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D M Klinman

Publications and source records attributed to D M Klinman.

At least 73 records · Page 4Linked to original sources
PubMed1997

DNA vaccines: safety and efficacy issues.

DNA technology has been harnessed to produce a variety of plasmid-based vaccines designed to prevent viral, bacterial and parasitic infections. The rapid adoption and implementation of this novel vaccine strategy carries with it important safety and efficacy concerns. This review will focus on whether DNA vaccines (1) are likely to induce systemic or organ-specific autoimmune disease, (2) have the potential to induce tolerance rather than immunity, and (3) are as effective in individuals with depressed immune function as they are in healthy adults.

Aging↗
PubMed1997

Toward clinical trials of DNA vaccines against malaria.

In mid 1997 the first malaria DNA vaccine will enter clinical trials. This single gene DNA vaccine encoding the Plasmodium falciparum circumsporozoite protein (PfCSP) will be studied for safety and immunogenicity. If these criteria are met, a multi-gene DNA vaccine designed to induce protective CD8+ T cell responses against P. falciparum infected hepatocytes will be subsequently assessed for safety, immunogenicity and capacity to protect immunized volunteers against experimental challenge with P. falciparum sporozoites. Our perspectives on malaria vaccine development in general, and on a multi-gene DNA vaccine in particular, have been recently reviewed. Herein, we review the rationale and experimental foundation for the anticipated P. falciparum DNA vaccine trials.

Animals↗
PubMed1996

Induction of neonatal tolerance by plasmid DNA vaccination of mice.

Plasmid DNA vaccines capable of preventing viral, bacterial, and parasitic infections are currently under development. Our labs have shown that a plasmid DNA vaccine encoding the circumsporozoite protein of the malaria parasite elicits protective immunity against live sporozoite challenge in adult BALB/c mice. We now find that the same DNA vaccine induces tolerance rather than immunity when administered to 2-5 d-old mice. Neonatally tolerized animals were unable to mount antibody, cytokine or cytotoxic responses when rechallenged with DNA vaccine in vitro or in vivo. Tolerance was specific for immunogenic epitopes expressed by the vaccine-encoded, endogenously produced antigen. Mice challenged with exogenous circumsporozoite protein produced antibodies against a different set of epitopes, and were not tolerized. These findings demonstrate important differences in the nature and specificity of the immune response elicited by DNA vaccines versus conventional protein immunogens.

Age Factors↗
PubMed1996

Rapid immune activation by CpG motifs in bacterial DNA. Systemic induction of IL-6 transcription through an antioxidant-sensitive pathway.

Unmethylated CpG dinucleotides (CpG motif) in bacterial DNA or synthetic oligodeoxynucleotides (CpG DNA) rapidly activate murine B cells to secrete IL-6 and IgM, as well as to proliferate. Within 30 min after CpG DNA stimulation in vivo, IL-6 mRNA levels were increased in liver, spleen, and thymus cells. Serum IL-6 protein was markedly increased within 1 h of stimulation. Treatment of a B cell line with CpG DNA led to an increase in the transcriptional activity of the IL-6 promoter. This CpG DNA-induced IL-6 production was not mediated via either a protein kinase C (PKC)-, protein kinase A (PKA)-, or nitric oxide (NO.)-dependent pathway but was inhibited by an antioxidant. In addition, the level of intracellular reactive oxygen species was increased within 20 min after CpG DNA, but not control non-CpG DNA, treatment. These results suggest that CpG DNA-induced IL-6 production is mediated through a reactive oxygen intermediate-dependent pathway. CpG DNA-mediated IL-6 production was enhanced by simultaneous signals delivered through the Ag receptor. The addition of neutralizing Abs against IL-6 to B cell cultures along with CpG oligodeoxynucleotides essentially abolished the CpG DNA-induced increased IgM secretion but had no significant effect on the B cell proliferation induced by the CpG motif. Our results suggest that the induction of IL-6 expression in response to CpG motifs in bacterial DNA may be an important immune defense mechanism that facilitates a rapid response to microbial infection.

Animals↗
PubMed1996

The natural history of disease expression in CD4 and CD8 gene-deleted New Zealand black (NZB) mice.

