RESUMEN

Glucagon-like peptide 1 (GLP-1) and its analogue exendin-4 (Ex4) have displayed potent glucose homeostasis-modulating characteristics in type 2 diabetes (T2D). However, there are few reports of effectiveness in type 1 diabetes (T1D) therapy, where there is massive loss of beta cells. We previously described a novel GLP-1 analogue consisting of the fusion of active GLP-1 and IgG heavy chain constant regions (GLP-1/IgG-Fc), and showed that in vivo expression of the protein, via electroporation-enhanced intramuscular plasmid-based gene transfer, normalized blood glucose levels in T2D-prone db/db mice. In the present study, GLP-1/IgG-Fc and Ex4/IgG-Fc were independently tested in multiple low-dose streptozotocin-induced T1D. Both GLP-1/IgG-Fc and Ex4/IgG-Fc effectively reduced fed blood glucose levels in treated mice and ameliorated diabetes symptoms, where as control IgG-Fc had no effect. Treatment with GLP-1/IgG-Fc or Ex4/IgG-Fc improved glucose tolerance and increased circulating insulin and GLP-1 levels. It also significantly enhanced islet beta-cell mass, which is likely a major factor in the amelioration of diabetes. This suggests that GLP-1/IgG-Fc gene therapy may be applicable to diseases where there is either acute or chronic beta-cell injury.

Asunto(s)

Diabetes Mellitus Tipo 1/terapia , Terapia Genética/métodos , Péptido 1 Similar al Glucagón/genética , Fragmentos Fc de Inmunoglobulinas/genética , Células Secretoras de Insulina/patología , Proteínas Recombinantes de Fusión/administración & dosificación , Animales , Glucemia/análisis , Tamaño de la Célula , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/patología , Electroporación , Exenatida , Expresión Génica , Péptido 1 Similar al Glucagón/sangre , Prueba de Tolerancia a la Glucosa , Inmunoglobulina G/genética , Inyecciones Intramusculares , Insulina/sangre , Ratones , Ratones Endogámicos , Péptidos/genética , Proteínas Recombinantes de Fusión/genética , Ponzoñas/genética

RESUMEN

Type I diabetes mellitus (T1D) is due to a loss of immune tolerance to islet antigen and thus, there is intense interest in developing therapies that can re-establish it. Tolerance is maintained by complex mechanisms that include inhibitory molecules and several types of regulatory T cells (Tr). A major historical question is whether gene therapy can be employed to generate Tr cells. This review shows that gene transfer of immunoregulatory molecules can prevent T1D and other autoimmune diseases. In our studies, non-viral gene transfer is enhanced by in vivo electroporation (EP). This technique can be used to perform DNA vaccination against islet cell antigens and when combined with appropriate immune ligands results in the generation of Tr cells and protection against T1D. In vivo EP can also be applied for non-immune therapy of diabetes. It can be used to deliver protein drugs such as glucagon-like peptide 1 (GLP-1), leptin or transforming growth factor beta (TGF-beta). These act in T1D or type II diabetes (T2D) by restoring glucose homeostasis, promoting islet cell survival and growth or improving wound healing and other complications. Furthermore, we show that in large animals EP can deliver peptide hormones, such as growth hormone releasing hormone (GHRH). We conclude that the non-viral gene therapy and EP represent a safe and efficacious approach with clinical potential.

Asunto(s)

Autoantígenos/genética , Enfermedades Autoinmunes/terapia , Diabetes Mellitus/terapia , Terapia Genética/métodos , Vacunas de ADN/administración & dosificación , Animales , Citocinas/genética , Citocinas/inmunología , Electroporación , Humanos , Leptina/genética , Ratones , Ratones Endogámicos NOD

RESUMEN

Glucagon-like peptide (GLP-1), a major physiological incretin, plays numerous important roles in modulating blood glucose homeostasis and has been proposed for the treatment of type 2 diabetes. The major obstacles for using native GLP-1 as a therapeutic agent are that it must be delivered by a parenteral route and has a short half-life. In an attempt to develop a strategy to prolong the physiological t(1/2) and enhance the potency of GLP-1, a fusion protein consisting of active human GLP-1 and mouse IgG(1) heavy chain constant regions (GLP-1/Fc) was generated. A plasmid encoding an IgK leader peptide-driven secretable fusion protein of the active GLP-1 and IgG(1)-Fc was constructed for mammalian expression. This plasmid allows for expression of bivalent GLP-1 peptide ligands as a result of IgG-Fc homodimerization. In vitro studies employing purified GLP-1/Fc indicate that the fusion protein is functional and elevates cAMP levels in insulin-secreting INS-1 cells. In addition, it stimulates insulin secretion in a glucose concentration-dependent manner. Intramuscular gene transfer of the plasmid in db/db mice demonstrated that expression of the GLP-1/Fc peptide normalizes glucose tolerance by enhancing insulin secretion and suppressing glucagon release. This strategy of using a bivalent GLP-1/Fc fusion protein as a therapeutic agent is a novel approach for the treatment of diabetes.

