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Targeting B7-H3 over-expressed tumor cells with anti-B7-H3 monoclonal antibodies inhibits tumor growth. Here we demonstrated the expression of B7 family homologue 3 (B7-H3) in a wide range of human tumor cells and further investigated whether B7-H3 could be served as a target for T-cell mediated immunotherapy against human cancers. The specific cytotoxic activity of activated T cell (ATC) armed with a novel anti-CD3 x anti-B7-H3 bispecific antibody (B7-H3Bi-Ab) against tumor cell was evaluated in vitro and in vivo. In contrast with unarmed ATC, an increase in cytotoxic activity of B7-H3Bi-armed ATC against tumor cells was observed at effector/target (E/T) ratios of 5:1, 10:1, and 20:1. Moreover, B7-H3Bi-armed ATC secreted more IFN-γ, TNF-α and IL-2 than unarmed ATC. Infusion of B7-H3Bi-armed ATC inhibited tumor growth in severe combined immunodeficiency (SCID) xenograft models, along with a significant survival benefit. Therefore, treatment with novel B7-H3Bi-armed ATC will be a promising strategy for current cancer immunotherapy.

Due to the association between inflammation and endothelial dysfunction in atherosclerosis, the blockage of the inflammatory process that occurs on the endothelial cells may be a useful way of preventing atherosclerosis. In the present study, human umbilical vein endothelial cells (HUVECs) were used to investigate the protective effects of quercetin and taraxasterol against H2O2-induced oxidative damage and inflammation. HUVECs were pretreated with quercetin or taraxasterol at concentrations ranging between 0 and 210 µM for 12 h, prior to being administered different concentrations of H2O2 for 4 h. Cell viability and levels of apoptosis were assessed through cell counting kit-8 (CCK-8) and terminal deoxynucleotidyl transferase dUTP nick end labeling assays, respectively, to determine the injury to the HUVECs. The viability loss in the H2O2-induced HUVECs was markedly restored in a concentration-dependent manner by pretreatment with quercetin or taraxasterol. This effect was accompanied by significantly decreased expression of vascular cell adhesion molecule 1 (VCAM-1) and cluster of differentiation (CD)80 for taraxasterol and that of CD80 for quercetin. In conclusion, the present study showed the protective effects of quercetin and taraxasterol against cell injury and inflammation in HUVECs and indicated that the effects were mediated via the downregulation of VCAM-1 and CD80 expression. This study has therefore served as a preliminary investigation on the anti-atherosclerotic and cardiovascular protective effects of quercetin and taraxasterol as dietary supplements.

Targeting HER2 overexpressed breast cancer cells with anti‑HER2 monoclonal antibodies inhibits tumor growth. Here we investigated whether HER2 can serve as a target for T cell-mediated immunotherapy of human colorectal carcinoma. Specific cytolytic activity of activated T cells (ATCs) armed with anti‑CD3 x anti‑HER2 bispecific antibody (HER2Bi-Ab) against HER2+ tumor cells was evaluated by bioluminescent signal generated by luciferase reporter on tumor cells in vitro and in vivo. In contrast to unarmed ATCs, increased cytotoxic activity of HER2Bi-armed ATCs against HER2+ tumor cells was observed. Moreover, HER2Bi-armed ATCs expressed higher level of activation marker CD69 and secreted significantly higher levels of IFN-γ than the unarmed ATC counterpart. In addition, compared with anti‑HER2 mAb (Herceptin®) or unarmed ATC, HER2Bi-armed ATCs showed significant suppression against colorectal carcinoma cells. In colorectal tumor cell xenograft mice, infusion of HER2Bi-armed ATCs successfully inhibited the growth of Colo205-luc cells. The HER2Bi-armed ATCs with anti-tumor effects may provide a promising immunotherapy for colorectal carcinoma in the future.

The efficacy of immunotherapy based on natural killer (NK) cells is hampered by intrinsic non-specific cytotoxicity and insufficient activation of NK cells. Here, we confer the T-cell receptor-like (TCR-like) specificity on NK cells, taking advantage of both the innate and adaptive immune arms of the immune response to generate enhanced anti-melanoma activity. The TCR-like antibody (Ab) GPA7 was selected against melanoma-associated gp100/human leukocyte antigen (HLA)-A2 complex and then fused to intracellular domain of CD3-ζ chain. This fusion construct was incorporated into NK-92MI cell line and expressed as a chimeric antigen receptor on the surface of the cell. The anti-tumour activity of the transgenic NK-92MI-GPA7-ζ cell line was assessed against melanoma in vitro and in vivo. The engineered NK-92MI-GPA7-ζ cells could recognize melanoma cells in the context of HLA-A2 and showed enhanced killing of both melanoma cell lines and primary melanoma. Furthermore, adoptively transferred NK-92MI-GPA7-ζ cells significantly suppressed the growth of human melanoma in a xenograft model in mice. Collectively, these results demonstrate that the TCR-like Ab, GPA7, could redirect NK cells to target the intracellular antigen gp100 and enhance anti-melanoma activity, providing a promising immunotherapeutic strategy to prevent and treat melanoma.

