In vivo priming by DNA injection occurs predominantly by antigen transfer.
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DNA vaccines can stimulate both humoral and cytolytic immune responses. Although bone marrow-derived elements present the expressed Ag, the mechanisms for acquiring immunogenic peptides have yet to be fully elucidated. APCs may become directly transfected by plasmid DNA or process extracellular proteins produced by other transfected cells. Using a transactivating plasmid system and bone marrow chimeras, we show that both mechanisms appear to be involved; however, the bulk of the immune response is dependent on expression of Ag by nonlymphoid tissues and transfer to APCs. These in vivo studies are the first to define the role of transfected nonlymphoid cells in generating Ag for presentation by bone marrow-derived APCs after needle injection with plasmid DNA. (+info)
Antibody production in autoimmune BXSB mice. I. CD40L-expressing B cells need fewer signals for polyclonal antibody synthesis.
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Male, but not female, BXSB mice develop severe lupus associated with multiple immune system defects. It was recently shown that one immunological abnormality found in male BXSB mice encompasses B cell expression of CD40 ligand (CD40L) by an expanded population of large B cells. The present study was undertaken to determine how the CD40L-expressing large B cells in male BXSB mice compared with size-matched B cells from female mice in terms of their ability to secrete antibody. It was shown that the large B cells from female mice, similar to the small B cells from either male or female mice, required CD40 signalling, immunoglobulin cross-linking and cytokines for optimal antibody synthesis. In contrast, large B cells from male BXSB mice produced high levels of antibody when stimulated with only two of the three signals, and made significantly more total IgM and IgG, and anti-ssDNA antibody than size-matched B cells from female mice when stimulated with IL-4/IL-5 alone, IL-4/IL-5 plus low levels of anti-IgD-dextran, or IL-4/IL-5 plus anti-CD40 MoAb. The ability of the large B cells from male mice to produce antibody under suboptimal stimulatory conditions correlated with their expression of CD40L, and was inhibited by CD40-immunoglobulin. Taken together, these findings suggested that large CD40L-expressing B cells from male BXSB mice may be able to bypass a need for CD40 signalling from T cells, thus contributing to autoimmune disease by promoting antibody production in the absence of cognate T cell help. (+info)
Keratinocytes from patients with lupus erythematosus show enhanced cytotoxicity to ultraviolet radiation and to antibody-mediated cytotoxicity.
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Keratinocyte cytotoxicity is an important component of the immunopathology of photosensitive lupus erythematosus, and antibody-dependent cell-mediated cytotoxicity (ADCC) has been shown to be an important mechanism by which autoantibodies, especially those specific for SS-A/Ro, can induce keratinocyte damage in models of photosensitive lupus. We provide further evidence that keratinocytes from patients with photosensitive lupus show significantly greater ultraviolet radiation (UVR)-induced cytotoxicity, and that ADCC of these targets is especially enhanced by autologous patient's serum or by anti-SS-A/Ro+ sera. Keratinocytes from normal uninvolved skin of 29 patients with cutaneous lupus erythematosus (LE) were grown in cell culture and tested as targets in cytotoxicity experiments in vitro. Cultured keratinocytes from patients with systemic lupus erythematosus (SLE) and subacute cutaneous lupus erythematosus (SCLE) showed significantly greater cytotoxicity following UVR treatment than did keratinocytes from normal adult controls or from neonatal foreskins (P < 0.01). The same cultures also showed greater UVR-induced binding of IgG from fractionated anti-SS-A/Ro+ preparations. ADCC experiments were also performed using keratinocytes cultured from patients with SLE, SCLE, discoid lupus erythematosus (DLE), and normal controls. When keratinocytes were incubated in autologous serum plus a standard mononuclear cell effector population, the percentage of ADCC observed was significantly greater in cultures containing keratinocytes and sera from the SLE and SCLE patients (P < 0.001). When cultured keratinocytes were added to different IgG antibody probes, plus standard mononuclear effector populations, greater ADCC was seen using the anti-SS-A/Ro probe and keratinocytes from patients with SLE or SCLE. With normal human neonatal keratinocyte targets, the anti-SS-A/Ro probe induced greater ADCC than that seen with anti-ssDNA or normal human serum. We have shown that keratinocytes from patients with some forms of lupus erythematosus (SLE and SCLE) show greater cytotoxicity in vitro when irradiated with UVR, and greater susceptibility to ADCC whether the antibody source is their own serum or an anti-SS-A/Ro probe. (+info)
A radioimmunoassay method for detection of DNA based on chemical immobilization of anti-DNA antibody.
