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D S Pisetsky

Publications and source records attributed to D S Pisetsky.

At least 55 records · Page 3Linked to original sources
PubMed1999

The influence of lipofectin on the in vitro stimulation of murine spleen cells by bacterial DNA and plasmid DNA vectors.

Lipofectin is a mixture of two cationic lipids, N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoyl phosphotidylethanolamine (DOPE), and has been commonly used to promote transfection of plasmid vectors in vitro and in vivo. In these experiments, the effect of lipofectin on in vitro immunostimulation by bacterial and plasmid DNA was tested to determine if these lipids can also influence immune properties of DNA. As a model, spleen cells from BALB/c and C3H/HeJ mice were cultured with DNA from either Escherichia coli DNA or the pEGFP-N1 plasmid at various ratios with lipofectin. As an index of immune stimulation, in vitro proliferation as well as production of interleukin-12 (IL-12) and interferon-gamma (IFN-gamma) were assessed. For both bacterial DNA and plasmid DNA, the presence of lipofectin led to a marked increase in the production of IFN-gamma under conditions in which increases in IL-12 production were limited. The IFN-gamma production was nevertheless dependent on IL-12, as shown by the effects of anti-IL-12 antibodies. Under these culture conditions, lipofectin did not significantly augment proliferation induced by DNA. These findings indicate that lipofectin can increase the in vitro immunostimulatory effects of bacterial and plasmid DNA, although the magnitude of the increase may vary among responses.

Animals↗
PubMed1999

Multivalent cross-linking of membrane Ig sensitizes murine B cells to a broader spectrum of CpG-containing oligodeoxynucleotide motifs, including their methylated counterparts, for stimulation of proliferation and Ig secretion.

We have previously reported that B cells that are activated by multivalent but not bivalent membrane Ig cross-linking ligands synergize with various B cell activators culminating in enhanced B cell proliferation. In this study we asked whether B cells that are activated by a multivalent mIg cross-linking agonist could respond to oligodeoxynucleotides (ODN) containing non-stimulatory motifs. Earlier reports have shown that ODN containing a CpG motif in which the cytosine is unmethylated and is flanked by two 5' purines and two 3' pyrimidines induce high levels of B cell activation, while ODN whose CpG are methylated or flanked by sequences other than the optimal two 5' purines and two 3' pyrimidines were non-stimulatory. In this manuscript we show that when B cells are stimulated in vitro with dextran-conjugated anti-IgD antibodies (anti-IgD-dex), as the multivalent mIg ligand, their proliferation is enhanced and they can be induced to secrete Ig in response to ODN containing various non-optimal motifs, both methylated and non-methylated. Furthermore we could induce synergistic levels of proliferation with concentrations of anti-IgD-dex that were in the picomolar concentration range and with concentrations of ODN that were 10- to 100-fold less than previously reported to be necessary for mitogenic activity. These data provided a model to explain how low concentrations of a multi-epitope-expressing microorganism in the context of mammalian (methylated) or microorganism (non-methylated) DNA can lead to dysregulated B cell proliferation and Ig secretion.

Animals↗
PubMed1998

Effects of bacterial DNA on cytokine production by (NZB/NZW)F1 mice.

Microbial DNA has multiple immune effects including the capacity to induce polyclonal B cell activation and cytokine production in normal mice. We recently described the accelerated induction of anti-DNA Abs in NZB/NZW mice immunized with Escherichia coli (EC) dsDNA; paradoxically these mice developed less renal disease than unimmunized mice or mice immunized with calf thymus DNA. We postulated that alterations in cytokine production induced by bacterial DNA may play a key role in renal protection. To determine the effect of bacterial DNA on cytokine production in NZB/NZW mice, we measured the serum cytokine levels, cell culture supernatant cytokine levels, and number of cytokine-producing splenocytes in NZB/NZW mice injected with EC DNA, calf thymus DNA, or an immune active oligonucleotide. There was a 10- to 25-fold increase in the number of cells secreting IFN-gamma compared with IL-4 in mice immunized with EC DNA. IL-12-secreting cells were also increased by bacterial DNA immunization. In parallel with the increase in IFN-gamma secreting cells, there was a significant rise in serum IFN-gamma levels in mice receiving EC DNA. These results indicate that EC DNA modulates systemic cytokine levels in NZB/NZW mice, selectively increasing IL-12 and IFN-gamma while decreasing IL-4 production. The cytokine response of NZB/NZW mice to bacterial DNA may be of significance in disease pathogenesis and relevant to the treatment of lupus-like disease.

