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D K Bishop

Publications and source records attributed to D K Bishop.

At least 37 records · Page 2Linked to original sources
PubMed1998

Xrcc3 is required for assembly of Rad51 complexes in vivo.

Rad51 is a member of a family of eukaryotic proteins related to the bacterial recombinational repair protein RecA. Rad51 protein localizes to multiple subnuclear foci in Chinese hamster ovary cells. Subnuclear Rad51 foci are induced by ionizing radiation or the DNA cross-linking agent cisplatin. Formation of these foci is likely to reflect assembly of a multimeric form of Rad51 that promotes DNA repair. Formation of damage-induced Rad51 foci does not occur in the Chinese hamster ovary cell line irs1SF, which is sensitive to DNA damaging agents. The Rad51 focus formation defect of irs1SF cells is corrected by a construct that encodes the repair protein Xrcc3. Xrcc3 is a human homolog of Rad51 previously isolated by virtue of its ability to correct the radiation sensitivity of irs1SF cells. Changes in the steady state level of Rad51 protein do not account for the irs1SF defect nor do they account for the appearance of foci following DNA damage. These results suggest that Xrcc3 is required for the assembly or stabilization of a multimeric form of Rad51 during DNA repair. Cell lines defective in two different components of DNA protein kinase formed Rad51 foci in response to damage, indicating DNA protein kinase is not required for damaged-induced mobilization of Rad51.

Animals↗
PubMed1998

Tolerance induction by anti-CD2 plus anti-CD3 monoclonal antibodies: evidence for an IL-4 requirement.

Anti-CD2 mAb plus anti-CD3 mAb induce alloantigen specific tolerance. We sought to determine whether Th2 cytokines are involved in the induction of tolerance in this model. Addition of anti-IL-4 mAb or anti-IL-10 mAb to anti-CD2 plus anti-CD3 treatment abrogated tolerance and resulted in graft survivals of 26+/-4 and 25+/-5 days, respectively. Splenocytes from the anti-IL-4 mAb and anti-IL-10 groups had greater proliferation in response to alloantigen than either tolerant or naive groups. Cytokine analysis of MLR supernatants showed increased IL-10 in the tolerant group and increased IFN-gamma in the anti-IL-4 mAb treated group. Donor-specific alloantibody responses in untreated immune animals had a predominantly Th1 (IgG2a) alloantibody response, while the tolerogenic regimen reduced the ratio of IgG2a:IgG1 titers. The addition of anti-IL-4 mAb to the tolerogenic regimen partly restored the Th1-related IgG2a response. Tolerance did not develop in IL-4 knockout animals treated with anti-CD2 plus anti-CD3 (mean graft survival, 27+/-5 days). Restoration of IL-4 to IL-4 knockout animals by gene transfer with plasmid DNA resulted in prolongation of survival to 46+/-7 days, while adoptive transfer of wild-type splenocytes into IL-4 knockout recipients resulted in indefinite graft survival (>60 days) and indefinite survival of second donor-type grafts. IL-10 gene transfer to IL-4 knockout recipients did not prolong graft survival (28+/-4). These results demonstrate that tolerance in this model is mediated at least in part by Th2-type cells that secrete IL-4, promote IL-10 and IgG1 production, and inhibit alloantigen reactivity.

Animals↗
PubMed1998

Rad52 associates with RPA and functions with rad55 and rad57 to assemble meiotic recombination complexes.

We show that the Saccharomyces cerevisiae recombination protein Rad52 and the single-strand DNA-binding protein RPA assemble into cytologically detectable subnuclear complexes (foci) during meiotic recombination. Immunostaining shows extensive colocalization of Rad52 and RPA and more limited colocalization of Rad52 with the strand exchange protein Rad51. Rad52 and RPA foci are distinct from those formed by Rad51, and its meiosis-specific relative Dmc1, in that they are also detected in meiosis during replication. In addition, RPA foci are observed during mitotic S phase. Double-strand breaks (DSBs) promote formation of RPA, Rad52, and Rad51 foci. Mutants that lack Spo11, a protein required for DSB formation, are defective in focus formation, and this defect is suppressed by ionizing radiation in a dose-dependent manner. DSBs are not sufficient for the appearance of Rad51 foci; Rad52, Rad55, and Rad57 are also required supporting a model in which these three proteins promote meiotic recombination by promoting the assembly of strand exchange complexes.

