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R C Duke

Publications and source records attributed to R C Duke.

At least 37 records · Page 2Linked to original sources
PubMed1995

HIV gp120-specific cell-mediated immune responses in mice after oral immunization with recombinant Salmonella.

Salmonella is of great interest as a potential human immunodeficiency virus vaccine vector because of its ability to elicit potent mucosal and systemic immune responses when administered orally. To determine whether such a vaccine could elicit an immune response in mice, plasmids expressing HIV gp120-LAI were introduced into attenuated S. typhimurium. Three serial doses of 10(10) recombinant organisms were administered orally to BALB/c mice at 2-week intervals. Immunized mice but not control mice demonstrated proliferative T cell responses to gp120-LAI, comparable in magnitude to the proliferative responses to Salmonella antigens. Immunized mice had detectable serum and intestinal Salmonella-specific IgA and serum Salmonella-specific IgG. However, no gp120-specific antibody was detected in either serum or intestinal washes. These results indicate that live recombinant Salmonella-based vaccine constructs can induce HIV-specific cellular immune responses in vivo.

AIDS Vaccines↗
PubMed1995

A role for CD95 ligand in preventing graft rejection.

Testis is a remarkable immune-privileged site, long known for its ability to support allogeneic and xenogeneic tissue transplants. Here we have investigated the molecular basis for testis immune privilege. Testis grafts derived from mice that can express functional CD95 (Fas or Apo-1) ligand survived indefinitely when transplanted under the kidney capsule of allogeneic animals, whereas testis grafts derived from mutant gld mice, which express non-functional ligand, were rejected. Further analysis of testis showed that CD95 ligand messenger RNA is constitutively expressed by testicular Sertoli cells, and that Sertoli cells from normal mice, but not gld mice, were accepted when transplanted into allogeneic recipients. CD95 ligand expression in the testis probably acts by inducing apoptotic cell death of CD95-expressing, recipient T cells activated in response to graft antigens. These findings indicate that CD95 ligand could be used to create immune-privileged tissue for a variety of transplant uses.

Animals↗
PubMed1995

Macrophage colony-stimulating factor complementary DNA: a candidate for gene therapy in metastatic melanoma.

BACKGROUND: At present, there is no highly effective treatment for metastatic melanoma. Innovative approaches aimed at inducing a more effective immune response against tumors have shown promising results in animal models. One approach involves the genetic modification of tumor cells so that they produce cytokines that stimulate an immune response. PURPOSE: The aim of this study was to determine the effectiveness of cytokine gene therapy for metastatic melanoma in a murine melanoma model. METHODS: B16F10 murine melanoma cells, which readily metastasize to the lungs, were transduced with a retroviral vector containing genes encoding neomycin resistance and human macrophage colony-stimulating factor (M-CSF). The presence of M-CSF messenger RNA in transduced cells was examined by coupled reverse transcription and polymerase chain reaction. Concentrations of soluble M-CSF in cell culture supernatants were determined by enzyme-linked immunosorbent assays (ELISAs). A clonal cell line, designated N+/CSF+, that expressed and secreted M-CSF was identified. Another clonal cell line, designated N+/CSF-, did not secrete M-CSF at levels detectable by ELISA. B16F10, N+/CSF-, and N+/CSF+ cells, individually or in combination, were injected intravenously or subcutaneously into C57BL/6 mice; we then evaluated the tumorigenicity and metastatic behavior of the cells, as well as the immune responses and survival of the mice. The immune responses assayed were the cytotoxic T lymphocyte (CTL) and peritoneal exudate cell (PEC) tumoricidal activities. RESULTS: Injection of B16F10 cells into the tail vein of C57BL/6 mice led to the establishment of lung metastases by week 2 and death by week 8. Injection of the N+/CSF+ or N+/CSF- cells led to the establishment of lung metastases that were detected at 2 and 3 weeks, respectively; however, these metastatic lesions were eliminated, and the animals had survival rates similar to those of the noninjected control mice. Injection of mice with a mixture of B16F10 and N+/CSF- cells resulted in the development of metastatic disease and 0% survival at 8 weeks, whereas mice that had been given an injection of a mixture of B16F10 and N+/CSF+ cells had an 80% survival rate at 8 weeks and survived at least two times longer (P = .007). The CTL and PEC tumoricidal activities in animals given an injection of N+/CSF+ cells suggested that monocytes and lymphocytes were responsible for the observed antitumor response. CONCLUSION: These findings suggest that the expression of M-CSF by genetically modified melanoma cells caused an effective antitumor immune response in host C57BL/6 mice and, thus, prolonged survival over that observed in the control mice.

Animals↗
PubMed1995

Co-ligation of mouse complement receptors 1 and 2 with surface IgM rescues splenic B cells and WEHI-231 cells from anti-surface IgM-induced apoptosis.