CD4 and CD8 gene-deleted New Zealand black (NZB) mice and, as controls, B6.CD4 -/-, B6.CD8 -/-, NZB, and B6 wild-type (wt) mice were studied for phenotypic and immunologic parameters to determine the contribution of CD4 and CD8 cell lineages in NZB mice. These studies suggest surprisingly that a number of abnormalities are not due to either CD4+ or CD8+ cell lineages but rather are most likely due to non-CD4+ and -CD8+ cell lineages and/or background genes. Such abnormalities include altered thymic architecture, decreased staining of MITS 33+ medullary thymocytes, and an increased frequency of splenic IgM secretory cells. In contrast, deletion of either CD4+ or CD8+ cells appears to differentially influence immunologic function. Deletion of CD8+ cells did not influence titers of spontaneously occurring anti-erythrocyte or anti-DNA autoantibodies. interestingly, 50% of NZB.CD4 -/- mice contained levels of anti-erythrocyte IgG and anti-ssDNA IgM autoantibodies; even without detectable CD4+ cells. Such deletion of CD4+ cells, while leading to marked decreases in the prototype cytokines that characterize Th1 and Th2 subsets in B6 mice, led to a marked increase in IFN-gamma and a moderate increase in IL-4 mRNA levels in NZB.CD4 -/- mice. These data suggest that whereas non-CD4+ and -CD8+ cell lineages and NZB background genes have a marked influence in the development of autoimmune abnormalities, CD4+ cells appear to play a major role in influencing the cytokine environment, whereas CD8+ cells appear to play a minor role.

Animals↗
PubMed1996

CpG motifs present in bacteria DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon gamma.

Bacterial infection stimulates the host to mount a rapid inflammatory response. A 6-base DNA motif consisting of an unmethylated CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines was shown to contribute to this response by inducing polygonal B-cell activation. This stimulatory motif is 20 times more common in the DNA of bacteria than higher vertebrates. The current work shows that the same motif induces the rapid and coordinated secretion of interleukin (IL) 6, IL-12, and interferon gamma (but not IL-2, IL-3, IL-4, IL-5, or IL-10) in vivo and in vitro. Stimulatory CpG DNA motifs induced B, T, and natural killer cells to secrete cytokine more effectively than did lipopolysaccharide. Thus, immune recognition of bacterial DNA may contribute to the cytokine, as well as the antibody production characteristic of an innate inflammatory response.

Animals↗
PubMed1996

Effect of HIV infection on the frequency of cytokine-secreting cells in human peripheral blood.

A selective decrease in type 1 cytokine secretion by in vitro stimulated peripheral blood cells is reportedly associated with disease progression in HIV-infected individuals. To examine whether a similar change in cytokine secretion occurs under physiologic conditions in vivo, sensitive and specific ELIspot assays were used to compare the phenotype and frequency of PBMC spontaneously producing interleukin (IL)-2, IL-4, IL-10, and interferon-gamma (IFN-gamma) in 83 HIV-infected subjects and 60 normal controls. Phenotypic analysis of freshly isolated cytokine-secreting cells showed that T cells were the primary source of IL-2, IL-4, and IFN-gamma while CD14+ macrophages/monocytes were the dominant source of IL-10 in vivo. The number of peripheral blood mononuclear cells (PBMC) spontaneously secreting both type 1 and type 2 cytokines was significantly reduced in HIV-infected patients versus controls. The magnitude of this decrease did not correlate with disease severity. Changes in IL-2-secreting cell number correlated with CD4 count, while changes in the frequency of IFN-gamma-secreting cells correlated with disease duration. These findings do not support the contention that a selective reduction in type 1 cytokine production correlates with disease progression.

Cytokines↗
PubMed1996

Contribution of interleukin-3 to antigen-induced Th2 cytokine production.

Short-term stimulation of mouse spleen cells in vitro with interleukin (IL)-3 induces the secretion of the Th2 cytokines IL-4 and IL-6. Non-B/non-T cells were the target of this IL-3 effect. However, during long-term antigen-dependent culture, T cells are the major source of IL-4 and IL-6. The addition of IL-3 to such cultures also led to a significant increase in IL-4 and IL-6 production. This Th2 cytokine secretion was amplified by the addition of irradiated non-B/non-T cells at the initiation of culture, and was inhibited by anti-IL-4 antibodies. These findings suggest that IL-3 induces the rapid release of IL-4 and IL-6 by non-B/non-T cells, thereby creating an immune milieu conducive to the development of antigen-specific IL-4 and IL-6-secreting Th2 cells.

Animals↗
PubMed1996

IFN-gamma is a potent inducer of Ig secretion by sort-purified murine B cells activated through the mIg, but not the CD40, signaling pathway.

