Interferon-gamma produced by interleukin-12-activated tumor infiltrating CD8+T cells directly induces apoptosis of mouse hepatocellular carcinoma.

BACKGROUND/AIMS: Interleukin-12 (IL-12), a cytokine with antitumor activity, was examined for the suppressive effect on hepatocellular carcinoma (HCC) in mouse model, and its mechanism of antitumor activity was analyzed. METHODS: Mice implanted with MIH-2 HCC cells were treated with recombinant mouse IL-12 (500 ng/mouse). Involvement of CD4(+), CD8(+), NK cells and interferon (IFN)-gamma on tumor suppression by IL-12 was examined by treatment of mice with each antibody. Interferon-gamma (IFN-gamma) production by tumor infiltrating cells was analyzed by immunofluorescence microscopy and flow cytometric analysis. Signal transduction for apoptosis induction was examined by immunoblot analysis. RESULTS: The growth of implanted MIH-2 tumors was significantly suppressed by IL-12 and the suppression was inhibited by depletion of CD8(+)T cells. IL-12 treatment caused numerous IFN-gamma-producing CD8(+)T cells to infiltrate into MIH-2 tumors. Antitumor activity of IL-12 was blocked by treating mice with anti-IFN-gamma mAb. CD8(+)T cells from IL-12-treated mice attached to MIH-2 cells and produced IFN-gamma in vitro. Cell attachment might be associated with intercellular adhesion molecule-1 induced by IFN-gamma. In vitro treatment with IFN-gamma induced apoptosis of MIH-2 cells via a mitochondria-dependent pathway. CONCLUSIONS: IL-12 suppressed HCC growth in mouse model. IFN-gamma produced by IL-12-activated tumor-infiltrating CD8(+)T cells directly induced apoptosis of HCC cells.

Autoimmune hepatic inflammation by vaccination of mice with dendritic cells loaded with well-differentiated hepatocellular carcinoma cells and administration of interleukin-12.

Vaccination of mice with dendritic cells loaded with Hepa1-6, well-differentiated hepatocellular carcinoma cell line (DC/Hepa1-6), induced cytotoxic T lymphocytes against Hepa1-6. Liver-specific inflammation was generated by vaccination of mice with DC/Hepa1-6 and subsequent administration of interleukin (IL)-12. Vaccination with DCs loaded with MC38 or B16 and administration of IL-12 did not generate significant liver-specific inflammation. Splenic T cells from DC/Hepa1-6-vaccinated mice showed proliferative response by stimulation with S-100 protein of the liver and showed cytotoxic activity to hepatocytes. Hepatic mononuclear cells from DC/Hepa1-6 + IL-12-treated mice also showed cytotoxic activity to hepatocytes. Adoptive transfer of splenocytes from DC/Hepa1-6-vaccinated mice produced hepatic inflammation in recipient mice that had been pretreated with IL-12. IL-12 upregulated the expression of adhesion molecules and chemokines in the liver. In conclusion, CTLs responsive to hepatocytes induced by DC/Hepa1-6 and enhanced expression of adhesion molecules and chemokines in the liver by IL-12 would produce autoimmune hepatic inflammation.

Antitumour activity mediated by CD4+ cytotoxic T lymphocytes against MHC class II-negative mouse hepatocellular carcinoma induced by dendritic cell vaccine and interleukin-12.

