Chang Geun Lee

Sustained E2 antibody response correlates with reduced peak viremia after hepatitis C virus infection in the chimpanzee.

Immune correlates of protection against hepatitis C virus (HCV) infection are not well understood. Here we investigated 2 naive and 6 immunized chimpanzees before and after intravenous challenge, 12 weeks after the last immunization, with 100 50% chimpanzee infectious doses (CID50) of heterologous genotype 1b HCV. Vaccination with recombinant DNA and adenovirus vaccines expressing HCV core, E1E2, and NS3‐5 genes induced long‐term HCV‐specific antibody and T‐cell responses and reduced peak viral load about 100 times compared with controls (5.91 ± 0.38 vs. 3.81 ± 0.71 logs, respectively). There was a statistically significant inverse correlation between peak viral loads and envelope glycoprotein 2 (E2)‐specific antibody responses at the time of challenge. Interestingly, one vaccinee that had sterilizing immunity against slightly heterologous virus generated the highest level of E2‐specific total and neutralizing antibody responses as well as strong NS3/NS5‐specific T‐cell proliferative responses. The other four vaccinees with low levels of E2‐specific antibody had about 44‐fold reduced peak viral loads but eventually developed persistent infections. In conclusion, vaccine‐induced E2‐specific antibody plays an important role in prevention from nonhomologous virus infection and may provide new insight into the development of an effective HCV vaccine. (HEPATOLOGY 2005;42:1429–1436.)

Efficient induction of T helper 1 CD4+ T-cell responses to hepatitis C virus core and E2 by a DNA prime-adenovirus boost.

Hepatitis C virus (HCV) is an important causative agent of liver disease, but currently there is no available prophylactic vaccine against HCV infection. Here, we investigated the HCV E2- and core-specific T-cell responses induced by DNA (D) and/or recombinant adenovirus (A) vaccines. In single (D versus A) or double immunizations (D-D versus A-A), the recombinant adenovirus vaccines induced higher levels of IFN-gamma secreting T-cell response and cytotoxic T lymphocytes (CTL) response than the DNA vaccines. However, a heterologous (D-A) regimen elicited the highest level of T helper 1 (Th1) CD4(+) T-cell responses. Furthermore, three E2-specific CTL epitopes were mapped using a peptide pool spanning the E2 protein sequence (a.a. 384-713) in BALB/c mice, and one of these (E2 405-414: SGPSQKIQLV) was shown to be immunodominant. Interestingly, no significant differences were found in the repertoire of E2-specific T-cell responses or in the immunodominance hierarchy of the three epitopes induced by D-D, D-A, A-A, and A-D, indicating that the breadth and hierarchy of T-cell responses is independent of these different vaccination regimens. In conclusion, the heterologous DNA prime-recombinant adenovirus boost regimen described offers an efficient promising strategy for the development of an effective T-cell-based HCV vaccine.

Mucosal immunity induced by adenovirus-based H5N1 HPAI vaccine confers protection against a lethal H5N2 avian influenza virus challenge

Development of effective vaccines against highly pathogenic avian influenza (HPAI) H5N1 viruses is a global public health priority. Considering the difficulty in predicting HPAI H5N1 pandemic strains, one strategy used in their design includes the development of formulations with the capacity of eliciting broad cross-protective immunity against multiple viral antigens. To this end we constructed a replication-defective recombinant adenovirus-based avian influenza virus vaccine (rAdv-AI) expressing the codon-optimized M2eX-HA-hCD40L and the M1-M2 fusion genes from HPAI H5N1 human isolate. Although there were no significant differences in the systemic immune responses observed between the intramuscular prime-intramuscular boost regimen (IM/IM) and the intranasal prime-intramuscular boost regimen (IN/IM), IN/IM induced more potent CD8(+) T cell and antibody responses at mucosal sites than the IM/IM vaccination, resulting in more effective protection against lethal H5N2 avian influenza (AI) virus challenge. These findings suggest that the strategies used to induce multi-antigen-targeted mucosal immunity, such as IN/IM delivery of rAdv-AI, may be a promising approach for developing broad protective vaccines that may be more effective against the new HPAI pandemic strains.

