Effects of oxygen tension and IGF-I on HIF-1α protein expression in mouse blastocysts.

PURPOSE: Hypoxia inducible factors (HIFs) are key regulators of oxygen homeostasis in response to reduced oxygenation in somatic cells. In addition, HIF-1α protein can be also induced by insulin-like growth factor I (IGF-I) treatment in various cell lines under normoxic condition. However, the expression and function of HIF-1α in embryogenesis are still unclear. Therefore, the objectives of this study were to examine the expression of HIF-1α in mouse blastocysts cultured under hypoxic and normoxic conditions, and to determine whether oxygen tension and IGF-I influence embryonic development through stimulation of HIF-1α expression. METHODS: Mouse embryos were cultured from the 1-cell to blastocyst stage under 5 % or 20 % O(2) in both the absence and presence of IGF-I. RESULTS: The embryonic development rates to the blastocyst stage were not affected by oxygen tension or IGF-I treatment. HIF-1α protein was localized to the cytoplasm of blastocysts, and its levels were independent of oxygen concentration or IGF-I treatment. Blastocysts cultured under 5 % O(2) exhibited significantly higher total cell numbers (83.4 ± 18.1) and lower apoptotic index (3.7 ± 1.5) than those cultured under 20 % O(2) (67.4 ± 15.6) (6.9 ± 3.5) (P<0.05). IGF-I reduced the apoptotic index in both oxygen conditions, but a significant decrease was detected in the 20 % O(2) group. CONCLUSIONS: HIF-1α may not be a major mediator that responds to change in oxygen tension within blastocysts, inconsistent with that of somatic cells. Supplementation of culture media with IGF-I has been shown to promote embryo development by an anti-apoptotic effect, instead of increasing HIF-1α protein expression.

CUX1 is a haploinsufficient tumor suppressor gene on chromosome 7 frequently inactivated in acute myeloid leukemia.

Loss of chromosome 7 and del(7q) [-7/del(7q)] are recurring cytogenetic abnormalities in hematologic malignancies, including acute myeloid leukemia and therapy-related myeloid neoplasms, and associated with an adverse prognosis. Despite intensive effort by many laboratories, the putative myeloid tumor suppressor(s) on chromosome 7 has not yet been identified.We performed transcriptome sequencing and SNP array analysis on de novo and therapy-related myeloid neoplasms, half with -7/del(7q). We identified a 2.17-Mb commonly deleted segment on chromosome band 7q22.1 containing CUX1, a gene encoding a homeodomain-containing transcription factor. In 1 case, CUX1 was disrupted by a translocation, resulting in a loss-of-function RNA fusion transcript. CUX1 was the most significantly differentially expressed gene within the commonly deleted segment and was expressed at haploinsufficient levels in -7/del(7q) leukemias. Haploinsufficiency of the highly conserved ortholog, cut, led to hemocyte overgrowth and tumor formation in Drosophila melanogaster. Similarly, haploinsufficiency of CUX1 gave human hematopoietic cells a significant engraftment advantage on transplantation into immunodeficient mice. Within the RNA-sequencing data, we identified a CUX1-associated cell cycle transcriptional gene signature, suggesting that CUX1 exerts tumor suppressor activity by regulating proliferative genes. These data identify CUX1 as a conserved, haploinsufficient tumor suppressor frequently deleted in myeloid neoplasms.

Effect of emotional intelligence on patient-physician interaction scores of clinical performance examination.

PURPOSE: The patient-physician interaction (PPI) is a critical part of the clinical encounter. Recent studies have emphasized the importance of the emotional intelligence (EI) of physician in the PPI. Despite emphasizing the EI, previous studies offer limited evidence regarding the effect of a student's EI on the PPI. The purpose of this study is to explore the differences in EI depending on the demographics of medical students and the correlation between EI and PPI scores. METHODS: The sample was 85 fourth-grade medical students. Prior to taking a 12-station clinical performance examination, the students completed questionnaires on their own perception of the EI, which included 5 domains and 50 items. The tool that was used to assess the level of EI was Moon's modified version of the EI test for adults. We investigated differences in EI depending on the demographics of medical students by ANOVA and noted a correlation between EI and PPI scores by stepwise multiple regression analysis. RESULTS: This study found that females or graduate entry students have higher EI scores and that 25 to 30-year-old students have higher EI scores than aged under 25 years. The PPI scores correlated positively with total EI scores (r=0.32) and 2 subdomains (perception and expression of emotion, r=0.26; empathy, r=0.33). Two subdomains were the best predictors of PPI score (R2=0.171). CONCLUSION: EI correlates significantly with PPI score and affects it. We conclude that EI is a key influence of the PPI. Further research is required to explore whether this is a consistent effect.

