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.

Fragrant environment with α-pinene decreases tumor growth in mice.

Stress is believed to be harmful to not only mental but also physical health. However, proving a link between stress and disease is difficult. A recent study reported that an environmental enrichment reduced cancer growth via the hypothalamic-pituitary-adrenal axis and leptin. Here, we report that mice kept in a fragrant environment enriched with α-pinene show reduced melanoma growth. Tumor volume of mice under the α-pinene environment was about 40% smaller than that in the control mice. α-Pinene had no inhibitory effect on melanoma cell proliferation in vitro, suggesting that this effect was not a direct effect of α-pinene. These results suggest that the provision of a fragrant environment may be an important factor in the therapeutic approach to cancer.

Flt3 ligand enhances anti-tumor effects of antibody therapeutics.

Fms-like tyrosine kinase 3 ligand ([Flt3 ligand], FL) stimulates proliferation and development of a wide range of hematopoietic cells including hematopoietic stem cells and myeloid and lymphoid progenitor cells. FL also has been shown to have anti-tumor effects in a variety of in vivo tumor models. In this study, the effect of FL against tumor growth was investigated in the COLO-205 human colon tumor xenograft model. FL was delivered in vivo by the "hydrodynamics-based gene delivery of naked DNA" method. In this experimental setting, FL and/or the therapeutic antibody anti-carcinoembryonic antigen (CEA) monoclonal antibody was administered. FL alone or anti-CEA antibody alone induced significant growth inhibition; furthermore, FL plus antibody treatment produced synergistic anti-tumor effects. This study is the first demonstration of a synergistic anti-tumor effect between FL and antibody therapeutics.

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.

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% (ß-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.

Identification of melanoma antigens using a Serological Proteome Approach (SERPA).

BACKGROUND: Melanoma is an intractable cancer with a poor prognosis and increasing prevalence worldwide. Specific biomarkers for early diagnosis have yet to be found. MATERIALS AND METHODS: Serum samples from melanoma patients and healthy volunteers were utilized for identifying melanoma marker proteins using a serological proteome approach. Specifically, G361 cell protein spots separated by 2-dimensional gel electrophoresis and transferred to a membrane were incubated with patient sera, and positive spots that reacted with more than 5 serum samples were identified using time of flight mass spectrometry. RESULTS: Only patient sera showed many spots reacted in G361 gels. A total of 13 positive spots were detected and 5 proteins were identified: eukaryotic elongation factor2 (EEF2), enolase1 (ENO1), aldolase A (ALDOA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and heterogeneous nuclear ribonucleoproteins (HNRNP) A2B1. The mRNAs of four proteins (EEF2, ENO1, ALDOA and HNRNPA2B1) were highly expressed in G361 cells compared with melanocytes. EEF2, ENO1 and ALDOA mRNAs were also frequently expressed in other melanoma cell lines. CONCLUSION: The autoantibody-based proteomic approach was effective for investigating melanoma biomarkers. This study might contribute to the development of a diagnostic device for the early detection of cancer.

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.

Characterization of cytomegalovirus pp65-HLA-A24 peptide-specific CTL lines from metastatic melanoma patients.

Because of advances in immunological technology for detecting a very small number of blood CTL cells, clinicians have been able to monitor cellular immunity against CMV and evaluate the status of CMV infections in highly advanced cancer patients or transplant recipients. Our previous study using healthy volunteer PBLs revealed a significant increase in CMV HLA-A24 tetramer+ CTLs after stimulation in vitro with autologous DCs. However, the efficiency of CMV A24 peptide-specific CTL expansion in highly advanced cancer patients has yet to be studied in detail. In the present study, we tried to characterize and expand HLA-A*2402 CMVpp65 peptide (QYDPVAALF)-specific tetramer+ CTLs from HLA-A*2402+ metastatic melanoma patients, and eventually demonstrated that expansion efficiency was closely related to both post-stimulation CMV tetramer frequency and anti-CMV IgG titer. This is a novel finding regarding in vitro CMVpp65-A24 peptide-specific CTL expansion based on metastatic cancer patient-derived PBLs. Interestingly, the current results using metastatic melanoma PBLs showed a much higher frequency of CMVpp65-A24 tetramer+ CTLs and expansion efficiency than in healthy volunteers. Finally, we were successful in cloning CMVpp65 HLA-A24 peptide-specific TCR cDNAs from in vitro expanded CTL lines derived from melanoma patients. Additionally, CMVpp65 HLA-A24 peptide-specific TCR cDNA was transduced into naive T cells from patients and functionally reconstructed. The results showed that cloned CMV-specific TCR genes were efficient in reconstituting specific anti-CMV activity and might be good tools for adoptive immunotherapy against CMV infections.