Adenosine Triphosphatases↗
PubMed1998

Enhanced T cell engraftment after retroviral delivery of an antiviral gene in HIV-infected individuals.

Intracellular expression of gene products that inhibit viral replication have the potential to complement current antiviral approaches to the treatment of AIDS. We previously have shown that a mutant inhibitory form of an essential viral protein, Rev M10, prolongs the survival of T cells transduced with a nonviral vector in HIV-infected individuals. Because these gene-modified cells were not observed in patients beyond 8 weeks, efforts were made to improve the duration of engraftment. In this study, we used retroviral vector delivery of Rev M10 to CD4(+) cells and analyzed relevant immune responses in a pilot study of three HIV-seropositive patients. DNA and RNA PCR analyses revealed that cells transduced with Rev M10 retroviral vectors survived and expressed the recombinant gene for significantly longer time periods than those transduced with a negative control vector in all three patients. Immune responses were not detected either to Rev M10 or to Moloney murine leukemia virus gp70 envelope protein. Rev M10-transduced cells were detected for an average of 6 months after retroviral gene transfer compared with approximately 3 weeks for the previously reported nonviral vector delivery. These findings suggest that retroviral delivery of an antiviral gene may potentially contribute to immune reconstitution in AIDS and could provide a more effective vector to prolong survival of CD4(+) cells in HIV infection.

Animals↗
PubMed1998

Alloantigen-reactive Th1 development in IL-12-deficient mice.

IL-12p70, a 70- to 75-kDa heterodimer consisting of disulfide-bonded 35-kDa (p35) and 40-kDa (p40) subunits, enhances Th1 development primarily by its ability to induce IFN-gamma production by NK and Th1 cells. Although homodimers of the p40 subunit of IL-12 are potent IL-12 receptor antagonists in some systems, we have reported that p40 homodimer may accentuate alloreactive CD8+ Th1 function. To test the role of endogenously produced p40 in alloimmunity, Th1 development was assessed in either IL-12 p35 knockout (p35-/-) mice, the cells of which are capable of secreting p40, or p40 knockout (p40-/-) mice. Compared with IL-12 wild-type controls, splenocytes obtained from both p35-/- and p40-/- mice produced markedly less IFN-gamma after in vitro stimulation with Con A or alloantigens. Interestingly, in vivo-sensitized Th1 were detected in both p35-/- and p40-/- cardiac allograft recipients. However, in vivo Th1 development was enhanced in p35-/- recipients compared with p40-/- animals, suggesting that endogenous p40 produced in p35-/- mice may stimulate alloreactive Th1. Indeed, neutralizing endogenous p40 with anti-IL-12 p40 mAb reduced Th1 development in p35-/- allograft recipients to that seen in p40-/- mice. To determine whether Th1 development that occurred in the absence of IL-12p70 and p40 required IFN-gamma, p40-/- allograft recipients were treated with anti-IFN-gamma mAb. Neutralizing IFN-gamma did not inhibit in vivo Th1 development in p40-/- recipients and resulted in a unique pathology of rejection characterized by vascular thromboses. Collectively, these data suggest that 1) endogenous p40 may substitute for IL-12p70 in alloantigen-specific Th1 sensitization in vivo and 2) in vivo alloreactive Th1 development may occur independent of IL-12 and IFN-gamma, suggesting an alternate Th1-sensitizing pathway.

Animals↗
PubMed1998

Lipid-mediated gene transfer of viral IL-10 prolongs vascularized cardiac allograft survival by inhibiting donor-specific cellular and humoral immune responses.

