ABSTRACT
Inflammatory bowel disease (IBD) is a poorly understood chronic immune disorder for which there is no medical cure. Milk and colostrum are rich sources of bioactives with immunomodulatory properties. Here we compared the therapeutic effects of oral delivery of bovine milk-derived iron-saturated lactoferrin (Fe-bLF), angiogenin, osteopontin (OPN), colostrum whey protein, Modulen IBD (Nestle Healthsciences, Rhodes, Australia), and cis-9,trans-11 conjugated linoleic acid (CLA)-enriched milk fat in a mouse model of dextran sulfate-induced colitis. The CLA-enriched milk fat significantly increased mouse body weights after 24d of treatment, reduced epithelium damage, and downregulated the expression of proinflammatory cytokines and nitrous oxide. Modulen IBD most effectively decreased the clinical score at d 12, and Modulen IBD and OPN most effectively lowered the inflammatory score. Myeloperoxidase activity that denotes neutrophil infiltration was significantly lower in mice fed Modulen IBD, OPN, angiogenin, and Fe-bLF. A significant decrease in the numbers of T cells, natural killer cells, dendritic cells, and a significant decrease in cytokine expression were observed in mice fed the treatment diets compared with dextran sulfate administered mice. The Fe-bLF, CLA-enriched milk fat, and Modulen IBD inhibited intestinal angiogenesis. In summary, each of the milk components attenuated IBD in mice, but with differing effectiveness against specific disease parameters.
Subject(s)
Colitis/drug therapy , Colostrum/chemistry , Milk Proteins/pharmacology , Milk/chemistry , Animals , Australia , Chronic Disease , Colitis/chemically induced , Cytokines/metabolism , Dairy Products , Dextran Sulfate/adverse effects , Disease Models, Animal , Glycolipids/pharmacology , Glycoproteins/pharmacology , Lactoferrin/pharmacology , Linoleic Acids, Conjugated/pharmacology , Lipid Droplets , Male , Mice , Mice, Inbred BALB C , Nitrous Oxide/metabolism , Osteopontin/pharmacology , Ribonuclease, Pancreatic/pharmacology , Whey Proteins/pharmacologyABSTRACT
BACKGROUND: Orally administered milk fat enriched in conjugated linoleic acid (CLA) and trans-vaccenic acid (VA) ('enriched milk fat'), produced by supplementing the diet of pasture-fed cows with fish and sunflower oil, has been shown previously to suppress the development of allergic airway disease in mice. OBJECTIVE: To investigate whether topical or oral application of enriched milk fat and its two major fatty acids cis-9, trans-11 CLA (c9,t11-CLA) and VA inhibit allergic dermatitis in mice. METHODS: Allergic dermatitis was induced in C57BL/6 mice by epicutaneous sensitization of tape-stripped skin with ovalbumin (OVA). Enriched milk fat and its two major fatty acids were either topically applied to the OVA-sensitized skin, or orally fed to mice by supplementation of the diet. Blood and skin tissues were collected for analysis after the third skin sensitization. RESULTS: Both topical and oral administration of enriched milk fat and its two major fatty acids led to significant suppression of allergic dermatitis as evidenced by reduced clinical and histological scores of affected skins, infiltration of inflammatory cells, and circulating allergen-specific IgE levels, compared with treatment with normal milk fat or the base control diet. C9,t11-CLA and VA individually inhibited multiple facets of allergic dermatitis when topically applied, and their combination produced a strong additive effect. CONCLUSION AND CLINICAL RELEVANCE: Enriched milk fat, and its two major fatty acids c9,t11-CLA and vaccenic acid attenuate allergic dermatitis in mice.
Subject(s)
Dermatitis, Allergic Contact/diet therapy , Fats/chemistry , Linoleic Acids/therapeutic use , Milk/chemistry , Oleic Acids/therapeutic use , Animals , Cattle , Dietary Supplements , Fats/administration & dosage , Fats/therapeutic use , Female , Linoleic Acids/administration & dosage , Linoleic Acids/chemistry , Mice , Mice, Inbred C57BL , Oleic Acids/administration & dosage , Oleic Acids/chemistry , Ovalbumin , Skin TestsABSTRACT
Angiostatin is a naturally occurring inhibitor of angiogenesis that is being developed as a drug to fight cancer. In this study we reveal that EL-4 tumors established in mice rapidly develop resistance to angiostatin gene therapy by upregulating hypoxia-inducible pathways. Angiostatin initially delayed tumor growth for 6 days by reducing blood vessel density. However, tumors quickly responded by upregulating the production of hypoxia-inducible factor-1alpha (HIF-1alpha) and its effector vascular endothelial growth factor (VEGF) in response to increasing tumor hypoxia, leading to restored angiogenesis and rapid tumor growth. Theoretically, blockade of HIF-1 should prevent resistance to anti-angiogenic therapy by preventing a tumor from responding to induced hypoxia. Antisense HIF-1alpha inhibited the expression of HIF-1alpha and of the HIF-1 effectors VEGF, glucose transporter-1 and lactate dehydrogenase. As a monotherapy, it was effective in eradicating small 0.1 cm diameter tumors, but only delayed the growth of large 0.4 cm diameter tumors. In contrast, timed injection of a combination of angiostatin and antisense HIF-1alpha plasmids completely eradicated large EL-4 tumors within 2 weeks, and prevented upregulation of hypoxia-inducible pathways induced by angiostatin. The data indicate that blocking hypoxia-inducible pathways by antisense HIF-1alpha can circumvent hypoxia-induced drug resistance and thereby augment the efficacy of anti-angiogenic therapies.