Application of two different synthetic sequential media for the human IVF-ET program: a prospective, randomized, and comparative study.

OBJECTIVE: Since IVF program was first established, various types of media and culture systems have been developed either in-house or commercially. The aim of this study was to compare the efficacy of in-house Maria Research Center (MRC) media to that of commercially available Sydney IVF media in human day 3 embryo transfer cycles. METHODS: Three hundred sixty nine couples were included in this prospective, randomized, and comparative study. All couples undergoing IVF treatment at the Maria Fertility Hospital were randomly assigned to either Sydney IVF (n=178) or MRC (n=191) media. RESULTS: No difference was observed between the MRC media and Sydney IVF media groups with respect to fertilization rate (74.4% vs. 75.5%). The clinical pregnancy and implantation rates of MRC media (47.1% and 20.0%, respectively) were also similar to those of Sydney IVF media (44.4% and 19.4%, respectively). However, the proportion of embryos with good quality on day 3 was significantly higher in the MRC media group than the Sydney IVF media group (50.2% vs. 43.2%) (p<0.05). CONCLUSION: MRC media were as effective as Sydney IVF media for sustaining embryo development and pregnancy rates. The present study implies that MRC media can be a suitable alternative to commercially available media for human IVF-ET program.

In vivo scintigraphic imaging of antitumor effects by combined radioiodine therapy and human sodium iodide symporter gene immunotherapy.

In a previous study, we demonstrated that pcDNA3.1/hNIS (human sodium iodide symporter) vaccination generated hNIS-associated CD8+IFN-gamma+ (interferon-gamma) T cells, which are known to be involved in antitumor immunity. However, the immune response induced was insufficient to control tumor growth in vivo, which required a novel approach to potentiate hNIS vaccination effects. In the present study, we administered 131I radioiodine therapy prior to hNIS vaccination in CT26/hNIS tumor-bearing mice to facilitate the vaccine-induced immune response. We characterized hNIS-associated cytotoxic T-cell immune response and the antitumor effects induced by this 131I + hNIS combination therapy. The survival rates of CT26/hNIS tumor cells were significantly reduced by 131I treatment compared with the parental CT26 cells in vitro. 131I + hNIS combination therapy stably suppressed tumor growth below or near the original tumor size level of initial treatment, achieving 100% survival rates. Specifically, 131I + hNIS therapy enhanced IFN-gamma production, hNIS-associated antitumor cytotoxic T-lymphocyte (CTL) response, and induced more dendritic cells but reduced T-regulatory cells in tumor masses. Collectively, these results suggest that combined therapy effectively enhances hNIS-associated antitumor immune response, leading to CT26/hNIS tumor growth inhibition and complete survival in Balb/C mice. These findings provide a novel and effective means of treating cancer.

MHC independent anti-tumor immune responses induced by Hsp70-enriched exosomes generate tumor regression in murine models.

The ideal cancer vaccine should work regardless of MHC types but currently the barrier generated by MHC specificity hampers the development of human cancer vaccines, requesting to identify strong immunogenic molecules that can induce anti-cancer immune responses without being affected by MHC polymorphism. Tumor-derived exosomes are small membrane vesicles containing tumor antigens as well as other immunologically important molecules such as MHC molecules and heat shock proteins (HSPs). Because of their potential immunogenicity, the plausible utility of tumor-derived exosomes as an MHC independent cancer vaccine was proposed. Here, we investigated whether Hsp70-enriched tumor exosomes can induce stronger immunogenicity as compared to normal tumor-derived exosomes in autologous as well as allogeneic murine models in vitro and in vivo. Western blotting showed that the exosomes of heat-treated tumor cells (HS Exo) contained higher amounts of Hsp70 than the exosomes of untreated cells (CNTL Exo). In both MHC type-identical and -irrelevant antigen-presenting cell models in vitro, HS Exo triggered the increased expressions of MHC class II molecules. Crucially, HS Exo performed greater therapeutic capability in regressing pre-established MHC type-identical and -irrelevant tumors than CNTL Exo in vivo. The analyses of anti-tumor function in allogeneic mouse model demonstrated that HS Exo elicited Th1-polarized immune responses defined by the increased productions of IgG2a and IFN-gamma. In summary, the Hsp70-enriched exosomes extracted from heat-treated tumors induced strong Th1 immune responses, resulting in eliminating cancer cells in allogeneic hosts in vivo. These results indicate that HS Exo is a potent MHC independent cell-free cancer therapeutic agent that can be developed for clinical trials.

