Ruen Liu

The enhanced anti-angiogenic and antitumor effects of combining flk1-based DNA vaccine and IP-10

The purpose of the present study was to evaluate the anti-vasculature effects and the anti-tumor effects of attenuated Salmonella typhimurium vaccine strain encoding murine vascular endothelial growth factor (VEGF) receptor-2 (flk1) in combination with plasmid DNA vector encoding the murine interferon-induced protein of 10kDa (IP-10 or CXCL10) gene. Mouse models of malignant melanoma (B16-F10) were treated with combining orally given attenuated S. typhimurium vaccine strain encoding flk1 with direct intratumoral injection of a non-viral plasmid DNA vector encoding the murine IP-10 gene. The volumes of tumors and survival of mice bearing B16-F10 tumors were observed. Cytolytic T lymphocyte (CTL) response was measured by cytotoxic assay, vessel density and tumor-cell proliferation were observed by immunostaining, and tumor apoptosis was determined by TUNEL staining. The results revealed the combination therapy groups showed more significantly inhibited tumor growth, apoptosis of tumor cells, and reduced neovascularization, cell proliferation, and developed a strong CTL response in these mice. In summary, the therapy of attenuated S. typhimurium vaccine strain encoding flk1 combined with the IP-10 gene has significant synergistic effect against tumors.

Improved therapeutic efficacy using vaccination with glioma lysate-pulsed dendritic cells combined with IP-10 in murine glioma.

The purpose of the present study was to evaluate the therapeutic efficacy of glioma lysate-pulsed DCs in combination with plasmid DNA vector encoding the murine interferon-induced protein of 10kDa (IP-10 or CXCL10) gene. Mouse models of brain glioma (GL261) were treated with combining glioma lysate-pulsed DCs with direct intratumoral injection of a nonviral plasmid DNA vector encoding the murine IP-10 gene. The survival of mice bearing GL261 glioma was observed. Enzyme-linked immuno-spot assay was used to determine the frequency of brain-infiltrating lymphocytes (BILs) capable of responding to GL261. Cytolytic T lymphocyte (CTL) response was measured by cytotoxic assay, vessel density and tumor cell proliferation were observed by immunostaining, and tumor apoptosis was determined by TUNEL staining. The results revealed that the combination therapy groups showed more significantly enhanced anti-tumor activity, attraction of lymphocytes into tumor tissues, apoptosis of tumor cells, and reduced neovascularization, cell proliferation, and developed a strong CTL response in these mice. In summary, the therapy of glioma lysate-pulsed DCs combined with the IP-10 gene has significant synergistic effect against glioma.

Adoptive transfer of pTRP2-specific CTLs expanding by bead-based artificial antigen-presenting cells mediates anti-melanoma response

Cytotoxic CD8(+) T cells are key effectors in the immunotherapy of malignant and viral diseases. However, the lack of efficient methods for their in vitro priming and expansion has become a bottleneck to the development of vaccines and adoptive transfer strategies. Synthetic artificial antigen-presenting cells (aAPCs) are now emerging as an attractive tool for eliciting and expanding CTL responses. This study reported a novel approach for targeting malignant melanoma with pTRP2-specific cytotoxic T lymphocytes (CTLs) expanded from the C57BL/6 splenocytes by multiple stimulations with aAPCs made by coating H-2K(b)-Ig/pTRP2 dimeric complexes, anti-CD28 antibody, 4-1BBL molecules and CD83 molecules to cell-sized latex beads. The induced CTLs exhibited specific lysis against RMA-S cells pulsed with the peptide pTRP2 and H-2K(b+) melanoma cells expressing TRP2, while a murine Lewis lung carcinoma cell line 3LL could not be recognized by the CTLs. The peptide-specific activity was inhibited by anti-H-2K(b) monoclonal antibody Y3. Adoptive Transfer of CTLs specific for malignant melanoma expanding by the aAPCs can mediate effective anti-melanoma response. These results suggested the bead-based aAPCs coated with an MHC-Ig/peptide complex, anti-CD28 antibody, 4-1BBL and CD83 could provide a useful tool for the reproducible expansion of specific CTLs for adoptive immunotherapy.

Vagoglossopharyngeal Neuralgia Treated by Microvascular Decompression and Glossopharyngeal Rhizotomy: Clinical Results of 21 Cases

Background: Microvascular decompression (MVD) and rhizotomy are all selected for treating vagoglossopharyngeal neuralgia (VGPN). Nonetheless, controversies still exist about their curative effect on VGPN. Here we evaluate the effectiveness of MVD together with rhizotomy of the glossopharyngeal nerve for the treatment of VGPN. Methods: This study was carried out on 21 patients who were diagnosed with VGPN between the years 2005 and 2010. Patients underwent MVD and glossopharyngeal rhizotomy through a retromastoid keyhole approach. Surgical technique, operation results and complications were our particular concern. Results: Eighteen (85.7%) of 21 patients experienced immediate and complete relief of pain after surgery. In the remaining 3 patients (14.3%), the pain faded away within the following week. No patient complained of dysphonia or dysphagia. All 21 patients reported no change in their outcome at follow-up. Conclusions: Intracranial vagoglossopharyngeal nerve MVD with glossopharyngeal rhizotomy is an effective and safe procedure to treat VGPN.