Subject(s)
Angiostatins/genetics , DNA, Antisense/genetics , Genetic Therapy/methods , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Lymphoma/therapy , Thymus Neoplasms/therapy , Angiostatins/biosynthesis , Animals , Cell Line, Tumor , DNA, Antisense/administration & dosage , Gene Expression Regulation, Neoplastic , Hypoxia-Inducible Factor 1, alpha Subunit/biosynthesis , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Lymphoma/genetics , Male , Mice , Mice, Inbred C57BL , Plasmids/administration & dosage , Plasmids/genetics , Thymus Neoplasms/geneticsABSTRACT
BACKGROUND: It has been argued that a reduction in the Western diet of anti-inflammatory unsaturated lipids, such as n-3 polyunsaturated fatty acids, has contributed to the increase in the frequency and severity of allergic diseases. OBJECTIVE: We investigated whether feeding milk fat enriched in conjugated linoleic acid and vaccenic acids (VAs) ('enriched' milk fat), produced by supplementing the diet of pasture-fed cows with fish and sunflower oil, will prevent development of allergic airway responses. METHODS: C57BL/6 mice were fed a control diet containing soybean oil and diets supplemented with milk lipids. They were sensitized by intraperitoneal injection of ovalbumin (OVA) on days 14 and 28, and challenged intranasally with OVA on day 42. Bronchoalveolar lavage fluid, lung tissues and serum samples were collected 6 days after the intranasal challenge. RESULTS: Feeding of enriched milk fat led to marked suppression of airway inflammation as evidenced by reductions in eosinophilia and lymphocytosis in the airways, compared with feeding of normal milk fat and control diet. Enriched milk fat significantly reduced circulating allergen-specific IgE and IgG1 levels, together with reductions in bronchoalveolar lavage fluid of IL-5 and CCL11. Treatment significantly inhibited changes in the airway including airway epithelial cell hypertrophy, goblet cell metaplasia and mucus hypersecretion. The two major components of enriched milk fat, cis-9, trans-11 conjugated linoleic acid and VA, inhibited airway inflammation when fed together to mice, whereas alone they were not effective. CONCLUSION: Milk fat enriched in conjugated linoleic and VAs suppresses inflammation and changes to the airways in an animal model of allergic airway disease.
Subject(s)
Fats/immunology , Hypersensitivity/immunology , Linoleic Acid/immunology , Lung Diseases, Obstructive/immunology , Lung Diseases, Obstructive/pathology , Milk/immunology , Oleic Acids/immunology , Allergens/immunology , Animals , Cell Survival , Chemokine CCL11/biosynthesis , Eosinophils/cytology , Eosinophils/immunology , Female , Hypersensitivity/metabolism , Hypersensitivity/pathology , Immunoglobulins/biosynthesis , Immunoglobulins/immunology , Interleukin-5/biosynthesis , Lung Diseases, Obstructive/metabolism , Male , Mice , Mice, Inbred C57BLABSTRACT
BACKGROUND: Integrin alphaVbeta3 plays a critical role in tumour angiogenesis and metastasis formation, and is recognized as a key therapeutic target in the treatment of cancer. AIM: To investigate whether antisense alphaV and beta3 gene therapy has utility in the treatment of hepatocellular carcinomas. METHODS: Antisense expression plasmids targeting integrin alphaV or beta3 were constructed, and examined by immunohistochemistry and Western blot analyses for their ability to inhibit alphaV and beta3 expression. The antisense alphaV and beta3 expression vectors, either alone or in combination, were injected into HepG2 hepatomas established subcutaneously in nude mice and tumour growth, angiogenesis and apoptosis were monitored. RESULTS: Antisense alphaV and beta3 downregulated the alphaV and beta3 subunits expressed by human umbilical vein endothelial cells, and the alphaV subunit expressed by HepG2 cells. Gene transfer of antisense alphaV and beta3 expression vectors downregulated alphaV and beta3 in HepG2 tumours established in nude mice, inhibited tumour vascularization and growth, and enhanced tumour cell apoptosis. Antisense alphaV suppressed tumour growth more strongly than antisense beta3; however antisense therapy that simultaneously targeted both integrin subunits was more effective than the respective monotherapies. Antisense alphaV and beta3 inhibited tumour angiogenesis to similar extents, by a process that is independent of vascular endothelial growth factor. CONCLUSIONS: Antisense gene therapy targeting alphaV integrins warrants consideration as an approach to treat hepatocellular carcinomas.
Subject(s)
DNA, Antisense/genetics , Genetic Therapy , Integrin alphaV/genetics , Integrin alphaVbeta3/genetics , Liver Neoplasms, Experimental/therapy , Xenograft Model Antitumor Assays , Animals , Apoptosis/drug effects , Cell Proliferation/drug effects , Down-Regulation/drug effects , Down-Regulation/genetics , Endothelial Cells/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Humans , Liver Neoplasms, Experimental/chemically induced , Liver Neoplasms, Experimental/genetics , Liver Neoplasms, Experimental/pathology , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Neovascularization, Pathologic , Plasmids , Transfection , Vascular Endothelial Growth Factor A/pharmacologyABSTRACT
The von Hippel-Lindau tumor suppressor protein (pVHL) suppresses tumor formation by binding the alpha subunits of hypoxia-inducible-factors responsible for stimulating tumor angiogenesis and glycolysis, and targeting them for ubiquitination and proteasomal destruction. Loss of pVHL leads to tumorigenesis and development of sporadic renal cell carcinomas and central nervous system hemangioblastomas. In the present study, we investigated whether engineered overexpression of pVHL in C6 glioma cells, which already express endogenous pVHL, would suppress the tumorigenicity of this particular tumor cell type. C6 cells overexpressing VHL displayed a reduced growth rate (70% inhibition) compared to the parental cell line when subcutaneously implanted in athymic (nu/nu) mice. Growth inhibition was associated with a 50% reduction in the number of tumor vessels and a 60% increase in tumor cell apoptosis, due in part to downregulation of HIF-1, VEGF, and the antiapoptotic factor Bcl-2, respectively. Gene transfer of VHL suppressed the growth of established C6 gliomas, and synergized with antisense HIF-1 to completely eradicate tumors. The data suggest that VHL gene therapy and/or agents that increase VHL expression could have utility in the treatment of gliomas, particularly when combined with agents that inhibit the expression or function of HIF-1.