The gene encoding the immunosuppressive cytokine viral interleukin-10 (vIL-10) was introduced into BALB/c (H-2d) vascularized cardiac allografts by perfusing the graft vasculature with DNA-liposome complexes, utilizing the experimental cationic lipid gamma AP DLRIE/DOPE and a plasmid encoding vIL-10 under the control of the HCMVie promoter. The DNA to lipid ratio and DNA dose were critical factors in obtaining optimal biologic effects. Gene transfer of vIL-10 with a 3:1 DNA to lipid weight ratio using 375 micrograms DNA significantly prolonged allograft survival in MHC-mismatched C57BL/6 (H-2b) recipients (16.00 days) compared with both unmodified allografts (8.14 days) and vIL-10 antisense controls (8.28 days). Enhanced graft survival was specific to vIL-10 expression since treatment with antisense plasmid or anti-vIL-10 monoclonal antibody (mAb) abrogated the effect. Prolonged survival was associated with a novel histology characterized by a moderate mononuclear infiltrate, edema, and diffuse fibrillar/collagen deposition in the interstitium. Despite these morphologic changes, myocytes remained viable and vessels were patent. Limiting dilution analysis revealed transient infiltration of IL-2 secreting, donor-reactive, helper T lymphocytes (HTL) and cytotoxic T lymphocytes (CTL) in vIL-10 expressing grafts on day 7, that decreased significantly by day 14. Similarly, vIL-10 gene transfer inhibited the accumulation of donor-specific HTL and CTL in the spleen, compared with antisense controls. Prolonged survival was also associated with a marked decrease in IgM and IgG alloantibody production, with little to no IgG isotype switching. These results show that viral IL-10 gene transfer inhibits graft rejection in a clinically relevant model by inhibiting donor-specific cellular and humoral immune responses.

Animals↗
PubMed1997

Type 1 versus type 2 cytokine release by Vbeta T cell subpopulations determines in vivo antitumor reactivity: IL-10 mediates a suppressive role.

We have previously reported that CD8+ tumor-draining lymph node (TDLN) cells activated with anti-CD3 and IL-2-mediated tumor regression in adoptive immunotherapy. In this study, we examined the TCR Vbeta repertoire usage of TDLN cells with respect to cytokine release profiles and therapeutic efficacy in vivo. The majority of the whole population of TDLN cells after activation with anti-CD3 were composed of Vbeta3+, -5+, -7+, -8+, and -11+ cells. Enrichment of Vbeta subsets of TDLN cells by in vitro activation with anti-Vbeta mAb revealed Vbeta8+ cells released high amounts of IFN-gamma and granulocyte/macrophage-CSF (GM-CSF) with minimal amounts of IL-10 in response to tumor and mediated tumor regression in vivo. In contrast, enriched populations of Vbeta5+, Vbeta7+, and Vbeta11+ cells released low amounts of IFN-gamma and GM-CSF with high levels of IL-10 and had no in vivo antitumor reactivity. In vitro depletion of specific Vbeta subsets from the whole TDLN pool confirmed that the profile of cytokines released correlated with in vivo antitumor function. Therapeutic efficacy mediated by TDLN cells required the release of IFN-gamma and GM-CSF since in vivo neutralization of both cytokines inhibited tumor regression. The administration of anti-IL-10 mAb abrogated the suppressed antitumor response manifested by adoptively transferred TDLN cells, which elaborated increased levels of IL-10. Our study documents that type 1 cytokine release (i.e., IFN-gamma and GM-CSF) promotes in vivo tumor Ag recognition, in contrast to type 2 release (i.e., IL-10), which suppresses this interaction, and discriminates the functional activity of Vbeta subpopulations of effector cells.

Adoptive Transfer↗
PubMed1997

Differential effects of IL-12 receptor blockade with IL-12 p40 homodimer on the induction of CD4+ and CD8+ IFN-gamma-producing cells.

The role of IL-12 role in regulating Th1/Th2 balance is attributed in part to the ability of this cytokine to induce IFNgamma production by NK and Th1 cells, which in turn promotes Th1 and inhibits Th2 development. In the present study, the requirement for IL-12 in the development of alloantigen-reactive Th1 was assessed by adding neutralizing anti-IL-12 Abs or the IL-12 receptor antagonist p40 homodimer to primary MLC. The resulting cell populations were assessed for Th1 development by measuring IFN-gamma production upon restimulation with alloantigens. While the addition of anti-IL-12 Abs to primary MLC did not influence subsequent cytokine production, addition of p40 homodimer markedly enhanced, rather than decreased, Th1 development. To determine which T cell population produced enhanced levels of IFN-gamma in response to p40 homodimer, CD4+ or CD8+ T cells were depleted from the MLC. While p40 homodimer was inhibitory to selected CD4+ Th1 development, it enhanced IFN-gamma production by CD8+ T cells. To test the in vivo relevance of these findings, mouse heterotopic cardiac allograft recipients were treated with either p40 homodimer, anti-CD8 mAb, or with both p40 homodimer and anti-CD8 mAb. Treatment of allograft recipients with p40 homodimer had no effect on the in vivo sensitization of IFN-gamma-producing cells and resulted in accelerated allograft rejection relative to unmodified recipients. However, p40 homodimer markedly prolonged allograft survival in mice depleted of CD8+ T cells. Hence, p40 homodimer stimulates CD8+ Th1 development in vitro but inhibits CD4+ T cell function both in vitro and in vivo.

