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D K Bishop

Publications and source records attributed to D K Bishop.

At least 73 records · Page 4Linked to original sources
PubMed1990

Differential distribution of antigen-specific helper T cells and cytotoxic T cells after antigenic stimulation in vivo. A functional study using limiting dilution analysis.

We have developed modified limiting dilution analysis (LDA) techniques that distinguish in vivo Ag-stimulated murine helper T lymphocytes (HTL) and CTL from unstimulated precursor T cells, even those with the same Ag specificity. We refer to these cells that are detectable in the modified LDA as "Ag-conditioned" T cells (cHTL and cCTL). We have used the modified LDA techniques in conjunction with conventional LDA techniques (which enumerate all Ag-specific T cells) to evaluate the in vivo distribution of Ag-conditioned cHTL and cCTL following in vivo sensitization to alloantigens via sponge matrix or skin allografts. In general, we observed the following regarding the distribution of cHTL and cCTL: 1) Ag-conditioned HTL and CTL were detectable only after in vivo sensitization with alloantigen: 2) not all Ag-reactive T cells became conditioned T cells after in vivo Ag deposition; 3) the percentage of Ag-reactive T cells that converted to conditioned T cells after Ag deposition varied among different lymphoid compartments; 4) a high percentage of cHTL, but a low percentage of cCTL, accumulated in regional lymph nodes and spleen; 5) cHTL accumulated in peripheral blood, whereas cCTL did not; 6) Ag-conditioned cHTL were detectable in various lymphoid tissues for greater than 60 days following Ag deposition, whereas cCTL were detectable for only 14 to 20 days; and 7) unlike the other lymphoid sites, the site of Ag deposition accumulated a high percentage of both Ag-stimulated cHTL and cCTL. Furthermore, cHTL and cCTL appeared to reside in phenotypically distinct T cell subsets in that in vivo treatment with anti-L3T4 mAb abrogated the accumulation of HTL, but not CTL, at the site of Ag deposition. These data demonstrate differential compartmentalization of Ag-conditioned cHTL and cCTL subsequent to in vivo Ag deposition. The implications of these findings regarding the monitoring of in vivo immune responses are discussed.

Animals↗
PubMed1990

Limiting dilution analysis of alloreactive T-cell status and distribution during allograft rejection.

We have developed limiting dilution analysis techniques that discriminate between precursor T cells and T cells that have responded to prior contact with alloantigens. We used these techniques to monitor the behavior of functional T-lymphocyte subsets in sponge matrix allograft recipients. Thus, we were able to measure at the graft site and/or in the peripheral blood (1) all donor-reactive cytotoxic T lymphocytes, (2) the subset of donor-reactive cytotoxic T lymphocytes that has responded to graft alloantigens in vivo, (3) all donor-reactive helper T lymphocytes, and (4) the subset of donor-reactive helper T lymphocytes that has responded to graft alloantigens in vivo. In this communication, we describe some aspects of the in vivo T-cell response to allograft implantation that were revealed by this study.

Animals↗
PubMed1990

Vascular endothelial differentiation in sponge matrix allografts.

These studies test the hypothesis that vascular endothelia in sponge allografts may develop a function and phenotype resembling the high endothelial venules (HEV) in lymph nodes, thus facilitating the lymphocytic infiltration that is characteristic of allograft rejection. Using limiting dilution analysis to quantitate helper-T-cell accumulation at graft sites, immunohistologic analysis of graft reactivity with the HEV-specific monoclonal antibody MECA 325, and ex vivo lymphocyte-endothelial adhesion assays with graft tissues, we obtained evidence to suggest that HEV-like endothelia may develop at a graft site but that the process whereby lymphocytes accumulate at a graft site is more complex than was initially expected.

Animals↗
PubMed1990

Relationship between leukocytic infiltration and endothelial specialization in murine cardiac allografts.

We have used immunologic and histologic techniques to analyze the rejection of heterotopic murine cardiac allografts. These studies were designed to test the hypothesis that lymph-node-like mechanisms of lymphocyte extravasation develop in immediately vascularized organ allografts during the process of rejection. The experimental results suggest that lymph-node-like mechanisms are not involved, and that an unexpected pattern of lymphocyte infiltration develops in these allografts.

Animals↗
PubMed1989

Lymphocyte entry into inflammatory tissues in vivo. Qualitative differences of high endothelial venule-like vessels in sponge matrix allografts vs isografts.