Animals↗
PubMed1998

The influence of DNA sequence on the immunostimulatory properties of plasmid DNA vectors.

To determine the influence of DNA sequence on immunostimulatory properties of vaccine vectors, we tested the induction of in vitro and in vivo immune responses by plasmids modified to contain extended runs of dG sequences. Studies with oligonucleotides indicate that dG sequences can directly stimulate B cells as well as enhance the activity of immunostimulatory CpG motifs because of interaction with the macrophage scavenger receptor (MSR); this receptor can bind a variety of polyanions including dG sequences. To modify vectors, we introduced stretches of 20-60 dG residues into the pCMV-beta and pSG5rab.gp vectors and measured the ability of these plasmids to induce IL-12 and IFN-gamma production by murine splenocytes. The induction of in vivo antibody responses to rabies glycoprotein was also assessed with the pSG5rab.gp vectors. In in vitro cultures, cytokine production induced by plasmids with and without dG sequences was similar. Furthermore, the addition of dG sequences to pSG5rab.gp vectors failed to enhance the anti-rabies glycoprotein response to immunization. To assess further mechanisms by which plasmids stimulate macrophages, we measured the effects of MSR ligands on in vitro cytokine induction. In in vitro cultures, poly(G), dG30, and fucoidan inhibited IL-12 induction by plasmids. IL-12 induction was also inhibited by mammalian DNA but was unaffected by polyanions that are not MSR ligands. Together, these results suggest that the addition of 20 to 60-base dG sequences to plasmids does not significantly affect their properties as immunostimulators or vaccines. Furthermore, these results suggest that MSR ligands can block cytokine induction by plasmid DNA whether or not the plasmid contains extended runs of dG.

Amino Acid Sequence↗
PubMed1998

The binding of anti-DNA antibodies to phosphorothioate oligonucleotides in a solid phase immunoassay.

Phosphorothioate oligonucleotides (S-oligos) are nucleic acid derivatives that are commonly used as antisense agents. These compounds, similar to bacterial DNA and CpG oligonucleotides, display a variety of immunological activities in vitro and in vivo. To assess further these activities, the antigenicity of a series of S-oligos was assessed in the solid phase using anti-DNA antibodies from sera of patients with systemic lupus erythematosus. By ELISA, S-oligos bound well to anti-DNA antibodies under the same conditions as calf thymus DNA antigen. The specificity for anti-DNA was established by competition assays showing cross-inhibition of binding by DNA and S-oligos. Reactivity with anti-DNA was observed with S-oligos varying in sequence, suggesting interaction with a conserved determinant not strictly dependent on the bases. Furthermore. in comparison with a phosphodiester oligomer of the same sequence, a phosphorothioate showed dramatically increased activity. These findings indicate that structural features associated with the S-oligo backbone promote specific binding to anti-DNA antibodies and influence the size requirement for antigenicity in the solid phase. These observations thus extend the immunological properties of S-oligos and suggest uses of these compounds in the diagnosis and treatment of autoimmune disease.

Animals↗
PubMed1998

The influence of base sequence on the immunological properties of defined oligonucleotides.

To assess the influence of base sequence on the immunostimulatory activities of DNA, cell binding and mitogenicity of a series of 30-mer phosphodiester oligonucleotides were tested using murine spleen cells. These compounds consisted of either a single base or a six base CpG motif in the context of 5' and 3' flanking sequences of each base. Among fluoresceinated oligonucleotides, (dG)30 had the highest binding of single base compounds tested while the presence of dG flanks increased binding of compounds with six base motifs, whether active on inactive. In assays of mitogenesis including incorporation of thymidine and uridine as well as expression of cell surface CD69, (dG)30 induced the highest responses among single base compounds. Among compounds with an active six base motif, the extent of proliferation varied with flanking sequence, with dG flanks producing the greatest stimulation in all assays tested. Together, these findings indicate that a variety of base sequences may affect the immunomodulatory properties of DNA, with the activity of dG sequences perhaps resulting from the formation of variant DNA structures.