HLA Tetramer ^ Based Artificial Antigen-Presenting Cells Efficiently Stimulate CTLs Specific for Malignant Glioma

Purpose: The interleukin-13 receptor α2 (IL-13Rα2) is a glioma-restricted cell-surface epitope not otherwise detected within the central nervous system. Here, we report a novel approach for targeting malignant glioma with IL-13Rα2–specific CTLs. Experimental Design: Artificial antigen-presenting cells (aAPC) were made by coating human leukocyte antigen (HLA)-A2/pIL-13Rα2345-354 tetrameric complexes, anti-CD28 antibody, and CD83 molecules to cell-sized latex beads, and used to stimulate IL-13Rα2–specific CTLs from the peripheral blood mononuclear cells of HLA-A2+ healthy donors. After multiple stimulations, the induced CTLs were analyzed for tetramer staining, IFN-γ production, and CTL reactivity. Results: Tetramer staining assay showed that the induced CTLs specifically bound HLA-A2/pIL-13Rα2345-354 tetramers. The CTLs specifically produced IFN-γ in response to the HLA-A2/pIL-13Rα2345-354-aAPCs and exhibited specific lysis against T2 cells pulsed with the peptide pIL-13Rα2345-354 and HLA-A2+ glioma cells expressing IL-13Rα2345-354, whereas HLA-A2− glioma cell lines that express IL-13Rα2345-354 could not be recognized by the CTLs. The peptide-specific activity was inhibited by anti–HLA class I monoclonal antibody. Conclusion: The induced CTLs specific for IL-13Rα2345-354 peptide could be a potential target of specific immunotherapy for HLA-A2+ patients with malignant glioma.

The Maximum Tolerated Dose of Gamma Radiation to the Optic Nerve during Gamma Knife Radiosurgery in an Animal Study

Background: The safety of gamma knife radiosurgery should be considered when treating pituitary adenomas. Objectives: To determine the maximum tolerated dose of radiation delivered by gamma knife radiosurgery to optic nerves. Methods: An animal model designed to establish prolonged balloon compression of the optic chiasm and parasellar region was developed to mimic the optic nerve compression caused by pituitary adenomas. Twenty cats underwent surgery to place a balloon for compression effect and 20 cats in a sham operation group received microsurgery without any treatment. The effects of gamma knife irradiation at 10–13 Gy on normal (sham operation group) and compressed (optic nerve compression group) optic nerves were investigated by pattern visual evoked potential examination and histopathology. Results: Gamma knife radiosurgery at 10 Gy had almost no effect. At 11 Gy, P100 latency was significantly prolonged and P100 amplitude was significantly decreased in compressed optic nerves, but there was little change in the normal optic nerves. Doses of 11 Gy and higher induced significant electrophysiological variations and degeneration of the myelin sheath and axons in both normal and compressed optic nerves. Conclusions: Compressed optic nerves are more sensitive to gamma knife radiosurgery than normal optic nerves. The minimum dose of gamma knife radiosurgery that causes radiation injury in normal optic nerves is 12 Gy; however, the minimum dose is 11 Gy in compressed optic nerves.

In vivo anti-melanoma efficacy of allo-restricted CTLs specific for melanoma expanded by artificial antigen-presenting cells

Cytotoxic CD8+ T cells are key effectors in the immunotherapy of malignant and viral diseases. However, autologous T cell responses to tumor antigens presented by self-MHC are usually weak and ineffective. Allo-restricted T cells represent a potent source of tumor-specific T cells for adoptive immunotherapy. This study reports in vivo anti-melanoma efficacy of the pTRP2-specific allo-restricted CTLs expanded from the BALB/c splenocytes by multiple stimulations with aAPCs made by coating H-2Kb-Ig/pTRP2 dimeric complexes, anti-CD28 antibody, 4-1BBL molecules and CD83 molecules to cell-sized latex beads. The induced allo-restricted CTLs exhibited specific lysis against RMA-S cells pulsed with the peptide pTRP2 and H-2Kb+ melanoma cells expressing TRP2, while a murine Lewis lung carcinoma cell line 3LL could not be recognized by the CTLs. The peptide-specific activity was inhibited by anti-H-2Kb monoclonal antibody Y3. Adoptive transfer of the allo-restricted CTLs specific for malignant melanoma expanded by the aAPCs can mediate effective anti-melanoma response in vivo. These results suggested that the specific allo-restricted CTLs expanded by aAPCs coated with an MHC-Ig/peptide complex, anti-CD28 antibody, 4-1BBL and CD83 could be a potential option of specific immunotherapy for patients with malignant melanoma.