When BALA/c mice with BNL hepatocellular carcinoma (HCC) were treated with dendritic cells fused with BNL cells (DC/BNL) and recombinant murine interleukin (IL)-12, tumour development was significantly suppressed, whereas treatment with either DC/BNL or IL-12 alone did not show a tumour-suppressive effect. Antitumour activity induced by DC/BNL + IL-12 was abrogated by depletion of CD4+ T cells, but not by depletion of CD8+ T cells or natural killer cells. Splenic CD4+ T cells and CD8+ T cells from DC/BNL-treated mice showed cytotoxic activity against BNL cells after 3 days of incubation with DC/BNL, although BNL cells do not express major histocompatibility complex (MHC) class II molecules even after treatment with interferon (INF)-gamma. Furthermore, CD4+ T cells killed syngeneic-irrelevant CT26 cells and even allogeneic Hepa1-6 cells. This cytotoxicity was blocked by concanamycin A, but not by an anti-Fas ligand (FasL) monoclonal antibody, indicating that cytotoxic activity was mediated by perforin. Immunofluorescence microscopy demonstrated that abundant CD4+ T cells and MHC class II-positive macrophages, but not CD8(+) T cells, had infiltrated tumour tissue in mice treated with DC/BNL + IL-12. Flow cytometric analysis of tumour-infiltrating cells in mice treated with DC/BNL + IL-12 showed increases in CD4+ T cells and MHC class II+ CD11b+ cells but not in CD8+ T cells or MHC class I+ CD11b+ cells. Our results suggest that, in BNL-bearing mice treated with DC/BNL + IL-12, tumour macrophages activated by INF-gamma produced by IL-12-stimulated T cells might present BNL tumour antigens and activate DC/BNL-primed CD4+ cytotoxic T lymphocytes (CTLs) in a MHC class II-dependent manner, leading to perforin-mediated bystander killing of neighbouring MHC class II-negative tumour cells.

Forphenicinol enhances the antitumor effects of cyclophosphamide in a model of squamous cell carcinoma.

We examined the interaction between forphenicinol (FPL) and cyclophosphamide (CPA) or ionizing radiation (IR) on the growth of murine squamous cell carcinoma tumors SCCVII. Primary tumors were established in C3H mice by injecting SCCVII tumor cells subcutaneously into the right hind limb. FPL (100 mg/kg for 8 days) and/or CPA (25 mg/kg twice) were administered by intraperitoneal injection. Tumors were irradiated to a total dose of 40 Gy (eight 5-Gy fractions). SCCVII tumor growth was inhibited by FPL (P=0.054), IR (P=0.003) and CPA (P<0.001) compared with control. The combination of FPL and CPA inhibited tumor growth additively compared with either treatment alone in both small- and large-volume tumors. FPL did not significantly enhance the antitumor effects of IR, however, when CPA+FPL were combined with IR, significant tumor growth inhibition was observed compared with FPL alone (P<0.001), CPA alone (P=0.002) and IR alone (P=0.002). Due to its low toxicity profile, FPL may be combined with CPA, IR and other cytotoxic therapies to potentially enhance the therapeutic ratio.

Synergistic anti-viral effect of oxanosine and ddI against human immunodeficiency virus.

The combined effect against human immunodeficiency virus (HIV) of oxanosine and 2'3'-dideoxyinosine (ddI) has been evaluated by the production of viral particles, the expression of viral antigens on cell surfaces, and the amount of viral genome integrated in the host cells. Oxanosine alone has no effect on HIV replication up to 100 microg/ml, however, in the presence of ddI, oxanosine revealed concentration dependent inhibition against HIV without cytotoxicity.

Vaccination of glioma patients with fusions of dendritic and glioma cells and recombinant human interleukin 12.

Despite aggressive treatment, the median survival of patients with high-grade malignant astrocytoma is about 1 year. The authors investigated the safety and clinical response to immunotherapy using fusions of dendritic and glioma cells combined with recombinant human interleukin 12 (rhIL-12) for the treatment of malignant glioma. Fifteen patients with malignant glioma participated in this study. Dendritic cells were generated from peripheral blood. Cultured autologous glioma cells were established from surgical specimens in each case. Fusion cells were prepared from dendritic and glioma cells using polyethylene glycol. All patients received fusion cells intradermally on day 1. rhIL-12 was injected subcutaneously at the same site on days 3 and 7. Response to the treatment was evaluated by clinical observations and radiologic findings. No serious adverse effects were observed. In four patients, magnetic resonance imaging showed a greater than 50% reduction in tumor size. One patient had a mixed response. These results show that administration of fusion cells and rhIL-12 safely induces clinical antitumor effects in some patients with malignant glioma.

Monoclonal antibody hNM01 in HIV-infected patients: a phase I study.

