RESUMEN

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.

Asunto(s)

Colitis/tratamiento farmacológico , Calostro/química , Proteínas de la Leche/farmacología , Leche/química , Animales , Australia , Enfermedad Crónica , Colitis/inducido químicamente , Citocinas/metabolismo , Productos Lácteos , Sulfato de Dextran/efectos adversos , Modelos Animales de Enfermedad , Glucolípidos/farmacología , Glicoproteínas/farmacología , Lactoferrina/farmacología , Ácidos Linoleicos Conjugados/farmacología , Gotas Lipídicas , Masculino , Ratones , Ratones Endogámicos BALB C , Óxido Nitroso/metabolismo , Osteopontina/farmacología , Ribonucleasa Pancreática/farmacología , Proteína de Suero de Leche/farmacología

RESUMEN

PURPOSE: Chemotherapy-induced diarrhoea (CID) has a significant impact. A medicinal food product (ReCharge) containing iron-saturated lactoferrin and anhydrous milk fat reduces the detrimental effects of chemotherapy on the gut in animals. We report results of a randomised blinded placebo-controlled phase IIb trial investigating the efficacy and safety of ReCharge in preventing CID. METHODS: Eligible patients were adults due to start the first cycle of a 2- or 3-week-cycle chemotherapy regimen, had an Eastern Cooperative Oncology Group (ECOG) status of 3 or less, had adequate haematological, liver and renal function and provided written informed consent. Patients (197) were randomised to ReCharge or placebo. They consumed 100-g study product for 2 weeks before and 6 weeks after starting chemotherapy, completed daily diaries for 8 weeks and attended clinic visits until 12 weeks (2-week cycles) or 14 weeks (3-week cycles). The primary outcome was days with CID. RESULTS: The mean number of days with diary-recorded CID was marginally but not statistically significantly lower on ReCharge than placebo (-2.0, 95 % CI (-4.7 to 0.7), p = 0.2). The proportion reporting diarrhoea in the previous cycle at the clinic visit was 30 % lower (p = 0.012) on ReCharge. Missing diary data may have contributed to the discrepancy. No significant differences were found in quality of life or other adverse events. CONCLUSIONS: We found no clear evidence that ReCharge reduced CID as measured by patient self-report diary. The converse finding of benefit as recorded at clinic visits and incomplete adherence to diary completion indicates that further research is required into methods for measuring CID.

Asunto(s)

Antidiarreicos/uso terapéutico , Antineoplásicos/efectos adversos , Diarrea/inducido químicamente , Diarrea/prevención & control , Helados , Adulto , Anciano , Antineoplásicos/uso terapéutico , Diarrea/tratamiento farmacológico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias/tratamiento farmacológico , Placebos , Calidad de Vida , Autoinforme

RESUMEN

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.

Asunto(s)

Dermatitis Alérgica por Contacto/dietoterapia , Grasas/química , Ácidos Linoleicos/uso terapéutico , Leche/química , Ácidos Oléicos/uso terapéutico , Animales , Bovinos , Suplementos Dietéticos , Grasas/administración & dosificación , Grasas/uso terapéutico , Femenino , Ácidos Linoleicos/administración & dosificación , Ácidos Linoleicos/química , Ratones , Ratones Endogámicos C57BL , Ácidos Oléicos/administración & dosificación , Ácidos Oléicos/química , Ovalbúmina , Pruebas Cutáneas

RESUMEN

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.

Asunto(s)

Angiostatinas/genética , ADN sin Sentido/genética , Terapia Genética/métodos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Linfoma/terapia , Neoplasias del Timo/terapia , Angiostatinas/biosíntesis , Animales , Línea Celular Tumoral , ADN sin Sentido/administración & dosificación , Regulación Neoplásica de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Linfoma/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Plásmidos/administración & dosificación , Plásmidos/genética , Neoplasias del Timo/genética

RESUMEN

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.

Asunto(s)

Grasas/inmunología , Hipersensibilidad/inmunología , Ácido Linoleico/inmunología , Enfermedades Pulmonares Obstructivas/inmunología , Enfermedades Pulmonares Obstructivas/patología , Leche/inmunología , Ácidos Oléicos/inmunología , Alérgenos/inmunología , Animales , Supervivencia Celular , Quimiocina CCL11/biosíntesis , Eosinófilos/citología , Eosinófilos/inmunología , Femenino , Hipersensibilidad/metabolismo , Hipersensibilidad/patología , Inmunoglobulinas/biosíntesis , Inmunoglobulinas/inmunología , Interleucina-5/biosíntesis , Enfermedades Pulmonares Obstructivas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL

RESUMEN

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.

