ovalbumin and Necrosis

Numerous alterations in CD8

Topics: Animals; Antigens; CD8-Positive T-Lymphocytes; Mice; Mice, Inbred C57BL; Mice, Transgenic; Necrosis; Ovalbumin; Up-Regulation

Ischemic diseases are a leading cause of mortality and can result in autoamputation of lower limbs. We explored the hypothesis that implantation of an antigen-releasing scaffold, in animals previously vaccinated with the same antigen, can concentrate T

Topics: Adjuvants, Immunologic; Allergens; Aluminum; Animals; Antigens; Female; Humans; Ischemia; Mice; Muscle, Skeletal; Myofibrils; Necrosis; Ovalbumin; T-Lymphocytes; Th2 Cells; Vaccines

Cross-priming of CD8+ T cells and generation of effector immune responses is pivotal for tumor immunity as well as for successful anticancer vaccination and therapy. Dead and dying cells produce signals that can influence Ag processing and presentation; however, there is conflicting evidence regarding the immunogenicity of necrotic cell death. We used a mouse model of sterile necrosis, in which mice were injected with sterile primary necrotic cells, to investigate a role of these cells in priming of CD8+ T cells. We discovered a molecular mechanism operating in Ag donor cells that regulates cross-priming of CD8+ T cells during primary sterile necrosis and thereby controls adaptive immune responses. We found that the cellular peptidases dipeptidyl peptidase 3 (DPP-3) and thimet oligopeptidase 1 (TOP-1), both of which are present in nonimmunogenic necrotic cells, eliminated proteasomal degradation products and blocked Ag cross-presentation. While sterile necrotic tumor cells failed to induce CD8+ T cell responses, their nonimmunogenicity could be reversed in vitro and in vivo by inactivation of DPP-3 and TOP-1. These results indicate that control of cross-priming and thereby immunogenicity of primary sterile necrosis relies on proteasome-dependent oligopeptide generation and functional status of peptidases in Ag donor cells.

Topics: Adaptive Immunity; Animals; Cell Line, Tumor; Cross-Priming; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Female; Metalloendopeptidases; Mice; Mice, Inbred C57BL; Models, Immunological; Necrosis; Ovalbumin; RNA, Small Interfering; T-Lymphocytes

Apoptotic cells are significantly more immunogenic than necrotic cells, even though both forms are identical in antigenic content. When a combination of apoptotic and necrotic cells are used to immunize, the phenotype conferred by apoptotic cells, i.e., high immunogenicity, is dominant. However, necrotic cells are not immunosuppressive or tolerogenic. Apoptotic and necrotic cells are taken up by antigen-presenting cells in an equivalent manner. The priming of naïve T cell response is also equivalent. However, the CD8+ T cells elicited by apoptotic cells expand, accumulate, and express effector function, while those primed by the necrotic cells do not. This dichotomy does not extend to CD4+ cells. Apoptotic and necrotic cells elicit equivalent CD4+ T cell priming, accumulation, and function. The deficit in CD8+ T cell function elicited by necrotic cells can be overcome to varying degrees by anti-CD40 antibody and ligands for TLR4 and TLR9; conversely, the immunogenicity of apoptotic cells can be abrogated by blocking anti-CD154 antibody. Our results indicate that immunization with apoptotic cells leads to engagement of CD40 on antigen-presenting cells; this is essential for their ability to elicit mature functional CD8+ cells. The necrotic cells fail to engage CD40, and this failure is the basis of their lack of immunogenicity. These differences have consequences for the understanding of mechanisms of cross-presentation and for efforts toward immunotherapy of cancers and autoimmune pathologies.