Mitotic catastrophe is the predominant response to histone acetyltransferase depletion

Histone acetylation induces chromatin opening by perturbing higher-order chromatin compaction and folding, suggesting that histone acetylation and deacetylation dynamics are central to chromosome condensation or decondensation. The condensation of chromosomes during mitosis is an essential prerequisite for successful chromosome segregation. In this study, we depleted three representative histone acetyltransferases (HATs; p300, CBP, and P/CAF) using shRNAs to explore their role in regulating mitotic progression and chromosome segregation. We showed that HAT depletion severely interfered with the normal timing of mitotic progression, and it reduced condensin subunit levels. The predominant response to HAT depletion, in both human primary and cancer cells, was a mitotic catastrophe following aberrant mitotic arrest. Alternatively, adaptation to HAT depletion, particularly in cancer cells, led to multinucleation and aneuploidy. Interestingly, mitotic catastrophe induced by HAT depletion appeared to be coupled to the signaling process of H2AX phosphorylation and foci formation, independently of DNA double-strand breaks and DNA damage. Taken together, our results provide novel molecular evidence that HAT proteins maintain mitotic chromatin assembly and integrity as a cellular determinant of mitotic cell death.

Enhanced delivery efficiency of recombinant adenovirus into tumor and mesenchymal stem cells by a novel PTD

Protein transduction domains (PTDs) are small peptides that facilitate the transduction of large molecules such as polyproteins, DNA and viruses into a eukaryotic cell. Here, we demonstrated that a novel PTD (HP4) derived from herring protamine appeared to enter C6Bu1 rat glioma cell lines more rapidly than other known PTDs such as Tat, Antp and Hph-1. Moreover, HP4 significantly enhanced in vitro transduction of recombinant adenoviruses (rAds) into various cancer cell lines, mesenchymal stem cells (MSCs) and dendritic cells, which are relatively resistant to rAd infection. Enhancement of rAd delivery into C6Bu1 and MSCs by HP4 is 20 and 7 times higher than that by Tat, respectively. The increase in the expression of rAd encoding IL-12N220L by HP4 is proportional to its antitumor effect in the ex vivo transduced mouse colon cancer model. Thus, these results suggest that HP4 could be utilized to improve the transduction efficiency of rAd, resulting in enhanced efficacy of rAd-mediated gene therapy, especially for ex vivo-transduced cell therapy.

DNA methylation and not allelic variation regulates STAT6 expression in human T cells

STAT6 transcription factor, which has been implicated in commitment to Th2, is known to be activated by IL-4 and IL-13. Accordingly, STAT6 is primarily responsible for the transcriptional effects of IL-4 and IL-13. STAT6-deficient mice are known to have defective IL-4-mediated functions, such as B cell proliferation, Th2 cell development and IgE secretion; therefore, they primarily contain the Th1 phenotype. However, the mechanism responsible for regulation of STAT6 expression transcriptionally and post-transcriptionally has yet to be elucidated. Here, we characterized the human STAT6 promoter gene and found that the transcriptional regulatory elements CCAAT and ATF were important for the STAT6 promoter activity. Direct sequencing analysis revealed that the 13 GT repeat allelic variation in noncoding exon 1 of the STAT6 gene appeared more frequently in 91 patients with asthma or rheumatoid arthritis than the 15 GT repeat variation, which is the dominant phenotype in healthy controls. However, it appears that this allelic variation did not affect the STAT6 transcriptional activity. Interestingly, treatment with a DNA methyltransferase inhibitor markedly increased the expression of STAT6 mRNA and protein in human primary T cells. In contrast, IFN-γ treatment significantly repressed the STAT6 transcriptional activity. Therefore, the present study provides insight into the molecular basis of STAT6 expression, and in particular, demonstrates that STAT6 expression is associated with DNA hypermethylation rather than promoter polymorphisms or allelic variations.

