ABSTRACT
Using a combination of local gene delivery and tolerizing DNA vaccination, we demonstrate that codelivery of the interleukin-4 (IL-4) gene and a DNA vaccine encoding the self-peptide proteolipid protein 139-151 (PLP139-151) provides protective immunity against experimental autoimmune encephalomyelitis (EAE). We provide evidence for a mechanism whereby IL-4 expressed from the naked DNA is secreted and acts locally on autoreactive T cells via activation of STAT6 to shift their cytokine profile to T helper 2. We also show that DNA vaccines can be used to reverse established EAE by covaccination with the genes for myelin oligodendrocyte glycoprotein and IL-4. This treatment strategy combines the antigen-specific effects of DNA vaccination and the beneficial effects of local gene delivery.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental/prevention & control , Genetic Therapy , Interleukin-4/genetics , Th1 Cells/physiology , Th2 Cells/physiology , Vaccines, DNA/immunology , Animals , Female , Humans , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , STAT6 Transcription Factor , Trans-Activators/metabolism , VaccinationABSTRACT
DNA vaccination is an effective means of protecting experimental animals against infectious pathogens and cancer and has more recently been used to prevent autoimmune disease. Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease characterized by T-cell-mediated destruction of the insulin-secreting beta cells in the pancreas. The NOD mouse is an animal model of IDDM in which several autoantigens, including insulin, have been identified. In this study we demonstrate that vaccination of NOD mice with DNA encoding an immunodominant peptide of insulin (residues 9-23 of the B chain) protects the animals from developing diabetes. Animals injected intramuscularly with a bacterial plasmid encoding the insulin B chain peptide show significantly lower disease incidence and delayed onset of disease when compared to controls. Protection appears to be mediated by insulin B (9-23)-specific down-regulation of IFN-gamma. Our results confirm that DNA vaccination has a protective effect on autoimmunity, the understanding of which will reveal new insights into the immune system and open doors for novel therapies.
Subject(s)
DNA/immunology , Diabetes Mellitus, Type 1/immunology , Insulin/immunology , Animals , Autoimmunity , Diabetes Mellitus, Type 1/prevention & control , Female , Immunization , Immunodominant Epitopes , Mice , Mice, Inbred NOD , Peptides/immunology , RatsABSTRACT
The protein huntingtin (htt), aggregated in neuronal nuclear inclusions, is pathognomonic of Huntington's disease (HD). Constructs, translated in vitro from the N terminus of htt, containing either polyQ23 from a normal individual, or polyQ41 or polyQ67 from an HD patient, were all soluble. Transglutaminase (TGase) crosslinked these proteins, and the aggregations did not have the staining properties of amyloid. More TGase-catalyzed aggregates formed when the polyglutamine domain of htt exceeded the pathologic threshold of polyQ36. Furthermore, shorter htt constructs, containing 135 aa or fewer, formed more aggregates than did larger htt constructs. TGase activity in the HD brain was increased compared with the control, with notable increases in cell nuclei. The increased TGase activity was brain specific. In lymphoblastoid cells from HD patients, TGase activity was decreased. TGase-mediated crosslinking of htt may be involved in the formation of the nonamyloidogenic nuclear inclusions found in the HD brain. The staining properties of nuclear inclusions in the HD brain revealed that they were not amyloid.
Subject(s)
Brain/metabolism , Huntington Disease/metabolism , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Transglutaminases/metabolism , Adult , Aged , Aged, 80 and over , Brain/ultrastructure , Cell Nucleus/metabolism , Cross-Linking Reagents , Dimerization , Enzyme Activation , Humans , Huntingtin Protein , Male , Middle Aged , Peptide Fragments/metabolismABSTRACT
Molecular mimicry refers to structural homologies between a self-protein and a microbial protein. A major epitope of myelin basic protein (MBP), p87-99 (VHFFKNIVTPRTP), induces experimental autoimmune encephalomyelitis (EAE). VHFFK contains the major residues for binding of this self-molecule to T cell receptor (TCR) and to the major histocompatibility complex. Peptides from papilloma virus strains containing the motif VHFFK induce EAE. A peptide from human papilloma virus type 40 (HPV 40) containing VHFFR, and one from HPV 32 containing VHFFH, prevented EAE. A sequence from Bacillus subtilis (RKVVTDFFKNIPQRI) also prevented EAE. T cell lines, producing IL-4 and specific for these microbial peptides, suppressed EAE. Thus, microbial peptides, differing from the core motif of the self-antigen, MBPp87-99, function as altered peptide ligands, and behave as TCR antagonists, in the modulation of autoimmune disease.