Asunto(s)

ADN sin Sentido/genética , Terapia Genética , Integrina alfaV/genética , Integrina alfaVbeta3/genética , Neoplasias Hepáticas Experimentales/terapia , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Células Endoteliales/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neovascularización Patológica , Plásmidos , Transfección , Factor A de Crecimiento Endotelial Vascular/farmacología

RESUMEN

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.

Asunto(s)

Glioma/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular , ADN sin Sentido/genética , ADN sin Sentido/metabolismo , Glioma/irrigación sanguínea , Glioma/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neovascularización Patológica/prevención & control , Ratas , Factores de Tiempo , Transfección , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/biosíntesis , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo

RESUMEN

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.

Asunto(s)

Inhibidores de la Angiogénesis/genética , ADN/administración & dosificación , Terapia Genética/métodos , Linfoma/terapia , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/genética , Inhibidores de la Angiogénesis/análisis , Animales , Apoptosis , Regulación de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Inyecciones Intralesiones , Linfoma/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Neoplasias Experimentales , Oligonucleótidos Antisentido/uso terapéutico , Factores de Transcripción/análisis , Proteínas Supresoras de Tumor/análisis , Ubiquitina-Proteína Ligasas/análisis , Factor A de Crecimiento Endotelial Vascular/análisis , Factor A de Crecimiento Endotelial Vascular/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau

RESUMEN

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.

Asunto(s)

Antígeno B7-1/genética , ADN/administración & dosificación , Terapia Genética/métodos , Linfoma/terapia , Neoplasias del Timo/terapia , Secuencia de Aminoácidos , Animales , Antígenos B7 , Secuencia de Bases , Linfocitos T CD4-Positivos/inmunología , Femenino , Inyecciones Intralesiones , Células Asesinas Naturales/inmunología , Activación de Linfocitos , Linfoma/inmunología , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Trasplante de Neoplasias , Neoplasias del Timo/inmunología

RESUMEN

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.

Asunto(s)

Apolipoproteínas E/deficiencia , Arterias/metabolismo , Arterias/patología , Arteriosclerosis/metabolismo , Arteriosclerosis/patología , Chaperonina 60/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Animales , Aorta/metabolismo , Aorta/patología , Western Blotting , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Regulación hacia Abajo , Inmunohistoquímica , Macrófagos/metabolismo , Ratones , Ratones Mutantes , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Linfocitos T/metabolismo , Regulación hacia Arriba

RESUMEN

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.

Asunto(s)

Antígeno B7-1/genética , Factores de Crecimiento Endotelial/metabolismo , Terapia Genética/métodos , Inmunoterapia/métodos , Linfocinas/metabolismo , Fragmentos de Péptidos/genética , Plasminógeno/genética , Neoplasias del Timo/terapia , Angiostatinas , Animales , Western Blotting , Terapia Combinada , Cartilla de ADN/química , Técnicas de Transferencia de Gen , Técnicas para Inmunoenzimas , Masculino , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/prevención & control , Fragmentos de Péptidos/metabolismo , Plasminógeno/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Linfocitos T/inmunología , Neoplasias del Timo/irrigación sanguínea , Neoplasias del Timo/metabolismo , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

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.

Asunto(s)

Anticuerpos Monoclonales/uso terapéutico , Moléculas de Adhesión Celular/antagonistas & inhibidores , Quimiotaxis de Leucocito/efectos de los fármacos , Diabetes Mellitus Tipo 1/terapia , Integrinas/antagonistas & inhibidores , Islotes Pancreáticos , Traslado Adoptivo , Factores de Edad , Animales , Antígenos CD/inmunología , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/inmunología , Diabetes Mellitus Tipo 1/etiología , Diabetes Mellitus Tipo 1/patología , Femenino , Inflamación/patología , Inflamación/prevención & control , Integrina alfa4 , Integrinas/inmunología , Islotes Pancreáticos/patología , Leucocitos/efectos de los fármacos , Leucocitos/fisiología , Ratones , Ratones Endogámicos NOD , Bazo/citología , Factores de Tiempo

RESUMEN

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.