Characterization of a MAGE-1-derived HLA-A24 epitope-specific CTL line from a Japanese metastatic melanoma patient.

A MAGE-1 HLA-A24 peptide-specific CTL line was characterized using a novel staining approach in the case of a metastatic melanoma patient who exhibited a remarkable clinical response in HLA-A24 peptide cocktail-pulsed dendritic cell (DCs) vaccine therapy. Briefly, pre- or post-vaccine peripheral blood mononuclear cells (PBMCs) from the vaccinated patient were stimulated several times with MAGE-1 A24 peptide-pulsed DCs and T2-A24 cells in vitro. Expanded MAGE-1 A24-specific CTL line was investigated in terms of immunological functions. The proportion of MAGE-1 A24 tetramer+ CTLs increased from 0.04% to 18.6%, and the absolute numbers of MAGE-1 tetramer+ CTLs increased up to 5,068-fold after stimulations. Expanded CTL line exhibited a strong cytotoxic activity against MAGE-1+ cancer cell line in the restriction of HLA. Finally, successful identification of MAGE-1 A24 peptide-specific T-cell receptor (TCR) cDNA from anti-TCR MoAbsorted CTL was obtained for the first time and the specific cytotoxicity in TCR gene-transduced naive T-cells was confirmed.

Novel approach to the characterization of melanoma associated-peptide-specific CTL lines from Japanese metastatic melanoma patients.

Melanoma-associated antigens, MART-1, tyrosinase, gp100 and MAGEs, are typical melanoma-specific tumor antigens which can potently induce immune responses in metastatic melanoma patients treated with peptide vaccines. In the present study, we established a dendritic cell (DC)-based HLA-A2 melanoma-associated peptide (MART-1 or gp100)-specific CTL induction method and characterized the CTLs using HLA-A2 tetramer staining in 6 cases of HLA-A2+ melanoma treated with DC vaccines. Peripheral blood mononuclear cells (PBMC) from patients were stimulated twice with MART-1 A2 peptide-pulsed DCs in the presence of a low dose of IL-2. To boost CTL populations, CTL lines were further stimulated twice with MART-1 A2 peptide-pulsed T2 cells. The frequency of MART-1 A2 tetramer-positive CTLs increased from 0.16% (prior to stimulation) to 2.15% (after DC stimulation), and reached 46.5% on average (after additional T2 stimulation) in 4 cases which showed a successful expansion. The absolute numbers of MART-1 A2 tetramer-positive CTLs increased from 187- to 619-fold (average, 415-fold) compared to prior to DC stimulation. CTL assays using MART-1-specific CTL lines demonstrated potent killing activity against MART-1 peptide-pulsed T2 cells or HLA-A2+ melanoma cell lines in accordance with the frequency of tetramer-positive CTLs. Finally, we were successful in identifying melanoma peptide-specific T-cell receptor (TCR) cDNAs in 2 cases for MART-1 and 1 case for gp100 using the anti-TCR MoAb-based sorting as a novel approach instead of a conventional cell cloning, and confirmed peptide-specific IFN-gamma production in TCR cDNA-transduced naïve T cells. The results showed that cloned TCR cDNAs were efficient in reconstituting tumor-specific cytotoxicity and good candidates for novel immunotherapy.

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.

Proteomic analysis of a highly metastatic gastric cancer cell line using two-dimensional differential gel electrophoresis.

Stomach cancer is still a major cause of death in Asian people despite a complete cure after the resection of early cancers, mainly because peritoneal dissemination is difficult to treat. In the present study, we used two-dimensional differential gel electrophoresis (2-D DIGE) to identify specific proteins differentially expressed between a highly metastatic stomach cancer cell line MKN-45-P and its parental cell line MKN-45. We detected 27 protein spots in at least 2 of 3 experiments which showed statistically significant differences in abundance. All 27 protein spots were identified using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) and database-searching software. A proteomic analysis revealed 13 different proteins with some isoforms sharing different biochemical characteristics, and that 8 proteins were up-regulated, and 5 were down-regulated. The 13 proteins were mainly involved in protein synthesis (transfer RNA synthetase), metabolism (flavoprotein subunit, pyruvate kinase, adenylate kinase), receptor and signal transduction (annexins I and A2), the cytoskeleton (keratin 5, cytokeratin 8) and cell cycling (ts11). These results suggested that a proteomic approach including 2-D DIGE would be an efficient way to identify the proteins responsible for specific biological functions. Moreover, these observations might be novel findings leading to the prediction of postoperative peritoneal recurrence.