Sponge matrix allografts and isografts become extensively encapsulated and neovascularized after s.c. implantation. Sponge allografts acquire alloantigen-reactive T lymphocytes, whereas sponge isografts fail to do so, even though these T cells are continuously circulating in the peripheral blood. We have investigated the possibility that the vascular endothelia regulates lymphocytic accumulation in sponge matrix implants. In normal lymph nodes, specialized high endothelial venules (HEV) regulate lymphocyte extravasation from the blood. We have now identified HEV-like vessels in sponge matrix allografts. These vessels are operationally defined as "HEV-like" in that they react with mAb MECA 325 which identifies murine HEV, and bind lymphocytes in ex vivo adhesion assays. In contrast, sponge isografts contain MECA 325 reactive vessels that are significantly smaller than those found in allografts. Further, vessels of sponge isografts do not readily bind lymphocytes in ex vivo adhesion assays. Immunohistologic analysis also revealed that the small MECA 325+ vessels present in sponge isografts are consistently found in close proximity to nerve bundles. Although this MECA 325 reactive vessel-nerve bundle association is also observed in sponge allografts, large MECA 325 reactive vessels are widely distributed in allografts. Our data suggest that small, poorly adhesive MECA 325 reactive vessels develop in sponge isografts and allografts, possibly under the influence of local nerve tissue. These vessels respond to regional alloimmune responses by developing into the larger HEV-like vessels capable of binding lymphocytes in sponge allografts. The value of this experimental system as an in vivo model to evaluate mechanisms involved in neovascularization and endothelial differentiation is discussed.

Animals↗
PubMed1989

Specificity of mismatch repair following transformation of Saccharomyces cerevisiae with heteroduplex plasmid DNA.

A method is described for genetic detection of mismatch repair products following transformation of Saccharomyces cerevisiae. The method is based on the detection of beta-galactosidase activity in clonal derivatives of cells transformed with heteroduplex plasmid DNA. Heteroduplex plasmid substrates were constructed by insertion of an oligonucleotide heteroduplex into the coding sequence of the Escherichia coli lacZ gene. The plasmid and oligonucleotides were designed so that one strand of the construct would code for a functional beta-galactosidase and the other strand would contain an in-frame nonsense codon. The frequencies of transformed clones containing only Lac+ cells, only Lac- cells, or a mixture of the two Lac phenotypes provided information on the efficiency of the repair reaction. With this method, plasmids carrying single-base substitution mismatches, a single-base frameshift mismatch (T/delta), or a 3-base-pair substitution mismatch (TGA/GAA) were tested. A/C, G/T, G/A, G/G, and T/delta mismatches were repaired with significantly greater efficiencies than C/C, A/A, T/T, and TGA/GAA. T/C was repaired with an intermediate efficiency. The frequencies of products obtained with G/G, G/A, and T/delta mismatches suggested modest inequality of repair in the two possible directions. Strains carrying the repair-deficient pms1-1 mutation were also tested. The efficiencies of repair of A/C, G/T, G/G, and A/A mismatches were reduced in pms1-1 cells compared with wild-type cells. In addition, a change in repair inequality was detected when transformation of the two strains with an A/C mismatch was compared.

DNA Repair↗
PubMed1989

Development and evaluation of a limiting dilution analysis technique that can discriminate in vivo alloactivated cytotoxic T lymphocytes from their naive CTL precursors.

We describe a permutation of the conventional limiting dilution analysis (LDA) technique that allows, for the 1st time, the differential enumeration of alloantigen-specific CTL that have been activated by alloantigens in vivo. This technique does not detect nonactivated CTL precursors, even those with similar alloantigen specificity. Data are presented to validate this limiting dilution analysis technique. Using this LDA technique, we demonstrate that large numbers of the donor-reactive CTL arrive in sponge matrix allografts (f = 1/3,599 cells), most or all of which are in an activated state (f = 1/4,385 cells). In contrast, alloactivated CTL constitute only a small fraction (f = 1/57,208 cells) of the donor-reactive CTL in the regional lymph node (f = 1/1,873 cells) of the same sponge allograft recipients. As expected, regional lymph nodes from sponge isograft recipients contain DBA/2-reactive CTL precursors (f = 1/1,873 cells), but no activated DBA/2-reactive CTL (f less than 1/385,529 cells). This LDA technique should be useful in studies regarding activation and redistribution of alloreactive CTL caused by allograft implantation.

