Sensitization of human osteosarcoma cells to Vγ9Vδ2 T-cell-mediated cytotoxicity by zoledronate.

Despite improvements in the treatment of osteosarcoma, there is a need for new therapeutic strategies, in particular for the treatment of recurrent tumors and metastases. Adoptive immunotherapy with Vγ9Vδ2 T lymphocytes represents an attractive strategy. We have investigated combining adoptive immunotherapy with Vγ9Vδ2 T cells and zoledronate to optimize osteosarcoma therapy. Vγ9Vδ2 T cells, from healthy volunteers and patients with osteosarcoma, cultures alone demonstrated moderate or poor cytotoxic activity against osteosarcoma cell lines, respectively. The addition of zoledronate further increased cytotoxicity in vitro. This enhancement was largely dependent on the granule exocytose and partly on TRAIL pathways, was TCR-mediated and partly NKG2D-mediated. These data suggest that combined treatment of human osteosarcoma with zoledronate and Vγ9Vδ2 T cells may be an effective complement to current chemotherapies.

Artesunate inhibits growth and induces apoptosis in human osteosarcoma HOS cell line in vitro and in vivo.

This paper aims to investigate the effects of artesunate (ART) on growth and apoptosis in human osteosarcoma HOS cell line in vitro and in vivo and to explore the possible underlying mechanisms. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The induction of apoptosis was detected by light and transmission electron microscopy and flow cytometry. Western blot analysis was used to investigate the related mechanisms. Nude mice were further employed to investigate the antitumour activity of ART in vivo. MTT assay results demonstrated that ART selectively inhibits the growth of HOS cells in a dose- and time-dependent manner. Based on the findings of light and transmission electron microscopy, Hoechst 33258 staining, and fluorescein isothiocyanate (FITC)-annexin V staining, the cytotoxicity of ART in HOS cells occurs through apoptosis. With ART treatment, cytosolic cytochrome c was increased, Bax expression was gradually upregulated, Bcl-2 expression was downregulated, and caspase-9 and caspase-3 were activated. Thus, the intrinsic apoptotic pathway may be involved in ART-induced apoptosis. Cell cycle analysis by flow cytometry indicated that ART may induce cell cycle arrest at G(2)/M phase. In nude mice bearing HOS xenograft tumours, ART inhibited tumour growth and regulated the expressions of cleaved caspase-3 and survivin, in agreement with in vitro observations. ART has a selective antitumour activity against human osteosarcoma HOS cells, which may be related to its effects on induction of apoptosis via the intrinsic pathway. The results suggest that ART is a promising candidate for the treatment of osteosarcoma.

IFN-γ enhances HOS and U2OS cell lines susceptibility to γδ T cell-mediated killing through the Fas/Fas ligand pathway.

Osteosarcoma is the second highest cause of cancer-related death in children and adolescents, partly due to dysfunction of the Fas/FasL signaling pathway, which leads to develop fatal metastasis. Since presenting no or low levels of Fas expression, resisting Fas ligand-induced apoptosis, and lack of FasL in the host environment, osteosarcoma cells always promote metastases growth and proliferate in the lungs. Therefore, agents, which up-regulate tumor cell surface Fas expression and function, in combination with immune cells, may be effective in treating osteosarcoma, especially lung metastases. The aim of this work was to investigate the effect of γδ T cells in combination with IFN-γ in treating osteosarcoma in vitro. In the present study, we found that IFN-γ up-regulated the expression of Fas in osteosarcoma cell lines, HOS and U2OS, resulting in an enhanced susceptibility of cells to γδ T cells lyses. Moreover, this cytotoxicity was prevented by treatment with FasL-blocking antibodies. These data suggest that adoptive transfer of γδ T cells in combination with IFN-γ may substantially increase anti-osteosarcoma activities and represent a novel strategy for osteosarcoma adjunct immunotherapy.

Identification of immunodominant B- and T-cell combined epitopes in outer membrane lipoproteins LipL32 and LipL21 of Leptospira interrogans.

Leptospirosis is a serious infectious disease caused by pathogenic Leptospira. B- and T-cell-mediated immune responses contribute to the mechanisms of Leptospira interrogans infection and immune intervention. LipL32 and LipL21 are the conserved outer membrane lipoproteins of L. interrogans and are considered vaccine candidates. In this study, we identified B- and T-cell combined epitopes within LipL32 and LipL21 to further develop a novel vaccine. By using a computer prediction algorithm, two B- and T-cell combined epitopes of LipL21 and four of LipL32 were predicted. All of the predicted epitopes were expressed in a phage display system. Four epitopes, LipL21 residues 97 to 112 and 176 to 184 (LipL21(97-112) and LipL21(176-184), respectively) and LipL32(133-160) and LipL32(221-247) of LipL32 were selected as antigens by Western blotting and enzyme-linked immunosorbent assay. These selected epitopes were also recognized by CD4(+) T lymphocytes derived from LipL21- or LipL32-immunized BALB/c (H-2(d)) mice and mainly polarized the immune response toward a Th1 phenotype. The identification of epitopes that have both B- and T-cell immune reactivities is of value for studying the immune mechanisms in response to leptospiral infection and for designing an effective vaccine for leptospirosis.

The construction of the eukaryotic expression plasmid pcDNA3.1/azurin and the increased apoptosis of U2OS cells transfected with it.