Development and validation of a questionnaire to evaluate medical students' evidence-based medicine competencies.

PURPOSE: The purpose of the study was to develop and validate a questionnaire to evaluate medical students' knowledge of, attitude towards and practice of evidence-based medicine (EBM). METHODS: The participants of the study were 418 medical students enrolled in the Kyung Hee University School of Medicine in Seoul, Korea. To examine construct validity of the questionnaire, an exploratory factor analysis (EFA) was performed with 118 participants; a confirmatory factor analysis (CFA) was conducted with the remaining 281 participants. We developed 41 items with a 4-point Likert scale. An EFA was performed to verify the emergence of four dimensions of EBM competencies. The principal axis factoring method and the direct oblimin rotation method were used. To confirm construct validity, a CFA was conducted with the remaining 281 participants. To evaluate model fitness, root mean square error of approximation (RMSEA) and comparative fit index (CFI) were used as fit indices. We conducted ANOVA with Scheffe as discriminant validation, and calculated Cronbach's alpha of 4 subscales as reliability checkup. RESULTS: After refinement procedure, factor analysis of the 32 items in therevised questionnaire yielded 4 factors. The Scree plot supported a 4 factor solution explaining 53.5% of the variance. The 4 components derived were: factor 1_knowledge on EBM (11 items; Cronbach's alpha=0.92); factor 2_ pursuit towards EBM (10; 0.88); factor 3_reluctance on EBM (7; 0.78); factor 4_practice of EBM (4; 0.75). The questionnaire could discriminate competence differences among 1-3 yr students. Satisfactory Cronbach's alpha scores were noted for each factor as well. CONCLUSION: The EBM competency questionnaire was validated.

Faculty observer and standardized patient accuracy in recording examinees' behaviors using checklists in the clinical performance examination.

PURPOSE: The purpose of the study was to examine the recording accuracy of faculty observers and standardized patients (SPs) on a clinical performance examination (CPX). METHODS: This was a cross-sectional study of a fourth-year medical students' CPX that was held at a medical school in Seoul, Korea. The CPX consisted of 4 cases and was administered to 118 examinees, with the participation of 52 SP and 45 faculty observers. For the study we chose 15 examinees per case, and analyzed 60 student-SP encounters in total. To determine the recording accuracy level, 2 SP trainers developed an answer key for each encounter. First, we computed agreement rates (P) and kappa coefficient (K) values between the answer key-SPs and the answer key-faculty observers. Secondly, we analyzed variance (ANOVA) with repeated measures to determine whether the mean percentage of the correct checklist score differed as a function of the rater, the case, or the interaction between both factors. RESULTS: Mean P rates ranged from 0.72 to 0.86, while mean K values varied from 0.39 to 0.59. The SP checklist accuracy was higher than that of faculty observersat the level of item comparison. Results from ANOVA showed that there was no significant difference between the percentage of correct scores by the answer key, faculty observers and SPs. There was no significant interaction between rater and case factors. CONCLUSION: Acceptable levels of recording accuracy were obtained in both rater groups. SP raters can replace faculty raters in a large-scale CPX with thorough preparation.

Suppression of E-protein activity interferes with the development of BCR-ABL-mediated myeloproliferative disease.

E-proteins are a class of helix-loop-helix (HLH) proteins, which play multiple roles throughout lymphoid development. The DNA binding activities of the E-proteins are regulated by a distinct class of antagonistic HLH proteins, named Id1-4. Here we demonstrate that Id2 deficient mice in a C57BL/6 genetic background exhibit increased cellularity in the granulocyte/myeloid progenitor compartment and show significantly higher numbers of maturing neutrophils. Within 6 months of age, Id2 deficient mice succumbed from overwhelming granulocytosis. The disease closely mimicked the distinctive features of human chronic myeloid leukemia: leukocytosis with maturing neutrophils, splenomegaly, hepatomegaly, and myeloid infiltration into peripheral tissues, including spleen, liver, and lungs. Strikingly, forced Id2 expression in murine bone marrow cells substantially delayed the onset of myeloproliferative disease (MPD). Collectively, these studies show that suppression of E-protein activity interferes with the development of BCR-ABL-mediated MPD.

