Sachiko Tai

Changes in the expression of CD106, osteogenic genes, and transcription factors involved in the osteogenic differentiation of human bone marrow mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are well known to possess multipotential differentiation and are becoming a good tool for clinical research. However, specific markers for their purification and the mechanism of their osteogenic differentiation remain to be elucidated. In the present study, we compared the expression of CD106, and osteogenic differentiation-related proteins and genes in human bone marrow (BM)-derived MSCs, before and after differentiation by FACS, histochemical staining, immunohistochemical staining, RT-PCR, and real-time PCR. It was found that MSCs were positive for CD13, CD29, CD44, CD73, CD90, CD105, and CD166, but negative for CD14, CD31, CD34, CD62E, CD45, and GlyA. Notably, CD106 was detected before osteogenic induction, but its expression was downregulated 10 fold after 2 weeks of osteogenic differentiation as determined by flow cytometry. The results of RT-PCR and real-time PCR revealed that the expression of CD106 mRNA in MSCs significantly decreased by 7.1-, 4.2-, and 5.1-fold, respectively after osteogenic, chondrogenic, and adipogenic differentiation. In contrast, other MSC-positive markers described above did not change significantly even after differentiation. Compared to levels in control cells, after 2 weeks of osteogenic differentiation, mRNA levels of alkaline phosphatase, bone sialoprotein, osteocalcin, and transcript factors RUNX2 and Osterix showed more than 2-fold, 5-fold, 1.5-fold, 2-fold, and 5-fold increase, respectively. Thus, we speculate that CD106 might be a useful surface marker for BMMSCs. Moreover, alkaline phosphatase, type I collagen, osteonectin, osteopontin, and biglycin were expressed in the early stages of osteogenic differentiation before bone sialoprotein and osteocalcin. The present study should help to provide a novel marker for isolating purified MSCs and characterizing osteogenic differentiation.

Identification of a New Series of STAT3 Inhibitors by Virtual Screening

The signal transducer and activator of transcription 3 (STAT3) is considered to be an attractive therapeutic target for oncology drug development. We identified a N-[2-(1,3,4-oxadiazolyl)]-4-quinolinecarboxamide derivative, STX-0119, as a novel STAT3 dimerization inhibitor by a virtual screen using a customized version of the DOCK4 program with the crystal structure of STAT3. In addition, we used in vitro cell-based assays such as the luciferase reporter gene assay and the fluorescence resonance energy transfer-based STAT3 dimerization assay. STX-0119 selectively abrogated the DNA binding activity of STAT3 and suppressed the expression of STAT3-regulated oncoproteins such as c-myc and survivin in cancer cells. In contrast, a truncated inactive analogue, STX-0872, did not exhibit those activities. Oral administration of STX-0119 effectively abrogated the growth of human lymphoma cells in a SCC-3 subcutaneous xenograft model without visible toxicity. Structure-activity relationships of STX-0119 derivatives were investigated using the docking model of the STAT3-SH2 domain/STX-0119.

Analysis of HLA‐A24‐restricted peptides of carcinoembryonic antigen using a novel structure‐based peptide‐HLA docking algorithm

Carcinoembryonic antigen (CEA) is a very common tumor marker because many types of solid cancer usually produce a variety of CEA and a highly sensitive measuring kit has been developed. However, immunological responses associated with CEA have not been fully characterized, and specifically a weak immunogenicity of CEA protein as a tumor antigen is reported in human leukocyte antigen (HLA)‐A24‐restricted CEA peptide‐based cancer immunotherapy. These observations demonstrated that immunogenic and potent HLA‐A24‐restricted CTL epitope peptides derived from CEA protein are seemingly difficult to predict using a conventional bioinformatics approach based on primary amino acid sequence. In the present study, we developed an in silico docking simulation assay system of binding affinity between HLA‐A24 protein and A24‐restricted peptides using two software packages, AutoDock and MODELLER, and a crystal structure of HLA‐A24 protein obtained from the Protein Data Bank. We compared the current assay system with HLA–peptide binding predictions of the bioinformatics and molecular analysis section (BIMAS) in terms of the prediction capability using MHC stabilization and peptide‐stimulated CTL induction assays for CEA and other HLA‐A24 peptides. The MHC stabilization score was inversely correlated with the affinity calculated in the docking simulation alone (r = −0.589, P = 0.015), not with BIMAS score or the IFN‐γ production index. On the other hand, BIMAS was not significantly correlated with any other parameters. These results suggested that our in silico assay system has potential advantages in efficiency of epitope prediction over BIMAS and ease of use for bioinformaticians. (Cancer Sci 2011; 102: 690–696)

Identification of cytomegalovirus (CMV)pp65 antigen-specific human monoclonal antibodies using single B cell-based antibody gene cloning from melanoma patients