Asunto(s)

Diabetes Mellitus Tipo 2/terapia , Terapia Genética/métodos , Péptido 1 Similar al Glucagón/genética , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/genética , Proteínas Recombinantes de Fusión/uso terapéutico , Animales , Reactores Biológicos , Glucemia/análisis , Células COS , Línea Celular , Chlorocebus aethiops , AMP Cíclico/sangre , Glucagón/sangre , Semivida , Insulina/sangre , Ratones , Ratones Obesos , Modelos Animales , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Transfección/métodos

RESUMEN

Lupus is a chronic autoimmune inflammatory disease with complex clinical manifestations. In humans, lupus, also known as systemic lupus erythematosus (SLE), affects between 40 and 250 individuals, mostly females, in each 100 000 of the population. There are also a number of murine models of lupus widely used in studies of the genetics, immunopathology, and treatment of lupus. Human patients and murine models of lupus manifest a wide range of immunological abnormalities. The most pervasive of these are: (1) the ability to produce pathogenic autoantibodies; (2) lack of T- and B-lymphocyte regulation; and (3) defective clearance of autoantigens and immune complexes. This article briefly reviews immunological abnormalities and disease mechanisms characteristic of lupus autoimmunity and highlight recent studies on the use of gene therapy to target these abnormalities.

Asunto(s)

Terapia Genética/métodos , Inmunoconjugados , Inmunoterapia/métodos , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/terapia , Abatacept , Animales , Complejo Antígeno-Anticuerpo , Antígenos CD , Antígenos de Diferenciación/genética , Autoanticuerpos/inmunología , Linfocitos B/inmunología , Antígeno CTLA-4 , Proteínas del Sistema Complemento/deficiencia , Citocinas/antagonistas & inhibidores , Citocinas/inmunología , Modelos Animales de Enfermedad , Técnicas de Transferencia de Gen , Predisposición Genética a la Enfermedad , Humanos , Células Asesinas Naturales/inmunología , Lupus Eritematoso Sistémico/genética , Ratones , Fagocitosis , Linfocitos T/inmunología , Factor de Crecimiento Transformador beta/genética

RESUMEN

Viral and nonviral gene therapy vectors have been successfully employed to deliver inflammatory cytokine inhibitors (anticytokines), or anti-inflammatory cytokines, such as transforming growth factor beta-1 (TGF-beta 1), which protect against experimental autoimmune diseases. These vectors carry the relevant genes into a variety of tissues, for either localised or systemic release of the encoded protein. Administration of cDNA encoding soluble IFN-gamma receptor (IFN-gamma R)/IgG-Fc fusion proteins, soluble TNF-alpha receptors, or IL-1 receptor antagonist (IL-1ra), protects against either lupus, various forms of arthritis, autoimmune diabetes, or other autoimmune diseases. These inhibitors, unlike many cytokines, have little or no toxic potential. Similarly, TGF-beta 1 gene therapy protects against numerous forms of autoimmunity, though its administration entails more risk than anticytokine therapy. We have relied on the injection of naked plasmid DNA into skeletal muscle, with or without enhancement of gene transfer by in vivo electroporation. Expression plasmids offer interesting advantages over viral vectors, since they are simple to produce, non-immunogenic and nonpathogenic. They can be repeatedly administered and after each treatment the encoded proteins are produced for relatively long periods, ranging from weeks to months. Moreover, soluble receptors which block cytokine action, encoded by gene therapy vectors, can be constructed from non-immunogenic self elements that are unlikely to be neutralised by the host immune response (unlike monoclonal antibodies [mAbs]), allowing long-term gene therapy of chronic inflammatory disorders.

Asunto(s)

Enfermedades Autoinmunes/terapia , Citocinas/antagonistas & inhibidores , Citocinas/genética , Terapia Genética , Animales , Humanos