Superparamagnetic iron oxide labeling of spinal cord neural stem cells genetically modified by nerve growth factor-β

This study established superparamagnetic iron oxide (SPIO)-labeled nerve growth factor-β (NGF-β) gene-modified spinal cord-derived neural stem cells (NSCs). The E14 rat embryonic spinal cord-derived NSCs were isolated and cultured. The cells of the third passage were transfected with plasmid pcDNA3-hNGFβ by using FuGENE HD transfection reagent. The expression of NGF-β was measured by immunocytochemistry and Western blotting. The positive clones were selected, allowed to proliferate and then labeled with SPIO, which was mediated by FuGENE HD transfection reagent. Prussian blue staining and transmission electron microscopy (TEM) were used to identify the SPIO particles in the cells. The distinctive markers for stem cells (nestin), neuron (β-III-tubulin), oligodendrocyte (CNPase) and astrocyte (GFAP) were employed to evaluate the differentiation ability of the labeled cells. The immunocytochemistry and western blotting showed that NGF-β was expressed in spinal cord-derived NSCs. Prussian blue staining indicated that numerous blue-stained particles appeared in the cytoplasma of the labeled cells. TEM showed that SPIO particles were found in vacuolar structures of different sizes and the cytoplasma. The immunocytochemistry demonstrated that the labeled cells were nestin-positive. After differentiation, the cells expressed β-III-tubulin, CNPase and GFAP. It was concluded that the SPIO-labeled NGF-β gene-modified spinal cord-derived NSC were successfully established, which are multipotent and capable of self-renewal.SummaryThis study established superparamagnetic iron oxide (SPIO)-labeled nerve growth factor-β (NGF-β) gene-modified spinal cord-derived neural stem cells (NSCs). The E14 rat embryonic spinal cord-derived NSCs were isolated and cultured. The cells of the third passage were transfected with plasmid pcDNA3-hNGFβ by using FuGENE HD transfection reagent. The expression of NGF-β was measured by immunocytochemistry and Western blotting. The positive clones were selected, allowed to proliferate and then labeled with SPIO, which was mediated by FuGENE HD transfection reagent. Prussian blue staining and transmission electron microscopy (TEM) were used to identify the SPIO particles in the cells. The distinctive markers for stem cells (nestin), neuron (β-III-tubulin), oligodendrocyte (CNPase) and astrocyte (GFAP) were employed to evaluate the differentiation ability of the labeled cells. The immunocytochemistry and western blotting showed that NGF-β was expressed in spinal cord-derived NSCs. Prussian blue staining indicated that numerous blue-stained particles appeared in the cytoplasma of the labeled cells. TEM showed that SPIO particles were found in vacuolar structures of different sizes and the cytoplasma. The immunocytochemistry demonstrated that the labeled cells were nestin-positive. After differentiation, the cells expressed β-III-tubulin, CNPase and GFAP. It was concluded that the SPIO-labeled NGF-β gene-modified spinal cord-derived NSC were successfully established, which are multipotent and capable of self-renewal.

Inhibition of tumor growth and angiogenesis by 2-(4-aminophenyl) benzothiazole in orthotopicglioma C6 rat model

In the present study antitumor effect of 2-(4-aminophenyl) benzothiazole (BTZ) was evaluated against human U251 and rat C6 glioma cell lines using MTT assay. It was observed that BTZ exhibited significant antitumor effect with IC50 of 3.5 and 4 µM against human U251 and rat C6 glioma cells respectively. To gain in-depth insights about the antitumor effect of BTZ, glioma xenograft rat model was prepared. The rats were treated with 10 mg and 15 mg/kg body weight doses of BTZ daily for 21 days after C6 cell administration. Treatment of the rats with BTZ reduced the tumor volume to 12% compared to 100% in the untreated rats. TUNEL assay showed a remarkable increase in the proportion of apoptotic cells in the BTZ treated rats than those in the untreated rats. The increase in the population of apoptotic cells was 23-fold compared to control. Immuno-histological staining revealed marked reduction (16%) in the proportion of CD31-stained vessels in the BTZ treated rats than those of the untreated rats. These changes were accompanied with decreased transcript levels of vascular endothelial growth factor (VEGF) and the VEGF receptor Flt1 as well as ERK1/2 and matrix metalloproteinase-2 (MMP2). Moreover, BTZ altered the expression of several cell cycle control proteins. While as pRb protein expression decreased, E2F1 remained unaltered and cyclin D1 protein and p53 expression was enhanced. Taken together, the results indicate that BTZ is a potent inhibitor of glioma cell proliferation in vivo and exerts its effects on cell cycle control and angiogenesis related proteins.