Animals↗
PubMed1989

Inflammation-induced endothelial cell adhesion to lymphocytes, neutrophils, and monocytes. Role of homing receptors and other adhesion molecules.

Adhesion to the vascular endothelium precedes or is a necessary prelude to leukocyte migration into the underlying tissue. Constitutive lymphocyte trafficking through lymphoid organs is controlled by tissue-specific interactions between molecules expressed on the surface of the lymphocyte (homing receptors) and ligands (vascular addressins) expressed on endothelial cells (HEV) within lymphoid tissues. Preliminary evidence suggests that lymphocytes may employ related but distinct interactions in their entry into some chronic sites of inflammation. Other leukocytes, such as neutrophils and monocytes, express molecules related or identical to lymphocyte homing receptors, and these molecules are exquisitely regulated by chemotactic factors and appear to be involved in the homing of these cells to inflamed tissues. In addition, inflammation in vivo induces increased endothelial cell adhesiveness for leukocytes that undoubtedly plays a key role in regulating leukocyte extravasation. Tissue- and inflammation-specific leukocyte/endothelial cell adhesion molecules constitute attractive targets for suppression or manipulation of the early stages of tissue inflammation.

Animals↗
PubMed1989

Limiting dilution analysis for alloreactive, TCGF-secretory T cells. Two related LDA methods that discriminate between unstimulated precursor T cells and in vivo-alloactivated T cells.

We have developed a rapid limiting dilution analysis technique for quantitating alloantigen-specific TCGF-secretory T lymphocytes (operationally defined as helper T lymphocytes or HTL) in murine lymphoid populations. A simple permutation of this technique allows the distinction between HTL that have encountered antigen in vivo and the large population of naive precursor HTL (pHTL) with the same alloantigen specificity. We present evidence to validate these LDA techniques, and show that lymphoid tissue from mice bearing sponge matrix allografts, but not isografts, contain HTL that have been activated in vivo. In addition, we demonstrate a requirement for restimulation with alloantigen for secretion of lymphokine by the in vivo-activated cells. We suggest that this differential LDA technique could serve as a valuable tool in evaluating in vivo immunity and/or the in vivo efficacy of immunosuppressive, as well as immunopotentiating, therapies.

Animals↗
PubMed1989

In vivo mechanisms of alloreactivity. VI. Evidence that alloantigen deposition initiates both local and systemic mechanisms that influence CTL accumulation at a graft site.

We have used sponge matrix allografts to investigate how alloantigen influences the pattern of CTL accumulation at a graft site. These studies employed two limiting dilution analysis techniques to monitor CTL accumulation. One technique quantitates the subpopulation of CTL that show evidence of in vivo contact with graft alloantigens (alloantigen-conditioned CTL or cCTL). The other quantitates all CTL with specificity for graft alloantigens, regardless of their differentiative status. Using these techniques, we have demonstrated that sponge allografts acquire three types of CTL: (a) donor-reactive CTL precursors (pCTL), (b) donor-reactive cCTL, and (c) pCTL with irrelevant antigen specificity. Sponge isografts rarely acquire LDA-detectable CTL, unless they receive a concurrent allogeneic stimulus at a distant anatomic site. In that instance, sponge isografts acquired pCTL, but not cCTL. This indicates that an ongoing immune response to alloantigens can influence the immunologic characteristics of an unrelated inflammatory response occurring elsewhere. We further observed that sponge isografts can be made to acquire alloreactive cCTL only when specific alloantigens are placed in the isograft. This indicates that specific grant alloantigen present at the graft site plays a necessary but undefined role in the acquisition or development of cCTL in sponge allografts. Collectively, these data demonstrate that alloantigen deposition initiates both local and systemic mechanisms that influence alloreactive CTL accumulation at a graft site.

Animals↗
PubMed1987

Adoptive transfer of immunity to Listeria monocytogenes. The influence of in vitro stimulation on lymphocyte subset requirements.