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High selectivity provided by biomolecules such as antibodies and enzymes has been exploited during the last two decades for development of biosensors. Of particular importance are efficient immobilization methods for biomolecules in order to preserve their biological activities. In this study, we have evaluated immobilization strategies for an anti-DNA antibody on a self-assembled monolayer of omega-functionalized thiols. The antibody was immobilized via peptide bond formation between the primary amines in the antibody and the carboxyl groups on the self-assembled monolayer. The peptide bond coupling was achieved by activating COOH groups on the surface through N-Hydroxysuccimide (NHS)-ester formation, followed by acylation of NH2 group in the antibody. DNA binding activity of the immobilized antibody was examined by counting beta emission from 35S-labeled DNA. (+info)
Pitfalls of formalin fixation for determination of antineutrophil cytoplasmic antibodies.
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Sera can produce nuclear or perinuclear immunofluorescence staining in neutrophils which may be caused by antibodies with differing antigenic specificities. These include perinuclear antineutrophil cytoplasmic antibodies (P-ANCA), granulocyte specific antinuclear antibody (GS-ANA), and antinuclear antibody (ANA). There is controversy over the value of formalin fixation of neutrophils in differentiating antibodies giving selective or preferential reaction with the nuclear or perinuclear area of neutrophils. In a comparative study of 77 sera, formalin fixation caused inconsistency, nonspecific effects, and false positivity owing to enhanced fluorescence. If formalin fixed neutrophils are used in the routine diagnostic laboratory, this will add confusion to the interpretation of the ANCA assay. (+info)
Mice transgenic for BAFF develop lymphocytic disorders along with autoimmune manifestations.
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The cause of many autoimmune and inflammatory diseases is unresolved, although dysregulated production of tumor necrosis factor (TNF) family members appears to be important in many cases. BAFF, a new member of the TNF family, binds to B cells and costimulates their growth in vitro. Mice transgenic for BAFF have vastly increased numbers of mature B and effector T cells, and develop autoimmune-like manifestations such as the presence of high levels of rheumatoid factors, circulating immune complexes, anti-DNA autoantibodies, and immunoglobulin deposition in the kidneys. This phenotype is reminiscent of certain human autoimmune disorders and suggests that dysregulation of BAFF expression may be a critical element in the chain of events leading to autoimmunity. (+info)
Some Bence-Jones proteins enter cultured renal tubular cells, reach nuclei and induce cell death.
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Eighteen monoclonal Bence-Jones proteins (BJPs) were examined for their effects on cultured LLC-PK1 (porcine kidney proximal tubule) cells as well as for their amidase and DNase activities. Five proteins were found to enter the cell and to gain access to the nucleus without degradation of epitopes. Intranuclear BJPs ultimately induced DNA fragmentation and cell death. BJPs with relatively high amidase activity were cytotoxic. On the other hand, three of four BJPs with DNase activity had a cytocidal effect on cultured cells; the remaining BJP, which had a relatively high DNase activity but a very low amidase activity, failed to enter the cell and was not cytotoxic in vitro. These results suggest that catalytic and cytotoxic activities of some BJPs may make a significant contribution, in a substantial proportion of myeloma patients, to the development and/or deterioration of the disease. (+info)
Increased serum levels of soluble L-selectin (CD62L) in patients with active systemic lupus erythematosus (SLE).
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The adhesion molecule L-selectin (CD62L) mediates lymphocyte recirculation and leucocyte rolling on vascular endothelium at sites of inflammation. Serum levels of soluble L-selectin (sL-selectin) were measured in patients with SLE in order to relate these levels to clinical activity and immunological parameters. An ELISA was used to detect the soluble form of human L-selectin (CD62L) in 42 patients with SLE and in 33 healthy individuals. The mean +/- s.e.m. values of sL-selectin were 1285 +/- 121 ng/ml for patients with SLE and 986 +/- 180 ng/ml for healthy blood donors, but there was no significant difference. When patients with active SLE were analysed, higher levels of circulating sL-selectin were found when compared with patients without activity (1497 +/- 167 ng/ml versus 941 +/- 150 ng/ml; P = 0.028). We found a significant correlation between the levels of sL-selectin and of dsDNA antibodies (r = 0.36, P = 0. 044) and between levels of sL-selectin and SLE Disease Activity Index (SLEDAI) score (r = 0.42, P = 0.003). Patients with active SLE studied cross-sectionally showed significant elevations of sL-selectin (CD62L) compared with controls. Thus, the levels of this soluble adhesion molecule correlated with active disease and levels of anti-dsDNA antibodies. (+info)