Subject(s)
Bacterial Proteins/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , Epitopes/immunology , Ligands , Peptide Fragments/immunology , T-Lymphocytes/immunology , Amino Acid Sequence , Animals , Cell Division/immunology , Encephalomyelitis, Autoimmune, Experimental/prevention & control , Female , Interferon-gamma/metabolism , Interleukin-4/metabolism , Mice , Mice, Inbred Strains , Molecular Sequence Data , Myelin Basic Protein/immunology , Receptors, Antigen, T-Cell/antagonists & inhibitorsABSTRACT
Usually we rely on vaccination to promote an immune response to a pathogenic microbe. In this study, we demonstrate a suppressive from of vaccination, with DNA encoding a minigene for residues 139-151 of myelin proteolipid protein (PLP139-151), a pathogenic self-Ag. This suppressive vaccination attenuates a prototypic autoimmune disease, experimental autoimmune encephalomyelitis, which presents clinically with paralysis. Proliferative responses and production of the Th1 cytokines, IL-2 and IFN-gamma, were reduced in T cells responsive to PLP139-151. In the brains of mice that were successfully vaccinated, mRNA for IL-2, IL-15, and IFN-gamma were reduced. A mechanism underlying the reduction in severity and incidence of paralytic autoimmune disease and the reduction in Th1 cytokines involves altered costimulation of T cells; loading of APCs with DNA encoding PLP139-151 reduced the capacity of a T cell line reactive to PLP139-151 to proliferate even in the presence of exogenous CD28 costimulation. DNA immunization with the myelin minigene for PLP-altered expression of B7.1 (CD80), and B7.2 (CD86) on APCs in the spleen. Suppressive immunization against self-Ags encoded by DNA may be exploited to treat autoimmune diseases.
Subject(s)
Autoantigens/genetics , Autoantigens/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/prevention & control , Immunosuppressive Agents/immunology , Lymphocyte Activation , T-Lymphocytes/immunology , Vaccines, DNA/immunology , Animals , Autoantigens/administration & dosage , Base Sequence , Encephalomyelitis, Autoimmune, Experimental/etiology , Female , Immunosuppressive Agents/administration & dosage , Injections, Intramuscular , Lymphocyte Activation/genetics , Mice , Mice, Inbred Strains , Molecular Sequence Data , Myelin Proteolipid Protein/administration & dosage , Myelin Proteolipid Protein/genetics , Myelin Proteolipid Protein/immunology , Peptide Fragments/administration & dosage , Peptide Fragments/genetics , Peptide Fragments/immunology , Th1 Cells/immunology , Vaccines, DNA/administration & dosageABSTRACT
The p53 molecule might serve as a common tumor-associated antigen, as the tumor suppressor gene p53 is mutated and the p53 protein is often over-expressed in tumor cells. We report that effective immunity to p53 can be induced through an idiotypic network by immunization of mice with a monoclonal antibody (PAb-240) specific for mutated p53, or with a peptide derived from the complementarity determining region (CDR) 3 of the variable domain of the light chain (VL) of this antibody. The immunized mice produced IgG antibodies to p53 and mounted a cytotoxic reaction to a tumor line bearing mutated p53. The idiotypically immunized mice were resistant to challenge with the tumor cells. Thus antibodies to p53 might serve as immunogens for activating resistance to some tumors. At the basic level, these findings indicate that a network of p53 immunity may be organized naturally within the immune system.
