Melba L. Andres

Acute Effects of Whole-Body Proton Irradiation on the Immune System of the Mouse

Abstract Kajioka, E. H., Andres, M. L., Li, J., Mao, X. W., Moyers, M. F., Nelson, G. A., Slater, J. M. and Gridley, D. S. Acute Effects of Whole-Body Proton Irradiation on the Immune System of the Mouse. The acute effects of proton whole-body irradiation on the distribution and function of leukocyte populations in the spleen and blood were examined and compared to the effects of photons derived from a 60Co γ-ray source. Adult female C57BL/6 mice were exposed to a single dose (3 Gy at 0.4 Gy/min) of protons at spread-out Bragg peak (SOBP), protons at the distal entry (E) region, or γ rays and killed humanely at six different times thereafter. Specific differences were noted in the results, thereby suggesting that the kinetics of the response may be variable. However, the lack of significant differences in most assays at most times suggests that the RBE for both entry and peak regions of the Bragg curve was essentially 1.0 under the conditions of this study. The greatest immunodepression was observed at 4 days postexposure. Flow cytometry and mitogenic stimulation analyses of the spleen and peripheral blood demonstrated that lymphocyte populations differ in radiosensitivity, with B (CD19+) cells being most sensitive, T (CD3+) cells being moderately sensitive, and natural killer (NK1.1+) cells being most resistant. B lymphocytes showed the most rapid recovery. Comparison of the T-lymphocyte subsets showed that CD4+ T helper/inducer cells were more radiosensitive than the CD8+ T cytotoxic/suppressor cells. These findings should have an impact on future studies designed to maximize protection of normal tissue during and after proton-radiation exposure.

Low-dose vaccinia virus-mediated cytokine gene therapy of glioma.

Recombinant viruses can produce cytokines in tumors mobilizing an immune response to tumor cells. In this study, the authors investigated gene expression, in vivo antitumor efficacy, and safety of attenuated recombinant vaccinia virus (rVV) carrying murine cytokine genes interleukin (IL)-2 (rVV–mIL-2), IL-12 (rVV–mIL-12), and both IL-2 and IL-12 (rVV-2–12) in an athymic nude mice model. Significant tumor inhibition (p < 0.05) was observed in a preestablished subcutaneously implanted C6 glioma model using rVVs at doses ranging from 102 to 107 plaque forming units (PFU). An antitumor effect did not depend on the dose of the rVV–mIL-2 and rVV–mIL-12 viruses. All constructed rVVs induced a high level of cytokine expression in vitro and in vivo. Most groups injected with high doses of recombinant viruses encoding cytokine genes (105 to 107 PFU) showed signs of cytokine toxicity, whereas in the low-dose treatment groups (102 to 103 PFU) toxicity was greatly reduced. The antitumor activity of rVV–mIL-12 was associated with increases in both the percentage and number of natural killer T cells in the spleen. Local detection of interferon-&ggr; and tumor necrosis factor-&agr; was also correlated with tumor growth arrest induced by the treatment. High-dose VV control vector per se induced tumor inhibition by activating Mac-1+ cells in blood, but the antitumor effect was less pronounced compared with rVV-carrying cytokine genes (p < 0.05). These results suggest that attenuated recombinant strains of VV at low doses may potentially be efficient vectors for cancer immunotherapy.

Evaluation of combined vaccinia virus-mediated antitumor gene therapy with p53, IL-2, and IL-12 in a glioma model.