Glioma is known to induce local and systemic immunosuppression, which inhibits antitumor T cell responses. The galectin-9-Tim-3-pathway negatively regulates T cell pathways in the tumor immunosuppressive environment. The present study assessed the expression of Tim-3 and galectin-9 in glioma patients, and evaluated the association between the expression of Tim-3 and galectin-9 with clinical characteristics. The present study identified that Tim-3 expression was significantly increased in peripheral blood T cells of glioma patients compared with those of healthy controls, and was additionally increased on tumor-infiltrating T cells. The expression of Tim-3 on tumor-infiltrating T cells was associated with the World Health Organization (WHO) grade of glioma, but negatively correlated with the Karnofsky Performance Status score of the glioma patients. Immunohistochemical analysis revealed that the expression of galectin-9 in tumor tissues was associated with Tim-3 expression on tumor-infiltrating T cells and the WHO grade of glioma. These findings suggest that the galectin-9-Tim-3 pathway may be critical in the immunoevasion of glioma and may be a potent target for immunotherapy in glioma patients.

The epidermal growth factor receptor (EGFR) is an attractive target for the immunotherapy of EGFR+ tumors. Adjuvant immunotherapy with cytokine-induced killer (CIK) cells may improve progression-free survival rates in patients suffering from cancer. In the present study, we examined the bispecific antibody anti-CD3 x anti-EGFR (EGFRBi-Ab) for its ability to redirect CIK cells to target EGFR-positive glioblastoma. The specific cytolytic activity of CIK cells armed with EGFRBi-Ab against U87MG-luc cells was evaluated by bioluminescent signal generated using luciferase reporter assay which did not alter the surface molecule expression or proliferation ability of U87MG cells. In contrast to unarmed CIK cells, increased cytotoxic activity of EGFRBi-armed CIK cells against the U87MG-luc target was observed at effector/target (E/T) ratios of 5:1, 10:1, and 20:1. Moreover, EGFRBi-armed CIK cells secreted significantly higher levels of IFN-γ, TNF-α, and IL-2 than their unarmed CIK counterpart cells. Furthermore, in glioblastoma xenograft mice, infusion of the EGFRBi-armed CIK cells successfully inhibited the growth of glioblastoma tumors. The in vitro and in vivo antitumor effects of EGFRBi-armed CIK cells support their clinical use for treatment of glioblastoma in the future.

Due to the association between inflammation and endothelial dysfunction in atherosclerosis, the blockage of the inflammatory process that occurs on the endothelial cells may be a useful way of preventing atherosclerosis. In the present study, human umbilical vein endothelial cells (HUVECs) were used to investigate the protective effects of quercetin and taraxasterol against H2O2-induced oxidative damage and inflammation. HUVECs were pretreated with quercetin or taraxasterol at concentrations ranging between 0 and 210 µM for 12 h, prior to being administered different concentrations of H2O2 for 4 h. Cell viability and levels of apoptosis were assessed through cell counting kit-8 (CCK-8) and terminal deoxynucleotidyl transferase dUTP nick end labeling assays, respectively, to determine the injury to the HUVECs. The viability loss in the H2O2-induced HUVECs was markedly restored in a concentration-dependent manner by pretreatment with quercetin or taraxasterol. This effect was accompanied by significantly decreased expression of vascular cell adhesion molecule 1 (VCAM-1) and cluster of differentiation (CD)80 for taraxasterol and that of CD80 for quercetin. In conclusion, the present study showed the protective effects of quercetin and taraxasterol against cell injury and inflammation in HUVECs and indicated that the effects were mediated via the downregulation of VCAM-1 and CD80 expression. This study has therefore served as a preliminary investigation on the anti-atherosclerotic and cardiovascular protective effects of quercetin and taraxasterol as dietary supplements.