Subject(s)
Glioma/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/genetics , Animals , Apoptosis , Cell Line, Tumor , Cell Proliferation , DNA, Antisense/genetics , DNA, Antisense/metabolism , Glioma/blood supply , Glioma/pathology , Hypoxia-Inducible Factor 1, alpha Subunit/biosynthesis , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Male , Mice , Mice, Nude , Neoplasm Transplantation , Neovascularization, Pathologic/prevention & control , Rats , Time Factors , Transfection , Vascular Endothelial Growth Factor A/biosynthesis , Vascular Endothelial Growth Factor A/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/biosynthesis , Von Hippel-Lindau Tumor Suppressor Protein/metabolismABSTRACT
The von Hippel-Lindau tumor suppressor protein (pVHL) suppresses tumor formation by binding the alpha subunits of hypoxia-inducible factors (HIFs) responsible for stimulating tumor angiogenesis and glycolysis, targeting them for ubiquitination and proteasomal destruction. Loss of pVHL leads to the development of sporadic renal cell carcinomas (RCCs). In the present study, we sought to determine whether engineered overexpression of pVHL in tumors other than RCC can inhibit tumor growth, either as a monotherapy, or in combination with antisense HIF-1alpha therapy. Intratumoral injection of subcutaneous EL-4 thymic lymphomas with an expression plasmid encoding pVHL resulted in the downregulation of HIF-1alpha and vascular endothelial growth factor (VEGF). There was a concomitant reduction in tumor angiogenesis and increased tumor cell apoptosis due in part to downregulation of Bcl-2 expression. VHL therapy resulted in the complete regression of small (0.1 cm diameter) tumors whereas, in contrast, large (0.4 cm diameter) EL-4 tumors were only slowed in their growth. Nevertheless, large tumors completely regressed in response to intratumoral injection of a combination of antisense HIF-1alpha and VHL plasmids. Combination therapy resulted in increased losses of HIF-1alpha, VEGF, and tumor blood vessels, and increased tumor cell apoptosis. These novel results suggest that synergistic therapies that simultaneously block the expression or function of HIF-1alpha, and enhance the expression or function of VHL may be beneficial in the treatment of cancer.
Subject(s)
Angiogenesis Inhibitors/genetics , DNA/administration & dosage , Genetic Therapy/methods , Lymphoma/therapy , Transcription Factors/genetics , Tumor Suppressor Proteins/genetics , Ubiquitin-Protein Ligases/genetics , Angiogenesis Inhibitors/analysis , Animals , Apoptosis , Gene Expression Regulation , Hypoxia-Inducible Factor 1, alpha Subunit , Immunohistochemistry , In Situ Nick-End Labeling , Injections, Intralesional , Lymphoma/pathology , Male , Mice , Mice, Inbred C57BL , Neoplasms, Experimental , Oligonucleotides, Antisense/therapeutic use , Transcription Factors/analysis , Tumor Suppressor Proteins/analysis , Ubiquitin-Protein Ligases/analysis , Vascular Endothelial Growth Factor A/analysis , Vascular Endothelial Growth Factor A/genetics , Von Hippel-Lindau Tumor Suppressor ProteinABSTRACT
Members of the B7 family costimulate the proliferation of lymphocytes during the initiation and maintenance of antigen-specific humoral and cell-mediated immune responses. While B7-1 and -2 are restricted to lymphoid tissues, and activate naïve T cells, recently identified members including B7-H2 and -H3 are widely expressed on nonlymphoid tissues, and regulate effector lymphocytes in the periphery. B7-H3 has properties that suggested it may display antitumor activity, including the ability to stimulate Th1 and cytotoxic T-cell responses. Here, we test this notion by determining whether intratumoral injection of an expression plasmid encoding a newly described mouse homologue of B7-H3 is able to eradicate EL-4 lymphomas. Intratumoral injection of a mouse B7-H3 pcDNA3 expression plasmid led to complete regression of 50% tumors, or otherwise significantly slowed tumor growth. Mice whose tumors completely regressed resisted a challenge with parental tumor cells, indicating systemic immunity had been generated. B7-H3-mediated antitumor immunity was mediated by CD8(+) T and NK cells, with no apparent contribution from CD4(+) T cells. In summary, the results indicate that B7-H3 interactions may play a role in regulating cell-mediated immune responses against cancer, and that B7-H3 is a potential therapeutic tool.