Asunto(s)

Antígeno B7-1/uso terapéutico , Proteínas Cromosómicas no Histona/fisiología , ADN sin Sentido/uso terapéutico , Terapia Genética , Inmunoterapia , Linfoma no Hodgkin/tratamiento farmacológico , Proteínas Asociadas a Microtúbulos , Proteínas de Neoplasias/fisiología , Neoplasias del Timo/terapia , Animales , Anticuerpos Monoclonales/administración & dosificación , Apoptosis , Antígeno B7-1/administración & dosificación , Proteínas Cromosómicas no Histona/antagonistas & inhibidores , Proteínas Cromosómicas no Histona/biosíntesis , Proteínas Cromosómicas no Histona/genética , Terapia Combinada , ADN sin Sentido/administración & dosificación , ADN sin Sentido/genética , Progresión de la Enfermedad , Femenino , Dosificación de Gen , Regulación Neoplásica de la Expresión Génica , Marcación de Gen , Genes Dominantes , Vectores Genéticos/administración & dosificación , Vectores Genéticos/uso terapéutico , Rechazo de Injerto/inmunología , Proteínas Inhibidoras de la Apoptosis , Inyecciones Intralesiones , Depleción Linfocítica , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/patología , Linfocitos Infiltrantes de Tumor , Linfoma no Hodgkin/inmunología , Linfoma no Hodgkin/patología , Linfoma no Hodgkin/terapia , Ratones , Ratones Endogámicos C57BL , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Survivin , Linfocitos T Citotóxicos/inmunología , Neoplasias del Timo/inmunología , Neoplasias del Timo/patología

RESUMEN

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.

Asunto(s)

Proteínas de Unión al ADN/genética , Terapia Genética/métodos , Linfoma de Células T/terapia , Neovascularización Patológica/terapia , Proteínas Nucleares/genética , Animales , Elementos sin Sentido (Genética)/genética , Antígeno B7-1/uso terapéutico , Terapia Combinada , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Técnicas de Transferencia de Gen , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Células Asesinas Naturales/inmunología , Linfoma de Células T/inmunología , Linfoma de Células T/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares/metabolismo , Plásmidos , Subgrupos de Linfocitos T/inmunología , Factores de Transcripción/genética

RESUMEN

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.

Asunto(s)

Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Antígeno B7-1/inmunología , Inmunoterapia/métodos , Neovascularización Patológica/terapia , Xantonas , Animales , Antígeno B7-1/genética , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Terapia Combinada , Femenino , Flavonoides/administración & dosificación , Dosificación de Gen , Terapia Genética , Vectores Genéticos/genética , Células Asesinas Naturales/inmunología , Subgrupos Linfocitarios/inmunología , Linfoma/inmunología , Linfoma/patología , Linfoma/terapia , Ratones , Ratones Endogámicos C57BL , Linfocitos T Citotóxicos/inmunología , Neoplasias del Timo/irrigación sanguínea , Neoplasias del Timo/inmunología , Neoplasias del Timo/patología , Neoplasias del Timo/terapia , Xantenos/administración & dosificación

RESUMEN

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.

Asunto(s)

Antígeno B7-1/genética , Citotoxicidad Inmunológica/genética , Proteínas HSP70 de Choque Térmico/genética , Inmunoterapia , Proteínas Protozoarias/genética , Animales , Presentación de Antígeno/genética , Presentación de Antígeno/inmunología , Antígeno B7-1/inmunología , Terapia Genética , Proteínas HSP70 de Choque Térmico/inmunología , Ratones , Ratones Endogámicos C57BL , Proteínas Protozoarias/inmunología , Ratas , Linfocitos T/inmunología , Transfección , Células Tumorales Cultivadas

RESUMEN

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.

Asunto(s)

Anticuerpos Monoclonales/uso terapéutico , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/prevención & control , Inmunoglobulinas/inmunología , Mucoproteínas/inmunología , Animales , Moléculas de Adhesión Celular , Enfermedad Crónica , Progresión de la Enfermedad , Encefalomielitis Autoinmune Experimental/patología , Molécula 1 de Adhesión Intercelular/inmunología , Leucocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Médula Espinal/inmunología , Médula Espinal/patología , Factores de Tiempo , Molécula 1 de Adhesión Celular Vascular/inmunología