RESUMEN

The general phosphodiesterase (PDE) inhibitor pentoxifylline (PTX), and the PDE type IV inhibitor rolipram (ROL), both increase intracellular cAMP levels and suppress inflammatory cytokine production by T cells and macrophages. We have previously shown that PTX and ROL protect from autoimmune diabetes in nonobese diabetic (NOD) mice. These drugs may mediate some of their anti-inflammatory effects by blocking nitric oxide (NO) production by macrophages. In this study, we investigated the effect of PDE inhibitors in blocking NO production by insulin-secreting NIT-1 insulinoma cells and mouse islet cells in vitro and in vivo. Insulinoma cells and islet cells produced NO when stimulated with a combination of inflammatory cytokines and lipopolysaccharide (LPS). We found that both PTX and ROL markedly suppressed this induced NO production. Islet cells express PDEs III and IV and, accordingly, the PDE III inhibitor cilostamide (CIL) also suppressed NO production, and a combination of ROL and CIL had a synergistic effect. This suppression appeared to be mediated, at least in part, by elevating cAMP level and was mimicked by other cAMP-elevating agents, ie, membrane-permeable cAMP analogs (dibutyryl cAMP and 8-bromo cAMP) and an adenylate cyclase stimulator (forskolin). PDE inhibitors suppressed the expression of inducible nitric oxide synthase (iNOS) mRNA. In vivo treatment with PTX or ROL prevented iNOS protein expression in the islets of NOD mice with cyclophosphamide-accelerated disease. Our findings suggest that PDE inhibitors can protect islets against autoimmunity.

Asunto(s)

3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Islotes Pancreáticos/metabolismo , Óxido Nítrico/biosíntesis , Inhibidores de Fosfodiesterasa/farmacología , Animales , Células Cultivadas , Técnicas de Cultivo , AMP Cíclico/biosíntesis , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Femenino , Islotes Pancreáticos/efectos de los fármacos , Ratones , Ratones Endogámicos NOD , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Pentoxifilina/farmacología , Quinolonas/farmacología , ARN Mensajero/biosíntesis , Rolipram/farmacología , Células Tumorales Cultivadas

RESUMEN

Immuno-gene therapy can be advantageously performed with nonviral approaches. Genes that encode regulatory cytokines or inflammatory cytokine inhibitors can be delivered intramuscularly and expressed for weeks or months. This type of gene transfer into muscle has been shown to ameliorate several autoimmune diseases and is relevant to the development of effective DNA vaccines in autoimmune diseases, infectious diseases and cancer.

Asunto(s)

Técnicas de Transferencia de Gen , Inmunoterapia/métodos , Músculo Esquelético , Animales , Presentación de Antígeno , Autoinmunidad/inmunología , Proteínas del Sistema Complemento/inmunología , Citocinas/genética , Citocinas/inmunología , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/prevención & control , Terapia Genética/métodos , Humanos , Tolerancia Inmunológica/inmunología , Inyecciones Intramusculares , Lupus Vulgar/inmunología , Lupus Vulgar/terapia , Vacunas de ADN/administración & dosificación

RESUMEN

We studied the effects of the phosphodiesterase inhibitors pentoxifylline (PTX) and rolipram (ROL) on nitric oxide (NO) production by macrophages and correlated this with cellular cAMP levels. The RAW 264.7 cell line or mouse peritoneal macrophages were activated with lipopolysaccharide (LPS) and interferon gamma (IFN gamma), with or without ROL, PTX, cAMP analogues, or Forskolin. In vivo, peritoneal macrophages were stimulated with staphylococcal enterotoxin B with or without administration of ROL. Nitrite levels in culture and the total cellular cAMP levels were measured. ROL and PTX suppressed NO production of LPS/IFN gamma-stimulated macrophages. ROL (IC(50) = 68-74 microM) was about 40 times more potent than PTX (IC(50) = 2.4-2.9 mM). The suppression paralleled increased total cellular cAMP level (EC(50) = 68-72 microM) and was mimicked by other cAMP elevating agents. ROL and PTX suppressed inducible NO synthase at the mRNA level. The inhibition of NO production of macrophages by ROL or PTX could be beneficial in NO-mediated inflammatory and/or autoimmune disorders.

Asunto(s)

Activación de Macrófagos/efectos de los fármacos , Óxido Nítrico/biosíntesis , Pentoxifilina/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Rolipram/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Bucladesina/farmacología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Colforsina/farmacología , AMP Cíclico/metabolismo , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , GMP Dibutiril Cíclico/farmacología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Enterotoxinas/farmacología , Inducción Enzimática/efectos de los fármacos , Femenino , Interferón gamma/farmacología , Interleucina-12/farmacología , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos NOD , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa/genética , Nitritos/análisis , ARN Mensajero/biosíntesis , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