IFN-gamma has been shown to either stimulate or inhibit Ig secretion. No studies have yet addressed the basis for these seemingly conflicting properties nor whether IFN-gamma acted directly at the level of the B cell to mediate its effects. Thus, we studied the ability of IFN-gamma to regulate Ig secretion in sort-purified, resting murine B cells that were >99% Ig+, activated either through membrane Ig using unconjugated or dextran-conjugated anti-IgD antibodies (alphadelta-dex) or through CD40 using soluble or membrane CD40 ligand (CD40L). B cells activated with alphadelta-dex proliferated but do not secrete Ig, even in the presence of IL-1 + IL-2. We demonstrate that IFN-gamma only when added subsequent to B cell stimulation with alphadelta-dex, but not unconjugated anti-IfD antibody, plus IL-1 + IL-2 induces up to 100-fold enhancements in Ig secretion and in the numbers of Ig-secreting cells. The predominant Ig isotype secreted is IgM, with IgG3 and IgG2a comprising the majority of non-IgM antibody. IFN-gamma must act in concert with IL-2 for stimulation of Ig secretion. Further, IFN-gamma synergizes with IL-3 + granulocyte-macrophage colony stimulating factor for induction of Ig synthesis. IFN-gamma also enhances IgA syntheses by transforming growth factor-beta-induced membrane IgA+ cells. By contrast, 125IIFN-gamma fails to stimulate Ig secretion in B cells activated with CD40L in the presence or absence of IL-1 + IL-2 or IL-4. However, the combination of CD40L and alphabeta-dex is strongly synergistic for IFN-gamma-induced Ig secretion. Thus, these data establish that IFN-gamma can act directly on the B cell to induce Ig synthesis without the participation of any other cell and demonstrates that the mode of activation of the B cell plays an important role in directing the action of IFN-gamma.

Animals↗
PubMed1995

Complexity of the cytokine and antibody response elicited by immunizing mice with Plasmodium yoelii circumsporozoite protein plasmid DNA.

The number, type, and location of cytokine- and Ab-secreting cells activated in mice immunized and boosted with plasmid DNA encoding the circumsporozoite protein of the malarial parasite Plasmodium yoelii (PyCSP) were monitored. The initial humoral response was localized to the draining lymph nodes and was characterized by production of IgG1 anti-PyCSP Abs and the Th2 cytokine IL-4. In contrast, the secondary response was dominated by IFN-gamma production (a Th1 cytokine) and the secretion of IgG2a anti-PyCSP Abs in the spleen. PyCSP DNA and mRNA were detected only in the quadriceps muscles (sites of plasmid injection), yet these sites lacked either cytokine- or Ab-secreting cells. These findings indicate that circulating lymphocytes encounter plasmid-encoded Ag in the muscle bed, initiate a humoral response in the draining lymph nodes, and then seed distal lymphoid organs. Profound differences were observed between the primary and secondary immune responses induced by plasmid immunization, which may influence vaccine efficacy.

Animals↗
PubMed1995

CpG motifs in bacterial DNA trigger direct B-cell activation.

Unmethylated CpG dinucleotides are more frequent in the genomes of bacteria and viruses than of vertebrates. We report here that bacterial DNA and synthetic oligodeoxynucleotides containing unmethylated CpG dinucleotides induce murine B cells to proliferate and secrete immunoglobulin in vitro and in vivo. This activation is enhanced by simultaneous signals delivered through the antigen receptor. Optimal B-cell activation requires a DNA motif in which an unmethylated CpG dinucleotide is flanked by two 5' purines and two 3' pyrimidines. Oligodeoxynucleotides containing this CpG motif induce more than 95% of all spleen B cells to enter the cell cycle. These data suggest a possible evolutionary link between immune defence based on the recognition of microbial DNA and the phenomenon of 'CpG suppression' in vertebrates. The potent immune activation by CpG oligonucleotides has implications for the design and interpretation of studies using 'antisense' oligonucleotides and points to possible new applications as adjuvants.

Animals↗
PubMed1995

IgE receptor-positive non-B/non-T cells dominate the production of interleukin 4 and interleukin 6 in immunized mice.

The phenotype and antigenic specificity of cells secreting interleukin (IL) 4, IL-6, and interferon gamma was studied in mice during primary and secondary immune responses. T lymphocytes were the major source of interferon gamma, whereas non-B/non-T cells were the dominant source of IL-4 and IL-6 in the spleens of immunized animals. Cytokine-secreting non-B/non-T cells expressed surface receptors for IgE and/or IgG types II/III. Exposing these cells to antigen-specific IgE or IgG in vivo (or in vitro) "armed" them to release IL-4 and IL-6 upon subsequent antigenic challenge. These findings suggest that non-B/non-T cells may represent the "natural immunity" analogue of CD4+ T helper type 2 cells and participate in a positive feedback loop involved in the perpetuation of T helper type 2 cell responses.