Animals↗
PubMed1997

Clinical and serologic manifestations of autoimmune disease in MRL-lpr/lpr mice lacking nitric oxide synthase type 2.

Nitric oxide (NO) is an important mediator of the inflammatory response. MRL-lpr/lpr mice overexpress inducible nitric oxide synthase (NOS2) and overproduce NO in parallel with the development of an autoimmune syndrome with a variety of inflammatory manifestations. In previous studies, we showed that inhibiting NO production with the nonselective nitric oxide synthase (NOS) inhibitor NG-monomethyl-arginine reduced glomerulonephritis, arthritis, and vasculitis in MRL-lpr/lpr mice. To define further the role of NO and NOS2 in disease in MRL-lpr/lpr mice, mice with targeted disruption of NOS2 were produced by homologous recombination and bred to MRL-lpr/lpr mice to the N4 generation. MRL-lpr/lpr littermates homozygous for disrupted NOS2 (-/-), heterozygous for disrupted NOS2 (+/-), or wildtype (+/+) were derived for this study. Measures of NO production were markedly decreased in the MRL-lpr/lpr (-/-) mice compared with MRL-lpr/lpr (+/+) mice, with intermediate production by the MRL-lpr/lpr (+/-) mice. There was no detectable NOS2 protein by immunoblot analysis of the spleen, liver, kidney, and peritoneal macrophages of the (-/-) animals, whereas that of (+/+) was high and (+/-) intermediate. The (-/-) mice developed glomerular and synovial pathology similar to that of the (+/-) and (+/+) mice. However, (-/-) mice and (+/-) mice had significantly less vasculitis of medium-sized renal vessels than (+/+) mice. IgG rheumatoid factor levels were significantly lower in the (-/-) mice as compared with (+/+) mice, but levels of anti-DNA antibodies were comparable in all groups. Our findings show that NO derived from NOS2 has a variable impact on disease manifestations in MRL-lpr/lpr mice, suggesting heterogeneity in disease mechanisms.

Animals↗
PubMed1997

Molecular properties of anti-DNA induced in preautoimmune NZB/W mice by immunization with bacterial DNA.

To elucidate the mechanism of Ag drive in the anti-DNA response, the Ab response to bacterial DNA has been analyzed in normal and autoimmune mice. Preautoimmune NZB/W mice immunized with Escherichia coli dsDNA produce Abs that resemble spontaneous autoantibodies and bind mammalian dsDNA. In contrast, normal mice, when immunized similarly, produce Abs that bind only bacterial dsDNA. To characterize further the responsiveness of NZB/W mice to bacterial DNA, we determined the molecular properties of mAbs from preautoimmune NZB/W mice immunized with E. coli DNA. Of nine Abs studied, all were IgM and all bound mammalian ssDNA, while four had appreciable reactivity with mammalian dsDNA. The induced anti-dsDNA resembled spontaneous anti-DNA from autoimmune mice in V gene utilization and V(H) CDR3 arginine content. These Abs lacked evidence of somatic mutation, however, indicating that affinity maturation via somatic mutation is not essential for dsDNA reactivity. The findings suggest that preautoimmune NZB/W mice have immunoregulatory defects that allow activation of mammalian dsDNA reactive B cells by bacterial DNA.

Amino Acid Sequence↗
PubMed1997

Specificity and immunochemical properties of antibodies to bacterial DNA.

