Takatomo Satoh

Region-specific chromatin decondensation and micronucleus formation induced by 5-azacytidine in human TIG-7 cells.

A human diploid lung fibroblast cell strain, TIG-7, has a heteromorphic chromosome 15 with an extra short arm carrying a homogeneously staining region (15p+hsr). We demonstrated previously that the 15p+hsr consists of an inactive and G+C-rich rDNA cluster characterized by fluorescence in situ hybridization (FISH) and various chromosome banding techniques. Thus, it was suggested that the region could contain highly methylated DNA. To observe methylation status on the target region directly under the microscope, we used a demethylating agent, 5-azacytidine (5-azaC), to induce decondensation of the chromatin containing methylated DNA. At 24 h after treatment with 0.5 µM 5-azaC, marked decondensation of the 15p+hsr was observed in almost all of the metaphases. Furthermore, we observed micronuclei, which were equivalent to the rDNA of the 15p+hsr demonstrated by FISH in the same preparation. In contrast, the DNA cross-linking agent mitomycin C (MMC) preferentially induced 15p+hsr-negative micronuclei. These findings indicated that chromatin decondensation and subsequent DNA strand breakage induced by the demethylating effect of 5-azaC led specifically to 15p+hsr-positive micronuclei.

Use of a Three-Dimensional Microarray System for Detection of Levofloxacin Resistance and the mecA Gene in Staphylococcus aureus

ABSTRACT We evaluated a novel three-dimensional microarray (PamChip microarray) system to detect the presence of levofloxacin-related resistance mutations and the mecA gene. The results were compared to those obtained for 27 Staphylococcus aureus isolates by conventional DNA sequencing or PCR methods. Hybridization and fluorescence detection were performed using an FD10 system designed for PamChip microarray under conditions optimized for each target/probe on the array. In dilution series analysis using multiplex PCR samples, the sensitivity of the microarray was about 10 times greater than that of conventional PCR methods. A high level of data reproducibility was also confirmed in those analyses. Various point mutations in quinolone resistance-determining regions detected by our system corresponded perfectly to the results obtained by conventional DNA sequencing. The results of the mecA gene detection using our system also corresponded to the PCR method; that is, signal/band was detected in all isolates of methicillin-resistant S. aureus, and no signal/band was detected in any isolate of methicillin-susceptible S. aureus. In conclusion, our novel three-dimensional microarray system provided rapid, specific, easy, and reproducible results for the simultaneous detection of levofloxacin resistance and the mecA gene in S. aureus.

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.