Recent studies have shown that complement receptors play important roles in both T-dependent and T-independent B lymphocyte responses to low doses of antigen (Ag) in vivo. Complement activation by either the classical or alternative pathway results in the covalent binding of C3 molecules to Ag in forms that ligate complement receptors type 1 (CR1) and 2 (CR2). We hypothesized that C3-bound Ag might cross-link CR2 and/or CR1 with surface (s)IgM and alter the signal that would be transduced through sIgM by Ag binding alone. One result of the altered signal could be the rescue of B lymphocytes from apoptosis that would otherwise be induced by the binding of certain types of Ag alone. We find that co-cross-linking of mouse CR2 and CR1 with sIgM rescues both resting B cells and WEHI-231.7 cells from apoptosis induced by sIgM ligation in a fashion similar to that found using soluble mouse CD40 ligand (mCD40L). Anti-CR2/CR1-mediated rescue requires co-cross-linking of the receptors with sIgM, and has an additive effect on mCD40L-mediated apoptosis rescue. Based on these results, it is likely that the CR2/CR1-derived signal is cooperative with T cell-derived signals such as CD40L and interleukin-4.

Animals↗
PubMed1994

Cytolysis mediated by ionophores and pore-forming agents: role of intracellular calcium in apoptosis.

Apoptosis is a term used to describe certain forms of physiological cell death that occur during embryogenesis, differentiation, and normal cell turnover. Previous reports concerning the effects of calcium ionophores on rodent thymocytes and the pore-forming proteins perforin and staphylococcal alpha-toxin on murine tumor cells led to the suggestion that simply raising intracellular calcium causes apoptotic cell death. This hypothesis was tested using two ionophores, A23187 and valinomycin, and two pore-forming agents, melittin and staphylococcal alpha-toxin, on four murine tumor cell lines. Although treatment with these agents could raise intracellular calcium, and in some instances cause DNA fragmentation, only valinomycin caused apoptosis. In contrast to previous reports, our results suggest that raising intracellular calcium and inducing internucleosomal DNA fragmentation is not sufficient to elicit apoptotic cell death in all cell types.

Animals↗
PubMed1992

Apoptosis and programmed cell death in immunity.

Death of some cells in the mammalian body is clearly programmed. In the immune system there are many examples of programmed cell death, during development of lymphocytes as well as at later stages, after interaction with antigen. Many of these examples display the morphology of apoptosis: They undergo shrinkage and zeiosis, the nucleus collapses, and chromatin is cleaved into nucleosomal fragments. The cell is rapidly recognized by phagocytes and disposed of without releasing its contents. In some but not all cases of apoptosis, new macromolecular synthesis is required. Cytotoxic T cells induce changes in their targets that are morphologically apoptotic. The mechanism of apoptosis is currently under active investigation.

Animals↗
PubMed1992

Apoptosis in cytotoxic T lymphocytes and their targets.

Cytotoxic T lymphocytes have been useful not only in understanding immune responses but also in providing valuable insights into the biology and mechanism of apoptosis. In this article two examples of apoptosis directly related to cytotoxic T lymphocyte biology are discussed. These are apoptosis of activated cytotoxic T cells as a result of antigen clearance and subsequent growth factor deprivation and apoptosis of target cells following interaction with cytotoxic T lymphocytes.

Animals↗
PubMed1989

Purified perforin induces target cell lysis but not DNA fragmentation.

Rapid and extensive target cell DNA fragmentation is a unique characteristic of CTL-mediated killing. We studied the role of the granule pore-forming protein (PFP/perforin/cytolysin) of CTL in mediating lysis and DNA fragmentation of target cells. Perforin was isolated from murine CTL by sequential application of perforin-enriched granule fractions to four chromatographic columns: DEAE-Sepharose, Q-Sepharose, Polyanion SI, and Superose 12. Purified perforin was eluted as a single band of 70 kD in SDS-PAGE. While purified perforin produced potent lysis of a variety of target cells tested, it did not induce any measurable amount of DNA fragmentation. In parallel experiments, intact CTL produced marked DNA fragmentation of the same target cell populations. Our results suggest that perforin alone is not responsible for the DNA fragmentation observed during CTL-mediated killing and that other, as yet unknown, mediators or mechanisms are likely to be involved in the induction of target cell nuclear damage.

Animals↗
PubMed1989

Self recognition by T cells. I. Bystander killing of target cells bearing syngeneic MHC antigens.

Activated CTL can kill any cell to which they bind or by which they are bound. This observation has been used to determine whether alloreactive CTL can recognize cells bearing self-MHC. When activated by their specific targets, 19 CTL clones of 4 different specificities and origins killed bystander targets bearing syngeneic but not third-party MHC antigens. Using target cells derived from MHC-recombinant animals, syngeneic bystander killing was shown to be restricted to a single self MHC-encoded molecule. These results provide the first clear demonstration that T cells, or more precisely CTL, are capable of self recognition in the absence of their specific antigen. Our findings support the model that T cell repertoire selection occurs as a result of positive selection during maturation in the thymus of precursor cells whose antigen receptors have low but real affinity for self-MHC.

Animals↗
PubMed1988

Cytotoxic lymphocyte-derived lytic granules do not induce DNA fragmentation in target cells.