Intramuscular (i.m.) injection of a plasmid encoding human carcinoembryonic antigen (CEA) elicited immunity against transplanted syngeneic (C57BL/6) CEA-positive Lewis lung carcinoma (CEA/LLC) cells, but tumors still appeared in all mice. In wild-type mice, coinjection of an IL-12 plasmid markedly enhanced anti-CEA humoral, T-helper-1 and cytotoxic T lymphocyte (CTL) responses, and resistance to a CEA/LLC tumor challenge such that 80% of mice remained tumor free. Injection of the IL- 12 plasmid alone was not protective. To analyze immune requirements, we immunized gene knockout (KO) mice of C57BL/6 background, deficient in either CD3, CD4, CD8, interferon gamma (IFNgamma), perforin or Fas ligand (FasL). Only CD3+ mice expressing both CD4 and CD8, which appear equally important, as well as IFNgamma and perforin, could fully resist a tumor challenge. IL-12 stimulated CTL activity, which was strictly CD3/CD8/perforin-dependent. FasL-KO mice had normal CTL activity and tumor resistance, indicating that only the perforin lytic pathway was involved. CD4-KO and IFNgamma-KO mice still generated CTLs. CEA-stimulated IFNgamma production occurred in both CD4- or CD8-KO mice and in both cases was augmented by IL-12. In IFNgamma-KO mice, IL-12 still enhanced anti-CEA antibody production but only moderately restored impaired DTH and tumor resistance. We conclude that the immune requirements for tumor rejection are stringent, involving multiple mechanisms which are all enhanced by IL-12.

Asunto(s)

Antígeno Carcinoembrionario/genética , Terapia Genética/métodos , Interleucina-12/genética , Neoplasias Experimentales/terapia , Vacunas de ADN/administración & dosificación , Animales , Formación de Anticuerpos , Complejo CD3/genética , Antígenos CD4/genética , Antígenos CD8/genética , Antígeno Carcinoembrionario/inmunología , Proteína Ligando Fas , Femenino , Humanos , Inyecciones Intramusculares , Interferón gamma/genética , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Trasplante de Neoplasias , Neoplasias Experimentales/inmunología , Perforina , Plásmidos/administración & dosificación , Proteínas Citotóxicas Formadoras de Poros , Linfocitos T Citotóxicos/inmunología , Células TH1/inmunología , Factores de Tiempo , Células Tumorales Cultivadas

RESUMEN

Gene therapy offers advantages for the immunotherapeutic delivery of cytokines or their inhibitors. After gene transfer, these mediators are produced at relatively constant, non-toxic levels and sometimes in a tissue-specific manner, obviating limitations of protein administration. Therapy with viral or nonviral vectors is effective in several animal models of autoimmunity including Type 1 diabetes mellitus (DM), experimental allergic encephalomyelitis (EAE), systemic lupus erythematosus (SLE), colitis, thyroiditis and various forms of arthritis. Genes encoding transforming growth factor beta, interleukin-4 (IL-4) and IL-10 are most frequently protective. Autoimmune/ inflammatory diseases are associated with excessive production of inflammatory cytokines such as IL-1, IL-12, tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma). Vectors encoding inhibitors of these cytokines, such as IL-1 receptor antagonist, soluble IL-1 receptors, IL-12p40, soluble TNFalpha receptors or IFNgamma-receptor/IgG-Fc fusion proteins are protective in models of either arthritis, Type 1 DM, SLE or EAE. We use intramuscular injection of naked plasmid DNA for cytokine or anticytokine therapy. Muscle tissue is accessible, expression is usually more persistent than elsewhere, transfection efficiency can be increased by low-voltage in vivo electroporation, vector administration is simple and the method is inexpensive. Plasmids do not induce neutralizing immunity allowing repeated administration, and are suitable for the treatment of chronic immunological diseases.

Asunto(s)

Enfermedades Autoinmunes/terapia , Citocinas/antagonistas & inhibidores , Citocinas/genética , Terapia Genética , Animales , Artritis Reumatoide/inmunología , Artritis Reumatoide/terapia , Enfermedades Autoinmunes/inmunología , Células Dendríticas , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/terapia , Vectores Genéticos , Humanos , Inmunoterapia

RESUMEN

IFN-gamma, a pleiotropic cytokine, is a key effector molecule in the pathogenesis of several autoimmune diseases, including lupus. Importantly, deletion of IFN-gamma or IFN-gammaR in several lupus-predisposed mouse strains resulted in significant disease reduction, suggesting the potential for therapeutic intervention. We evaluated whether intramuscular injections of plasmids with cDNA encoding IFN-gammaR/Fc can retard lupus development and progression in MRL-Fas(lpr) mice. Therapy significantly reduced serum levels of IFN-gamma, as well as disease manifestations (autoantibodies, lymphoid hyperplasia, glomerulonephritis, mortality), when treatment was initiated at the predisease stage, particularly when IFN-gammaR/Fc expression was enhanced by electroporation at the injection site. Remarkably, disease was arrested and even ameliorated when this treatment was initiated at an advanced stage. This therapy represents a rare example of disease reversal and makes application of this nonviral gene therapy in humans with lupus (and perhaps other autoimmune/inflammatory conditions) highly promising.