A possible usage of a CDK4 inhibitor for breast cancer stem cell-targeted therapy

Cancer stem cells (CSCs) are one of the main reasons behind cancer recurrence due to their resistance to conventional anti-cancer therapies. Thus, many efforts are being devoted to developing CSC-targeted therapies to overcome the resistance of CSCs to conventional anti-cancer therapies and decrease cancer recurrence. Differentiation therapy is one potential approach to achieve CSC-targeted therapies. This method involves inducing immature cancer cells with stem cell characteristics into more mature or differentiated cancer cells. In this study, we found that a CDK4 inhibitor sensitized MDA-MB-231 cells but not MCF7 cells to irradiation. This difference appeared to be associated with the relative percentage of CSC-population between the two breast cancer cells. The CDK4 inhibitor induced differentiation and reduced the cancer stem cell activity of MDA-MB-231 cells, which are shown by multiple marker or phenotypes of CSCs. Thus, these results suggest that radiosensitization effects may be caused by reducing the CSC-population of MDA-MB-231 through the use of the CDK4 inhibitor. Thus, further investigations into the possible application of the CDK4 inhibitor for CSC-targeted therapy should be performed to enhance the efficacy of radiotherapy for breast cancer.

Functional Interaction between BubR1 and Securin in an Anaphase-Promoting Complex/CyclosomeCdc20–Independent Manner

Activation of the mitotic checkpoint requires the precise timing and spatial organization of mitotic regulatory events, and ensures accurate chromosome segregation. Mitotic checkpoint proteins such as BubR1 and Mad2 bind to Cdc20, and inhibit anaphase-promoting complex/cyclosome(Cdc20)-mediated securin degradation and the onset of anaphase. BubR1 mediates the proper attachment of microtubules to kinetochores, and links the regulation of chromosome-spindle attachment to mitotic checkpoint signaling. Therefore, disruption of BubR1 activity results in a loss of the checkpoint control, chromosome instability, and/or early onset of malignancy. In this study, we show that BubR1 directly interacts with securin in vitro and in vivo. In addition, the BubR1 interaction contributes to the stability of securin, and there is a significant positive correlation between BubR1 and securin expressions in human cancer. Importantly, BubR1 competes with Cdc20 for binding to securin, and thereby the interaction between BubR1 and securin is greatly increased by the depletion of Cdc20. Our findings may identify a novel regulation of BubR1 that can generate an additional anaphase-inhibitory signal through the Cdc20-independent interaction of BubR1 with securin.

Effect of Low dose Radiation on Differentiation of Bone Marrow Cells into Dendritic Cells

Low dose radiation has been shown to be beneficial to living organisms using several biological systems, including immune and hematopoietic systems. Chronic low dose radiation was shown to stimulate immune systems, resulting in controlling the proliferation of cancer cells, maintain immune balance and induce hematopoietic hormesis. Since dendritic cells are differentiated from bone marrow cells and are key players in maintaining the balance between immune activation and tolerance, it may be important to further characterize whether low dose radiation can influence the capacity of bone marrow cells to differentiate into dendritic cells. We have shown that bone marrow cells from low dose-irradiated (γ-radiation, 0.2Gy, 15.44mGy/h) mice can differentiate into dendritic cells that have several different characteristics, such as expression of surface molecules, cytokine secretion and antigen uptake capacity, when compared to dentritic cells differentiated from the control bone marrow cells. These differences observed in the low dose radiation group can be beneficial to living organisms either by activation of immune responses to foreign antigens or tumors, or maintenance of self-tolerance. To the best of our knowledge, this is the first report showing that total-body low dose radiation can modulate the capacity of bone marrow cells to differentiate into dendritic cells.

Cyclin-dependent kinase 4 signaling acts as a molecular switch between syngenic differentiation and neural transdifferentiation in human mesenchymal stem cells.

Multipotent mesenchymal stem/stromal cells (MSCs) are capable of differentiating into a variety of cell types from different germ layers. However, the molecular and biochemical mechanisms underlying the transdifferentiation of MSCs into specific cell types still need to be elucidated. In this study, we unexpectedly found that treatment of human adipose- and bone marrow-derived MSCs with cyclin-dependent kinase (CDK) inhibitor, in particular CDK4 inhibitor, selectively led to transdifferentiation into neural cells with a high frequency. Specifically, targeted inhibition of CDK4 expression using recombinant adenovial shRNA induced the neural transdifferentiation of human MSCs. However, the inhibition of CDK4 activity attenuated the syngenic differentiation of human adipose-derived MSCs. Importantly, the forced regulation of CDK4 activity showed reciprocal reversibility between neural differentiation and dedifferentiation of human MSCs. Together, these results provide novel molecular evidence underlying the neural transdifferentiation of human MSCs; in addition, CDK4 signaling appears to act as a molecular switch from syngenic differentiation to neural transdifferentiation of human MSCs.