Therapeutic Potential of Ex Vivo Expanded γδ T Cells against Osteosarcoma Cells.

Immunotherapy is an attractive therapeutic strategy for the treatment of osteosarcoma (OS). The unique features of γδ T cells have made them popular for cancer immunotherapy. Here, we expanded γδ T cells using human peripheral blood mononuclear cells (PBMCs) and investigated their therapeutic potential against OS cells. PBMCs from healthy donors were cultured for 10 days with CON medium (unstimulated control); EX media, CON with recombinant human interleukin-2 (rhIL-2) and zoledronate; and EX28 media, CON with rhIL-2, zoledronate, and CD3/CD28 activator. The expanded γδ T cells were isolated by magnetic cell separation or fluorescence-activated cell sorting, cultured with two OS cell lines (KHOS/NP and MG-63) at various cell ratios with or without doxorubicin or ifosfamide, and analyzed for cytotoxicity and cytokine secretion. The number of CD3+γδTCR+Vγ9+ triple-positive γδ T cells and concentrations of IFN-γ and TNF-α were highest in the rhIL-2 (100 IU) and zoledronate (1 µM) supplemented culture conditions. The CD3/CD28 agonist did not show any additional effects on γδ T cell expansion. The expanded γδ T cells exhibited potent in vitro cytotoxicity against OS in a ratio- and time-dependent manner. The γδ T cells may enhance the effect of chemotherapeutic agents against OS and may be a new treatment strategy, including chemo-immunotherapy, for OS.

Development of a Bioluminescent Human Osteosarcoma Model in Humanized NSG Mice: A Pilot Study.

BACKGROUND/AIM: Osteosarcoma is the most common type of bone cancer, but current therapeutic interventions remain largely insufficient. The development of new treatment strategies is needed, and moreover, optimal rodent models are necessary for testing the efficacy of new treatment modalities of osteosarcoma. Humanized mice carry human hematopoietic and immune systems, and are considered an ideal tool to study human diseases including cancer immunology. Herein, we performed a preliminary study toward developing an in vivo bioluminescent osteosarcoma model using humanized immunodeficient (NSG) mice. MATERIALS AND METHODS: To establish the xenograft and orthotopic mouse model, NSG mice engrafted with human CD34+ hematopoietic stem cells were injected with luciferase-expressing KHOS/NP cells at two different time points. Bioluminescence images were obtained to monitor in vivo tumor growth and metastasis. Influence of the degree of human cell engraftment on tumor growth and metastatic behavior was analyzed and compared between the two groups. RESULTS: KHOS/NP-luc cells injected in humanized NSG mice formed macroscopic tumors. The percentage of human CD45+ cells in these models was similar, but the percentage of human CD45+CD3+ and their subset was higher in the late-injection group compared to that of the early-injection group. The rate of KHOS/NP tumor growth was higher in the early-injection group than in the late-injection group. In the present study, human hematopoietic cell engraftment was not influenced by KHOS/NP cell injection, but KHOS/NP osteosarcoma showed more aggressive behavior in the early-injection group than that in the late-injection group, forming larger tumor volumes and earlier metastases. CONCLUSION: The results indicated that tumor growth and progression in humanized NSG mice may have been influenced by higher levels of human cell engraftment, especially T cells. Although there exist some limitations to our study, our preliminary results can provide the basis for the development of a humanized osteosarcoma mouse model.

Effects of Aster scaber Seed Oil Containing trans-Δ3 Fatty Acids on Lipid Profiles of Hamsters and Rats.

The effects of Aster scaber seed oil (ASO) on lipid profiles were studied in rats and hamsters. ASO contained considerable amounts of Δ3t-16:1 (11.4%), Δ3t, 9c-18:2 (4.6%), and Δ3t, 9c, 12c-18:3 (11.3%). Young rats and hamsters were fed diets containing ASO, soybean oil (SBO), or olive oil (OLO) as fat sources for 4 weeks in separate experiments with or without cholesterol. In the rat study, there were no significant differences in the concentrations of serum total cholesterol, high-density lipoprotein (HDL)-cholesterol, and triacylglycerol among the groups. The serum but not liver malondialdehyde (MDA) level was significantly lower in the ASO-fed group than it was in the other groups. The biochemical and growth parameters revealed no significant biological damages in the ASO-fed animals. In the hamster study, dietary cholesterol-dependent effects were evident in the serum lipids profiles, whereas the fat-induced effect was only observed in the ratio of serum low-density lipoprotein (LDL)-/HDL-cholesterol. Furthermore, fat- and cholesterol-induced effects were evident in the ratio of serum LDL-/HDL-cholesterol. Significant interactions between dietary fat and cholesterol were observed as evident from the concentration of serum cholesterol and triacylglycerol, as well as the activity of serum cholesterol ester transfer protein. These results suggest that dietary ASO containing trans-Δ3 fatty acids appeared to improve the serum LDL-/HDL-cholesterol ratio more than the SBO did, especially when hamsters were simultaneously fed cholesterol-supplemented diet.

Kinetic study for the optimization of ginsenoside Rg3 production by heat treatment of ginsenoside Rb1.