Subject(s)
B7-1 Antigen/genetics , DNA/administration & dosage , Genetic Therapy/methods , Lymphoma/therapy , Thymus Neoplasms/therapy , Amino Acid Sequence , Animals , B7 Antigens , Base Sequence , CD4-Positive T-Lymphocytes/immunology , Female , Injections, Intralesional , Killer Cells, Natural/immunology , Lymphocyte Activation , Lymphoma/immunology , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Neoplasm Transplantation , Thymus Neoplasms/immunologyABSTRACT
In the study, we investigate whether the expressions of heat shock protein (hsp)60 (a potential autoantigen) and the stress-inducible form of cytoprotector hsp70 are correlated with the development of atherosclerotic lesions in the aortic tree of apolipoprotein E-deficient (apoE(-/-)) mice. The apoE(-/-) mouse model is advantageous because the stress-inducible form of hsp70 is not constitutively expressed in mice, unlike primates; hence, tissues under stress can be clearly defined. Both mammalian hsps were detected newly expressed (before mononuclear cell infiltration) on aortic valves and endothelia at lesion-prone sites of 3-week-old apoE(-/-) mice. In 8- and 20-week-old mice, they were strongly and heterogeneously expressed in early to advanced fibrofatty plaques, with levels correlating with lesion severity. Expression was markedly downregulated in advanced collagenous, acellular, calcified plaques of 40- and 69-week-old mice and was absent in control aortas of normocholesterolemic wild-type (apoE(+/+)) mice. Western blot analysis of tissue homogenates confirmed the temporal expression of the hsps. Double immunostaining revealed that both hsps were expressed by lesional endothelial cells, macrophages, smooth muscle cells, and CD3(+) T lymphocytes. This study provides evidence that hsp60 and hsp70 are temporally expressed on all major cell types in lesion-prone sites during atherogenesis, suggesting that few cells escape the toxic environment of the atherosclerotic plaque.
Subject(s)
Apolipoproteins E/deficiency , Arteries/metabolism , Arteries/pathology , Arteriosclerosis/metabolism , Arteriosclerosis/pathology , Chaperonin 60/metabolism , HSP70 Heat-Shock Proteins/metabolism , Animals , Aorta/metabolism , Aorta/pathology , Blotting, Western , Disease Models, Animal , Disease Progression , Down-Regulation , Immunohistochemistry , Macrophages/metabolism , Mice , Mice, Mutant Strains , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/pathology , T-Lymphocytes/metabolism , Up-RegulationABSTRACT
Tumors must develop an adequate vascular network to meet their increasing demands for nutrition and oxygen. Angiostatin, a multiple kringle (1-4)-containing fragment of plasminogen, is an effective natural inhibitor of tumor angiogenesis. Here we show that gene transfer of angiostatin into small (0.1 cm in diameter) solid EL-4 lymphomas established in syngeneic C57BL/6 mice led to reduced tumor angiogenesis and weak inhibition of tumor growth. In contrast, when angiostatin gene therapy was preceded by in situ gene transfer of the T-cell costimulator B7.1, large (0.4 cm in diameter) tumors were rapidly and completely eradicated, whereas B7.1 and angiostatin monotherapies were ineffective. Combined gene transfer of B7.1 and angiostatin generated potent systemic antitumor immunity that was effective in eradicating a systemic challenge of 10(7) EL-4 cells. Gene transfer of angiostatin expression plasmids led to overexpression of angiostatin in tumors, increased apoptosis of tumor cells, and decreased density of tumor blood vessels, which may allow the immune system to overcome tumor immune resistance. The latter effects were not the result of a decrease in vascular endothelial growth factor expression, as tumoral vascular endothelial growth factor expression increased slightly after angiostatin gene transfer, presumably in response to increasing hypoxia. These results suggest that combining immunogene therapy with a vascular attack by angiostatin is a particularly effective approach for eliciting antitumor immunity.
Subject(s)
B7-1 Antigen/genetics , Endothelial Growth Factors/metabolism , Genetic Therapy/methods , Immunotherapy/methods , Lymphokines/metabolism , Peptide Fragments/genetics , Plasminogen/genetics , Thymus Neoplasms/therapy , Angiostatins , Animals , Blotting, Western , Combined Modality Therapy , DNA Primers/chemistry , Gene Transfer Techniques , Immunoenzyme Techniques , Male , Mice , Mice, Inbred C57BL , Neovascularization, Pathologic/prevention & control , Peptide Fragments/metabolism , Plasminogen/metabolism , Polymerase Chain Reaction/methods , T-Lymphocytes/immunology , Thymus Neoplasms/blood supply , Thymus Neoplasms/metabolism , Tumor Cells, Cultured , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
Leukocytes infiltrate the pancreatic islets of nonobese diabetic mice, causing beta-cell destruction and autoimmune Type I diabetes. Here, we completely blocked adoptive transfer of diabetes and reduced spontaneous disease incidence from 71% to 17% by simultaneously administering a combination of antibodies directed against alpha4, beta2, and beta7 integrins and their ligands VCAM-1, MAdCAM-1, and ICAM-1 for 52 and 28 days, respectively. CD4 and CD8 T cells and macrophages were excluded from islets and remained entrapped in a peri-islet location as inactive exiles, no longer expressing normal levels of interferon-gamma, interleukin-4, and iNOS. Only IL-10 expression was retained, which could aid immunosuppression. Infiltrating leukocytes retained a peri-islet location, even 215 days following suspension of antibody treatment, potentially forming a barrier to the entry of active, autoantigen-reactive T cells. Combination treatment was effective against spontaneous disease when administered from 7 days of age but ineffective when initiated late in the prediabetic period (day 40 or 70). Nevertheless, anti-alpha4 subunit mAb monotherapy alone was very effective, reducing insulitis to levels similar to those obtained with combinational antibody treatment, suggesting that alpha4 integrins are major receptors contributing to leukocyte infiltration. Treatment with anti-alpha4 integrin antibody retained some therapeutic benefit when administered from days 7, 40, or 70 of age. The results have implications for the treatment of diabetes and provide a unique insight into the fate of disease-forming leukocytes following anti-CAM therapy.