BACKGROUND: hNM01 is a humanized monoclonal antibody that binds to the V(3) region of the HIV-1 envelope protein gp120. This binding leads to the activation of complement and the disruption of the viral envelope. OBJECTIVES AND STUDY DESIGN: The aim of this study was to investigate the clinical responses of the individuals when treated with the humanized antibody NMO1. In this phase I study, four HIV-1 infected patients with CD4 counts between 50 and 500 cells/mul received a total of four doses of hNM01 in an intrapatient dose escalation fashion: day 1-0.2 mg/kg, day 15-1 mg/kg, day 29-5 mg/kg, and day 43-5mg/kg. Patients were required to have virus that reacted to hNM01 by a virion capture assay and to have a viral load > or =15,000 copies/mL. RESULTS AND CONCLUSION: The antibody was well-tolerated; no significant adverse events were observed even at the highest dose tolerated. None of the patient developed either human anti-hNM01 (anti-idiotype) or human anti-rat antibodies. The mean elimination half-life was 153 h (6.4 days). During hNMO1 therapy effects were observed on CD4 cell counts and plasma viral loads and further dose finding trials are necessary to better determine the therapeutic activity of hNM01 in HIV-infected individuals.

Anti-tumor effects of dendritic and tumor cell fusions are not dependent on expression of MHC class I and II by dendritic cells.

Vaccination of fusion cells (FCs) made from dendritic and tumor cells elicits anti-tumor effects. We investigated whether major histocompatibility complex (MHC) class I and II play an important role in the induction of anti-tumor immunity by FCs. Immunization with fusion cells composed of syngeneic, allogeneic, or MHC I(-/-)II(-/-) DCs and B16 cells inhibited tumor growth. Elispot assay showed a higher population of interferon-gamma secreting T lymphocytes in mice immunized with fusion cells. These data suggest that anti-tumor effects of DCs and tumor cell fusions are not dependent on the expression of MHC class I and II on DCs.

Inhibition of spontaneous development of liver tumors by inoculation with dendritic cells loaded with hepatocellular carcinoma cells in C3H/HeNCRJ mice.

We attempted to prevent spontaneous development of liver tumors by s.c. inoculation with DCs loaded with syngeneic HCC cells in C3H/HeNCrj mice. A new cell line, MIH-2, was established from an HCC that had developed spontaneously in a C3H/HeNCrj mouse. Bone marrow-derived DCs were loaded with irradiated MIH-2 cells by treatment with PEG. Fluorescence microscopy and flow-cytometric analysis showed that about 45% of PEG-treated DCs and MIH-2 cells (DC/MIH-2) were DCs loaded with MIH-2 cells. Thirteen-month-old mice received inoculations of DC/MIH-2 (9 x 10(5)/mouse) 4 times at 6-day intervals and were killed at 16 months of age to assess liver tumors. The incidence of liver tumors in these mice was significantly lower than that in mice not receiving inoculations (p < 0.05) but similar to that in 13-month-old mice (the age at which inoculation started), indicating that inoculation inhibited the development of new tumors. Splenocytes from inoculated mice, but not those from uninoculated mice, showed cytotoxic activity against MIH-2 cells. Cytotoxic activity was not elicited by CD4(+) T cells, CD8(+) T cells, or DX5(+) cells isolated from splenocytes but was elicited by adherent cells, identified as CD11b(+) macrophages. CD4(+) T cells, but not CD8(+) T cells, from inoculated mice produced IFN-gamma by incubation with DC/MIH-2. Cytotoxicity by splenocytes was attenuated by anti-IFN-gamma antibody. Immunization with DCs loaded with syngeneic HCC cells induces CD4(+) T cells that produce IFN-gamma by response to antigen of HCC, which would lead to macrophage activation to kill liver tumor cells at an early stage.

Prevention of gastrointestinal tumors based on adenomatous polyposis coli gene mutation by dendritic cell vaccine.

Here we describe the effect of immunization with dendritic cells loaded with syngeneic tumor cells (DC/Ts) by polyethylene glycol treatment, on tumor development in adenomatous polyposis coli (APC) gene mutant mouse models, APC1309 and APC(Min-/+), in which adenomatous polyps of the gastrointestinal tracts develop with a high incidence. Treatment with DC/Ts prevented the development of gastrointestinal tumors, and coadministration of DC/Ts and IL-12 caused a further reduction in tumor incidence. Splenocytes from APC1309 mice treated with DC/Ts and IL-12 showed no cytotoxic activity toward the tumor cells, but serum antibody specific to them was detected. IgG from the treated mice exhibited cytotoxic activity against the tumor cells in vitro. Predominance of Th2 cell response over Th1 response was also suggested by ELISPOT assays in the treated mice. Depletion in vivo of CD4(+) T cells, not CD8(+) T cells, by the intraperitoneal administration of corresponding mAb's decreased the antitumor effect of DC/T inoculation. Immunofluorescence microscopic studies showed that Ig was attached to tumor cells in mice treated with DC/Ts and IL-12. These findings indicate that DC/T vaccination prevents tumor development through APC gene mutation and that its preventive effects are mediated by humoral antitumor immunity.