Asunto(s)

ADN Complementario/uso terapéutico , Fragmentos Fc de Inmunoglobulinas/uso terapéutico , Lupus Eritematoso Sistémico/terapia , Receptores de Interferón/inmunología , Animales , Cromatina/inmunología , Predisposición Genética a la Enfermedad , Terapia Genética/métodos , Hiperplasia , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/genética , Inmunoglobulina G/uso terapéutico , Inyecciones Intramusculares , Interferón gamma/inmunología , Riñón/patología , Lupus Eritematoso Sistémico/mortalidad , Tejido Linfoide/efectos de los fármacos , Tejido Linfoide/patología , Ratones , Ratones Endogámicos MRL lpr , Pruebas de Neutralización , Proteínas Recombinantes de Fusión/uso terapéutico , Receptor de Interferón gamma

RESUMEN

Intramuscular (i.m.) injections of a plasmid encoding human carcinoembryonic antigen (CEA) elicited both humoral and cellular immune responses in mice, but only partial inhibition of the growth of transplanted syngeneic CEA-positive P815 tumor cells (CEA/P815). Coinjection of the CEA vector with a vector encoding either interferon-gamma (IFN gamma) or IL-12 promoted IgG2a isotype anti-CEA antibody production, anti-CEA/P815 CTL activity and greater resistance to CEA/P815 tumor challenge. As well, CEA/P815-stimulated IFN gamma secretion in vitro was increased, but IL-4 diminished, consistent with a T-helper type 1 (Th1) response. In contrast, coinjection of the CEA vector with an IL-4 vector increased IgG1 production, but reduced CTL activity and resistance to tumor challenge. The latter treatment inhibited CEA/P815-dependent IFN gamma production but enhanced IL-4 secretion, consistent with a Th type 2 (Th2) response. Antitumor immunity was enhanced when the CEA and IL-12 plasmids were coinjected at the same muscle site, but not at separate sites despite increased serum IL-12 levels. Though the tumor cells expressed neomycin phosphotransferase, mice immunized with vectors encoding that protein (without CEA) were not protected against tumor growth, and produced no CTLs except for low levels when coinjected with an IL-12 vector. Thus, we show that immunity elicited by DNA vaccination against CEA can be biased to a protective type (high Th1 and CTL activity) or nonprotective type (high Th2 and low CTL activity) by i.m. coinjection of cytokine-expressing plasmids. IL-12 appears to act locally, but not systemically, through an adjuvant effect.

Asunto(s)

Antígeno Carcinoembrionario/genética , Citocinas/genética , Terapia Genética/métodos , Inmunoterapia Activa/métodos , Neoplasias/terapia , Linfocitos T Colaboradores-Inductores/inmunología , Análisis de Varianza , Animales , Formación de Anticuerpos/genética , Células COS , Femenino , Técnicas de Transferencia de Gen , Humanos , Interferón gamma/genética , Interleucina-12/genética , Ratones , Ratones Endogámicos DBA , Trasplante de Neoplasias , Linfocitos T Citotóxicos/inmunología , Células TH1/inmunología , Células Th2/inmunología , Células Tumorales Cultivadas , Vacunas de ADN/administración & dosificación

RESUMEN

The importance of transforming growth factor-beta-1 (TGF-beta1) in immunoregulation and tolerance has been increasingly recognized. It is now proposed that there are populations of regulatory T cells (T-reg), some designated T-helper type 3 (Th3), that exert their action primarily by secreting this cytokine. Here, we emphasize the following concepts: (1) TGF-beta1 has multiple suppressive actions on T cells, B cells, macrophages, and other cells, and increased TGF-beta1 production correlates with protection and/or recovery from autoimmune diseases; (2) TGF-beta1 and CTLA-4 are molecules that work together to terminate immune responses; (3) Th0, Th1 and Th2 clones can all secrete TGF-beta1 upon cross-linking of CTLA-4 (the functional significance of this in autoimmune diseases has not been reported, but TGF-beta1-producing regulatory T-cell clones can produce type 1 inflammatory cytokines); (4) TGF-beta1 may play a role in the passage from effector to memory T cells; (5) TGF-beta1 acts with some other inhibitory molecules to maintain a state of tolerance, which is most evident in immunologically privileged sites, but may also be important in other organs; (6) TGF-beta1 is produced by many cell types, is always present in the plasma (in its latent form) and permeates all organs, binding to matrix components and creating a reservoir of this immunosuppressive molecule; and (7) TGF-beta1 downregulates adhesion molecules and inhibits adhesion of leukocytes to endothelial cells. We propose that rather than being passive targets of autoimmunity, tissues and organs actively suppress autoreactive lymphocytes. We review the beneficial effects of administering TGF-beta1 in several autoimmune diseases, and show that it can be effectively administered by a somatic gene therapy approach, which results in depressed inflammatory cytokine production and increased endogenous regulatory cytokine production.