BALB/c mice develop specific and relatively long lasting immunity after exposure to sublethal numbers of viable Listeria monocytogenes. This immunity can be passively transferred to naive recipients with maximal protection conferred by spleen cells obtained from donors 6 days after immunization. Immunity that can be directly transferred to syngeneic recipients is surprisingly short lived. Cell recipients lose immunity as early as 72 hr after transfer, and recipients express no detectable immunity after 1 wk. This short lived immunity requires both L3T4+ and Lyt-2+ T cell populations for full expression. Both the level of immunity transferred and the duration of the protective response expressed in recipients are dramatically increased if the spleen cell population is cultured in vitro with concanavalin A before cell transfer. Recipients of concanavalin A-activated cells express antigen-specific levels of immunity increased 100- to 1000-fold compared with syngeneic recipients of directly transferred immune spleen cells. In addition, this elevated level of adoptively transferred immunity remains constant for at least 8 wk. Transfer of this culture-enhanced immunity requires only an Lyt-2+ T cell population and is not influenced by cells of the L3T4+T cell subpopulation. Both direct as well as culture-enhanced transfer of immunity require major histocompatibility complex-compatible recipients. These findings suggest that two phenotypically distinct T cell subpopulations function in the development of the immune response to L. monocytogenes and that only one cell subpopulation is required for expression of immunity to this intracellular parasite.

Animals↗
PubMed1986

Repair of heteroduplex plasmid DNA after transformation into Saccharomyces cerevisiae.

Purified heteroduplex plasmid DNAs containing 8- or 12-base-pair insertion mismatches or AC or CT substitution mismatches were used to transform Saccharomyces cerevisiae. Two insertion mismatches, separated by 943 base pairs, were repaired independently of each other at least 55% of the time. This suggested that repair tracts were frequently shorter than 1 kilobase. The two insertion mismatches were repaired with different efficiencies. Comparison of the repair efficiency of one mismatched site with or without an adjacent mismatch suggests that mismatches promote their own repair and can influence the repair of neighboring mismatches. When two different plasmids containing single-insertion mismatches were transformed into S. cerevisiae cells, a slight preference towards insertion was detected among repair products of one of the two plasmids, while no repair preference was detected among transformants with the second plasmid.

Base Sequence↗
PubMedDate not supplied

Analysis of retinoid-mediated immunosuppression in vivo. Effects of Ro23-6457 on cellular alloimmune responses.

We have investigated the immunosuppressive effects of a synthetic retinoid, Ro23-6457, on the in vivo development of cellular alloimmunity. We initially observed that treatment of C57B1/6 mice with 10 mg/kg/day Ro23-6457 drug could prolong the survival of DBA/2 cardiac allografts, thus verifying its immunosuppressive potential in murine experimental models. We next used the sponge matrix model of allograft rejection and limiting dilution analysis (LDA) of cytotoxic T lymphocyte (CTL) frequency to dissect this phenomenon further. In this experimental system we observed the following effects of Ro23-6457: (1) dose-dependent decrease in the number of LDA-detectable, donor-reactive CTL accumulating in sponge matrix allografts; (2) failure to interfere with in vitro assays of cellular alloimmunity, including LDA; and (3) antigen non-specific depression of LDA-detectable CTL in lymph nodes, spleen and especially in peripheral blood. For peripheral blood CTL, the drug eliminated LDA-detectable CTL, an effect that was reversible and could not be attributed to the activation of suppressor cells. Since Ro23-6457 has little effect on the number of peripheral blood Thy1.2+ cells, it appears that this drug does not physically eliminate CTL, but makes them temporarily hyporesponsive to antigen stimulation, and thus undetectable in functional assays like LDA.

Animals↗
PubMedDate not supplied

The role of heteroduplex correction in gene conversion in Saccharomyces cerevisiae.

Two different models have been proposed to explain the relative frequencies of the non-mendelian allelic segregations which are detected by tetrad analysis after meiosis in fungi. The first model maintains that 6:2 type tetrads result from correction of heteroduplexes containing mismatched sites and 5:3 type tetrads result from failure to correct mismatched sites. The second model suggests that 6:2 segregations result from the filling-in of double-strand gaps using information obtained from both strands of a homologous duplex. In this model 5:3 type tetrads result if the allele is included in the heteroduplex regions flanking the gap and the resulting mismatched nucleotides are not corrected. We have studied the correction of heteroduplex plasmid DNA in pms1 mutant strains of Saccharomyces cerevisiae, which are known to exhibit higher frequencies of 5:3 type tetrads and lower frequencies of 6:2 tetrads than wild-type strains. Our results suggest that the pms1 mutation causes a defect in mismatch correction, supporting the hypothesis that meiotic gene conversion in wild-type yeast cells often results from the correction of heteroduplex DNA.

DNA Repair↗