Antitumor effects of fusions composed of dendritic cells and fibroblasts transfected with genomic DNA from tumor cells.

Based on several previous studies indicating that transfection of genomic DNA can stably alter the character of the cells that take up the exogenous DNA, we investigated antitumor immunity conferred by fusions of syngeneic dendritic cells (DCs) and allogeneic fibroblasts (NIH3T3) transfected with genomic DNA from B16 tumor cells. Fusion cells (FCs) composed of dendritic and genetically engineered NIH3T3 cells were prepared with polyethylene glycol, and fusion efficiency was 30.3%. Prior immunization with FCs prevented tumor formation upon challenge with B16 tumor cells. Efficacy was reduced when studies were performed in mice depleted of NK cells. Vaccination with FCs containing DCs and fibroblasts transfected with denatured DNA did not inhibit tumor growth. Cytotoxic T cell (CTL) activity of spleen cells from immunized mice against both Yac-1 and tumor cells was also stimulated by administration of FCs compared with the activity observed for cells obtained from naïve mice. These data demonstrate the therapeutic efficacy of fusion cell-based vaccine therapy using syngeneic DCs and allogeneic fibroblasts transfected with tumor-derived genomic DNA.

Potential role of Helicobacter pylori in hepatocarcinogenesis.

Helicobacter species can induce carcinoma in the liver of certain mice. Furthermore, Helicobacter pylori (H. pylori) exhibits hepatotoxicity in vitro. These reports indicate that H. pylori may play a role in hepatocarcinogenesis. The aim of this study was to assess the presence of H. pylori in human hepatocellular carcinoma (HCC) to determine if H. pylori may affect the development of this disease. Liver specimens from 15 HCC patients dissected into tumor and non-tumor tissues were examined for H. pylori by PCR using two sets of primers for 16S rRNA and urease B. DNA sequencing analysis was performed to confirm that PCR products with 16S rRNA primers were derived from H. pylori DNA. The specimens were also examined for H. pylori by immunohistochemistry using anti-H. pylori antibody. H. pylori was found in 13 of 15 tumor tissues, not in the non-tumor tissues. By contrast, Escherichia coli and Bacteroides fragilis, frequent colonizers of gut, were not detected by PCR in the HCC tumors. Ten cirrhotic liver tissue specimens and seven normal liver tissue specimens were also negative for H. pylori DNA by PCR. The nucleotide sequence of the amplified fragment shared 100% identity with the 16S rRNA gene of H. pylori. H. pylori was also detected in HCC tissue by immunohistochemical analysis. The presence of H. pylori in human HCC tissue was demonstrated by PCR and immunohistochemical analysis. These findings suggest that H. pylori might contribute to the development of HCC. Further study is needed to prove the pathogenetic role of H. pylori in the development of human HCC.

Immunotherapy using fusions of autologous dendritic cells and tumor cells showed effective clinical response in a patient with advanced gastric carcinoma.