In our previous study, we demonstrated that azurin could selectively trigger apoptosis in human osteosarcoma cell line U2OS cells. However, the rate of apoptosis (35.8 +/- 3.2%) is not very high, and azurin is too expensive to obtain readily. To solve these problems, we constructed a eukaryotic expression plasmid containing the azurin gene with an influenza virus haemagglutinin 9 peptide HA epitope tag, and transfected the recombinant plasmid pcDNA3.1(+)/azurin into U2OS cells. RT-PCR and Western blot analysis validated the successful transfection and the expression of the azurin-HA protein. Conspicuous apoptosis of the transfected cells was detected by flow cytometry (FCM) and the DNA ladder test. The apoptosis rate reached 64.3 +/- 13.1%. The transcriptional levels of the Bax and p53 genes increased significantly in U2OS cells transfected with pcDNA3.1(+)/azurin, but the Bcl-2 mRNA level decreased. There was no difference in the levels of Bcl-xl mRNA and Survivin mRNA. We propose that the transfection of the recombinant plasmid pcDNA3.1(+)/azurin can significantly induce apoptosis in U2OS cells. This is closely associated with the up-regulation of the transcriptional level of the Bax and p53 genes, and the down-regulation of that of the Bcl-2 gene.

[Identification on the immunogenic activity of recombinant rLTB/CTB-LipL41/ 1 to Leptospira interrogans and detection of lipL41 gene with its production].

OBJECTIVE: To construct prokaryotic expression systems of ltB/ctB-lipL41/1 fusion genes, identify immunogenic and adjuvant activities of the products as well as to understand the frequencies of lipL41 gene that carrying and expressing in L. interrogans wild strains and specific antibody levels in sera from patients with leptospirosis. METHODS: Polymerase chain reaction (PCR) with linking primer was applied to construct the fusion genes ltB-lipL41/1 and ctB-lipL41/1. By routine molecular biological techniques, prokaryotic expression systems of the two fusion genes were constructed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to examine expression of the target recombinant proteins rLTB-rLipL41/1 and rCTB-rLipL41/1. Both western blot and Ganglioside-enzyme linked immunosorbent assay (GM-ELISA) were used while immunogenic and adjuvant activities of rLTB-rLipL41/1 and rCTB-rLipL41/1 were measured. PCR and MAT were performed to detect lipL41 gene and expression of the gene in 97 wild strains of L. interrogans, respectively. Antibodies against product of lipL41 gene in serum samples from 228 leptospirosis patients were detected by ELISA. RESULTS: In comparison with reported corresponding sequences, the similarities of ltB-lipL41/1 and ctB-lipL41/1 fusion genes to nucleotide and putative amino acid sequence were 99.6%-99.9% and 99.8%-100%, respectively. Expression outputs of both rLTB-rLipL41/1 and rCTB-rLipL41/1, mainly presenting with inclusion body, consisting approximate 10% of the total bacterial proteins. Both rLTB-rLipL41/1 and rCTB-rLipL41/1 could combine rabbit anti-rLipL41/1 serum as well as bovine GM1, respectively. 87.6% of the L. interrogans wild strains(85/97) having lipL41 gene while 84.5% (82/97) of the wild strains with rLipL41/1 or rLipL41/2 antiserum were positive for MAT with titers of 1:4-1:128. 84.6% (193/ 228), 78.5% (179/228) from the patients' serum samples were positive for rLipL41/1 and rLipL41/2 antibodies, respectively. CONCLUSION: ltB-lipL41/1 and ctB-lipL41/1 fusion genes and their prokaryotic expression systems were successfully constructed in this study. The two expressed fusion proteins showed qualified immunogenic and adjuvant activities. lipL41 gene was extensively distributed and frequently expressed in different serogroups of L. interrogans. rLTB-rLipL41/1 or rCTB-rLipL41/1 seemed to have had good potential to serve as an antigen in L. interrogans genus-specific vaccine.

Influence factors of serum fibrosis markers in liver fibrosis.

AIM: To analyze the factors which influence the serum levels of hyaluronic acid (HA), type III pro-collagen (PCIII), laminin (LN) and type IV collagen (C-IV) in liver fibrosis. METHODS: The serum specimens from 141 chronic hepatitis patients were assayed for fibrosis indexes including HA, PCIII, LN and C-IV with radioimmunoassay (RIA) and liver function indexes by an automatic biochemistry analyzer. The patients were then divided into consistent group and inconsistent group. The patients'clinical manifestations were recorded, routine blood pictures were done by a blood counter and analyzer (AC-900). Liver biopsy specimens were examined path-morphologically. The inner diameters of portal vein, splenic vein and thickness of spleen were all measured by ultrasonography. RESULTS: Sixteen patients (14.16%) had serum fibrosis indexes inconsistent with histological stage of their hepatic fibrosis. Their serum fibrosis indexes did not correlate with the stage of hepatic fibrosis (P>0.05), but were positively correlated with the grade of inflammation (chi2=12.07, P<0.05). At the same time, serum albumin (ALB) and the ratio of albumin and globulin (A/G) were significantly increased (t=3.06, P<0.01), (t=3.70, P<0.01). Serum levels of glutamic-pyruvic transaminase (ALT), glutamic-oxaloacetic transaminase (AST), gamma-glutamyl transferase (GGT) and globulin (GLB) were all significantly decreased (t=2.45, P<0.05), (t=2.33, P<0.05), (t=2.08, P<0.05), (t=3.03, P<0.01). Weary degree also decreased more obviously (chi2=7.52, P<0.05), but other clinical manifestations, routine blood indexes, serum levels of alkaline phosphatase (AKP), total bilirubin (TBIL), total protein (TP), width of main portal vein, width of splenic vein and thickness of spleen had no significant change (P>0.05). CONCLUSION: Serum fibrosis indexes can be influenced by the grade of inflammation, some liver function indexes and clinical manifestations. Comprehensive analysis is necessary for its proper interpretation.