Our previous studies have shown that vaccinia virus (VV) expressing p53, interleukin-2 (IL-2), and interleukin-12 (IL-12) results in an effective inhibition of subcutaneous glioma growth in mice. We propose that combination therapy of tumors with virus-mediated p53 and cytokine genes offers the prospect of synergistic antitumor response. In this work, the antitumor efficacy of VV-mediated combination of p53, IL-2, and IL-12 genes was evaluated in a nude mouse model. To minimize cytokine-associated toxicity, a virus dose as low as 10 plaque-forming units of VV expressing IL-2 and IL-12 per animal was used alone and together with 2×107 plaque-forming units of VV expressing p53. Intratumoral treatment of established C6 glioma with recombinant viruses rVV-p53, rVV-mIL2, rVV-mIL12, and rVV-2-12 induced the prolonged expression of p53, IL-2, IL-12, and both cytokines simultaneously. The combination of rVV-p53/rVV-mIL12 or rVV-p53/rVV-2-12 resulted in significant tumor inhibition compared to single modality treatment (P<.05). rVV-p53/rVV-2-12 therapy was associated with significant elevation of natural killer, Mac-1+, and NKT cells in blood and interferon-γ and tumor necrosis factor-α expression in tumors. The difference in the inhibition of tumor growth between the rVV-p53/rVV-mIL2 combination and rVV-p53 was statistically insignificant. These data demonstrate that gene therapy based on VV-mediated combination of p53, IL-2, and IL-12 treatment may be a promising adjunctive strategy for glioma treatment. Cancer Gene Therapy (2000) 7, 1437–1447

Acute effects of iron-particle radiation on immunity. Part II: Leukocyte activation, cytokines and adhesion.

Abstract Gridley, D. S., Dutta-Roy, R., Andres, M. L., Nelson, G. A. and Pecaut, M. J. Acute Effects of Iron-Particle Radiation on Immunity. Part II: Leukocyte Activation, Cytokines and Adhesion. Radiat. Res. 165, 78–87 (2006). The effects of high-linear energy transfer (LET) radiation on immune function have not been clearly established. The major goal of this study was to evaluate leukocyte responses after whole-body exposure to high-LET radiation. C57BL/6 mice were exposed to 0, 0.5, 2 and 3 Gy 56Fe26+ particles (1055 MeV/nucleon, 148.2 keV/μm) and killed humanely 4 days after exposure. Spontaneous synthesis of DNA in blood and spleen cells was increased significantly in groups receiving either 2 or 3 Gy (P < 0.001). In contrast, a significant depression in the response of T lymphocytes to phytohemagglutinin (PHA) and concanavalin A (ConA) was noted (P < 0.005); the response to lipopolysaccharide (LPS), a B-cell mitogen, was similar among groups. A cytometric bead array assay revealed that the level of tumor necrosis factor α (Tnfa) secreted by splenocytes increased significantly with increasing 56Fe-particle dose (P < 0.05); interferon γ, interleukin2 (Il2), Il4 and Il5 were unaffected. Flow cytometry analysis showed that 2 and 3 Gy markedly reduced splenic mononuclear cells expressing the activation markers CD25 and CD71, both with and without the T-cell marker CD3 (P < 0.05); proportions also varied significantly. Similar patterns were noted in mononuclear and granular cells with adhesion markers CD11b and, to a lesser extent, CD54 (P < 0.05). The results show that a single, acute exposure to high-LET radiation induced changes that can profoundly alter leukocyte functions. The implications of the data are discussed in relation to low-LET radiation, altered gravity, and space flight.

Liposome-Encapsulated Tumor Necrosis Factor-α Enhances the Effects of Radiation Against Human Colon Tumor Xenografts

Recent reports have shown that tumor necrosis factor-alpha (TNF-alpha) can augment the effects of radiation against certain tumor types. However, the high concentrations of intravenous infusion of TNF-alpha needed to cause tumor regression can induce many systemic side effects. The aims of this study were to determine if TNF-alpha encapsulated in sterically stabilized (Stealth, ALZA Corporation, Mountain View, CA), PEGylated liposomes (SL) augments the antitumor effects of radiation and to compare its efficacy and possible toxicity with free TNF-alpha in the LS174T human colon tumor xenograft model. Nude mice were injected subcutaneously (s.c.) with LS174T cells and treated intravenously (i.v.) with Stealth-liposomal TNF-alpha (SL-TNF-alpha) with and without radiation or TNF-alpha with or without radiation when tumor size was approximately 200 mm(3). In phase 1, a significant decrease (p = 0.047) in tumor growth was observed with radiation at day 21 but not with SL-TNF-alpha or free TNF-alpha alone. By the end of phase 1 (day 27) with continued treatments, the SL-TNF-alpha plus radiation group had significantly smaller tumors (p = 0.044) than those in the free TNF-alpha plus radiation group. In phase 2, where a similar tumor growth reduction pattern was observed, the addition of TNF-alpha to radiation, either as free protein or within SL, increased lymphocyte activation and natural killer (NK) cell numbers in both blood and spleen. The effect was generally more pronounced with SL-TNF-alpha. Systemic toxicity, based on hematologic analyses and body weight, was absent or minimal. Collectively, the data show that pretreatment with SL-TNF-alpha can enhance more effectively, and possibly more safely, the effects of radiation against human colon tumor xenografts than can free TNF-alpha and that the increased antitumor action may involve upregulation of lymphocytes.