Topics: Amino Acid Sequence; Animals; Antigen-Presenting Cells; Apoptosis; CD4-Positive T-Lymphocytes; CD40 Antigens; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cross-Priming; Cytokines; Cytoprotection; Freezing; Gamma Rays; Immune Tolerance; Immunization; Immunosuppressive Agents; Lipopolysaccharides; Mice; Molecular Sequence Data; Necrosis; Oligodeoxyribonucleotides; Ovalbumin; Peptides; Phenotype; T-Lymphocytes, Cytotoxic; Toll-Like Receptor 9

DNGR-1 (CLEC9A) is a receptor for necrotic cells required by DCs to cross-prime CTLs against dead cell antigens in mice. It is currently unknown how DNGR-1 couples dead cell recognition to cross-priming. Here we found that DNGR-1 did not mediate DC activation by dead cells but rather diverted necrotic cell cargo into a recycling endosomal compartment, favoring cross-presentation to CD8(+) T cells. DNGR-1 regulated cross-priming in non-infectious settings such as immunization with antigen-bearing dead cells, as well as in highly immunogenic situations such as infection with herpes simplex virus type 1. Together, these results suggest that DNGR-1 is a dedicated receptor for cross-presentation of cell-associated antigens. Our work thus underscores the importance of cross-priming in immunity and indicates that antigenicity and adjuvanticity can be decoded by distinct innate immune receptors. The identification of specialized receptors that regulate antigenicity of virus-infected cells reveals determinants of antiviral immunity that might underlie the human response to infection and vaccination.

Topics: Alphavirus Infections; Animals; Antigen Presentation; Antigens, Surface; CD8-Positive T-Lymphocytes; Cells, Cultured; Cross-Priming; Dendritic Cells; Endocytosis; Fibroblasts; Herpes Simplex; Humans; Lectins, C-Type; Lung; Mice; Mice, Inbred C57BL; Myeloid Cells; Necrosis; Ovalbumin; Protein Transport; Receptors, Immunologic; Recombinant Proteins; Toll-Like Receptor 3

Terpenoids are ubiquitous natural compounds that have been shown to improve vaccine efficacy as adjuvants. To gain an understanding of the structural features important for adjuvanticity, we studied compounds derived from a diterpene phytol and assessed their efficacy. In a previous report, we showed that phytol and one of its derivatives, PHIS-01 (a phytol-derived immunostimulant, phytanol), are excellent adjuvants. To determine the effects of varying the polar terminus of PHIS-01, we designed amine and mannose-terminated phytol derivatives (PHIS-02 and PHIS-03, respectively). We studied their relative efficacy as emulsions with soluble proteins, ovalbumin, and a hapten-protein conjugate phthalate-KLH. Immunological parameters evaluated consisted of specific antibody responses in terms of titers, specificities and isotype profiles, T cell involvement and cytokine production. Our results indicate that these new isoprenoids were safe adjuvants with the ability to significantly augment immunogen-specific IgG1 and IgG2a antibody responses. Moreover, there was no adverse phthalate cross-reactive anti-DNA response. Interestingly, PHIS-01 and PHIS-03 influenced differentially T-helper polarization. We also observed that these compounds modulated the immune response through apoptotic/necrotic effects on target tumor cells using murine lymphomas. Finally, unlike squalene and several other terpenoids reported to date, these phytol derivatives did not appear arthritogenic in murine models.

Topics: Adjuvants, Immunologic; Animals; Apoptosis; Cytokines; Diterpenes; Emulsions; Female; Haptens; Hemocyanins; Immunity, Humoral; Immunoglobulin Class Switching; Immunoglobulin G; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Necrosis; Ovalbumin; Phthalic Acids; Phytol; T-Lymphocytes, Helper-Inducer; Vaccines