Animals↗
PubMed1997

Inhibition of the alloantibody response by CD95 ligand.

We investigated the effect of Fas/APO1-ligand (CD95L) gene transfer on allogeneic immune responses in vivo. A colon carcinoma cell line from BALB/c mice, CT26, was stably transfected with a vector encoding mouse CD95L and was inoculated into C57BL/6 mice. CD95L expression markedly reduced allogeneic cytotoxic T lymphocyte and helper T lymphocyte activity directed toward CT26. Strikingly, expression of CD95L on these allogeneic tumors completely inhibited the generation of alloantibodies of both IgM and IgG subclasses. Thus, CD95L inhibited alloantibody production and conferred localized immune suppression through this mechanism. These results provide insight into the role of CD95L in regulating the alloantibody response and the generation of local immune responses.

Animals↗
PubMed1997

Saccharomyces cerevisiae recA homologues RAD51 and DMC1 have both distinct and overlapping roles in meiotic recombination.

BACKGROUND: Rad51 and Dmc1 are Saccharomyces cerevisiae homologues of the Escherichia coli recombination protein RecA. Mutant analysis has shown that both proteins are required for normal meiotic recombination, for timely and efficient formation of synaptonemal complex and for normal progression out from meiotic prophase. RESULTS: We have further characterized rad51 and dmc1 single mutants. A dmc1 mutation confers more severe defects in double strand break (DSB) resolution, crossover recombination and meiotic progression than does a rad51 mutant; in contrast, during return to growth, a rad51 mutation confers more severe defects in viability and intrachromosomal recombination than does a dmc1 mutation. Analysis of a rad51 dmc1 double mutant, in parallel with single mutants, shows that the double mutant is more defective with respect to the formation of crossovers during meiosis and, especially strikingly, with respect to interhomologue and intrachromosomal recombination during return to growth. Consistent with the observation of DMC1-dependent recombination in a rad51 mutant, subnuclear complexes of Dmc1 protein were detected for the first time in this mutant. In contrast to the effects on recombination, the effect of the double mutant on meiotic progression was similar to that of the rad51 single mutant. CONCLUSION: Rad51 and Dmc1 each make unique contributions to meiotic recombination. However, the two proteins are capable of substituting for one another under some circumstances, implying that they most likely share at least one recombination function. Recombination and cell cycle phenotypes are all consistent with the possibility that a dmc1 mutation causes an arrest of the post-DSB recombination complexes at a later, more stable stage than does a rad51 mutation.

Cell Cycle Proteins↗
PubMed1996

Immune response in human melanoma after transfer of an allogeneic class I major histocompatibility complex gene with DNA-liposome complexes.

Analysis of the antitumor immune response after gene transfer of a foreign major histocompatibility complex class I protein, HLA-B7, was performed. Ten HLA-B7-negative patients with stage IV melanoma were treated in an effort to stimulate local tumor immunity. Plasmid DNA was detected within treated tumor nodules, and RNA encoding recombinant HLA-B7 or HLA-B7 protein was demonstrated in 9 of 10 patients. T cell migration into treated lesions was observed and tumor-infiltrating lymphocyte reactivity was enhanced in six of seven and two of two patients analyzed, respectively. In contrast, the frequency of cytotoxic T lymphocyte against autologous tumor in circulating peripheral blood lymphocytes was not altered significantly, suggesting that peripheral blood lymphocyte reactivity is not indicative of local tumor responsiveness. Local inhibition of tumor growth was detected after gene transfer in two patients, one of whom showed a partial remission. This patient subsequently received treatment with tumor-infiltrating lymphocytes derived from gene-modified tumor, with a complete regression of residual disease. Thus, gene transfer with DNA-liposome complexes encoding an allogeneic major histocompatibility complex protein stimulated local antitumor immune responses that facilitated the generation of effector cells for immunotherapy of cancer.

