Immunotherapy for Tumor Metastasis by Artificial Antigen-Presenting Cells via Targeted Microenvironment Regulation and T-Cell Activation.

Effective expansion of T-cells without ex vivo stimulation and maintenance of their antitumor functions in the complex tumor microenvironment (TME) are still daunting challenges in T-cell-based immunotherapy. Here, we developed biomimetic artificial antigen-presenting cells (aAPCs), ultrathin MnOx nanoparticles (NPs) functionalized with T-cell activators (anti-CD3/CD28 mAbs, CD), and tumor cell membranes (CMs) for enhanced lung metastasis immunotherapy. The aAPCs, termed CD-MnOx@CM, not only efficiently enhanced the expansion and activation of intratumoral CD8+ cytotoxic T-cells and dendritic cells after homing to homotypic metastatic tumors but also regulated the TME to facilitate T-cell survival through catalyzing the decomposition of intratumoral H2O2 into O2. Consequently, the aAPCs significantly inhibited the development of lung metastatic nodules and extended the survival of a B16-F10 melanoma metastasis model, while minimizing adverse events. Our work represents a new biomaterial strategy of inhibiting tumor metastasis through targeted TME regulation and in situ T-cell-based immunotherapy.

Crucial Role of Lateral Size for Graphene Oxide in Activating Macrophages and Stimulating Pro-inflammatory Responses in Cells and Animals.

Graphene oxide (GO) is increasingly used in biomedical applications because it possesses not only the unique properties of graphene including large surface area and flexibility but also hydrophilicity and dispersibility in aqueous solutions. However, there are conflicting results on its biocompatibility and biosafety partially due to large variations in physicochemical properties of GO, and the role of these properties including lateral size in the biological or toxicological effects of GO is still unclear. In this study, we focused on the role of lateral size by preparing a panel of GO samples with differential lateral sizes using the same starting material. We found that, in comparison to its smaller counterpart, larger GO showed a stronger adsorption onto the plasma membrane with less phagocytosis, which elicited more robust interaction with toll-like receptors and more potent activation of NF-κB pathways. By contrast, smaller GO sheets were more likely taken up by cells. As a result, larger GO promoted greater M1 polarization, associated with enhanced production of inflammatory cytokines and recruitment of immune cells. The in vitro results correlated well with local and systemic inflammatory responses after GO administration into the abdominal cavity, lung, or bloodstream through the tail vein. Together, our study delineated the size-dependent M1 induction of macrophages and pro-inflammatory responses of GO in vitro and in vivo. Our data also unearthed the detailed mechanism underlying these effects: a size-dependent interaction between GO and the plasma membrane.

Mineral trioxide aggregate stimulates macrophages and mast cells to release neutrophil chemotactic factors: role of IL-1beta, MIP-2 and LTB(4).

OBJECTIVE: In the present study, the role of macrophages and mast cells in mineral trioxide aggregate (MTA)-induced release of neutrophil chemotactic factor was investigated. STUDY DESIGN: MTA suspension (50 mg/mL) was plated over inserts on macrophages or mast cells for 90 minutes. Untreated cells served as controls. Cells were washed and cultured for 90 minutes in RPMI without the stimuli. Macrophages and mast cell supernatants were injected intraperitoneally (0.5 mL/cavity), and neutrophil migration was assessed 6 hours later. In some experiments, cells were incubated for 30 minutes with dexamethasone (DEX, 10 muM/well), BWA4C (BW, 100 muM/well) or U75302 (U75, 10 muM/well). The concentration of Leukotriene B(4) (LTB(4)) in the cell-free supernatant from mast cells and macrophage culture was measured by ELISA. RESULTS: Supernatants from MTA-stimulated macrophages and mast cells caused neutrophil migration. The release of neutrophil chemotactic factor by macrophages and mast cells was significantly inhibited by DEX, BW, or U75. Macrophages and mast cells expressed mRNA for interleukin-1 (IL-1)beta and macrophage inflammatory protein-2 (MIP-2) and the pretreatment of macrophages and mast cells with DEX, BW, or U75 significantly altered IL-1beta and MIP-2 mRNA expression. LTB(4) was detected in the MTA-stimulated macrophage supernatant but not mast cells. CONCLUSIONS: MTA-induces the release of neutrophil chemotactic factor substances from macrophages and mast cells with participation of IL-1beta, MIP-2, and LTB(4).

Expression of the inflammatory marker cyclooxygenase-2 in dental pulp cells cultured with mineral trioxide aggregate or calcium silicate cements.

INTRODUCTION: Mineral trioxide aggregate (MTA) and calcium silicate (CS) cements exhibit acceptable physical and chemical properties. The aim of the present study was to evaluate the effects of MTA and CS cements on inflammatory reactions in primary cultured human dental pulp cells. METHODS: The mitochondrial colorimetric assay was used to evaluate pulp cell survival rates. Fluorescent immunohistochemistry was used to observe focal adhesion kinase (FAK) and cyclooxygenase-2 (COX-2) distributions in the cells. Reverse transcription-polymerase chain reaction was used to assess COX-2 expression. RESULTS: The results showed that MTA and CS are biocompatible with pulp cells (P>.05). FAK was well-distributed in pulp cells in contact with both cements. Both MTA and CS cements induced pulp cell inflammation as evidenced by increased COX-2 expression. CONCLUSIONS: The present study demonstrated that MTA and CS cements are biocompatible with primary cultured pulp cells. Both cements can induce inflammatory COX-2 expression in the pulp cells.