BACKGROUND: Ginsenoside Rg3 is a promising anticancer agent. It is usually produced by heat treatment of ginseng, in which ginsenoside Rb1 is the major ginsenoside. A kinetic study was conducted to optimize ginsenoside Rg3 production by the heat treatment of ginsenoside Rb1. METHODS: Ginsenoside Rb1 was heated using an isothermal machine at 80°C and 100°C and analyzed using HPLC. The kinetic parameters were calculated from the experimental results. The activation energy was estimated and used to simulate the process. The optimized parameters of ginsenoside Rg3 production are suggested based on the simulation. RESULTS: The rate constants were 0.013 h(-1) and 0.073 h(-1) for the degradation of ginsenosides Rb1 and Rg3 at 80°C, respectively. The corresponding rate constants at 100°C were 0.045 h(-1) and 0.155 h(-1). The estimated activation energies of degradation of ginsenosides Rb1 and Rg3 were 69.2 kJ/mol and 40.9 kJ/mol, respectively. The rate constants at different temperatures were evaluated using the estimated activation energies, and the kinetic profiles of ginsenosides Rb1 and Rg3 at each temperature were simulated based on the proposed kinetic model of consecutive reaction. The optimum strategies for producing ginsenoside Rg3 from ginsenoside Rb1 are suggested based on the simulation. With increased temperature, a high concentration of ginsenoside Rg3 is formed rapidly. However, the concentration decreases quickly after the reaching the maximal concentration value. CONCLUSION: The optimum temperature for producing ginsenoside Rg3 should be the highest temperature technically feasible below 180°C, in consideration of the cooling time. The optimum reaction time for heat treatment is 30 min.

Developmental expression of Pitx2c in Xenopus trigeminal and profundal placodes.

Cranial placodes are thickenings of the embryonic head ectoderm that contribute to the paired sense organs and to the cephalic peripheral nervous system. Here we report the spatiotemporal expression pattern of transcription factor Pitx2c during Xenopus laevis cranial placode formation, focusing more specifically on key stages of trigeminal and profundal placode development. We also compare its expression to five genes that have been associated with development of these sensory placodes, namely Foxi1c, Islet1, NeuroD, Pax3, and Six1. We show that while initially expressed in both the trigeminal and profundal placodes, Pitx2c is later restricted to the prospective profundal ganglion, where it is co-expressed with Islet1, NeuroD and Pax3. This combination of factors defines a molecular signature for the characterization of the profundal versus trigeminal ganglia in Xenopus.

Effect of Korean red ginseng extract on liver damage induced by short-term and long-term ethanol treatment in rats.

Korean red ginseng (KRG) is prepared by the process of steaming the roots of Panax ginseng. In this study, the feeding effects of KRG-water extract (KRGE) on ethanol-induced liver damage were elucidated by measuring serum biomarkers in rats. Serum γ-glutamyltranspeptidase (γ-GT) activity and the concentration of malondialdehyde (MDA) were significantly increased by short-term and long-term ethanol treatment in rats, whereas the activities of serum glutamate pyruvate transaminase (GPT) and glutamate oxaloacetate transaminase (GOT) did not respond. Pretreatment with KRGE maintained the activity of serum GPT, and the MDA concentration induced by short-term ethanol ingestion remained within the normal range. However, co-feeding of KRGE to rats decreased the concentration of MDA but failed to modulate the serum γ-GT activity induced by long-term ethanol treatment. Our studies suggest that in rats, it appears that KRGE does not sufficiently reverse the physiological response evoked by long-term ethanol ingestion to maintain normal conditions, in view of the serum biomarker γ-GT, regardless of KRGE's favorable antioxidant activity.

Hypolipidaemic effects of dietary whole soybean curd (jeondubu) in rats.

BACKGROUND: The characteristic of whole soybean curd is that it includes the soybean residue that is discarded as waste in the manufacture of usual soybean curd (known as tofu). In this study the effect of dietary whole soybean curd on lipid profiles in rats was compared with that of usual soybean curd. Rats were fed for 4 weeks with diets differing only in the source of protein, namely casein, whole soybean curd or usual soybean curd. RESULTS: There were no significant differences in growth parameters due to diet differences. However, the two groups fed with curds had significantly lower levels of serum cholesterol and triglyceride than the group fed with casein, the greatest reduction in lipid profiles being observed in the group fed with whole soybean curd. The serum high-density lipoprotein cholesterol/total cholesterol ratio was higher in rats fed with whole soybean curd. CONCLUSION: The results suggest the possibility that whole soybean curd may have more beneficial effects in controlling serum lipid profiles than usual soybean curd that is normally consumed.

Enhancing phage display of antibody fragments using gIII-amber suppression.

The effect of utilizing Ex12 helper phage, a mutant M13K07 helper having two amber codons at the gIII (gIII-amber), in combination with Escherichia coli host strains belonging to the supE genotype on improving the phage display of antibody fragments was investigated. Because of an inefficient read-through of the UAG codons, Ex12 helper phage produced approximately 10% of the intracellular wt pIII in the supE host cells compared to M13K07. The phage progenies rescued from the supE XL-1 Blue MRF' strain carrying the recombinant phagemid, pCMTG-SP112, by Ex12 helper phage displayed both antibody-DeltapIII fusion and wt pIII at a ratio of 1:1.5, and achieved a 50-fold greater display of the antibody-DeltapIII compared to those obtained by a conventional phage rescue using M13K07. Additionally observed were a 100-fold increase in antigen-binding functionality and a drastic improvement on antigen-specific panning efficiency by the phage progenies. Our approach permits the display of at least one antibody fragment as well as more than one copy of wt pIII on the surface of recombinant phages, and this would make the phagemid-based phage display technology more practical and reliable.