Molecular host-pathogen interaction in brucellosis: current understanding and future approaches to vaccine development for mice and humans.

Brucellosis caused by Brucella spp. is a major zoonotic disease. Control of brucellosis in agricultural animals is a prerequisite for the prevention of this disease in human beings. Recently, Brucella melitensis was declared by the Centers for Disease Control and Prevention to be one of three major bioterrorist agents due to the expense required for the treatment of human brucellosis patients. Also, the economic agricultural loss due to bovine brucellosis emphasizes the financial impact of brucellosis in society. Thus, vaccination might efficiently solve this disease. Currently, B. abortus RB51 and B. melitensis REV.1 are used to immunize cattle and to immunize goats and sheep, respectively, in many countries. However, these genetically undefined strains still induce abortion and persistent infection, raising questions of safety and efficiency. In fact, the REV.1 vaccine is quite virulent and apparently unstable, creating the need for improved vaccines for B. melitensis. In addition, Brucella spp. may or may not provide cross-protection against infection by heterologous Brucella species, hampering the acceleration of vaccine development. This review provides our current understanding of Brucella pathogenesis and host immunity for the development of genetically defined efficient vaccine strains. Additionally, conditions required for an effective Brucella vaccine strain as well as the future research direction needed to investigate Brucella pathogenesis and host immunity are postulated.

Virulence criteria for Brucella abortus strains as determined by interferon regulatory factor 1-deficient mice.

Interferon regulatory factor 1-deficient (IRF-1(-/-)) mice infected with virulent Brucella abortus 2308 at 5 x 10(5) CFU developed acute hepatitis similar to many natural hosts but, unlike natural hosts, IRF-1(-/-) mice were unable to resolve infection and died. In contrast, IRF-1(-/-) mice survived when infected at 5 x 10(5) CFU with several attenuated Brucella strains (S19, RB51, cbp, and cyd). The survival of infected IRF-1(-/-) mice is likely a function of the level of virulence of each Brucella strain and the extent of retained immunity. Further, these findings suggest that adaptive immunity may be important to the survival of IRF-1(-/-) mice since attenuated Brucella strains can protect IRF-1(-/-) mice against lethal challenge with virulent Brucella: Using the IRF-1(-/-) mouse model, the following set of criteria were identified to define Brucella virulence: (i) the day of death for 50% of mice infected with 5 x 10(5)CFU of Brucella, (ii) the extent of liver toxicity, and (iii) the minimum immunizing dose of Brucella to protect against challenge with virulent S2308. Thus, IRF-1(-/-) mice are important to determining the level of Brucella virulence, to evaluating Brucella mutants for attenuation, and to investigating adaptive immunity in brucellosis.

Susceptibility of IFN regulatory factor-1 and IFN consensus sequence binding protein-deficient mice to brucellosis.

IFN-gamma is a key cytokine controlling Brucella infection, and the diverse functions of this cytokine are mediated by IFN regulatory factors (IRFs) such as IRF-1, IRF-2, and IFN consensus sequence binding protein (ICSBP). However, the roles of these three IRFs in Brucella infection have not been investigated. The infection of each IRF-deficient mouse strain provides an opportunity to determine not only the significance of each IRF molecule but also the crucial immune components necessary for host defense during in vivo infection, because respective IRF-deficient mouse strains contain unique immunodeficient phenotypes. Brucella abortus S2308-infected IRF-1-/- mice were dead within 2 wk postinfection, while IRF-2-/- mice contained less splenic Brucella CFU than wild-type mice at the early stage of infection. Infected ICSBP-/- mice maintained a plateau of splenic Brucella CFU throughout the infection. Additional infection of IL-12p40-, NO synthase 2-, and gp91(phox)-deficient mice indicates that these immune components are crucial for Brucella immunity and may contribute to the susceptibility of IRF-1-/- and ICSBP-/- mice. Immunologic and histopathological analyses of infected IRF-1-/- mice indicate that the absence of IL-12p40 induction and serious hepatic damage are involved in the death of IRF-1-/- mice. These results indicate that 1) IRF-1 and ICSBP are essential transcriptional factors for IFN-gamma-mediated protection against Brucella; 2) IL-12, reactive nitrogen intermediates, and reactive oxygen intermediates are crucial immune components against Brucella, and their absence may contribute to the susceptibility of IRF-1-/- and ICSBP-/- mice; and 3) hepatic damage caused by Brucella virulence contributes to the death of IRF-1-/- mice.