5,6-Di-methylxanthenone-4-acetic acid (DMXAA) is a small molecule in the flavanoid class that has antitumor activity. Although classified as a "vascular disrupting agent," we have recently conducted studies showing that DMXAA has remarkable efficacy in a range of tumors, working primarily as an immune modulator that activates tumor-associated macrophages and induces a subsequent CD8(+) T-cell-mediated response. To more completely analyze the effect of DMXAA on CD8(+) T-cell generation, we treated mice bearing tumors derived from EG7 thymoma cells that express the well-characterized chicken ovalbumin neotumor antigen. Treatment with DMXAA led to cytokine release, tumor cell necrosis, and ultimately reduction in tumor size that was lymphocyte dependent. Within 24 h of administration, we observed dendritic cell activation in tumor-draining lymph nodes (TDLN). This was followed by a rapid and marked increase in the number of tetramer-specific CD8(+) T cells in the spleens of treated animals. In contrast, the vascular disrupting agent combretastatin A4-phosphate, which caused a similar amount of immediate tumor necrosis, did not activate dendritic cells, nor induce an effective antitumor response. Using in vitro systems, we made the observation that DMXAA has the ability to directly activate mouse dendritic cells, as measured by increased expression of costimulatory molecules and proinflammatory cytokine release via a pathway that does not require the Toll-like receptor adaptor molecule MyD88. DMXAA thus has the ability to activate tumor-specific CD8(+) T cells through multiple pathways that include induction of tumor cell death, release of stimulatory cytokines, and direct activation of dendritic cells.

Topics: Animals; Antineoplastic Agents; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Chickens; Cytokines; Dendritic Cells; Mice; Mice, Inbred C57BL; Myeloid Differentiation Factor 88; Necrosis; Neoplasm Transplantation; Ovalbumin; T-Lymphocytes, Cytotoxic; Xanthones

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end-labeling (TUNEL) staining is a widely accepted method for the detection of DNA fragmentation in nuclei of apoptotic cells. Tumor necrosis factor (TNF)-alpha is closely associated with changes in condylar cartilage and modulates apoptosis in various tissues including cartilage. The aim of this study was to investigate the relationship between apoptotic chondrocytes and TNF-alpha in a rabbit model of arthritis.. Unilateral temporomandibular joint (TMJ) arthritis was induced in 20 adult New Zealand White rabbits. From 1 day to 6 weeks after the induction of arthritis, immunohistochemical analysis for TNF-alpha and TUNEL was performed.. In condylar cartilage, TNF-alpha-positive cells and TUNEL-positive cells were localized together. TNF-alpha-positive chondrocytes seemed to precede TUNEL-positive cells.. The results of the present study suggest that TNF-alpha may be involved in apoptosis and/or apoptotic necrosis of chondrocytes as TMJ arthritis progresses from the acute to chronic stage.

Topics: Animals; Antigens; Apoptosis; Arthritis, Experimental; Cartilage; Cell Nucleus; Cell Proliferation; Chondrocytes; Disease Models, Animal; Disease Progression; DNA Fragmentation; Hypertrophy; Immunohistochemistry; In Situ Nick-End Labeling; Male; Mandibular Condyle; Necrosis; Ovalbumin; Rabbits; Temporomandibular Joint Disorders; Time Factors; Tumor Necrosis Factor-alpha

Saponin is the major component in the formation of immune stimulating complex (ISCOM), a potent adjuvant able to induce both humoral and cellular immune reactions. The immunogenicity induced by saponin, however, has been unclear. The objective of this study was to investigate the apoptotic and necrotic effects induced by saponin in ELA mouse lymphoma cells, expected to be a possible mechanism of the cytotoxic T-lymphocyte (CTL) effect elicited by the ISCOM.. EL4 cells were treated with saponin, and viability of the cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase release assays. Fluorescence microscopy was used to detect the morphological changes by staining saponin-treated cells with Hoechst 33342. Extent of apoptosis and necrosis was determined by Annexin V-FITC/propidium iodide staining, followed by flow cytometric analysis. Dendritic cells were cultured with either saponin-protein complexes or saponin-treated cells and analyzed by flow cytometry.. Treatment of EL4 cells with saponin resulted in concentration-dependent cytotoxicity and the appearance of the hypodiploid DNA peak. Cells treated with saponin showed highly condensed chromatin when stained with fluorescent DNA-binding dye Hoechst 33342. Analysis of EL4 cells by flow cytometry after Annexin V/propidium iodide staining demonstrated that saponin induced both apoptosis and necrosis. Pretreatment of EL4 cells with zVAD-fmk, a broad-range caspase inhibitor, did not prevent cell death induced by saponin, indicating the non-caspase-dependent cell death. Dendritic cells were shown to phagocytose both the antigen-saponin complexes and the saponin-induced dead cells.. Results obtained in this study demonstrated that saponin induced both apoptosis and necrosis in ELA cells. These events are critical for antigen processing and presentation.