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Cell Adhesion Molecules/antagonists & inhibitors , Chemotaxis, Leukocyte/drug effects , Diabetes Mellitus, Type 1/therapy , Integrins/antagonists & inhibitors , Islets of Langerhans , Adoptive Transfer , Age Factors , Animals , Antigens, CD/immunology , Cell Adhesion/drug effects , Cell Adhesion Molecules/immunology , Diabetes Mellitus, Type 1/etiology , Diabetes Mellitus, Type 1/pathology , Female , Inflammation/pathology , Inflammation/prevention & control , Integrin alpha4 , Integrins/immunology , Islets of Langerhans/pathology , Leukocytes/drug effects , Leukocytes/physiology , Mice , Mice, Inbred NOD , Spleen/cytology , Time FactorsABSTRACT
BACKGROUND: Survivin, a member of the inhibitor of apoptosis (IAP) protein family, is detectable in most types of cancer, and its presence is associated with a poor prognosis. We determined the effects of gene-based therapies that inhibit survivin function in a mouse tumor model. METHODS: Using five to six mice per treatment group, we injected tumors derived from mouse EL-4 thymic lymphoma cells with plasmids encoding antisense survivin, a dominant-negative mutant survivin, and the T-cell costimulator B7-1. Expression of endogenous survivin and the proteins encoded by the injected plasmids were examined by immunohistochemical staining of tumor sections and by western blot and flow cytometry analyses of isolated tumor cells. Tumor growth, the generation of antitumor cytotoxic T-lymphocyte (CTL) activity, apoptosis, and the contribution of leukocyte subsets to antitumor activity were measured. All statistical tests were two-sided. RESULTS: Large (1.0-cm diameter) tumors had approximately 10-fold more survivin than small (0.2-cm diameter) tumors. At 28 days after injection, antisense and dominant-negative mutant survivin plasmids statistically significantly inhibited the growth of both small (P =.006 and P =.0018, respectively) and large (P<.001 for both plasmids) EL-4 tumors compared with tumors injected with empty plasmid. The growth of large tumors was further inhibited by intratumoral injection with antisense survivin and B7-1 (P =.004); thus, inhibition of survivin expression renders large tumors susceptible to B7-1-mediated immunotherapy. Mice whose tumors were completely eradicated by injection of B7-1 remained tumor free for 26 days after re-injection with EL-4 cells (when the experiment ended). Compared with tumors injected with empty plasmid, tumors injected with survivin-based plasmids had increased apoptosis, and animals bearing such tumors generated more antitumor CTLs. CONCLUSION: Intratumoral injection of plasmids that block survivin expression and stimulate the generation of tumor-specific CTLs may be beneficial for the treatment of large lymphomas.
Subject(s)
B7-1 Antigen/therapeutic use , Chromosomal Proteins, Non-Histone/physiology , DNA, Antisense/therapeutic use , Genetic Therapy , Immunotherapy , Lymphoma, Non-Hodgkin/drug therapy , Microtubule-Associated Proteins , Neoplasm Proteins/physiology , Thymus Neoplasms/therapy , Animals , Antibodies, Monoclonal/administration & dosage , Apoptosis , B7-1 Antigen/administration & dosage , Chromosomal Proteins, Non-Histone/antagonists & inhibitors , Chromosomal Proteins, Non-Histone/biosynthesis , Chromosomal Proteins, Non-Histone/genetics , Combined Modality Therapy , DNA, Antisense/administration & dosage , DNA, Antisense/genetics , Disease Progression , Female , Gene Dosage , Gene Expression Regulation, Neoplastic , Gene Targeting , Genes, Dominant , Genetic Vectors/administration & dosage , Genetic Vectors/therapeutic use , Graft Rejection/immunology , Inhibitor of Apoptosis Proteins , Injections, Intralesional , Lymphocyte Depletion , Lymphocyte Subsets/immunology , Lymphocyte Subsets/pathology , Lymphocytes, Tumor-Infiltrating , Lymphoma, Non-Hodgkin/immunology , Lymphoma, Non-Hodgkin/pathology , Lymphoma, Non-Hodgkin/therapy , Mice , Mice, Inbred C57BL , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Neoplasm Transplantation , Survivin , T-Lymphocytes, Cytotoxic/immunology , Thymus Neoplasms/immunology , Thymus Neoplasms/pathologyABSTRACT
Solid tumors meet their demands for nascent blood vessels and increased glycolysis, to combat hypoxia, by activating multiple genes involved in angiogenesis and glucose metabolism. Hypoxia inducible factor-1 (HIF-1) is a constitutively expressed basic helix-loop-helix transcription factor, formed by the assembly of HIF-1alpha and HIF-1beta (Arnt), that is stablized in response to hypoxia, and rapidly degraded under normoxic conditions. It activates the transcription of genes important for maintaining oxygen homeostasis. Here, we demonstrate that engineered down-regulation of HIF-1alpha by intratumoral gene transfer of an antisense HIF-1alpha plasmid leads to the down-regulation of VEGF, and decreased tumor microvessel density. Antisense HIF-1alpha monotherapy resulted in the complete and permanent rejection of small (0.1 cm in diameter) EL-4 tumors, which is unusual for an anti-angiogenic agent where transient suppression of tumor growth is the norm. It induced NK cell-dependent rejection of tumors, but failed to stimulate systemic T cell-mediated anti-tumor immunity, and synergized with B7-1-mediated immunotherapy to cause the NK cell and CD8 T cell-dependent rejection of larger EL-4 tumors (0.4 cm in diameter) that were refractory to monotherapies. Mice cured of their tumors by combination therapy resisted a rechallenge with parental tumor cells, indicating systemic antitumor immunity had been achieved. In summary, whilst intensive investigations are in progress to target the many HIF-1 effectors, the results herein indicate that blocking hypoxia-inducible pathways and enhancing NK-mediated antitumor immunity by targeting HIF-1 itself may be advantageous, especially when combined with cancer immunotherapy.