Radiation-enhanced Endostatin Gene Expression and Effects of Combination Treatment

Targeting cells that support tumor growth by administering potent angiogenesis inhibitors is currently an area of intense interest. In the present study, a unique plasmid vector for the mouse endostatin gene, pXLG-mEndo, was constructed and evaluated with and without radiation using the Lewis lung carcinoma (LLC) cell line. The physical properties of the expressed endostatin protein were validated by PCR, gel electrophoresis, and Western blot. Enzyme-linked immunosorbent and immunocytochemical analyses for the therapeutic gene demonstrated that transfected LLC cells secreted the protein into the medium. Exposure of the cells to 2 gray (Gy) γ-rays reduced the time to reach the maximum expression level of the endostatin gene and also increased the amount of secreted endostatin protein (P<0.001). Biological activity of the endostatin was demonstrated by the inhibition of tube formation by human umbilical vein endothelial cells (HUVEC). Based on 3 H-thymidine incorporation, endostatin expression significantly depressed DNA synthesis in HUVEC and LLC cells compared to controls transfected with parental vector or no vector (P>0.005). In addition, radiation increased the efficiency of endostatin-mediated inhibition of both cell types over a 3-day period post-exposure (P<0.05 or less). Intratumoral injection of 100 μg pXLG-mEndo combined with 10 Gy radiation significantly delayed LLC tumor growth, especially when each modality was delivered twice (P<0.05 or less compared to all other groups). No toxicity was observed. These findings are very promising and suggest that endostatin therapy with a plasmid vector, such as pXLG-mEndo, may enhance the efficacy of radiotherapy for lung cancer.

Changes in the activation and reconstitution of lymphocytes resulting from total-body irradiation correlate with slowed tumor growth

Alterations in cytokine secretion, activation marker expression, and immune cell concentrations were investigated at sequential time points following delivery of total-body irradiation (TBI) to C57BL/6 mice (n = 64) in the Lewis lung tumor model. Significantly slower tumor growth was observed when a 3-Gy dose of TBI was administered 2 h prior to tumor implantation (p < 0.05). The antitumor effect was correlated with an increased CD4:CD8 T cell ratio and heightened leukocyte blastogenesis. TBI was also found to induce an expansion of natural killer (NK) cells in the blood and spleen of tumor-bearing animals 10 days after irradiation (2.8 × 106 NK cells/spleen in test mice compared to 8.9 × 105 NK cells/spleen in normal control animals). However, no significant differences were found in NK cell levels within the tumor tissue. Enhanced production of interleukin (IL)-12 and IL-18 from spleen supernatants was consistent with an augmentation of the NK cell response. Significant reductions in transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor, both of which are associated with immune suppression, were also noted. Furthermore, TBI induced changes in expression of CD25 and CD71 activation markers, suggesting that radiation may alter tumor surveillance. Taken together, the relative percentages and activation status of immune cell compartments support the conclusion that these TBI-induced changes function to slow tumor progression.