Asunto(s)

Enfermedades Autoinmunes/tratamiento farmacológico , Inmunoconjugados , Factor de Crecimiento Transformador beta/farmacología , Abatacept , Adyuvantes Inmunológicos/farmacología , Animales , Antígenos CD , Antígenos de Diferenciación/metabolismo , Enfermedades Autoinmunes/inmunología , Antígeno CTLA-4 , División Celular/efectos de los fármacos , Citocinas/metabolismo , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/inmunología , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Inmunosupresores/farmacología , Mediadores de Inflamación/metabolismo , Ratones , Ratones Noqueados , Receptores de Factores de Crecimiento Transformadores beta/inmunología , Transducción de Señal , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo

RESUMEN

We report on long-term delivery of an interferon-gamma (IFN gamma) inhibitory protein by intramuscular (i.m.) gene therapy. IFN gamma is a cytokine that plays an important role in many inflammatory disorders, including autoimmune insulin-dependent diabetes mellitus (IDDM) in NOD mice and (in various strains) multiple low-dose streptozotocin (STZ)-induced diabetes (MDSD). By cDNA insertion into plasmid VICAL VR-1255 we constructed an expression vector encoding a soluble IFN gamma receptor/IgG1 heavy chain (all murine) fusion protein (IFN gamma R/IgG1). This protein is secreted as a homodimer and neutralizes IFN gamma in vitro. We show that i.m. injections of this vector as naked DNA in mice results in secretion of IFN gamma R/IgG1, with serum levels exceeding 100 ng/ml for months after treatment. These levels are sufficient to neutralize IFN gamma in vivo, and to prevent either MDSD or cyclophosphamide (CYP)-accelerated diabetes in NOD mice, which are both characterized by systemic release of IFN gamma. In these diseases gene therapy considerably reduces inflammation in the islets of Langerhans (insulitis). Also, circulating IFN gamma R/IgG1 blocked IFN gamma-enhanced nitric oxide production by peritoneal macrophages. The fusion protein is constructed from non-immunogenic self elements, avoiding a neutralizing immune response and making it suitable for prolonged therapy of numerous inflammatory disorders.

Asunto(s)

Diabetes Mellitus Tipo 1/prevención & control , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Inmunoglobulina G/genética , Interferón gamma/metabolismo , Receptores de Interferón/genética , Animales , Línea Celular , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/prevención & control , Diabetes Mellitus Tipo 1/patología , Femenino , Vectores Genéticos/uso terapéutico , Cadenas Pesadas de Inmunoglobulina/genética , Inyecciones Intramusculares , Islotes Pancreáticos/patología , Activación de Macrófagos , Ratones , Ratones Endogámicos NOD , Plásmidos , Receptores de Interferón/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo

RESUMEN

Antiinflammatory cytokines such as transforming growth factor beta1 (TGF-beta1) and interleukin 4 (IL-4) can protect from autoimmune diseases. To study the immunoregulatory effects of these cytokines in vivo, we used a method of gene therapy that permits continuous cytokine delivery over a period of weeks. We injected naked plasmid DNA expression vectors encoding either TGF-beta1 (pVR-TGF-beta1) or an IL-4-IgG1 chimeric protein (pVR-IL-4-IgG1) intramuscularly. This resulted in production of TGF-beta1 or IL-4-IgG1, respectively, and protection from myelin basic protein (MBP)-induced experimental allergic encephalomyelitis (EAE). TGF-beta1 gene delivery had pronounced downregulatory effects on T cell proliferation and production of interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha), on in vitro restimulation with MBP. IL-4-IgG1 vector administration also suppressed these responses, although much less than TGF-beta1, and enhanced secretion of endogenous IL-4. Therapy resulted in a significant decrease in the severity of histopathologic inflammatory lesions. In the CNS, treatment with either vector suppressed IL-12 and IFN-gamma mRNA expression, while IL-4 and TGF-beta1 mRNA levels were increased compared with control mice. Thus, cytokine plasmid treatment appeared to inhibit MBP-specific pathogenic Thl responses, while enhancing endogenous secretion of protective cytokines. We demonstrate that gene therapy with these vectors is an effective therapeutic strategy for EAE.