Immunotherapy using tumor antigen-loaded dendritic cells is a new approach for the treatment of various types of malignant tumors. Here, we describe a patient with advanced gastric carcinoma who received immunotherapy using fused autologous dendritic cells and carcinoma cells (fusions) and showed effective clinical responses to the treatment. A 74-year-old man showed massive ascitic effusion due to peritonitis carcinomatosa after surgical operation for gastric carcinoma. A gastric carcinoma cell line was established from the patient's tumor tissue. Dendritic cells were obtained by cultivation of the adherent cell fraction of the patient's peripheral blood mononuclear cells (PBMCs) with granulocyte macrophage-colony stimulating factor, interleukin-4, and tumor necrosis factor-alpha. The cells were mixed with irradiated tumor cells and treated with 50% polyethyleneglycol (PEG) for the generation of fusions, as described previously. The PEG-treated cells were injected subcutaneously every 2 weeks. Low-grade fever was observed after the first and second treatments. After the third treatment, ascitic effusion and leg edema decreased markedly, without any other treatments. Serum levels of carcinoembryonic antigen (CEA) and carbohydrate antigen (CA) 19-9 decreased to levels lower than those at the initiation of treatment. PBMCs collected after the fifth treatment elicited cytotoxic activity against autologous tumor cells. Although treatment was continued in the same way, recurrence of the disease was observed about 5 months after the start of the treatment. This is the first report of immunotherapy utilizing fusions of autologous dendritic cells and tumor cells resulting in effective clinical responses in advanced gastric carcinoma, without severe adverse effects.

Mechanistic effect of NMSO3 on replication of human immunodeficiency virus.

NMSO3, a sulphated sialyl lipid, was evaluated for its efficacy against human immunodeficiency virus type 1 (HIV-1). The compound exhibited concentration-dependent inhibition of HIV-1 replication in primary infection cell culture systems. Substantial inhibition was observed at concentrations of NMSO3 that showed little cytotoxicity. NMSO3 also exhibited anti HIV-1 activity in chronically HIV-1 infected cultures. The production of progeny viruses was completely abolished without cytotoxicity by continuous addition of NMSO3 to chronically infected U937 cells. Furthermore, in attempting to define the inhibitory mechanism of NMSO3, we investigated its effect on several steps of the HIV-1 replication cycle. NMSO3 competes with gp120 for binding to CD4 receptors on cells and inhibits the entry of HIV-1. By epitope analysis of the human CD4 molecule, NMSO3 inhibits the binding of antibodies, which recognize the D1 domain of CD4. Moreover, semi-quantitative reverse transcribed polymerase chain reaction (RT-PCR) showed that the integrated provirus is transcriptionally inactive in NMSO3-treated cells, supporting the lack of progeny in the culture supernatant of chronically HIV-1-infected cells treated with NMSO3. These findings indicate that NMSO3 has a unique mechanism of action against HIV-1 in both primary and chronic infection, and may be a valuable compound for the treatment of HIV-1 infection.

Effects of glucocorticoids on antitumor effects of immunizations with fusions of dendritic and tumor cells.

BACKGROUND: A pitfall of immunotherapy with dendritic cells (DCs) for patients with malignant tumor is that certain patients have already been treated with Dexamethasone (DEX) prior to vaccination. Therefore, it is important to investigate whether DEX has negative effects on induction of antitumor immune response in dendritic cell-therapy. MATERIALS AND METHODS: We analyzed surface phenotypes and phagocytosis of DCs incubated with or without DEX. We also investigated the effects of DEX on in vivo antitumor effects of immunization with fusions of dendritic and glioma cells. RESULTS: Treatment with DEX did not affect the expression of surface markers, including H-2Kk, I-Kk, CD80 and CD86, on DCs. Likewise, DEX did not affect phagocytosis. Immunization with FCs inhibited tumor growth in vivo, and DEX did not suppress antitumor effects induced by FCs. CONCLUSION: These data suggest that vaccination with FCs can be used to treat cancer patients receiving DEX.

Shedding of growth-suppressive gangliosides from glomerular mesangial cells undergoing apoptosis.

BACKGROUND: Apoptosis of glomerular mesangial cells is a common feature in several types of glomerular diseases. However, its pathophysiologic significance is not known. We recently identified gangliosides as a major growth-inhibitory substance in the conditioned medium of mesangial cells. In this report, we tested whether biologically distinct forms of cell fate, apoptosis and necrosis, could modulate ganglioside shedding from mesangial cells. METHODS: Mesangial cells were exposed to low (10 to 40 mJ/cm2) and high (400 mJ/cm2) doses of ultraviolet light to induce apoptosis and necrosis, respectively. Conditioned media were collected and examined for its growth-inhibitory activity for mesangial cells. Ganglioside shedding was analyzed using metabolic labeling and thin-layer chromatography (TLC). RESULTS: Shedding of gangliosides as well as growth-inhibitory activity in the conditioned medium predominantly increased when mesangial cells were undergoing apoptosis in contrast to that of viable or necrotic mesangial cells. The inhibitory substance in the conditioned medium from apoptotic mesangial cells completely fulfilled the characteristic criteria of gangliosides. This substance was less than 3 kD and was sensitive to neuraminidase digestion. Shedding of gangliosides from mesangial cells reduced significantly when apoptosis was inhibited by overexpression of antiapoptotic gene, Bcl-XL. In addition, ganglioside shedding also increased when mesangial cells were exposed to other inducers of apoptosis for mesangial cells (i.e., H2O2 and staurosporin). CONCLUSION: These results provide the novel link between masangial cell apoptosis and increased release of gangliosides that potentially suppress mesangial cell proliferation and thus indicate a mechanism for the negative regulation of mesangial cell growth by apoptosis.

