Myung-Shin Lee

Centrifugal enhancement of Kaposi's sarcoma-associated virus infection of human endothelial cells in vitro

In order to improve the efficiency of infection of primary human endothelial cells in vitro of Kaposis sarcoma-associated herpesvirus (KSHV), the effect of low speed centrifugation was investigated. The recombinant KSHV, BAC36, was used to examine the centrifugal enhancement of KSHV. Infectivity was estimated by green fluorescent protein (GFP) expression and real-time RT-PCR. The enhancement of infectivity was dependent upon the time and force of centrifugation in human umbilical vein endothelial cells (HUVECs). Centrifugation enhanced the infectivity of KSHV by up to 70 fold compared to non-centrifugal control infection for the same period of time; viral mRNA expression was also enhanced by centrifugation. HUVECs that were centrifuged before infection with KSHV displayed no enhancement in infectivity; therefore, enhancement is believed to occur during centrifugation. In addition, the mechanisms of infection including the initial viral attachment to cells, lipid rafts, and clathrin-mediated and caveolae endocytosis appear to be similar in KSHV infection with and without centrifugal enhancement. These results show that low speed centrifugation could be a useful tool for improving the efficiency of KSHV infection in vitro.

Production and Characterization of Monoclonal Antibody to Botulinum Neurotoxin Type B Light Chain by Phage Display

A monoclonal antibody to the light chain of botulinum neurotoxin type B (BoNT/B) was generated and its protective activity was evaluated in vivo. A chimeric rabbit/human Fab library was generated using bone marrow and spleen cDNAs of rabbits immunized with the BoNT/B light chain, and three monoclonal antibodies specific to the catalytic domain of BoNT/B were isolated. One of these clones, BCXRH1, was specific to a conformation-dependent epitope, and partially neutralized the BoNT/B complex in vivo.

Prognostic Significance of CREB-Binding Protein and CD81 Expression in Primary High Grade Non-Muscle Invasive Bladder Cancer: Identification of Novel Biomarkers for Bladder Cancer Using Antibody Microarray.

High-grade (HG) bladder cancers (BCs) are genetically unstable and have an unpredictable course. The identification of prognostic factors in HG non-muscle invasive BC (NMIBC) is crucial for improving patients’ quality of life and preventing BC-specific mortality. Here, we used an antibody microarray (AbM) to identify novel candidate biomarkers in primary HG NMIBC and validated the prognostic significance of the candidate biomarkers. Three pairs of tissue samples from primary HG NMIBC and normal urothelium were analyzed using an AbM kit containing 656 antibodies, and differentially expressed proteins were identified. Among the 42 upregulated and 14 downregulated proteins with statistical significance in BC tissues, CREB-binding protein and CD81 were selected as representative upregulated and downregulated candidate biomarkers, respectively. We then validated the expression of these candidate biomarkers in primary human urothelial cells and BC cell lines by western blotting and immunofluorescence assays, and the results were consistent with the AbM expression profiles. Additionally, Kaplan-Meier survival using immunohistochemical data from an independent primary HG NMIBC cohort comprising 113 patients showed that expression of the 2 biomarkers was significantly associated with recurrence-free and progression-free survival. In multivariate analysis, the 2 biomarkers remained significant predictors for recurrence-free survival. Taken together, our findings suggest that expression of CREB-binding protein and CD81 in BC tissue specimens may have prognostic value in patients with primary HG NMIBC.

Kaposi’s sarcoma-associated herpesvirus infection of human bone-marrow-derived mesenchymal stem cells and their angiogenic potential

Kaposi’s sarcoma (KS) is a vascular tumor, and KS spindle cells express endothelial-cell-specific markers. Generally, it is believed that KS originates from endothelial cells. However, as various mesodermal-derived tissue markers are also expressed in KS spindle cells, the exact origin of KS still needs to be elucidated. Here, Kaposi’s sarcoma-associated herpesvirus (KSHV) was used to infect human mesenchymal stem cells derived from bone marrow (hMSC-bm), and we investigated the angiogenic properties of these cells, which are one of the most important pathologic features of KS. KSHV-infected hMSC-bm showed latent infection and increased tube formation activity in vitro. In addition, the expression of endothelial-cell-specific markers and a growth factor that affects the angiogenesis of endothelial cells was induced in KSHV-infected cells. This study suggests that human mesenchymal stem cells might have important roles in KS pathogenesis.

A novel protein encoded by Kaposi's sarcoma-associated herpesvirus open reading frame 36 inhibits cell spreading and focal adhesion kinase activation.

Objective: Kaposi’s sarcoma-associated herpesvirus (KSHV) is a γ-herpesvirus implicated in the development of Kaposi’s sarcoma, primary effusion lymphoma and some forms of multicentric Castleman’s disease. The KSHV open reading frame (ORF) 36 encodes a viral serine/threonine protein kinase. The aim of this study was to characterize the cellular function of the ORF36 protein. Methods: The expression kinetics of the ORF36 protein and its localization were determined. The wild-type ORF36 and its mutant proteins were subjected to in vitro kinase assay. Cell morphology change by ORF36 protein was studied. The focal adhesion kinase (FAK) tyrosine phosphorylation and cleavage were determined when ORF36 was expressed. Results: ORF36 protein expressed in the late phase during the KSHV reactivation. The C-terminal domain of ORF36 protein was important for kinase activity. Moreover, the ORF36 protein altered cell morphology to a round shape, similar to the phenotype of FAK-deficient cells. The kinase activity of ORF36 protein was required for the inhibition of cell spreading. Interestingly, ORF36 protein colocalized with FAK, suppressed its tyrosine phosphorylation and promoted FAK cleavage. Conclusion: Our results collectively demonstrate that the KSHV ORF36 protein is a viral protein kinase that inhibits cell spreading and FAK activation.