Genetically modified T cells to recognize tumor-associated antigens by transgenic TCRs or chimeric antigen receptors (CAR) have been successfully applied in clinical trials. However, the disadvantages of either TCR mismatching or the requirement of a surface tumor antigen limit their wider applications in adoptive T cell therapy. A TCR-like chimeric receptor, specific for the melanoma-related gp100/HLA-A2 complex was created by joining a TCR-like antibody GPA7 with the endodomains of CD28 and CD3-ζ chain. This TCR-like CAR, GPA7-28z, was subsequently introduced into human T cells. Retargeted T cells expressing GPA7-28z could exhibit efficient cytotoxic activities against human melanoma cells in vitro in the context with HLA-A2. Furthermore, infusion of GPA7-28z-transduced T cells suppressed melanoma progression in a xenograft mouse model. Redirecting human T cells with TCR-like CARs would be a promising alternative approach to TCR-mediated therapy for melanoma patients, which is also feasible for targeting a variety of other tumor antigens.

To exploit the biological and pharmacological properties of immunoglobulin constant domain Fc fragment and increase the killing efficacy of T cells, a single chain variable fragment specific to CD3 was fused with Fcab (Fc antigen binding), a mutant Fc fragment with specificity against Human epidermal growth factor receptor 2 (HER2) developed by F-star. The bispecific fusion named as FcabCD3 was expressed by transient transfection in HEK-293T cells and purified by affinity chromatography. Specific cytolytic activity of retargeted T cells to kill HER2 positive SKBR3 cell line was evaluated in vitro. FcabCD3 was able to retarget T cells to kill both Herceptin insensitive Colo205-luc cell line and HER2 low expression MDA-MB-231-luc cell line. Furthermore, FcabCD3 was effective in eliminating the Colo205 tumor established on BALB/c nu/nu mice.

Enteropeptidase can cleave trypsinogen on the sequence of Asp-Asp-Asp-Asp-Lys and plays an important role in food digestion. The RANKL–RANK signalling pathway plays a pivotal role in bone remodelling. In this study, we reported that enteropeptidase can inhibit the RANKL–RANK signalling pathway through the cleavage of RANK. A surrogate peptide blocking assay indicated that enteropeptidase could specifically cleave RANK on the sequence NEEDK. Osteoclast differentiation assay and NF-κB activity assay confirmed that enteropeptidase could inhibit osteoclastogenesis in vitro through the cleavage of RANK. This is the first study to prove that the RANKL–RANK signalling pathway can be inhibited by cleavage of RANK instead of targeting RANKL. Structured summary of protein interactions EP cleaves hRANK by cleavage assay (View interaction) EP cleaves mRANK by cleavage assay (View interaction)

Human influenza is a seasonal disease associated with significant morbidity and mortality. The most effective means for controlling infection and thereby reducing morbidity and mortality is vaccination with a three inactivated influenza virus strains mixture, or by intranasal administration of a group of three different live attenuated influenza vaccine strains. Comparing to the inactivated vaccine, the attenuated live viruses allow better elicitation of a long-lasting and broader immune (humoral and cellular) response that represents a naturally occurring transient infection. The cold-adapted (ca) influenza A/AA/6/60 (H2N2) (AA ca) virus is the backbone for the live attenuated trivalent seasonal influenza vaccine licensed in the United States. Similarly, the influenza A components of live-attenuated vaccines used in Russia have been prepared as reassortants of the cold-adapted (ca) H2N2 viruses, A/Leningrad/134/17/57-ca (Len/17) and A/Leningrad/134/47/57-ca (Len/47) along with virulent epidemic strains. However, the mechanism of temperature-sensitive attenuation is largely elusive. To understand how modification at genetic level of influenza virus would result in attenuation of human influenza virus A/PR/8/34 (H1N1,A/PR8), we investigated the involvement of key mutations in the PB1 and/or PB2 genes in attenuation of influenza virus in vitro and in vivo. We have demonstrated that a few of residues in PB1 and PB2 are critical for the phenotypes of live attenuated, temperature sensitive influenza viruses by minigenome assay and real-time PCR. The information of these mutation loci could be used for elucidation of mechanism of temperature-sensitive attenuation and as a new strategy for influenza vaccine development.