Subject(s)
DNA-Binding Proteins/genetics , Genetic Therapy/methods , Lymphoma, T-Cell/therapy , Neovascularization, Pathologic/therapy , Nuclear Proteins/genetics , Animals , Antisense Elements (Genetics)/genetics , B7-1 Antigen/therapeutic use , Combined Modality Therapy , DNA-Binding Proteins/metabolism , Down-Regulation , Gene Transfer Techniques , Hypoxia-Inducible Factor 1 , Hypoxia-Inducible Factor 1, alpha Subunit , Killer Cells, Natural/immunology , Lymphoma, T-Cell/immunology , Lymphoma, T-Cell/pathology , Male , Mice , Mice, Inbred C57BL , Nuclear Proteins/metabolism , Plasmids , T-Lymphocyte Subsets/immunology , Transcription Factors/geneticsABSTRACT
The promise of cancer immunotherapy is that it will not only eradicate primary tumors but will generate systemic antitumor immunity capable of destroying distant metastases. A major problem that must first be surmounted relates to the immune resistance of large tumors. Here we reveal that immune resistance can be overcome by combining immunotherapy with a concerted attack on the tumor vasculature. The functionally related antitumor drugs 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and flavone acetic acid (FAA), which cause tumor vasculature collapse and tumor necrosis, were used to attack the tumor vasculature, whereas the T-cell costimulator B7.1 (CD80), which costimulates T-cell proliferation via the CD28 pathway, was used to stimulate antitumor immunity. The injection of cDNA (60-180 microg) encoding B7.1 into large EL-4 tumors (0.8 cm in diameter) established in C57BL/6 mice, followed 24 h later by i.p. administration of either DMXAA (25 mg/kg) or FAA (300 mg/kg), resulted in complete tumor eradication within 2-6 weeks. In contrast, monotherapies were ineffective. Both vascular attack and B7.1 immunotherapy led to up-regulation of heat shock protein 70 on stressed and dying tumor cells, potentially augmenting immunotherapy. Remarkably, large tumors took on the appearance of a wound that rapidly ameliorated, leaving perfectly healed skin. Combined therapy was mediated by CD8+ T cells and natural killer cells, accompanied by heightened and prolonged antitumor cytolytic activity (P < 0.001), and by a marked increase in tumor cell apoptosis. Cured animals completely rejected a challenge of 1 x 10(7) parental EL-4 tumor cells but not a challenge of 1 x 10(4) Lewis lung carcinoma cells, demonstrating that antitumor immunity was tumor specific. Adoptive transfer of 2 x 10(8) splenocytes from treated mice into recipients bearing established (0.8 cm in diameter) tumors resulted in rapid and complete tumor rejection within 3 weeks. Although DMXAA and B7.1 monotherapies are complicated by a narrow range of effective doses, combined therapy was less dosage dependent. Thus, a broad range of amounts of B7.1 cDNA were effective in combination with 25 mg/kg DMXAA. In contrast, DMXAA, which has a very narrow range of high active doses, was effective at a low dose (18 mg/kg) when administered with a large amount (180 microg) of B7.1 cDNA. Importantly, combinational therapy generated heightened antitumor immunity, such that gene transfer of B7.1 into one tumor, followed by systemic DMXAA treatment, led to the complete rejection of multiple untreated tumor nodules established in the opposing flank. These findings have important implications for the future direction and utility of cancer immunotherapies aimed at harnessing patients' immune responses to their own tumors.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , B7-1 Antigen/immunology , Immunotherapy/methods , Neovascularization, Pathologic/therapy , Xanthones , Animals , B7-1 Antigen/genetics , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Combined Modality Therapy , Female , Flavonoids/administration & dosage , Gene Dosage , Genetic Therapy , Genetic Vectors/genetics , Killer Cells, Natural/immunology , Lymphocyte Subsets/immunology , Lymphoma/immunology , Lymphoma/pathology , Lymphoma/therapy , Mice , Mice, Inbred C57BL , T-Lymphocytes, Cytotoxic/immunology , Thymus Neoplasms/blood supply , Thymus Neoplasms/immunology , Thymus Neoplasms/pathology , Thymus Neoplasms/therapy , Xanthenes/administration & dosageABSTRACT
Heat shock proteins (hsps) chaperone cytosolic peptides, forming complexes that stimulate antitumor immunity. Hsps facilitate signal 1 in the two-signal model of T-cell costimulation, whereas cell adhesion molecules such as B7.1 provide secondary (signal 2) costimulatory signals. B7.1 gene transfer into tumors in situ has been shown to eradicate small (<0.3 cm in diameter) tumors in mice, and induce systemic antitumor immunity, but is ineffective against larger tumors. We examine whether mammalian hsps, as facilitators of T-cell costimulation, also exhibit this ability, and whether simultaneously stimulating both signal 1 (hsp-facilitated antigen presentation) and signal 2 (B7.1-mediated costimulation) enhances antitumor immunity compared to that achieved with either monotherapy. Prophylactic vaccination of mice with an hsp preparation from an EL-4 lymphoma weakly retarded tumor growth, to the same extent as that achieved with a single EL-4-derived peptide (AQHPNAELL), previously shown to induce antitumor immunity establishing that a preparation of EL-4 hsp-peptide complexes has antitumor activity. Here we show that injection of rat hsp70.1 into mouse tumors in situ causes the complete eradication of tumors, and generates potent systemic antitumor immunity mediated by CD4+ and CD8+ T cells. Unexpectedly, simultaneous gene transfer of hsp70.1 and B7.1 compromised the efficacy of hsp-mediated tumor rejection--a problem which could be partially overcome by the timed delivery of hsp70.1 and B7.1. Thus, gene transfer of hsp70 into tumors can be employed to generate potent systemic antitumor immunity, but further consideration is required if this approach is to be successfully combined with immunotherapies employing other T-cell costimulators.