Awakening dormant haematopoietic stem cells.

Haematopoietic stem cells (HSCs) in mouse bone marrow are located in specialized niches as single cells. During homeostasis, signals from this environment keep some HSCs dormant, which preserves long-term self-renewal potential, while other HSCs actively self renew to maintain haematopoiesis. In response to haematopoietic stress, dormant HSCs become activated and rapidly replenish the haematopoietic system. Interestingly, three factors - granulocyte colony-stimulating factor, interferon-alpha and arsenic trioxide - have been shown to efficiently activate dormant stem cells and thereby could break their resistance to anti-proliferative chemotherapeutics. Thus, we propose that two-step strategies could target resistant leukaemic stem cells by priming tumours with activators of dormancy followed by chemotherapy or targeted therapies.

High affinity anti-inorganic material antibody generation by integrating graft and evolution technologies: potential of antibodies as biointerface molecules.

Recent advances in molecular evolution technology enabled us to identify peptides and antibodies with affinity for inorganic materials. In the field of nanotechnology, the use of the functional peptides and antibodies should aid the construction of interface molecules designed to spontaneously link different nanomaterials; however, few material-binding antibodies, which have much higher affinity than short peptides, have been identified. Here, we generated high affinity antibodies from material-binding peptides by integrating peptide-grafting and phage-display techniques. A material-binding peptide sequence was first grafted into an appropriate loop of the complementarity determining region (CDR) of a camel-type single variable antibody fragment to create a low affinity material-binding antibody. Application of a combinatorial library approach to another CDR loop in the low affinity antibody then clearly and steadily promoted affinity for a specific material surface. Thermodynamic analysis demonstrated that the enthalpy synergistic effect from grafted and selected CDR loops drastically increased the affinity for material surface, indicating the potential of antibody scaffold for creating high affinity small interface units. We show the availability of the construction of antibodies by integrating graft and evolution technology for various inorganic materials and the potential of high affinity material-binding antibodies in biointerface applications.

Differential effects on innate versus adaptive immune responses by WF10.

Oxidative compounds that are physiologically generated in vivo can induce natural defense mechanisms to enhance the elimination of pathogens and to limit inflammatory tissue damage in the course of inflammation. Here, we have investigated WF10, a chlorite-based non-toxic compound for its functional activities on human PBMC in vitro. WF10 exerts potent immune-modulatory effects through generating endogenous oxidative compounds such as taurine chloramine. Proliferation and IL-2 production of anti-CD3 stimulated PBMC were inhibited by WF10, as was the nuclear translocation of the transcription factor NFATc. In PBMC and monocytes, however, WF10 induced pro-inflammatory cytokines like IL-1beta, IL-8, and TNF-alpha. In the monocytic cell line THP-1, the activation of the transcription factors AP-1 and NFkappaB by WF10 was demonstrated. Inhibition of NFAT regulated genes in activated lymphocytes in concert with the induction of several myeloid cell associated pro-inflammatory genes in monocytes represents a novel mechanism of immune modulation.

Evidence of an immunologic mechanism behind the therapeutical effects of arsenic trioxide (As(2)O(3)) on myeloma cells.

Exposure of RPMI 8226, Karpas 707 and U266 human myeloma-like lines to low doses of As(2)O(3) was followed by a marked increase in lymphokine activated killers (LAK)-mediated killing and up- modulation of CD38 and CD54, two molecules involved in cell-cell interactions. Moreover, simultaneous exposure of effectors and targets to As(2)O(3) yielded the most effective condition for lysis. The expression of CD31 (CD38 ligand) and CD11a (CD54 ligand) was also up-regulated by LAK, suggesting that increased adhesion was responsible for the improved killing. Similar results were obtained using freshly isolated myeloma cells. These findings indicate that As(2)O(3) may be useful to boost the immune system against myelomas.

Antibody-conjugated magnetoliposomes for targeting cancer cells and their application in hyperthermia.

Magnetoliposomes for hyperthermia treatment of cancer were prepared by coating phospholipid on to magnetite particles. The optimum phospholipid composition for dispersibility was a phosphatidylcholine/phosphatidylethanolamine ratio of 2:1. The average size of the magnetoliposomes, which were aggregates of 10 nm core magnetite particles, was about 80 nm. The magnetoliposomes were coated with hydrazide pullulan to stabilize the phospholipid capsules and provide an anchor for the immobilization of antibodies. By this method, 90-180 molecules of a monoclonal antibody were immobilized on to a magnetoliposome particle. When the antibody-conjugated magnetoliposomes were incubated with cancer cells, they were adsorbed on to the cell surface and incorporated by the cells about 12 times more effectively than the control after 4 h. The amount of incorporated magnetite was 0.61-3.6 pg/cell, depending on the antigen-antibody system. The heating properties of the magnetoliposomes were also measured and found to vary with the size of the core magnetite.