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Background Human influenza is a seasonal disease associated with significant morbidity and mortality. Anti-flu Traditional Chinese Medicine (TCM) has played a significant role in fighting the virus pandemic. In TCM, dandelion is a commonly used ingredient in many therapeutic remedies, either alone or in conjunction with other natural substances. Evidence suggests that dandelion is associated with a variety of pharmacological activities. In this study, we evaluated anti-influenza virus activity of an aqueous extract from dandelion, which was tested for in vitro antiviral activity against influenza virus type A, human A/PR/8/34 and WSN (H1N1). Results Results obstained using antiviral assays, minigenome assay and real-time reverse transcription-PCR analysis showed that 0.625-5 mg/ml of dandelion extracts inhibited infections in Madin-Darby canine kidney (MDCK) cells or Human lung adenocarcinoma cell line (A549) of PR8 or WSN viruses, as well as inhibited polymerase activity and reduced virus nucleoprotein (NP) RNA level. The plant extract did not exhibit any apparent negative effects on cell viability, metabolism or proliferation at the effective dose. This result is consistent with the added advantage of lacking any reported complications of the plant's utility in traditional medicine over several centuries. Conclusion The antiviral activity of dandelion extracts indicates that a component or components of these extracts possess anti-influenza virus properties. Mechanisms of reduction of viral growth in MDCK or A549 cells by dandelion involve inhibition on virus replication.

Background Acquired immunodeficiency syndrome (AIDS), which is caused by the human immunodeficiency virus (HIV), is an immunosuppressive disease that results in life-threatening opportunistic infections. The general problems in current therapy include the constant emergence of drug-resistant HIV strains, adverse side effects and the unavailability of treatments in developing countries. Natural products from herbs with the abilities to inhibit HIV-1 life cycle at different stages, have served as excellent sources of new anti-HIV-1 drugs. In this study, we aimed to investigate the anti-HIV-1 activity of aqueous dandelion extract. Methods The pseudotyped HIV-1 virus has been utilized to explore the anti-HIV-1 activity of dandelion, the level of HIV-1 replication was assessed by the percentage of GFP-positive cells. The inhibitory effect of the dandelion extract on reverse transcriptase activity was assessed by the reverse transcriptase assay kit. Results Compared to control values obtained from cells infected without treatment, the level of HIV-1 replication and reverse transcriptase activity were decreased in a dose-dependent manner. The data suggest that dandelion extract has a potent inhibitory activity against HIV-1 replication and reverse transcriptase activity. The identification of HIV-1 antiviral compounds from Taraxacum officinale should be pursued. Conclusions The dandelion extract showed strong activity against HIV-1 RT and inhibited both the HIV-1 vector and the hybrid-MoMuLV/MoMuSV retrovirus replication. These findings provide additional support for the potential therapeutic efficacy of Taraxacum officinale. Extracts from this plant may be regarded as another starting point for the development of an antiretroviral therapy with fewer side effects.

The expression of a soluble protein on cell surface is often desirable for study of a functional protein, wide application of a protein or investigation of protein-protein interaction. The expression of a soluble protein on the surface of a cell is often achieved by genetically linking a protein to the extra-cellular fragment of a transmembrane partner. In this study, the myc epitope was linked with N terminal of transmembrane proteins either A2TM or deltaLNGFR amplified by overlapping PCR. The plasmids expressing fusion protein were transfected into 293FT cells and the expression of target proteins was evaluated by fluorescent microscope, flow cytometry and Western blotting. The results of flow cytometry revealed that both A2TM and deltaLNGFR were expressed on the cell surface, but A2TM could only be detected with high copy number. Western blotting showed that the expression level of deltaLNGFR was very high and protein was heavily glycosylated, by contrast the expression of A2TM was hardly detected. The results indicate that glycosylated deltaLNGFR is a good candidate partner for the expression of a soluble protein on the cell surface.

Dendritic cells (DCs) are crucial for the induction and maintenance of tumor-specific immune responses. Studies have shown that tumor-associated DCs are immunosuppressed in some human tumors. However, phenotype and function of DCs in retinoblastoma (RB) remain unclear. RB cell supernatant (RBcs) was used to treat DCs in vitro to explore the effect of RB cells on DCs. DCs were generated from peripheral blood mononuclear cells of healthy donors. On day 5 of culture, DCs were treated with RBcs for 24 h, and then purified using magnetic beads. The maturation of DCs was induced by TNF-α or LPS. After treatment with RBcs, expression of co-stimulatory molecules CD80 and CD86 was elevated in DCs, accompanied by increased production of IL-12p70, TNF-α, IL-6, IL-1β, and IL-8 but decreased production of IL-10. RBcs neither inhibited DC maturation nor promoted DC apoptosis. Moreover, RBcs-exposed DCs stimulated allogenetic T cell proliferation and T cell-derived cytokine production. These results indicate that RBcs can improve DCs’ antigen presenting function and capability to activate T cells, suggesting that RB cells may have an immunostimulatory effect on DCs, and DC-based immunotherapy may be adopted in the treatment of RB.