Recently, because of highly advanced protein engineering technology, beyond the chimeric antibody, highly humanized and fully human antibody development is becoming crucial in the medical field. In the last decade, investigational approaches using clinical samples for fully human antibody production have been performed, but there are still problems with efficiency and accuracy, which should be solved. In the present study, based on novel IgG antibody-measuring ELISA and antibody gene copy number-quantitative PCR, a human single B cell RT-PCR-mediated IgG monoclonal antibody (mAb) gene cloning method was established, and CMVpp65-specific human mAbs were successfully identified. Quantitative PCR for the human IgG mRNA copy number per cell demonstrated that the detection range was 10-250copies/cell. CMVpp65(+)surfaceIgG(+) B cells were collected from melanoma patients who showed high titers of serum anti-CMVpp65 IgG antibody. RT-PCR was successful in 64% (IGH) and 84% (β-actin) of 88 single B cells. Finally, both IGH and IGL gene amplifications in the same cell were successful in 21 single cells, and 18 IgG antibody genes specific for CMVpp65 antigen were cloned. Four of 13 recombinant human single-chain fragment variable (scFv) antibodies showed strong responses to full-length CMVpp65 protein. These results suggested that the current fully human mAb production procedure through antibody-titer screening by ELISA, single B cell RT-PCR-based antibody gene cloning, and the making of scFv recombinant antibody is an efficient method of therapeutic antibody development.

Characterization of beta-tricalcium phosphate as a novel immunomodulator

Calcium phosphate (CaP) ceramics including hydroxyapatite (HA) and beta-tricalcium phosphate (β-TCP) have been widely used for bone substitution in orthopedic, maxillofacial and dental surgery, as well as in tumor resections. CaP particles are also known to cause inflammatory responses, which are thought to be an unfavorable characteristic of prosthetic coating materials. On the other hand, the immunostimulatory effect of β-TCP induces an anti-tumor effect in xenograft tumor models in athymic mice. To date, in depth analysis of the biological effects of β-TCP has not been studied in mice. In the present study, in vivo biological effects of β-TCP were investigated by subcutaneously injecting β-TCP particles into mice. This induced extensive migration of immune cells to the area surrounding the injection. In addition, we found that in vitro treatment with β-TCP in murine monocyte/macrophage cells (J774A.1) induced up-regulation of surface expression of CD86, and increased production of TNF-α, MIP-1α, and sICAM-1. Furthermore, conditioned medium from J774A.1 cells treated with β-TCP facilitated migration of murine splenocytes in a transwell migration assay. These findings clarify that β-TCP induces an immunostimulatory effect in mice, and suggest a potential for β-TCP as a novel adjuvant for cancer therapy.

Effects of beta-tricalcium phosphate particles on primary cultured murine dendritic cells and macrophages

Beta-tricalcium phosphate (β-TCP) is widely used for bone substitution in clinical practice. Particles of calcium phosphate ceramics including β-TCP act as an inflammation mediators, which is an unfavorable characteristic for a bone substituent or a prosthetic coating material. It is thought that the stimulatory effect of β-TCP on the immune system could be utilized as an immunomodulator. Here, in vitro effects of β-TCP on primary cultured murine dendritic cells (DCs) and macrophages were investigated. β-TCP particles enhanced expression of costimulatory surface molecules, including CD86, CD80, and CD40 in DCs, CD86 in macrophages, and MHC class II and class I molecules in DCs. DEC205 and CCR7 were up-regulated in β-TCP-treated DCs. Production of cytokines and chemokines, including CCL2, CCL3, CXCL2, and M-CSF, significantly increased in DCs; CCL2, CCL3, CCL4, CCL5, CXCL2, and IL-11ra were up-regulated in macrophages. The results of the functional assays revealed that β-TCP caused a prominent reduction in antigen uptake by DCs, and that conditioned medium from DCs treated with β-TCP facilitated the migration of splenocytes in the transwell migration assay. Thus, β-TCP induced phenotypical and functional maturation/activation of DCs and macrophages; these stimulating effects may contribute to the observed in vivo effect where β-TCP induced extensive migration of immune cells. When compared to lipopolysaccharide (LPS), an authentic TLR ligand, the stimulatory effect of β-TCP on the immune systems is mild to moderate; however, it may have some advantages as a novel immunomodulator. This is the first report on the direct in vitro effects of β-TCP against bone marrow-derived DCs and macrophages.

Innate Immunity-Based Immunotherapy of Cancer

The immune system protects against invading pathogens and transformed cells, including cancer. Mammalian immune system is divided into two major categories, i.e., innate and adaptive immunity. Innate immunity consists of cellular and biochemical defense mechanisms that respond in the early phase after harmful events, such as encounters with microbes or transformed cells. The cellular components of innate immunity include dendritic cells (DCs), macrophages and monocytes, polynuclear cells (e.g. neutrophils and mast cells), natural killer (NK) cells, ┛├ T cells and natural killer T (NKT) cells. Adaptive immunity consists of T and B lymphocytes and their humoral mediators, including cytokines and antibodies, and achieves excellent antigen specificity by somatic rearrangement of the antigen receptor genes of each lymphocyte lineage; T cell receptor for T lymphocytes and immunoglobulin for B lymphocytes, respectively. Furthermore, another excellent characteristic of adaptive immunity is a “memory system” to maintain antigenspecific lymphocytes in a functionally quiescent or slowly cycling state for many years. The memory system enables host organisms to respond to the second and subsequent exposure to the same or related antigens in a more rapid and effective manner.