Maturation of dendritic cells induced by Candida beta-D-glucan.

We investigated whether Candida beta-D-glucan (CSBG) alters the maturation of dendritic cells (DCs). DC phenotypes were analyzed using FACScan. The expression of surface molecules, including major histocompatibility complex (MHC) classes I and II, as well as CD80 and CD86, increased on DCs that were stimulated with lipopolysaccaride (DCs/LPS), in comparison with unstimulated bone marrow-derived DCs (BM-DCs). Furthermore, the level of surface molecule expression on DCs stimulated with CSBG (DCs/CSBG) was between that of DCs and DCs/LPS. Phagocytosis was assessed by the uptake of FITC-dextran. There were no differences in the uptake of dextran among DCs/LPS and DCs/CSBG. The ability of BM-DCs to uptake dextran was higher than that of DCs/LPS and DCs/CSBG. We analyzed the concentration of IL-12 secreted by DCs using ELISA. BM-DCs secreted a low concentration of IL-12, while DCs/LPS and DCs/CSBG secreted higher levels of IL-12 than BM-DCs. There were no remarkable differences in the concentrations of IL-12 produced by DCs/LPS and DCs/CSBG. This data suggests that CSBG may augment DC maturation.

Intratumoral injection of dendritic and irradiated glioma cells induces anti-tumor effects in a mouse brain tumor model.

Malignant astrocytoma is the most common primary brain tumor in adults. The median survival of patients with malignant astrocytomas (high-grade astrocytomas) is about 1-2 years, despite aggressive treatment that includes surgical resection, radiotherapy and cytotoxic chemotherapy. Therefore, novel therapeutic approaches are needed to prolong survival. We investigated antitumor immunity conferred by the intratumoral injection of dendritic (DC) and irradiated glioma cells (IR-GC) in a mouse brain tumor model. Intratumorally injected DC migrated to the lymph nodes and elicited systemic immunity against autologous glioma cells. In a treatment model, intratumoral injection of DC and IR-GC prolonged the survival of brain tumor-bearing mice. Efficacy was reduced when studies were performed in mice depleted of CD8(+) T cells. Administration of DC or IR-GC alone had no effect on survival of brain tumor-bearing mice. CTL activity against glioma cells from immunized mice was also stimulated by coadministration of DC and IR-GC compared with the controls. These results support the therapeutic efficacy of intratumoral injection of DC and IR-GC.

Adenoviral transfer of antisenses or ribozyme to O6-methylguanine-DNA methyltransferase mRNA in brain-tumor model resistant to chloroethyl-nitrosourea.

Chloroethyl-nitrosourea is a potent chemotherapeutic agent for brain tumors. However, acquired resistance to this drug has become a serious problem for the treatment of patients. Previously, we established an animal model resistant to nitrosourea (Anticancer Res 19: 5313-5318, 1999). In this study, we evaluate the efficacies of antisense sequences and ribozyme transduction by an adenoviral vector utilizing this model. Adenoviral vectors encoding antisense sequences or ribozyme to MGMT mRNA were constructed, then MGMT-expressing glioma cells were infected with these viruses and 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU) sensitivities were quantified. The adenoviral transfer of antisense RNA and ribozyme down-regulated the transcription and expression of MGMT in vitro. It also conferred sensitivity to nitrosourea in vitro and in vivo. However, the effect was minimal. These data suggest that incomplete depletion of MGMT is not sufficient to overcome the resistance and that additional optimization will be required for the complete reversion of drug resistance.