ABSTRACT
Objective: To evaluate the anti-tumor activity of mouse multi-subtype heat shock protein/peptide (mHSP/P) vaccine in combination with a programmed death ligand 1 (PD-L1) inhibitor in mouse sarcoma. Methods: Immunohistochemical staining and en-zyme-linked immunosorbent assay (Elisa) was used to quantitatively identify the expression of heat shock proteins (HSP70, HSP90, Grp94) in the sarcoma cell line MCA207. From the protein suspension prepared, mHSP/P and Grp94/peptide (Grp94/P) sarcoma vac-cines were isolated using chromatography and were identified by Western blot (WB). Flow cytometry was used to determine their cy-totoxic effects. The levels of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) produced upon mHSP/P and Grp94/P stimulation were measured by Elisa. The effect of sarcoma vaccines on the growth and survival of sarcoma was evaluated in mice. The expression of PD-L1 on the surface of MCA207 sarcoma cells was evaluated by immunofluorescent staining. The effect of IFN-γ treatment on the expression of PD-L1 was determined by WB. Animal experiments explored the effects of PD-L1 inhibitor in combination with mHSP/P treatment on tumors. Results: Tumor tissue carries a variety of HSP subtypes (HSP70, HSP90, Grp94). We successfully isolated sarco-ma tissue-derived mHSP/P and Grp94/P tumor vaccines, which were identified by WB; flow cytometry analysis demonstrated their cy-totoxicity. The levels of IFN-γ and TNF-α cytokines upon mHSP/P stimulation were significantly higher than that observed upon Grp94/P stimulation (P<0.05). The expression of PD-L1 on the surface of sarcoma cells increased with IFN-γ treatment. Animal experiments demonstrated that PD-L1 inhibitor in combination with mHSP/P significantly increased the immune response against tumor (P<0.05). Conclusions: Tumor-derived mHSP/P and Grp94/P can be used as tumor vaccines in animal models. The mHSP/P can elicit a stronger anti-tumor immune response than Grp94/P. IFN-γ stimulates the expression of PD-L1 in sarcoma cells, which results in immune eva-sion. The PD-L1 inhibitor in combination with mHSP/P increased the anti-tumor effect in the tumor microenvironment.
ABSTRACT
BACKGROUND: Platelet-rich plasma containing various high-concentration growth factors can promote new bone formation and accelerate bone healing. But its effects on distraction osteogenesis remain unclear. OBJECTIVE: To observe the effects of autologous platelet-dch plasma on premaxillary distraction osteogeaesis in rabbits. DESIGN, TIME AND SETTING: A randomized controlled animal experiment was performed at the Laboratory of Physiology, North Campus,Sun Yat-sen University between July and August 2007. MATERIALS: Sixteen healthy rabbits, the same number for male and female, aged 3-5 months,weighing 1. 4-1.7 kg,were randomly and evenly divided into an experimental group and a control group. Premaxillary incisor band,outside front distraction mask, and distraction rubber band were self-made.Coagulant was prepared by dissolving 1000 u bovine thrombin in 1 mL of 10% calcium chloride.METHODS: Titanium nails (1.5 mm in diameter) were separately inserted on two sides of left premaxillary suture in rabbits. A self-made distraction device was used. Gelatum-like substance [Vplatelet-rich plasma: V coagulant=9:1] was injected into the left premaxillary suture of experimental group rabbits immediately prior to distraction.In each group, one-week sustained distraction was performed in 4 rabbits,and three-week sustained distraction was performed in another rabbits. MAIN OUTCOME MEASURES: The increase of nail-nail distance on the two sides and histological results following distraction osteogenesis.RESULLTS: Rabbit premaxillary bone moved anterior in the two groups. The experimental group showed greater increase of nail-nail distance, faster bone formation and mineralization, more blood vessels, and thicker and more mature bone trabecula in the distraction interspace in comparison with the control group. CONCLUSION: Platelet-rich plasma will help bone tissue regeneration and promote premaxillary distraction osteogenesis.