Activation of NLRP3 Inflammasome Complexes by Beta-Tricalcium Phosphate Particles and Stimulation of Immune Cell Migration in vivo.

Beta-tricalcium phosphate (ß-TCP) serves as a bone substitute in clinical practice because it is resorbable, biocompatible, osteointegrative, and osteoconductive. Particles of ß-TCP are also inflammatory mediators although the mechanism of this function has not been fully elucidated. Regardless, the ability of ß-TCP to stimulate the immune system might be useful for immunomodulation. The present study aimed to determine the effects of ß-TCP particles on NLR family pyrin domain containing 3 (NLRP3) inflammasome complexes. We found that ß-TCP activates NLRP3 inflammasomes, and increases interleukin (IL)-1ß production in primary cultured mouse dendritic cells (DCs) and macrophages, and human THP-1 cells in caspase-1 dependent manner. In THP-1 cells, ß-TCP increased also IL-18 production, and NLRP3 inflammasome activation by ß-TCP depended on phagocytosis, potassium efflux, and reactive oxygen species (ROS) generation. We also investigated the effects of ß-TCP in wild-type and NLRP3-deficient mice in vivo. Immune cell migration around subcutaneously injected ß-TCP particles was reduced in NLRP3-deficient mice. These findings suggest that the effects of ß-TCP particles in vivo are at least partly mediated by NLRP3 inflammasome complexes.

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

Beta-tricalcium phosphate (ß-TCP) is widely used for bone substitution in clinical practice. Particles of calcium phosphate ceramics including ß-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 ß-TCP on the immune system could be utilized as an immunomodulator. Here, in vitro effects of ß-TCP on primary cultured murine dendritic cells (DCs) and macrophages were investigated. ß-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 ß-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 ß-TCP caused a prominent reduction in antigen uptake by DCs, and that conditioned medium from DCs treated with ß-TCP facilitated the migration of splenocytes in the transwell migration assay. Thus, ß-TCP induced phenotypical and functional maturation/activation of DCs and macrophages; these stimulating effects may contribute to the observed in vivo effect where ß-TCP induced extensive migration of immune cells. When compared to lipopolysaccharide (LPS), an authentic TLR ligand, the stimulatory effect of ß-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 ß-TCP against bone marrow-derived DCs and macrophages.

Characterization of beta-tricalcium phosphate as a novel immunomodulator.

Calcium phosphate (CaP) ceramics including hydroxyapatite (HA) and beta-tricalcium phosphate (ß-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 ß-TCP induces an anti-tumor effect in xenograft tumor models in athymic mice. To date, in depth analysis of the biological effects of ß-TCP has not been studied in mice. In the present study, in vivo biological effects of ß-TCP were investigated by subcutaneously injecting ß-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 ß-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 ß-TCP facilitated migration of murine splenocytes in a transwell migration assay. These findings clarify that ß-TCP induces an immunostimulatory effect in mice, and suggest a potential for ß-TCP as a novel adjuvant for cancer therapy.

Use of a three-dimensional microarray system for detection of levofloxacin resistance and the mec A gene in Staphylococcus aureus.

We evaluated a novel three-dimensional microarray (Pam Chip microarray) system to detect the presence of levofloxacin-related resistance mutations and the mec A 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 FD 10 system designed for Pam Chip 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 mec A 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 mec A gene in S. aureus.

Three-dimensional microarray compared with PCR-single-strand conformation polymorphism analysis/DNA sequencing for mutation analysis of K-ras codons 12 and 13.

BACKGROUND: We developed a rapid, precise, and accurate microarray-based method that uses a three-dimensional platform for detection of mutations. METHODS: We used the PamChip microarray to detect mutations in codons 12 and 13 of K-ras in 15 cell lines and 81 gastric or colorectal cancer tissues. Fluorescein isothiocyanate-labeled PCR products were analyzed with the microarray. We confirmed the microarray results with PCR-single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. RESULTS: We could correctly identify wild-type, heterozygous, and homozygous mutant genotypes with the PamChip microarray in <3.5 h. The array data were consistent with those of PCR-SSCP analysis and DNA sequencing. All 15 cell lines and 80 of 81 clinical cancer specimens (98.8%; 95% confidence interval, 96.4-100%) were genotyped accurately with the microarray, a rate better than that of direct DNA sequencing (38.9%) or SSCP (93.8%). Only one clinical specimen was misdiagnosed as homozygous for the wild-type allele. Densitometric analysis of SSCP bands indicated that the content of the mutant allele in the specimen was approximately 16%. The PamChip microarray could detect mutant alleles representing more than 25% of the SSCP band proportions. Therefore, the limit for detection of mutant alleles by the PamChip microarray was estimated to be 16-25% of the total DNA. CONCLUSIONS: The PamChip microarray is a novel three-dimensional microarray system and can be used to analyze K-ras mutations quickly and accurately. The mutation detection rate was nearly 100% and was similar to that of PCR-SSCP together with sequencing analysis, but the microarray analysis was faster and easier.