DNA is a structurally heterogeneous molecule that elicits antibody production in both normal and aberrant immunity. In the prototypic autoimmune disease systemic lupus erythematosus, anti-DNA antibodies occur prominently and are serological markers for diagnosis and prognosis. These antibodies target conserved sites on both single-stranded (ss) and double-stranded (ds) DNA from essentially all species. In contrast, sera of normal human subjects (NHS) contain antibodies that selectively bind to DNA from certain bacteria. The NHS antibodies differ from lupus anti-DNA in their exclusive binding to foreign DNA as well as in their isotype, light chain distribution, and mode of DNA interaction. The properties of these antibodies suggest that they arise as a specific response to bacterial DNA introduced during infection or colonization. This conclusion is supported by studies demonstrating that bacterial DNA is a potent immunogen in normal mice and can induce antibodies specific for determinants on bacterial ss and dsDNA. Furthermore, immunization of normal animals with bacterial DNA elicits antibodies that bind mammalian as well as bacterial ssDNA; this immunization can also provoke glomerulonephritis. In addition to its immunogenicity, bacterial DNA is mitogenic, stimulating polyclonal activation of murine B cells; bacterial DNA can also induce cytokine production in the mouse. The range of immunological activities of bacterial DNA suggests an important role of this molecule in stimulating host defense in normal individuals and provoking autoimmunity in individuals predisposed to SLE.

Animals↗
PubMed1997

Systemic lupus erythematosus. Diagnosis and treatment.

Systemic lupus erythematosus is a multisystem inflammatory disease characterized by antinuclear antibody production. The diagnosis of this disease is established on the basis of a constellation of clinical and serologic features. Therapy is directed to specific disease manifestations and involves an array of anti-inflammatory and immunosuppressive agents that are administered judiciously to limit toxicity.

Anti-Inflammatory Agents↗
PubMed1997

Specificity and immunochemical properties of antibodies to bacterial DNA in sera of normal human subjects and patients with systemic lupus erythematosus (SLE).

To elucidate the mechanisms of anti-DNA production, we assessed the binding of sera of normal human subjects (NHS) and patients with SLE to a panel of bacterial and mammalian DNA. Using single-stranded DNA as antigens in an ELISA, NHS showed significant binding to some but not all bacterial DNA, while lacking reactivity to calf thymus DNA. Among bacterial DNA, the highest levels of binding were observed with DNA from Micrococcus lysodeikticus and Staphylococcus aureus. In contrast, SLE sera showed high levels of binding to all DNA tested. To evaluate further immunochemical properties of the anti-DNA antibodies, the subclass distribution of these responses was evaluated by subclass-specific reagents. While NHS showed a predominance of IgG2 antibodies to bacterial DNA, SLE sera had a predominance of IgG1 antibodies to these antigens. Together, these results provide further evidence for the antigenicity of bacterial DNA and suggest that NHS and SLE anti-DNA differ in the patterns of epitope recognition as well as mechanisms of induction.

Animals↗
PubMed1997

Deficient expression of antibodies specific for bacterial DNA by patients with systemic lupus erythematosus.

Antibodies to DNA occur commonly in the sera of normal human subjects and bind to nonconserved sites exclusive to DNA from certain bacterial species. These antibodies are primarily IgG2 and differ from anti-DNA antibodies in systemic lupus erythematosus (SLE) sera, which are primarily IgG1 and bind conserved sites on all DNA. To investigate the immune response to bacterial DNA in SLE, antibodies to single-stranded DNA from Micrococcus lysodeikticus (MC) were measured in normal human subjects and in SLE sera by enzyme-linked immunosorbent assay either directly or following absorption by cellulose bearing calf thymus (CT) DNA; isotype-specific reagents were used to assess the IgG2 response. In these assays, SLE sera had a marked deficiency in antibodies specific for MC single-stranded DNA as demonstrated by the reduction of antibody levels by CT DNA cellulose absorption. Furthermore, IgG2 anti-DNA in SLE sera were cross-reactive with MC and CT single-stranded DNA, whereas IgG2 anti-DNA in normal human subjects' sera were specific for the bacterial DNA antigen. Together, these findings indicate that SLE patients have a marked deficiency in the production of antibodies specific for bacterial DNA antigen. This deficiency could predispose to anti-DNA autoantibody production and result from tolerance defects that distort the array of specificities in the B-cell repertoire.

Adolescent↗