Adult↗
PubMed1996

A meiotic recombination checkpoint controlled by mitotic checkpoint genes.

In budding yeast, meiotic recombination occurs at about 200 sites per cell and involves DNA double-strand break (DSB) intermediates. Here we provide evidence that a checkpoint control requiring the mitotic DNA-damage checkpoint genes RAD17, RAD24 and MEC1 ensures that meiotic recombination is complete before the first meiotic division (MI). First, RAD17, RAD24 and MEC1 are required for the meiotic arrest caused by blocking the repair of DSBs with a mutation in the recA homologue DMC1. Second, mec1 and rad24 single mutants (DMC1+) appear to undergo MI before all recombination events are complete. Curiously, the mitosis-specific checkpoint gene RAD9 is not required for meiotic arrest of dmc1 mutants. This shows that although mitotic and meiotic control mechanisms are related, they differ significantly. Rad17 and Rad24 proteins may contribute directly to formation of an arrest signal by association with single-strand DNA in mitosis and meiosis.

Cell Cycle Proteins↗
PubMed1996

IL-12 antagonism induces T helper 2 responses, yet exacerbates cardiac allograft rejection. Evidence against a dominant protective role for T helper 2 cytokines in alloimmunity.

IL-12 promotes Th1 development and inhibits the generation of Th2 by inducing IFN-gamma production. In several experimental models, Th2 are preferentially induced in the absence of IL-12. It was proposed that the preferential induction of Th2 by IL-12 antagonism would inhibit Th1-driven rejection responses, thereby promoting allograft acceptance. To test this possibility, mouse cardiac allograft recipients were treated with either anti-IL-12 Abs, or with the IL-12 receptor antagonist p40 homodimer. Unmodified rejection is characterized by a Th1-dominated response, with Th2 cytokines being absent or only weakly expressed within the allograft. Though both forms of IL-12 antagonism induced Th2 cytokine expression within the allograft, these treatments surprisingly exacerbated graft rejection relative to control animals. Interestingly, IL-12 antagonism did not inhibit IFN-gamma gene expression or in vivo sensitization of IFN-gamma-producing cells. Similar observations were made when IL-12 p40 knockout mice were used as allograft donors and recipients, verifying that IL-12 was not required for Th1 development. Further, IL-12 antagonism was associated with strong expression of p40 and weak expression of p35 within the graft. Neither p35 nor p40 mRNAs were detectable in control allografts. These data indicate that while IL-12 antagonism does induce Th2 cytokine expression within cardiac allografts, Th2 cytokines do not play a dominant protective role in the rejection process. Further, the Th2-inducing activity of IL-12 antagonism is not related to decreased IFN-gamma production, but may reflect altered regulation of IL-12 itself.

Animals↗
PubMed1996

Allogeneic class I MHC requirement for alloantigen-reactive helper T-lymphocyte responses in vivo. Evidence for indirect presentation of alloantigen.

The mechanisms by which host T cells recognize transplant-associated alloantigens in vivo have not been established. Two alloantigen presentation pathways may be used: (1) allogeneic class I and class II MHC molecules may be recognized directly by host CD8+ and CD4+ cells, respectively, or (2) allogeneic MHC molecules may be processed as foreign peptide and presented by host antigen-presenting cells to CD4+ cells in the context of self class II proteins. In this study, the sponge matrix allograft model was used to examine the relative contributions of these alloantigen presentation pathways to CD4+ T-cell activation in vivo. Limiting dilution analysis was used to quantify the localization of interleukin-2-producing helper T lymphocytes (HTL) following implantation of sponge allografts. Allografts either were disparate at both class I and class 11, or were derived from beta2-microglobulin knockout (beta2M-/-) mice, which express class II but are deficient in class I. Two measures of in vivo HTL function were monitored: (1) the accumulation of HTL within the allograft (a process that is dependent upon antigen-driven cytokine production), and (2) the development of cytolytic alloantibodies. After implantation of sponge allografts expressing both class I and class II, HTL were readily detectable in the allograft, and cytolytic alloantibodies were present in the serum. When mice were implanted with beta2M-/- sponge allografts, HTL failed to infiltrate these class I-deficient allografts, and alloantibodies were not detectable in the sera of recipients of beta2M-/- sponge allografts. This in vivo requirement for class I expression was not reflected by traditional in vitro measures of HTL function; cells obtained from lymphoid tissues mounted a mixed lymphocyte response and produced interleukin-2 when stimulated with beta2M-/- splenocytes in vitro. One possible interpretation of these data is that in vivo HTL functions are dependent upon the presence of class I-reactive CD8+ T cells. However, HTL readily infiltrated grafts expressing both class I and class II when recipients depleted of CD8+ T cells, and alloantibodies were produced. These observations support the idea that indirect presentation of allogeneic class I molecules plays a critical role in regulating CD4+ HTL functions associated with allograft rejection in vivo.