RESUMEN

Beta cell destruction in NOD mice can be accelerated by adoptive transfer of diabetic spleen cells into irradiated adult NOD mice. Here mice receiving diabetic spleen cells were examined at days 0, 7, 14, 21 and at onset of diabetes for the resulting insulitis and the number of intra-islet CD4 and CD8 cells and macrophages. The progression of insulitis and the number of intra-islet CD4 and CD8 cells and macrophages were correlated with the expression and co-localization of inducible nitric oxide synthase, interferon-gamma and interleukin-4 by dual-label light and confocal immunofluorescence microscopy. Diabetes developed in 7/8 mice by 27 days following cell transfer. The insulitis score increased slightly by day 7 but rose sharply at day 14 (p = 0.001) and was maintained until diabetes. The mean number of intra-islet CD4 and CD8 cells and macrophages showed a similar trend to the insulitis scores and were present in almost equal numbers within the islets. Immunolabelling for inducible nitric oxide synthase was observed at day 7 in only some cells of a few islets but increased sharply from day 14. It was restricted to islets with insulitis and was co-localized in selective macrophages. Weak intra-islet interleukin-4 labelling was observed at days 7 and 14 but became more pronounced at day 21 and at onset of diabetes, being present in selective CD4 cells. Intra-islet labelling for interferon-gamma was first observed at day 21, but became more intense at onset of diabetes and was co-localized in a proportion of macrophages. Both cytokines were expressed in islets with advanced insulitis. Interferon-gamma staining was also observed within endothelial cells located in the exocrine pancreas. We conclude that transfer of diabetic spleen cells results in a rapid influx of CD4 and CD8 cells and macrophages within the pancreas of recipient mice. During the period of heightened insulitis, selective immune cells begin to express inducible nitric oxide synthase and the opposing cytokines, interferon-gamma and interleukin-4. Expression of these molecules becomes more pronounced immediately prior to and during the onset of diabetes.

Asunto(s)

Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Interferón gamma/biosíntesis , Interleucina-4/biosíntesis , Islotes Pancreáticos/inmunología , Macrófagos/inmunología , Óxido Nítrico Sintasa/biosíntesis , Traslado Adoptivo , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Trasplante de Células , Glucagón/biosíntesis , Cobayas , Inmunohistoquímica/métodos , Incidencia , Insulina/biosíntesis , Insulina/inmunología , Islotes Pancreáticos/patología , Recuento de Linfocitos , Macrófagos/citología , Ratones , Ratones Endogámicos NOD , Óxido Nítrico Sintasa de Tipo II , Conejos , Ratas , Ovinos , Somatostatina/biosíntesis , Bazo/citología , Bazo/inmunología

RESUMEN

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.

Asunto(s)

Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Cadenas beta de Integrinas , Integrinas/metabolismo , Traslado Adoptivo , Animales , Anticuerpos Monoclonales/administración & dosificación , Antígenos CD/inmunología , Encéfalo/inmunología , Encéfalo/metabolismo , Encéfalo/patología , Células Cultivadas , Enfermedades Autoinmunes Desmielinizantes SNC/metabolismo , Sinergismo Farmacológico , Encefalomielitis Autoinmune Experimental/complicaciones , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Integrina alfa4 , Integrinas/antagonistas & inhibidores , Integrinas/genética , Integrinas/inmunología , Activación de Linfocitos/efectos de los fármacos , Linfocitos/citología , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de la Mielina , Glicoproteína Asociada a Mielina/inmunología , Glicoproteína Asociada a Mielina/farmacología , Glicoproteína Mielina-Oligodendrócito , Parálisis/etiología , Parálisis/prevención & control

RESUMEN

Expression of large numbers of different costimulatory integrin ligands (CILs) attributes dendritic cells with an ability to induce primary anti-tumor immune responses. Here, we show that optimized gene transfer of the xenogeneic (human) CILs VCAM-1, MAdCAM-1 and ICAM-1 causes rapid and complete rejection of established mouse EL-4 tumors, and generates prolonged systemic anti-tumor immunity; whereas human E-cadherin weakly slows tumor growth. In each case the immune response was mediated by CD8+ T cells and NK cells, accompanied by augmented tumor-specific cytolytic T cell (CTL) activity involving both the perforin and Fas-ligand pathways. Adoptive transfer of splenocytes from cured mice rapidly cleared established tumors in recipients. The mechanism for CIL-mediated immunity is unknown, but may involve CTL-facilitated tumor lysis, since CTLs were generally twice as efficient at killing CIL-transfected tumor cells than parental tumor cells. Optimized CIL-based gene therapy may provide an approach to complement or replace conventional DC adoptive cell therapy for suppressing tumor growth.

Asunto(s)

Células Dendríticas/inmunología , Terapia Genética/métodos , Células Asesinas Naturales/inmunología , Neoplasias/terapia , Animales , Antígenos Heterófilos/inmunología , Moléculas de Adhesión Celular/inmunología , Citotoxicidad Inmunológica , Técnicas de Transferencia de Gen , Humanos , Inmunoterapia/métodos , Integrinas/inmunología , Ligandos , Ratones , Neoplasias/inmunología , Células Tumorales Cultivadas