Topics: Adjuvants, Immunologic; Amino Acid Chloromethyl Ketones; Animals; Antigens; Apoptosis; Caspase Inhibitors; Cell Line, Tumor; Cell Survival; Dendritic Cells; DNA; Flow Cytometry; L-Lactate Dehydrogenase; Male; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Necrosis; Ovalbumin; Phagocytosis; Plant Bark; Saponaria; Saponins

Therapeutic use of IL-2 can generate antitumor immunity; however, a variety of different mechanisms have been reported. We injected IL-2 intratumorally (i.t.) at different stages of growth, using our unique murine model of mesothelioma (AE17; and AE17 transfected with secretory OVA (AE17-sOVA)), and systematically analyzed real-time events as they occurred in vivo. The majority of mice with small tumors when treatment commenced displayed complete tumor regression, remained tumor free for >2 mo, and survived rechallenge with AE17 tumor cells. However, mice with large tumors at the start of treatment failed to respond. Timing experiments showed that IL-2-mediated responses were dependent upon tumor size, not on the duration of disease. Although i.t. IL-2 did not alter tumor Ag presentation in draining lymph nodes, it did enhance a previously primed, endogenous, tumor-specific in vivo CTL response that coincided with regressing tumors. Both CD4(+) and CD8(+) cells were required for IL-2-mediated tumor eradication, because IL-2 therapy failed in CD4(+)-depleted, CD8(+)-depleted, and both CD4(+)- and CD8(+)-depleted C57BL/6J animals. Tumor-infiltrating CD8(+) T cells, but not CD4(+) T cells, increased in association with a marked reduction in tumor-associated vascularity. Destruction of blood vessels required CD8(+) T cells, because this did not occur in nude mice or in CD8(+)-depleted C57BL/6J mice. These results show that repeated doses of i.t. (but not systemic) IL-2 mediates tumor regression via an enhanced endogenous tumor-specific CTL response concomitant with reduced vasculature, thereby demonstrating a novel mechanism for IL-2 activity.

Topics: Adjuvants, Immunologic; Animals; Antigen Presentation; Antigens, CD; Antigens, Neoplasm; Antineoplastic Agents; B7-2 Antigen; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Death; Cell Division; Cell Line, Tumor; Cytotoxicity, Immunologic; Egg Proteins; Female; Graft Rejection; Growth Inhibitors; Histocompatibility Antigens Class I; Immunodominant Epitopes; Immunotherapy, Active; Injections, Intralesional; Injections, Intraperitoneal; Interleukin-2; Lymphocytes, Tumor-Infiltrating; Membrane Glycoproteins; Mesothelioma; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Nude; Mice, Transgenic; Necrosis; Neoplasm Transplantation; Neovascularization, Pathologic; Ovalbumin; Peptide Fragments; Survival Rate; T-Lymphocyte Subsets