Subject(s)
B7-1 Antigen/genetics , Cytotoxicity, Immunologic/genetics , HSP70 Heat-Shock Proteins/genetics , Immunotherapy , Protozoan Proteins/genetics , Animals , Antigen Presentation/genetics , Antigen Presentation/immunology , B7-1 Antigen/immunology , Genetic Therapy , HSP70 Heat-Shock Proteins/immunology , Mice , Mice, Inbred C57BL , Protozoan Proteins/immunology , Rats , T-Lymphocytes/immunology , Transfection , Tumor Cells, CulturedABSTRACT
A role for alpha4 and beta7 integrins in mediating leucocyte entry into the central nervous system in the multiple sclerosis (MS)-like disease experimental autoimmune encephalomyelitis (EAE) has been demonstrated. However, the individual contributions of their respective ligands mucosal addressin cell adhesion molecule-1 (MAdCAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-cadherin expressed on the blood-brain barrier has not been determined. In the present paper, it is shown that an antibody directed against MAdCAM-1, the preferential ligand for alpha4beta7, effectively prevented the development of a progressive, non-remitting, form of EAE, actively induced by injection of myelin oligodendrocyte glycoprotein peptide (MOG(35-55)) autoantigen. Combinational treatment with both anti-MAdCAM-1, VCAM-1, and intercellular adhesion molecule-1 (ICAM-1) (ligand for integrin lymphocyte function-associated antigen (LFA)-1) mAbs led to more rapid remission than that obtained with anti-MAdCAM-1 antibody alone. However, neither MAdCAM-1 monotherapy, nor combinational antibody blockade was preventative when administered late in the course of disease progression. In conclusion, MAdCAM-1 plays a major contributory role in the progression of chronic EAE and is a potential therapeutic target for the treatment of MS. Critically, antivascular addressin therapy must be given early in the course of disease prior to the establishment of irreversible damage if it is to be effective, as a single treatment modality.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/prevention & control , Immunoglobulins/immunology , Mucoproteins/immunology , Animals , Cell Adhesion Molecules , Chronic Disease , Disease Progression , Encephalomyelitis, Autoimmune, Experimental/pathology , Intercellular Adhesion Molecule-1/immunology , Leukocytes/immunology , Mice , Mice, Inbred C57BL , Spinal Cord/immunology , Spinal Cord/pathology , Time Factors , Vascular Cell Adhesion Molecule-1/immunologyABSTRACT
A role for alpha4 integrins in different forms of the multiple sclerosis-like disease experimental autoimmune encephalomyelitis (EAE) has been demonstrated, but the individual contributions of alpha4beta1, alpha4beta7, and the related alphaEbeta7 integrin have not been determined. The P7 integrins alpha4beta7 and alphaEbeta7 play a central role in chronic inflammation, mediating the trafficking, entry, and/or adhesion of lymphocytes in the inflamed pancreas and gut, and their ligands MAdCAM-1, VCAM-1 and E-cadherin are expressed on brain endothelial cells and/or on microvessels in the inflamed central nervous system. Here, we show that an antibody directed against the beta7 subunit greatly attenuates a non-remitting form of EAE, induced by adoptive transfer of myelin oligodendrocyte peptide (MOG35-55)-stimulated T cells. Combinational treatment with both anti-beta7 and alpha4 integrin subunit antibodies led to more rapid and complete remission than that obtained with anti-alpha4 antibody alone, potentially implicating a role for alphaEbeta7 in disease progression. Remission correlated with the down-regulation of the vascular addressins VCAM-1. MAdCAM-1, and ICAM-1 on cerebral blood vessels. Attenuated forms of disease were induced by adoptive transfer of either wild-type encephalitogenic T cells to beta7-deficient gene knockout mice, or of beta7-/-encephalitogenic T cells to wild-type recipients. The former finding indicates that beta7 + ve recruited cells contribute to disease progression. Thus alpha4beta1, alpha4beta7, and alphaEbeta7 integrins may all play a contributory role in the progression of chronic forms of demyelinating disease, and together with their ligands could represent potential targets for improved treatment of some forms of multiple sclerosis.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/metabolism , Integrin beta Chains , Integrins/metabolism , Adoptive Transfer , Animals , Antibodies, Monoclonal/administration & dosage , Antigens, CD/immunology , Brain/immunology , Brain/metabolism , Brain/pathology , Cells, Cultured , Demyelinating Autoimmune Diseases, CNS/metabolism , Drug Synergism , Encephalomyelitis, Autoimmune, Experimental/complications , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Integrin alpha4 , Integrins/antagonists & inhibitors , Integrins/genetics , Integrins/immunology , Lymphocyte Activation/drug effects , Lymphocytes/cytology , Lymphocytes/drug effects , Lymphocytes/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Myelin Proteins , Myelin-Associated Glycoprotein/immunology , Myelin-Associated Glycoprotein/pharmacology , Myelin-Oligodendrocyte Glycoprotein , Paralysis/etiology , Paralysis/prevention & controlABSTRACT