The role of Kaposi’s sarcoma-associated herpesvirus infection in the proliferation of human bladder cancer cells

Existing evidence suggests a possible role of viruses in human bladder cancer development. Recently, Kaposi’s sarcoma-associated herpesvirus (KSHV) was reported to be the most frequently detected virus in bladder cancer tissue from Croatian patients on screening with the Lawrence Livermore Microbial Detection Array. In the current study, to investigate the functional roles of KSHV in bladder cancer, five bladder cancer cell lines were infected with KSHV and their tumour progression-associated changes investigated. Four KSHV-infected bladder cancer cell lines were established; two invasive bladder cancer cell lines showed higher proliferation rates than uninfected cells. Additionally, these KSHV-infected invasive bladder cancer cells showed a greater number of colonies, which were also significantly larger than those of uninfected cells, in a soft agar colony formation assay. cDNA microarray analysis showed that various genes associated with cell proliferation and cancer development were upregulated in these KSHV-infected bladder cancer cells. Taken together, we suggest that KSHV infection affects the proliferation of a subset of invasive bladder cancer cells and may therefore play a role in their oncogenic progression. Further studies are required to elucidate the exact mechanism used by KSHV to promote bladder cancer progression.

Latent Kaposi’s sarcoma-associated herpesvirus infection in bladder cancer cells promotes drug resistance by reducing reactive oxygen species

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the major etiologic agent of Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman’s disease. Recent studies have indicated that KSHV can be detected at high frequency in patient-derived bladder cancer tissue and might be associated with the pathogenesis of bladder cancer. Bladder cancer is the second most common cancer of the genitourinary tract, and it has a high rate of recurrence. Because drug resistance is closely related to chemotherapy failure and cancer recurrence, we investigated whether KSHV infection is associated with drug resistance of bladder cancer cells. Some KSHV-infected bladder cancer cell lines showed resistance to an anti-cancer drug, cisplatin, possibly as a result of down-regulation of reactive oxygen species. Additionally, drug resistance acquired from KSHV infection could partly be overcome by HDAC1 inhibitors. Taken together, the data suggest the possible role of KSHV in chemo-resistant bladder cancer, and indicate the therapeutic potential of HDAC1 inhibitors in drug-resistant bladder cancers associated with KSHV infection.

Expression of DcR3 and its effects in kaposi's sarcoma-associated herpesvirus-infected human endothelial cells.

Objective: Kaposi’s sarcoma-associated herpesvirus (KSHV) is classified as a gamma-herpesvirus and it causes Kaposi’s sarcoma in patients infected with the human immunodeficiency virus (HIV). Decoy receptor 3 (DcR3) is known as a decoy receptor for Fas ligand, LIGHT and TL1A and it can neutralize the biological effect of TL1A by inhibiting the TL1A-DR3 interaction in human endothelial cells. The present study examined the expression of DcR3 in human endothelial cells and its effect during the early stages of KSHV infection. Methods: The expression of DcR3 was assessed using real-time RT-PCR and ELISA in human umbilical cord vein endothelial cells (HUVECs) infected with KSHV. Cell proliferation and apoptosis of KSHV-infected HUVECs were assessed after treatment of infected cells with an anti-DcR3 antibody or recombinant human TL1A. Results: DcR3 expression was induced during the early phase of KSHV infection. Inhibition of DcR3 with anti-DcR3 antibodies or recombinant human TL1A-induced apoptosis in KSHV-infected HUVECs. Conclusion: The expression of DcR3 plays an important role in the prevention of apoptosis in HUVECs during the early phases of KSHV infection, thus ensuring the successful establishment and maintenance of the viral infection.

Generation of a naïve/synthetic antibody specific to botulinum neurotoxin via motif-grafting

In this study, we describe a new approach to the production of naïve/synthetic human antibodies against the botulinum neurotoxin (BoNT). First, peptides that bind to BoNT serotype A (BoNT/A) were screened from a phage display of a combinatorial peptide library. One peptide, designated ANT 12-2 (TLPSPLALLTVH), was determined to interact with BoNT/A, as well as with other serotypes of BoNT. This peptide specifically reacted with the native form of BoNT/A, but not with its formalin-inactivated form. Next, a hybrid naïve/synthetic human Fab library was generated via the grafting of a peptide motif from ANT 12-2 into HCDR3 with randomized flanking residues. Through biopanning, the Fab clone, ANTHU-1, which harbors the HCDR3 sequence of VRIQRSPLALLSWGDV, was selected and confirmed in order to retain the same BoNT-binding characteristics as ANT 12-2.

Kaposi’s sarcoma-associated herpesvirus infection of endothelial progenitor cells impairs angiogenic activity in vitro

A recent study reported that endothelial progenitor cells (EPCs0 are one of the reservoirs of Kaposi’s sarcoma associated herpesvirus (KSHV). Although EPCs are closely linked to angiogenesis and vasculogenesis, little is known about the angiogenic potential of KSHV in EPCs. In this study, we used EPCs isolated from human umbilical cord blood to show that early infection by KSHV in vitro impaired the neovascularization of EPCs in matrigel. Our results suggest that KSHV may disrupt the angiogenic potential of EPCs and that the disseminated infection of KSHV could be associated with EPC dysfunction.