Modification of a transplantable colon tumor and immune responses in mice fed different sources of protein, fat and carbohydrate

The effects of different sources of dietary protein, fat and carbohydrate on tumor development and on tests relating to cell-mediated immunity were investigated in male BALB/c mice after subcutaneous injection of 8 X 10(4) 1,2-dimethylhydrazine (DMH)-induced colon tumor (no. 51) cells. Results indicated that mice fed the milk protein source (especially at the low protein level) had smaller tumors, a higher spleen cell proliferative response to stimulation by phytohemagglutinin (PHA), and greater cytotoxic T-cell activity against the tumor cells than those fed the comparable diets containing protein from the other sources. Peripheral blood lymphocytes only from the milk-fed mice, regardless of tumor presence, exhibited a relatively low response to PHA stimulation, thereby suggesting a dietary effect on the migration pattern of PHA-responsive lymphocytes. The level of protein significantly affected both T-cell and natural killer cell cytotoxicity. The tumor-bearing mice fed the diet containing sucrose (table sugar) had a significantly lower spleen cell response to PHA stimulation than those fed the comparable diet containing dextrin. The level or source of fat did not significantly affect any of the parameters tested in this system.

Radiation Enhances the Anti-tumor Effects of Vaccinia-p53 Gene Therapy in Glioma

The overall goal of this study was to analyze the effect and mechanism of radiation in combination with vaccinia viruses (VV) carrying the p53 gene against glioma. Comparison of two alternative treatments of cultured C6 (p53+) and 9L (p53−) rat glioma cells showed significantly reduced survival for both cell lines, especially 9L, when radiation was applied prior to virus versus radiation alone. High p53 protein expression mediated by VV-TK-p53 was measured in infected cells. Single modality treatment of C6 cells with psoralen and UV (PUV)-inactivated VV-TK-p53 (PUV-VV-TK-53) or radiation significantly decreased survival compared with PUV-inactivated L-15 (PUV-L-15) control virus. However, no difference was observed between radiation and combination treatments of C6 cells. In contrast, radiation followed by PUV-VV-TK-53 resulted in dramatic reduction of 9L cell viability, compared to single modality treatment. Flow cytometry analysis of Annexin-V-stained 9L cells showed that radiation and PUV-VV-TK-53 caused a significant decrease in live cells (17.2%) as compared to other treatments and control (61.6–98.3%). Apoptosis was observed in 37.2% of cells, while the range was 0.7–7.8% in other treatment groups; maximal p53 level was measured on day 7 post-infection. In athymic mice bearing C6 tumors, VV-TK-53 plus radiation in both single and multiple therapies resulted in significantly smaller tumors by day 30 compared to the agents given only once. Immunohistochemical analysis of tumor sections demonstrated p53 protein expression over 20 days after VV-TK-53 treatment. Analysis of blood and spleen cells of mice given multiple combination treatments showed significant splenomegaly, leukocytosis, and increased DNA synthesis and response to mitogen. Multiple combination treatments were also associated with significantly elevated natural killer and B cells in the spleen. There were no overt toxicities, although depression in red blood cell and thrombocyte parameters was noted. Collectively, the data demonstrate that radiation significantly improves the efficacy of VV-mediated tumor suppressor p53 therapy and may be a promising strategy for glioma treatment. Furthermore, the results support the conclusion that the mechanisms underlying the enhanced anti-tumor effect of combination treatment include apoptosis/necrosis and upregulation of innate immune defenses.

Modification of herpes 2-transformed cell-induced tumors in mice fed different sources of protein, fat and carbohydrate

The effects of different sources of protein (milk, soy, wheat, fish and beef), fat (corn oil and butter), and carbohydrate (dextrin and sucrose) on tumor development and on spleen characteristics were investigated in BALB/c mice injected subcutaneously with 5 X 10(5) herpes simplex virus Type 2-transformed cells (H238 cells). Low or high levels of protein and fat were used. Several weeks post-injection results indicated that a high level of fat significantly enhanced tumor incidence. A high fat level was also associated with a lower spleen weight and a smaller proportion of mature granulocytes in the spleen. Butter, compared to corn oil, significantly restricted tumor volume. Among the most highly significant findings was the low tumor incidence in mice fed protein from either a milk or a fish source.