Subject(s)
Antibodies, Anti-Idiotypic/immunology , Antibodies, Monoclonal/immunology , Neoplasms, Experimental/immunology , Tumor Suppressor Protein p53/immunology , Animals , Antibodies, Anti-Idiotypic/administration & dosage , Antibodies, Monoclonal/administration & dosage , Antibody Formation/drug effects , Epitopes/administration & dosage , Epitopes/chemistry , Epitopes/immunology , Female , Graft Rejection/genetics , Graft Rejection/immunology , Immunity/drug effects , Immunoglobulin Light Chains/genetics , Immunoglobulin Light Chains/immunology , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/immunology , Mice , Mice, Inbred BALB C , Mutation/genetics , Neoplasm Transplantation/immunology , Neoplasms, Experimental/physiopathology , Oligopeptides/administration & dosage , Oligopeptides/chemical synthesis , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/immunology , Tumor Suppressor Protein p53/genetics , VaccinationABSTRACT
The transformation of an unpatterned epithelium into a patterned one is a fundamental issue in morphogenesis. This transformation occurs in a dramatic fashion in the developing eye imaginal disc, the primordium of the Drosophila compound eye. Molecular and developmental analyses reveals that the sine oculis (so) locus encodes a homeodomain-containing protein that is expressed and required in the unpatterned epithelium prior to morphogenesis. In mutants, cells undergo apoptosis. These findings argue that so plays an essential role in controlling the initial events of pattern formation in the eye disc. So is also expressed and required for the development of the rest of the fly visual system, including the optic lobes (i.e., those regions of the brain that process visual information). So is expressed in the optic lobe primordium prior to its invagination from the embryonic ectoderm; in so mutants, the optic lobe primordium fails to invaginate.
Subject(s)
Drosophila Proteins , Drosophila/genetics , Eye Proteins/genetics , Eye/growth & development , Gene Expression , Genes, Homeobox , Homeodomain Proteins , Mutation , Alleles , Amino Acid Sequence , Animals , Apoptosis , Base Sequence , Cell Differentiation , Cell Division , DNA/chemistry , DNA/metabolism , Eye/cytology , Eye/ultrastructure , Eye Proteins/chemistry , Introns , Microscopy, Electron, Scanning , Molecular Sequence Data , Morphogenesis , Nervous System/growth & development , Nervous System Physiological Phenomena , Protein Structure, Secondary , Sequence Deletion , Sequence Homology, Amino Acid , Transcription, Genetic , Vision, Ocular/geneticsABSTRACT
The Drosophila anachronism (ana) locus controls the proliferation of neuroblasts, neuronal stem cells that give rise to the central nervous system. In ana mutants, quiescent postembryonic central brain and optic lobe neuroblasts enter S phase precociously. ana encodes a novel secreted protein of 474 amino acids that is expressed not in the affected neuroblasts, but rather in a subclass of neighboring glial cells. These studies argue for an important role for glia in negatively regulating proliferation of neuronal precursor cells, thereby controlling the timing of postembryonic neurogenesis.
Subject(s)
Drosophila Proteins , Drosophila/genetics , Glycoproteins/genetics , Neuroglia/metabolism , Amino Acid Sequence , Animals , Base Sequence , Brain/embryology , Cell Differentiation/drug effects , Cell Division/drug effects , Cells, Cultured/drug effects , DNA Mutational Analysis , Drosophila/embryology , Glycoproteins/pharmacology , Molecular Sequence Data , Mutation , Neurons/drug effects , Stem Cells/drug effectsABSTRACT
We have examined the distribution of immunoreactivity for the monoclonal antibody Cat-301 in visual cortex of the macaque monkey. Remarkably, those portions of striate cortex (V1) and extrastriate cortex that are most immunoreactive for Cat-301 are anatomically interconnected and are dominated by inputs arising from the magnocellular layers of the LGN (which are themselves highly immunoreactive). In particular, we found that a band of Cat-301 labeled neurons known to exist in layer 4 of V1 is centered on the boundary between layers 4C alpha and 4B and thus includes portions of both the primary target of the magnocellular LGN and its subsequent relay through layer 4B. We also demonstrated consistently strong Cat-301 immunoreactivity in all three extrastriate targets of layer 4B: areas V3, MT, and the cytochrome-oxidase (CO) enriched thick stripes of V2. In V2, there was a close correlation between Cat-301 labeling and clusters of cells projecting to MT but not to V4. This was true even in regions where the CO pattern was equivocal or irregular, indicating that Cat-301 is a more reliable marker than CO for the thick-stripe subregions of V2. Finally, we found strong Cat-301 immunoreactivity in at least parts of areas V3A, the MST complex, and the posterior parietal complex, but not in area V4 or inferotemporal cortex. The molecular specificity revealed by this single marker thus correlates with functionally specific subdivisions at each hierarchical level over nearly the entire known extent of the visual pathway in macaques. This supports the notion that these subdivisions form an anatomically, physiologically, and now molecularly distinct pathway known as the M-stream.