Animals↗
PubMed1995

Activation of IL-4 and IL-6 secreting cells by antigen.

The number, antigenic specificity and phenotype of cells secreting IL-4 and IL-6 in mice immunized with ovalbumin or keyhole limpet haemocyanin (KLH) in Freund's adjuvant (FA) was studied. The frequency of cells producing either of these cytokines began to rise 6 days post immunization, peaked at 11-14 days post-immunization, and fell to background by 21 days. The number of spleen cells secreting IL-6 was higher than the number producing IL-4 at all time points. Boosting elicited an anamnestic response characterized by a significant increase in the number of cytokine secreting cells within 4 days. Cytokine production was induced in multiple strains of normal mice, and was critically dependent on the use of Complete FA in addition to antigen. Immunization induced IL-4 and IL-6 production in vivo while 'priming' additional cells to release these cytokines when reexposed to soluble antigen in vitro. The latter response was antigen specific and was dominated by non-B/non-T cells. Those cells may serve to boost the immune response in cases of persistent or repeated antigenic challenge.

Animals↗
PubMed1995

Phenotype and frequency of cells secreting IL-2, IL-4, IL-6, IL-10, IFN and TNF-alpha in human peripheral blood.

The phenotype and frequency of cells in normal human peripheral blood spontaneously secreting IL-2, IL-4, IL-6, IL-10, IFN and TNF-alpha ex vivo was determined using ELIspot assays. CD4+ T cells were the dominant source of IL-2 and IL-4 while multiple cell types (primarily CD8+ lymphocytes) produced IFN. Fewer than 0.05% of mononuclear cells were spontaneously secreting these T cell derived factors. By comparison, IL-6, IL-10 and TNF-alpha were produced by 0.7-20% of PBMC. The primary sources of the latter cytokines were CD14+ macrophages/monocytes. A significant positive correlation was found in the frequency of cells secreting IL-6, IL-10 and TNF-alpha ex vivo, suggesting that the release of such factors was coordinately regulated. No such correlation was found among IL-2, IL-4 and IFN secreting cells, indicating that the production of predominantly T cell derived cytokines was regulated independently.

Antigens, CD↗
PubMed1995

Activation of interleukin 4- and interleukin 6-secreting cells by HIV-specific synthetic peptides.

Peptides were synthesized in which the type-specific determinant of the V3 loop region of gp120 (SP10) was expressed C terminal to a conserved T helper epitope (T1) on the same molecule. These T1-SP10 peptides can stimulate both cell-mediated and humoral immune responses. The current work used a novel approach to study the nature and specificity of the response elicited by these peptides. Cytokine-specific ELIspot assays were used to examine the number, kinetics and fine specificity of cells induced to secrete IL-4 and IL-6 in mice immunized with T1-SP10 peptides. Results indicate that the peptides activated cytokine-secreting cells in a dose-dependent manner in vivo. In vitro restimulation experiments demonstrated that both the SP10 and T1 regions contributed to this activation. Consistent with previous studies, mice sequentially immunized with peptides expressing different V3 loop regions generated B cell responses that were larger and more cross-reactive than those induced by a single peptide. Sequential immunizations had less effect on the number or specificity of the cytokine-producing cells.

Amino Acid Sequence↗
PubMed1995

Mouse monoclonal antibodies to human immunodeficiency virus glycoprotein 120 generated by repeated immunization with glycoprotein 120 from a single isolate, or by sequential immunization with glycoprotein 120 from three isolates.

Mouse hybridomas were isolated that produce monoclonal antibodies (MAbs) to several regions of the HIV envelope, gp120, which may be used to map immunogenic regions in animal and human immunity. One series of MAbs was generated by repeated immunization with recombinant gp120 (rgp120) from a single isolate, and a second series by sequential immunization with rgp120 from three isolates. All MAbs bound to rgp120IIIB, but only one bound well to cell surface-expressed gp160. Synthetic peptides spanning much of the length of gp120 were used to map MAb reactivity. Two MAbs were mapped to the C1 region, one to the C2 region, and three to the C5 region by this approach. Six distinct epitopes were detected by competitive binding analysis. One MAb binding the C1 region blocks binding of CD4 to rgp120IIIB, binds by ELISA to rgp120MN (an isolate not used during immunization), and binds to cell surface-expressed gp160. These hybridomas will be made available to the scientific community through a hybridoma culture facility.

AIDS Vaccines↗