Here we report that an activator (AIF4-) of heterotrimeric GTP-binding proteins (G-proteins) and inhibitors (lovastatin and C3 exoenzyme) of small GTP-binding proteins regulate the induction of alpha4beta7-mediated adhesion of TK-1 T lymphoma cells (alpha4+beta7+beta1-) to the mucosal addressin cell adhesion molecule MAdCAM-1. Activation of cell adhesion by AIF4- was abrogated by lovastatin, thereby establishing a link between heterotrimeric G-proteins and small GTP-binding proteins in the regulation of alpha4beta7-mediated cell adhesion. Increased numbers of cells bound MAdCAM-1-coated microspheres following activation with AIF4-, discounting an obligatory role for cell spreading in alpha4beta7-mediated cell adhesion. MAdCAM-1-Fc dimers triggered ligand-induced clustering of alpha4beta7 in response to AIF4- and Mn2+-induced activation of integrins. Hence alpha4beta7 cluster formation may be responsible, at least in part, for inducing cell adhesion in response to both extracellular and intracellular signals that impact on integrin function. Electroporation of constitutively active V14RhoA and V12Rac1 recombinant proteins into TK-1 cells revealed that both RhoA and Rac1 induce alpha4beta7 adhesion to MAdCAM-1. Activation is hierarchical since Rac1 is unable to directly activate alpha4beta7, but induces cell adhesion via RhoA, whereas the transient induction of cell adhesion mediated by RhoA is dependent on the activities of protein tyrosine kinases and protein kinase(s) C.
Subject(s)
Immunoglobulins/immunology , Mucoproteins/immunology , T-Lymphocytes/immunology , rac GTP-Binding Proteins/immunology , rho GTP-Binding Proteins/immunology , 3T3 Cells , Aluminum Compounds/pharmacology , Animals , Cell Adhesion/immunology , Cell Adhesion Molecules , Complement C3/physiology , Electroporation , Fluorides/pharmacology , Genistein/pharmacology , Integrins/analysis , Integrins/immunology , Integrins/metabolism , Lymphoma, T-Cell , Mice , Receptors, Lymphocyte Homing/immunology , T-Lymphocytes/metabolism , Tetradecanoylphorbol Acetate/analogs & derivatives , Tetradecanoylphorbol Acetate/pharmacology , Tumor Cells, Cultured , rac GTP-Binding Proteins/genetics , rac GTP-Binding Proteins/metabolism , rho GTP-Binding Proteins/genetics , rho GTP-Binding Proteins/metabolismABSTRACT
The integrins are a large family of cell adhesion molecules consisting of noncovalently associated alphabeta heterodimers. We have cloned and sequenced the cDNA of a novel human integrin alpha-subunit, designated alpha11. The alpha11 cDNA encodes a mature protein with a large 1120-residue extracellular domain that contains an I-domain of 207 residues and is linked by a transmembrane domain to a short cytoplasmic domain of 24 amino acids. The deduced alpha11 protein shows the typical structural features of integrin alpha-subunits and is similar to a distinct group of alpha-subunits from collagen-binding integrins. However, it differs from most integrin alpha-chains by an incompletely preserved cytoplasmic GFFKR motif. The human ITGA11 gene was localized to bands q22.3-q23 on chromosome 15, and its transcripts were found in a variety of tissues, but predominantly in bone, cartilage, cardiac muscle, and skeletal muscle. Expression of a 5.5-kb alpha11 mRNA was detectable in small intestine.
Subject(s)
Integrin alpha Chains , Integrins/genetics , Amino Acid Sequence , Base Sequence , Chromosome Mapping , Chromosomes, Human, Pair 15/genetics , Cloning, Molecular , DNA Primers/genetics , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Female , Humans , Integrins/chemistry , Male , Molecular Sequence Data , Phylogeny , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Collagen , Tissue DistributionABSTRACT
Human mucosal lymphocyte antigen-1 (HML-1, alphaEbeta7) and E-cadherin, two members of unrelated cell adhesion superfamilies, have evolved to play cooperative roles in gut mucosal immunity. Human E-cadherin is self-ligand mediating intercellular adhesion of epithelial cells, as well as adhesion of intra-epithelial lymphocytes to intestinal enterocytes via an interaction with HML-1. Herein we report that both dimeric and monomeric forms of recombinant mouse E-cadherin-human immunoglobulin Fc chimera self-associate and support attachment of E-cadherin+ mouse colon epithelial cells. Both forms also support the adhesion of mouse MTC-1 T cells via M290, thereby establishing M290 as the functional mouse homologue of HML-1 and revealing that E-cadherin homophilic and heterophilic binding sites are distinct. Adhesion of MTC-1 cells to E-cadherin-Fc was inhibited by arginine-glycine-aspartate (RGD) peptides and vice versa cells bound to immobilized RGD polymer in an M290-dependent fashion, where adhesion was inhibitable with soluble E-cadherin-Fc. Hence, E-cadherin and RGD integrin ligands antagonize cell binding by one another, either by inducing integrin cross-talk or by binding to shared or overlapping sites within M290. Binding of E-cadherin-Fc by HML-1 costimulated the CD3-induced proliferation of purified CD4+ T cells, suggesting that E-cadherin expressed on dendritic cells may play a T cell costimulatory role in addition to facilitating dendritic cell-keratinocyte adhesion.