. WEHI-3-specific immune stimulation occurred in WEHI-3 lysate-pulsed DC treated group, which had no correlation with tumor regression. CONCLUSION: The data suggest the possible anti-tumor effect of cultured DCs as an adjuvant therapy for minimal residual disease state of lung cancer. The significance of immune modulation in DC therapy including the possible involvement of NK cell as well as antigen-specific cytotoxic T cell activity induction was discussed.

Comparison of Monocyte Selection Method by Immunomagnetic Adsorption or Adherence for the Generation of Dendritic cells / 대한수혈학회지

BACKGROUND: Dendritic cells (DCs) are the most potent stimulators of immune response including antitumor response. DCs are currently being pursued clinically in the development of cancer vaccines; therefore there are demands for large-scale and clinical-grade generation of DCs. In the present study, to find out the most efficient separation method of DC precursors, we compared two separation methods, namely, based on magnetic based selection and plastic adherence selection. METHODS: MNCs were collected by leukapheresis from healthful donors and separated by CD14 + immunomagnetic adsorption or plastic adherence. DC precursors separated using the two methods were differenciated in the same condition. Matured DCs were compared in terms of yield, viability, the expression of surface markers and ability to induce immune reaction. RESULTS: This study demonstrated that mature DCs from CD14 + monocytes separated using CD14 + immunomagnetic adsorption had higher expression of surface markers of DCs, yield (1.9 +/-0.5% vs. 0.5 +/-0.2%), viability (94.7 +/-2.5% vs. 72.8 +/-7.5%) and better functionality in inducing immune reaction than those from plastic adherent cells. CONCLUSION: These results demonstrated that CD14 + immunomagnetic adsorption was found to be more effective than the adherent selection for the generation of DCs. This study will allow researcher to facilitate choosing the appropriate protocol to obtain DCs.

Dendritic Cell Based Cancer Immunotherapy: in vivo Study with Mouse Renal Cell Carcinoma Model

BACKGROUND: As a potent antigen presenting cell and a powerful inducer of antigen specific immunity, dendritic cells (DCs) are being considered as a promising anti-tumor therapeutic module. The expected therapeutic effect of DCs in renal cell carcinoma was tested in the mouse model. Established late-stage tumor therapeutic (E-T) and minimal residual disease (MRD) model was considered in the in vivo experiments. METHODS: Syngeneic renal cell carcinoma cells (RENCA) were inoculated either subcutaneously (E-T) or intravenously (MRD) into the Balb/c mouse. Tumor cell lysate pulsed-DCs were injected twice in two weeks. Intraperitoneal DC injection was started 3 week (E-T model) or one day (MRD model) after tumor cell inoculation. Two weeks after the final DC injection, the tumor growth and the systemic immunity were observed. Therapeutic DCs were cultured from the bone marrow myeloid lineage cells with GM-CSF and IL-4 for 7 days and pulsed with RENCA cell lysate for 18 hrs. RESULTS: Compared to the saline treated group, tumor growth (E-T model) or formation (MRD model) was suppressed in pulsed-DC treated group. RENCA specific lymphocyte proliferation was observed in the RENCA tumor-bearing mice treated with pulsed-DCs. Primary cytotoxic T cell activity against RENCA cells was increased in pulsed-DC treated group. CONCLUSION: The data suggest the possible anti-tumor effect of cultured DCs in established or minimal residual disease/metastasis state of renal cell carcinoma. Systemic tumor specific immunity including cytotoxic T cell activity was modulated also in pulsed-DC treated group.

Internet Survey on the Sexual Life and Attitude of Sexual Life of Young Women / 대한남성과학회지

PURPOSE: To obtain basic information for studies of female sexual dysfunction, we investigated the sexual activities and attitudes of young Korean women. MATERIALS AND METHODS: We conducted a questionnaire survey via the Internet. From July 2004 to August 2004, we sent e-mail to 43,000 women who registered with an internet research company. The recipients of the e-mail were asked to join our study if they had stable sexual activities more than once a month for the most recent 6 months. RESULTS: A total of 508 subjects completed the questionnaire, a response rate of 24.7%. Among 423 questionnaires analyzed, 176 women were single and 247 were married. The mean frequency of coitus per month was 5.5 3.9. About 40% of the women had experienced masturbation, and 112 women (26.5%) had masturbated regularly, even though they had stable sexual activities with their partner(s). Among the 423 subjects, 349 had no plan to have a baby; however, only 287 subjects (67.8%) had used a contraceptive method(s). Among those who used contraceptive methods, 183 subjects (63.8%) used methods with low success rates, such as menstrual timing or extra-vaginal ejaculation. Most of the women answered that they have a positive attitude toward sex (3.4 0.8 point on a 5 point scale) and their sex life has great importance in their life. CONCLUSIONS: Via an Internet survey, we investigated the sexual life and attitudes of young Korean women. An Internet survey requires less manpower, a shorter study period, and less research funds than classical survey methods such as mail or interview surveys. The study results obtained will be useful as basic data for studies of female sexual function in Korean women.

Regulation of Gene Expression in Murine Renal Cell Carcinoma Cells Growing in Ectopic or Orthotopic Organs of Syngenic Mice / 대한암학회지

PURPOSE: The biologic behavior of tumor cells is partially controlled by the microenvironment. We investigated the expression levels of several genes involved in metastasis and drug response in RENCA cells growing in ectopic (skin) and orthotopic (kidney) sites. MATERIALS AND METHODS: Murine renal carcinoma cells were injected into kidney (orthotopic) and subcutis (ectopic) of syngeneic mice. Mice were treated with doxorubicin (DXR) (8 mg/kg) on days 8 and 15 after tumor cell implantation. Drug response was measured both in vivo and ex vivo by measuring tumor size and MTT assay. We also performed an in situ mRNA hybridization to estimate the expression levels of mdr (multidrug resistance), EGFR (epidermal growth factor receptor) and type IV collagenase. RESULTS: RENCA cells growing in the kidney of syngeneic mice produced metastatic lesions in the lung (57% of mice), while the same cells growing in the subcutis did not. Tumors growing in the kidney were more resistant to DXR than tumors growing in the subcutis. MTT assays revealed that tumor cells derived from kidney were more resistant to DXR than those cells from subcutis. In situ hybridization analyses showed that transcripts of EGFR and type IV collagenase genes in kidney tumors were higher than those of subcutaneous tumors but mdr expression showed no difference between the two tumors. CONCLUSION: These results demonstrate that the organ environment influences the drug responsive ness and the expression of EGFR and type IV collagenase genes in murine renal cell carcinoma cells.