Asunto(s)

Citocinas/genética , Encefalomielitis Autoinmune Experimental/prevención & control , Técnicas de Transferencia de Gen , Terapia Genética , Vectores Genéticos/administración & dosificación , Plásmidos/genética , Animales , Citocinas/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Inyecciones Intramusculares , Interleucina-4/genética , Interleucina-4/metabolismo , Activación de Linfocitos , Ratones , Proteína Básica de Mielina/efectos adversos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/inmunología , Linfocitos T/inmunología , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/inmunología

RESUMEN

Transplantation of microencapsulated islets of Langerhans is impaired by a pericapsular host reaction that eventually induces graft failure. We are studying the role of cytokines in the pathogenesis of this reaction, using the model of alginate-polylysine microcapsule implantation in rat epididymal fat pads. The objectives were: (1) to develop a method to measure, by semiquantitative PCR, TGF-beta1 gene expression in fat pad pericapsular infiltrates, and (2) to use this method to evaluate TGF-beta1 gene expression 14 days after microcapsule implantation. TGF-beta1 mRNA level was significantly higher in pericapsular infiltrate cells than in nonimplanted tissue cells and saline-injected tissue cells (p < 0.0001 and p < 0.01, respectively). There was no significant difference between the TGF-beta1 mRNA levels of the two types of controls (p = 0.0945). These results suggest that TGF-beta1 plays a role in the pathogenesis of the pericapsular reaction. The method developed can be used to study the role of other fibrogenic cytokines potentially involved. This will shed light on the mechanisms underlying the pericapsular reaction and will serve as a basis for the development of strategies to control this reaction.

Asunto(s)

Alginatos/toxicidad , Materiales Biocompatibles/toxicidad , Citocinas/biosíntesis , Polilisina/análogos & derivados , Tejido Adiposo/enzimología , Tejido Adiposo/inmunología , Animales , Cápsulas , Recuento de Células , Citocinas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Reacción Huésped-Injerto , Inmunidad Celular/efectos de los fármacos , Masculino , Ensayo de Materiales , Polilisina/toxicidad , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta/biosíntesis , Factor de Crecimiento Transformador beta/genética

RESUMEN

BACKGROUND: Interferon gamma (IFNgamma) is an inflammatory cytokine that promotes autoimmune insulitis and diabetes in NOD mice, while interleukin-4 (IL-4) is protective. We constructed plasmids encoding either an IFNgamma receptor/IgG1 (IFNgammaR/IgG1) chimeric protein which inhibits IFNgamma, or an IL-4/IgG1 chimeric protein with IL-4 activity, for therapeutic gene transfer into NOD mice. METHODS: Murine IFNgammaR/IgG1 and IL-4/IgG1 cDNA segments were cloned into the VICAL VR1255 expression plasmid. Naked plasmid DNA was injected i.m. into young NOD mice, which were then observed for development of insulitis and diabetes. RESULTS: After transient transfection of COS-7 cells, IFNgammaR/IgG1 and IL-4/IgG1 fusion proteins are secreted in vitro as disulfide-linked homodimers, with the expected biological activity. Intramuscular injection of these vectors results in the production of the respective fusion proteins locally in muscle. In serum, the IFNgammaR/IgG1 protein is present at >200 ng/ml over 130 days after the last of five DNA injections, but IL-4/IgG1 is undetectable in our assays (<10 pg/ml) at all time points. Both vectors protect NOD mice from autoimmune insulitis and diabetes, but the IL-4/IgG1 vector is more effective. Neutralization of IFNgamma with IFNgammaR/IgG1 was most protective when treatment was begun early (3 weeks of age). CONCLUSION: Gene therapy by i.m. injection of these plasmids protects NOD mice from autoimmunity, and the IL-4/IgG1 vector is more effective despite low circulating protein levels. These chimeric proteins consist of nonimmunogenic self elements and are suitable for long-term therapy of autoimmune disorders.

Asunto(s)

Enfermedades Autoinmunes/prevención & control , Diabetes Mellitus Tipo 1/prevención & control , Terapia Genética , Vectores Genéticos/administración & dosificación , Inmunoglobulina G/genética , Interferón gamma/fisiología , Interleucina-4/genética , Islotes Pancreáticos/patología , Pancreatitis/prevención & control , Receptores de Interferón/genética , Proteínas Recombinantes de Fusión/genética , Animales , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Células COS , Línea Celular , Chlorocebus aethiops , ADN Recombinante/administración & dosificación , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Femenino , Genes Sintéticos , Vectores Genéticos/genética , Inmunoglobulina G/fisiología , Inyecciones Intramusculares , Interferón gamma/farmacología , Interleucina-4/fisiología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Endogámicos NOD , Pancreatitis/genética , Pancreatitis/inmunología , Receptores de Interferón/fisiología , Proteínas Recombinantes de Fusión/fisiología , Organismos Libres de Patógenos Específicos , Transfección , Receptor de Interferón gamma