When target cells are exposed to CTL, they very quickly sustain nuclear damage, including DNA cleavage, and then they lyse. Nuclear damage of this type is not seen when cells are killed by antibody and C. The role of nuclear damage in the T cell-mediated killing process as well as the mechanism by which the killer cell induces this damage are unknown; however, accumulating evidence suggests that cytolysis may depend on induction of nuclear damage. The exocytosed contents of CTL granules are thought by many workers to mediate target cell lysis. We have now determined whether lytic granules also induce nuclear damage (DNA fragmentation) in cells which they lyse. They do not. In addition, no DNA fragmentation was detected in nuclei incubated with lytic granules or activated CTL. In summary, our results suggest that target cell DNA fragmentation induced by CTL is mediated neither by lytic granules nor by a CTL-derived endonuclease and support the view that the target cell is itself responsible for the internal damage it sustains.

Animals↗
PubMed1986

Differences in target cell DNA fragmentation induced by mouse cytotoxic T lymphocytes and natural killer cells.

Fragmentation of YAC-1 target cell DNA during cytolysis mediated by mouse natural killer (NK) cells and cytotoxic T lymphocytes (CTL) was compared. Cleavage of nuclear chromatin was always an extensive and early event in CTL-mediated cytolysis, whereas with NK cell-mediated killing the degree of DNA fragmentation showed an unexpected relationship to the effector:target (E:T) ratio. At low NK:YAC-1 ratios, DNA fragmentation and 51Cr release were equivalent and increased proportionately until a ratio of about 50:1 was reached; at higher ratios, 51Cr release increased as expected but DNA fragmentation decreased dramatically. Comparison of time course data at E:T ratios producing similar rates of 51Cr release showed that the target cell DNA fragmentation observed in NK killing was not nearly as rapid nor as extensive as that observed with CTL effectors. These results suggest that NK cells induce target cell injury via two different mechanisms. One mechanism would involve lysis mediated by cell-to-cell contact, while the other may induce DNA fragmentation via a soluble mediator. In support of this notion, cell-free culture supernatants containing NK cytotoxic factor (NKCF) induced DNA fragmentation in YAC-1 cells. The DNA fragments induced by NK cells and NKCF-containing supernatants consisted of oligonucleosomes indistinguishable from those induced by CTL. The results presented here show distinct differences in target cell DNA fragmentation induced by CTL and NK cells, and suggest that these two effectors use different mechanisms to achieve the same end. CTL seem to induce DNA fragmentation in their targets by direct signaling, whereas NK cells may do so by means of a soluble factor.

Animals↗
PubMed1986

IL-2 addiction: withdrawal of growth factor activates a suicide program in dependent T cells.

IL-2-dependent T effector cells usually die when deprived of growth factor. Cell death (as measured by plasma membrane breakdown) requires protein synthesis because it is inhibited by cycloheximide or emetine. When the DNA in several IL-2-dependent cell lines was examined following removal of IL-2, it was found that extensive chromatin cleavage precedes plasma membrane breakdown by several hours. The DNA fragments observed were not randomly generated but consisted of oligonucleosomes. This suggests that IL-2 deprivation led to activation of an endonuclease with specificity for linker DNA. DNA fragmentation, like cell death, did not occur in the presence of protein synthesis inhibitors. The protein(s) synthesized in response to IL-2 deprivation may, therefore, be the endonuclease or its activator; none of the IL-2-dependent T cells examined contain detectable endogenous endonuclease prior to IL-2 removal. DNA fragments were also found in vivo in lymph node cells draining a site of antigen administration. These results suggest that one aspect of the termination of immune responses involves activation of a cell suicide program in the expanded effector T cell clones. In this program an endonuclease is activated and chromatin is cleaved; as a result macromolecular synthesis begins to wind down so that repair synthesis stops; within a few hours, the cell lyses.

Animals↗
PubMed1984

Glucocorticoid activation of a calcium-dependent endonuclease in thymocyte nuclei leads to cell death.

Dexamethasone and corticosterone kill mouse thymocytes, as measured by eosin uptake, after several hours of in vitro incubation. This killing requires RNA and protein synthesis, because it is inhibited by cycloheximide, emetine, or actinomycin D. An earlier event than cell death is the extensive fragmentation of nuclear DNA into oligonucleosomal subunits; this fragmentation also requires RNA and protein synthesis. The DNA cleavage results from the action of an endonuclease that preferentially cleaves DNA in the linker regions between nucleosomes. This endonuclease is found constitutively in the nuclei of thymocytes and some other cells, and requires calcium and magnesium ions for its activation; if isolated fresh thymocyte nuclei are incubated with these ions, as much as 77% of their DNA is cleaved within 90 min. Thus, the protein for which synthesis is necessary for glucocorticoid-induced thymocyte death is not the endonuclease itself, but is in some way involved in its activation; we suggest that it may be part of a system for transporting calcium into the nucleus. The endonuclease is inhibited by zinc, which also prevents thymocyte death. It appears that glucocorticoids cause thymocyte death by activating an enzyme that rapidly and extensively degrades DNA. This "death from within" is biochemically and morphologically different from toxic or accidental cell death, such as that induced by azide, heat, or antibody and complement treatment. Although mature T cells also contain the endogenous endonuclease, they lack the glucocorticoid-inducible mechanism for activating it, and are thus glucocorticoid-resistant.

Animals↗