Dendritic cells (DCs) primed with tumor antigens can effectively mediate the regression of a variety of established solid malignancies in both murine and human models. Despite such clinical efficacy, the optimal means of DC priming is unknown. The goal of this study was to compare three methods of tumor preparation: irradiation, boiling, or freeze thaw lysis for DC priming. Mouse bone marrow-derived DCs were loaded with defined ratios of E.G7 tumor cells expressing a model tumor antigen, OVA. Sensitized DCs were used for stimulation of OVA-specific CTLs derived from OT-1 T-cell receptor transgenic mice. IFN-gamma release, determined by ELISA at 24 and 48 h, was used to assess the expression of antigens by DCs. DCs loaded with irradiated tumors were effective stimulators for OT-1 CTLs, whereas DCs stimulated with freeze-thawed or boiled tumors did not stimulate IFN-gamma production. Freeze-thaw lysis appeared to inhibit CTL activity in vitro and in two of three cases, this effect was not overcome by the addition of OVA. The ability to load irradiated tumor cells was reproduced in two analogous human melanoma models using melanoma cell lines expressing gp100 and CTL clones specific for a gp100 melanoma antigen. Consistent with the in vitro data, only DC/irradiated tumor vaccines were effective in preventing or delaying outgrowth of E.G7 and a poorly immunogenic murine squamous cell carcinoma (SCCVII), on local tumor challenge. These data demonstrate that the method of tumor cell preparation clearly influences the ability of DCs to present antigen to T cells. Correlation of in vitro data with the generation of protective immunity in vivo suggests the utility of irradiated tumor-primed DCs as a means to generate protective immunity in patients with solid malignancies.

Topics: Animals; Antigen Presentation; Antigens, Neoplasm; Apoptosis; Cancer Vaccines; Carcinoma, Squamous Cell; Dendritic Cells; Freezing; Heating; Humans; Immunotherapy, Adoptive; Melanoma; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Necrosis; Neoplasms, Experimental; Ovalbumin; T-Lymphocytes, Cytotoxic; Thymoma; Tumor Cells, Cultured

Minimally invasive synovectomy techniques have been unsuccessful due to lack of selectivity. The purpose of this study was to evaluate the potential of photodynamic therapy to destroy diseased synovium in an antigen-induced arthritis model.. Three sets of experiments evaluated the biodistribution and treatment effects of Photofrin (PF) in rabbits with bilateral knee antigen-induced arthritis. The first set of experiments evaluated the biodistribution of PF in articular tissues of 30 rabbits from 6-72 hours after systemic injection of 2 mg/kg. In the second series of experiments, light was delivered to the knee joint via cleaved optical fibers, whereas for the third, light was delivered via a 600 microm diffusion tip fiber. Tissues were harvested at 2 and 4 weeks posttreatment.. The biodistribution experiments demonstrated maximal uptake in inflamed synovium at 48 hours and a lack of uptake in normal tissues. With bare cleaved fibers, necrosis was observed in one specimen at 2 weeks and was absent in all specimens at 4 weeks. In the third experiment, synovial necrosis was observed in 3 of 7 specimens at 2 weeks and 3 of 8 at 4 weeks. No damage to articular cartilage or periarticular tissues was seen with either mode of light delivery.. These studies indicate that selective destruction of synovium can be achieved with PF and suggest that optimization of light delivery techniques will play an important role in development of this new technique.

Topics: Analysis of Variance; Animals; Arthritis, Rheumatoid; Cartilage, Articular; Dihematoporphyrin Ether; Disease Models, Animal; Hyperplasia; Knee Joint; Microscopy, Fluorescence; Necrosis; Ovalbumin; Photochemotherapy; Rabbits; Synovial Membrane; Synovitis

Topics: Animals; Antibody Formation; Cells, Cultured; Child; Child, Preschool; Deoxyguanosine; Epithelium; Female; Humans; Immunity, Cellular; Infant; Lymphoid Tissue; Mice; Necrosis; Ovalbumin; Rats; Rats, Inbred Strains; Rats, Mutant Strains; T-Lymphocytes; Thymus Gland

Antigen induced arthritis (AIA) was elicited in 28 adult rabbits using ovalbumin. All animals developed an intensive monarthritis manifested by local heat and swelling. Two, 7 and 21 days later, respectively, 0.3 ml of 1 per cent OsO4 was injected into the arthritic knees of three groups of 9 rabbits. The rabbits were killed 1, 3 and 6 months later. No modifying influence on the severe to complete cartilage destruction was evident. One group of 8 rabbits received only OsO4 and the cartilage of these animals remained intact after 6 months.