RESUMEN

Nonobese diabetic (NOD) mice develop insulitis and diabetes through an autoimmune process. Since TGF-beta1 down-regulates many immune responses, we hypothesized that TGF-beta1 could prevent disease in NOD mice and that there would be several advantages to cytokine delivery by a somatic gene therapy approach. We opted for i.m. injection of a naked plasmid DNA expression vector encoding murine TGF-beta1 (pCMV-TGF-beta1). Treatment with pCMV-TGF-beta1 resulted in the retention and expression of the vector in muscle cells, associated with a considerable elevation in the plasma levels of TGF-beta1, that was not observed in control vector-treated mice. The levels of TGF-beta1 produced were sufficient to exert immunosuppressive effects. Delayed-type hypersensitivity responses were suppressed, and autoimmunity-prone NOD mice were protected from insulitis and diabetes in models of cyclophosphamide-accelerated and natural course disease. In pCMV-TGF-beta1-treated mice, pancreatic IL-12 and IFN-gamma mRNA expression was depressed, and the ratio of IFN-gamma to IL-4 mRNA was decreased, as determined by semiquantitative reverse-transcription PCR. In contrast, NOD mice injected with a vector encoding the proinflammatory cytokine IFN-gamma developed diabetes earlier. Intramuscular administration of cytokine-encoding plasmid vectors proved to be an effective method of cytokine delivery in these mice, and altered autoimmune disease expression.

Asunto(s)

Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/prevención & control , Terapia Genética , Factor de Crecimiento Transformador beta/genética , Animales , Autoinmunidad/genética , Diabetes Mellitus Tipo 1/inmunología , Técnicas de Transferencia de Gen , Ratones , Ratones Endogámicos NOD

RESUMEN

Interleukin (IL)-12, interferon (IFN)-gamma, and other inflammatory cytokines play an important role in the pathogenesis of autoimmune insulitis and diabetes in NOD mice, and inhibition of these cytokines is likely to be beneficial. In this study, we found that Pentoxifylline (PTX) and Rolipram (phosphodiesterase [PDE] inhibitors that induce increased intracellular cAMP) can block inflammatory cytokine production. Inhibition of IL-12 and IFN-gamma secretion was demonstrated in macrophages activated with lipopolysaccharide or T-cells stimulated through the CD3/T-cell receptor complex, respectively. Moreover, strong inhibition of IL-12 was demonstrated in vivo in superantigen-immunized mice. Rolipram was inhibitory at concentrations as low as 10(-8) to 10(-7) mol/l, and on a molar basis, it was 100-fold more effective than PTX. Tumor necrosis factor-alpha was also inhibited, but IL-4 was less sensitive to suppression. In NOD mice, both PTX and Rolipram reduced the severity of insulitis and prevented diabetes, with or without cyclophosphamide administration (which precipitates onset of disease). This protection of NOD mice was still apparent over 10 weeks after withdrawal of the drug treatment. It appears that blocking the activity of type IV PDE is sufficient to mediate the effects reported in this study, since Rolipram inhibits only this isoform, unlike PTX (a general inhibitor). PTX and Rolipram may be effective in the treatment of autoimmune diabetes or other conditions characterized by excessive production of inflammatory cytokines.

Asunto(s)

3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Diabetes Mellitus Tipo 1/prevención & control , Pentoxifilina/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Pirrolidinonas/farmacología , Animales , Anticuerpos Monoclonales/inmunología , Complejo CD3/inmunología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Ciclofosfamida/farmacología , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/fisiopatología , Femenino , Inmunosupresores/farmacología , Técnicas In Vitro , Interferón gamma/antagonistas & inhibidores , Interleucina-12/antagonistas & inhibidores , Interleucina-4/antagonistas & inhibidores , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Páncreas/efectos de los fármacos , Páncreas/inmunología , Cavidad Peritoneal/citología , Rolipram , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Superantígenos/inmunología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores

RESUMEN

In this study we compare the effects of cyclosporin A (CsA), FK520 (an agent similar to FK506), and rapamycin (RAPA) on peripheral T-cell deletion induced by either superantigens or anti-TCR alpha beta mAb, and on anergy induced by superantigens in mice. CsA enhanced T-cell deletion and blocked anergy induction (in residual T cells), while FK520 and RAPA had no effects on these processes. CsA also enhanced apoptosis of stimulated T cells in vitro, where cell death occurred without prior proliferation and in the absence of phagocytes. Our data suggest that CsA exerts these effects through a calcineurin-independent pathway, and this may be relevant to the development of tolerance in some models.

Asunto(s)

Inmunosupresores/farmacología , Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Linfocitos T CD4-Positivos/inmunología , Anergia Clonal/efectos de los fármacos , Supresión Clonal/efectos de los fármacos , Ciclosporina/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Polienos/farmacología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Sirolimus , Superantígenos/inmunología , Linfocitos T/efectos de los fármacos , Tacrolimus/análogos & derivados , Tacrolimus/farmacología