Rapid analysis of gene expression changes caused by liver carcinogens and chemopreventive agents using a newly developed three-dimensional microarray system.

We investigated changes of gene expression in livers of rats treated with carcinogens and tumor promoters using a novel three-dimensional microarray system developed by Olympus Optical Co., Ltd., to assess the feasibility of predicting modifying effects on hepatocarcinogenesis on the basis of changes in the patterns. For this purpose, two genotoxic carcinogens, two nongenotoxic carcinogens (promoters) and seven candidate chemopreventive agents were examined. Six-week-old male F344 rats were treated for 2 weeks with the 11 chemicals (0.05% phenobarbital, 0.3% clofibrate, 0.01% N-diethylnitrosamine (DEN), 0.01% 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 1% catechol, 1% caffeic acid, 0.05% nobiletin, 0.05% garcinol, 0.05% auraptene, 0.05% zermbone and 0.05% 1'-acetoxychavicol acetate (ACA). Test chemicals were mixed in food with the exception of DEN, which was administered in drinking water. RNAs from liver were then analyzed using two kinds of customized microarrays (PamChip(\xa8) microarray A spotted for 28 genes of drug-metabolizing enzymes in duplicate, and PamChip microarray B spotted for 131 genes which are known to be up- or down-regulated in hepatocarcinoma cells). Hybridization and subsequent analysis were usually completed within 2 h and the data obtained were highly reproducible. Carcinogens were classified into genotoxic and nongenotoxic substances by clustering analysis. We could also divide test chemicals into carcinogens and chemopreventive agents from their effects on gene expression. In this study, we have thus shown that it is feasible to predict the modifying effects of chemicals on the basis of changes of gene expression patterns after only 2 weeks of exposure, using our novel three-dimensional microarrays.

Chromosomal instability in B-lymphoblasotoid cell lines from Werner and Bloom syndrome patients.

Werner's syndrome (WS) and Bloom's syndrome (BS) are rare autosomal genetic diseases that predispose to cancer and are associated with genomic instability. To characterize the genomic instability of WS and BS, we analyzed and compared the cytogenetics of B-lymphoblastoid cell lines (LCLs) from WS and BS patients and healthy donors. Although, similar spontaneous frequencies of micronuclei (MN) and sister chromatid exchanges (SCE) were observed in LCLs from WS patients and healthy donors, they were much higher in BS-LCLs. We also examined the cells' cytotoxic and cytogenetic formation (MN) response to camptothecin (CAM), etoposide (ETO), 4-nitroquinoline 1-oxide (4NQO), and mitomycin C (MMC). Compared to healthy donor LCLs, BS-LCLs but not WS-LCLs tended to be resistant to cytotoxicity and sensitive to MN induction by 4NQO and MMC. Spectrum karyotyping analysis revealed that most WS- and BS-LCLs generated "variegated translocation mosaicism" at high frequencies during cell culture. These findings support the idea that the basis of genomic instability in WS is different from that in BS.

Application of mFISH for the analysis of chemically-induced chromosomal aberrations: a model for the formation of triradial chromosomes.

Using a human lymphoblastoid cell line WTK-1, we applied multicolor fluorescence in situ hybridization (mFISH) technique to analyze mitomycin C (MMC)-induced chromatid exchanges, focusing especially on the triradial chromosomes. It was found that the triradial chromosomes were formed with a specific rearrangement, "recipient and donor" relationship. The exchange sites of the recipient chromosomes were on single chromatid breaks and distributed randomly throughout the interstitial, pericentromeric, and terminal regions. In counterpart, donor chromosomes exchanged on isochromatid breaks of their telomeric and/or subtelomeric regions with the single chromatid breaks of recipient chromosomes. More than 80% of the scored triradial chromosomes were formed with such rearrangements, and few acentric chromosome fragments derived from the donor chromosomes could be detected in the metaphases observed. We therefore suggest that biological mechanisms of breakages between the recipient and donor chromosomes are different: the former due to direct DNA-damage by MMC, but the latter due to indirect DNA-damage depending on telomeric specific structure/function.