Topics: Animals; Arthritis; Cartilage; Knee Joint; Necrosis; Osmium; Osmium Tetroxide; Ovalbumin; Rabbits; Time Factors

A technique is described for producing large demyelinating lesions of the spinal cord in the guinea pig. Guinea pigs were pretreated by immunization with ovalbumin and water-soluble adjuvant (N-acetyl-muramyl L-alanyl D-isoglutamine, MDP) in water-in-oil emulsion (Freund's incomplete adjuvant). They were given a large dose (10 mg) of ovalbumin i.p. one month later. After a few weeks the animals were sensitized with guinea-pig basic protein in Freund's complete adjuvant. Five out of 11 animals developed large, distinctive, sharply demarcated, symmetrical demyelinating lesions within 30 days. These lesions occurred in the dorsal and anterior columns, root entry zones and subpial region of the spinal cord. Histology showed a considerable amount of free lipids. There were also infiltrative lesions of classical experimental allergic encephalomyelitis (EAE) of normal severity in the same animals. The demyelinating lesions resembled those seen in multiple sclerosis in their location and extent in the spinal cord and in the presence of free lipids. Control experiments indicated that pretreatment with ovalbumin/MDP and the second injection of ovalbumin was necessary for all the demyelination; moreover guinea pigs immunized with basic protein in Freund's complete adjuvant or Freund's incomplete adjuvant plus MDP without pretreatment only developed classical EAE with minimal or no demyelination.

Topics: Adjuvants, Immunologic; Animals; Encephalomyelitis, Autoimmune, Experimental; Female; Guinea Pigs; Lymph Nodes; Male; Multiple Sclerosis; Myelin Sheath; Necrosis; Ovalbumin; Spinal Cord

Topics: Adipose Tissue; Allergy and Immunology; Animals; Beta-Globulins; Guinea Pigs; Hypersensitivity; Hypersensitivity, Delayed; Macrophages; Muscles; Necrosis; Ovalbumin; Pathology; Research; Skin Tests

A short term model in which circulating antigen-antibody complexes and host complement localized in vessel walls of guinea pigs was analyzed. Localization was accomplished by subjecting the animals to anaphylactic shock. The circulating macromolecules, such as antigen-antibody complexes, appeared to localize by being trapped in the vessel wall along the basement membrane that acted as a filter during a state of increased permeability of the vessel. It was suggested that this point of localization between the endothelial cell and the basement membrane may well represent the earliest focus of inflammation in diseases caused by the deposition of injurious macromolecules such as soluble antigen-antibody complexes from the blood stream. Complexes localized in the vessel walls did not provoke Arthus-type vasculonecrotic reactions even though in both these vessels and in cutaneous Arthus reactions antibody, antigen, and host complement (C'3c) were deposited in the vessel walls. The possibility was presented that since circulating macromolecules and probably complexes deposited in (a) relatively small amounts, and (b) in a position beneath endothelial cells, they were not strongly chemotactic toward circulating polymorphonuclear leukocytes. Vasculonecrotic reactions, therefore, were not observed. It was brought out that this may be similar to the situation in glomerulonephritis induced by localized immune complexes, in which severe necrosis is not observed. In the Arthus vascular reaction, host complement was found microscopically accumulated with the immune reactants in affected vessel walls.

Topics: Anaphylaxis; Animals; Antibodies; Antigen-Antibody Complex; Antigen-Antibody Reactions; Arthus Reaction; Blood Vessels; Cattle; Complement System Proteins; Electrons; Fluorescent Antibody Technique; gamma-Globulins; Glomerulonephritis; Guinea Pigs; Immune Sera; Immunoelectrophoresis; Inflammation; Microscopy; Microscopy, Electron; Necrosis; Neutrophils; Ovalbumin; Pathology; Rabbits; Research; Serum Albumin; Serum Albumin, Bovine; Vascular Diseases