Animals↗
PubMed1996

Restricted V beta usage by T cells infiltrating rejecting human lung allografts.

TCR expression was evaluated in lung transplant patients to determine whether T cells infiltrating rejecting lung allografts employed restricted V beta elements. Serial bronchoalveolar lavage (BAL) specimens were obtained from six lung transplant recipients at approximately 3 wk, 6 wk, and 3 mo post-transplant. T cell lines were established by culturing lavage cells with irradiated donor splenocytes in the presence of low dose IL-2 for 3 wk, and TCR V beta usage was determined by quantitative reverse transcriptase-PCR. Patients were grouped into three categories based on TCR V beta profiles and the clinical status of the allograft. 1) In one patient, BAL-derived T cells expressed heterogeneous V beta repertoires at all time points evaluated. This patient did not experience graft rejection during the 16-mo period of observation, though respiratory infections were diagnosed. 2) In three patients, V beta usage by BAL-derived T cells was restricted during allograft rejection episodes, but was heterogeneous in the absence of rejection and during respiratory infections. In one of these patients, similar V beta repertoires were employed by BAL cells during multiple rejection episodes. 3) In two patients, restricted V beta usage by BAL-derived T cells was observed before and during rejection episodes. Collectively, these data illustrate that human lung allograft rejection, but not pulmonary infection, is associated with T cells expressing a limited number of V beta families. Restricted V beta usage by graft-reactive T cells may allow for the selective elimination of these cells using TCR-specific reagents, thereby promoting allograft-specific tolerance.

Bronchoalveolar Lavage Fluid↗
PubMed1996

Lysis of adult ventricular myocytes by cells infiltrating rejecting murine cardiac allografts.

BACKGROUND: Immunologic mechanisms that mediate myocardial cell injury during rejection are not fully understood. We therefore investigated whether cells that infiltrate rejecting cardiac allografts are capable of directly injuring myocytes and whether this injury resembles that produced by cytotoxic T lymphocytes (CTLs) that are generated in a mixed lymphocyte reaction (MLR). METHODS AND RESULTS: Heart-infiltrating cells (HICs) were isolated from murine heterotopic BALB/c cardiac allografts undergoing rejection 6 to 8 days after transplantation into C57BL/6 mice. An in vitro model system of cultured adult murine ventricular myocytes was developed to facilitate investigation of cell-mediated myocyte injury. Isolated adult myocytes were incubated with either HICs or MLR effector cells, and myocyte death was quantified by counting the number of rod-shaped myocytes excluding trypan blue. The frequency of donor-reactive CTLs was similar in the HIC and MLR populations, as assessed by limiting dilution analysis. However, HICs were less efficient at killing donor-strain myocytes than were MLR cells. CTL-mediated cell lysis occurred by 6 hours, whereas myocyte injury produced by HICs was more gradual, with considerable cytotoxicity occurring between 12 and 24 hours. Furthermore, whereas MLR cells lysed only donor-strain myocytes, HIC lysed donor, third-party, and syngeneic myocytes. Treatment of MLR cells and HICs with anti-CD8 antibody plus complement produced a much greater inhibition of MLR cytotoxicity than of HIC cytotoxicity. CONCLUSIONS: These data demonstrate that only a small component of myocyte injury mediated by allograft-infiltrating cells can be ascribed to CTLs within the infiltrating cell population. These findings suggest that cell types associated with a delayed-type hypersensitivity response, as well as CTLs, cause myocyte injury during cardiac rejection.

Animals↗