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Biomedical subjects

D G Kilburn

Publications and source records attributed to D G Kilburn.

At least 109 records · Page 6Linked to original sources
PubMed1984

Mode of action and substrate specificities of cellulases from cloned bacterial genes.

Three recombinant plasmids, pEC1, pEC2, and pEC3, each containing a unique Cellulomonas fimi chromosomal DNA insert, expressed Cm-cellulase activities in Escherichia coli C600 (Whittle, D. J., Kilburn, D. H., Warren, R. A. J., and Miller, R. C., Jr. (1982) Gene (Amst.) 17, 139-145; Gilkes, N. R., Kilburn, D. G., Langsford, M. L., Miller, R. C., Jr., Wakarchuk, W. W., Warren, R. A. J., Whittle, D. J., and Wong, W. K. R. (1984) J. Gen. Microbiol. 130, 1377-1384). Viscometric and chemical analyses showed that the enzymes encoded by pEC2 and pEC3 behaved as endoglucanases, whereas that encoded by pEC1 behaved as an exoglucanase. The activities of the exoglucanase and the pEC2-encoded endodglucanase were additive on Cm-cellulose as substrate. The pEC1-encoded enzyme also hydrolyzed xylan and p-nitrophenyl cellobioside. Two substrate-bound Cm-cellulases were isolated from the residual cellulose in a C. fimi culture by guanidine hydrochloride elution, affinity chromatography, and polyacrylamide gel electrophoresis. Both were glycoproteins of apparent Mr = 58,000 and 56,000, respectively. The 56-kDa enzyme appeared to be identical with the pEC1-encoded product, suggesting that they arise from the same gene.

Actinomycetales↗
PubMed1984

Quantitation of helper factor activity. II. Inhibition of antibody binding.

A sensitive direct assay has been developed to quantitate antigen specific helper factors (ThF). The assay depends on the ability of ThF to block the binding of free antibody to immobilized antigen. It has been used to follow the course of purification of ThF produced in short-term cultures of in vitro primed helper cells and purified by affinity chromatography on antibody and antigen immunoabsorbents. The identity of the ThF was confirmed by assaying its biological activity and by its reactivity with anti-Ia antibody and with a monoclonal anti-ThF.

Animals↗
PubMed1984

The use of cytotoxic T cells in the regulation of tumour growth in syngeneic mice.

Normal C57BL/6 (B6) spleen cells were cultured with syngeneic EL4 tumour cells, expanded in IL2-containing medium, and tested for anti-tumour activity in vitro and in vivo. The activated cells were highly cytotoxic for EL4 and to a lesser degree killed syngeneic B6 blasts and allogeneic (D2) P815 tumour cells. B6 or BDF1 mice that received these cultured cells by IP injection cleared 125IUdR-labelled EL4 cells faster than untreated mice. However, this enhanced clearance was evident only 7-12 days after injection. Since the injected cells had a short half-life (less than 10% remaining after 48 h) the effect of these cells in vivo was most probably due to the activation of the host's immune system. Mice that received cultured cells survived significantly longer than untreated mice following a lethal dose of EL4 cells. Cultured cells were much more effective in prolonging survival when used in conjunction with cyclophosphamide (CY). In animals receiving either cultured cells with or without CY or CY alone tumour clearance was markedly enhanced 7-12 days after injection. When challenged with a small dose of EL4 tumour cells (1 X 10(4) SC per mouse) three of ten B6 mice treated with B6 anti-EL4 cultured cells were able to survive indefinitely. The frequency of CTL precursors to EL4 from the spleen cells of these surviving animals was about five-fold higher than that of normal spleen cells. Furthermore, CTL derived from primed spleen cells were more specific for EL4 than those derived from normal spleen cells.

Animals↗
PubMed1984

Induction of CTL responses to alloantigens by a Db-specific T helper clone.

A T cell helper clone was derived 2 yr ago from a mixed lymphocyte culture. This clone, referred to as clone 9, was propagated in interleukin 2 (IL 2)-containing medium in the presence of irradiated stimulator and irradiated syngeneic spleen cells. Clone 9 was of H-2d origin and was found to be Thy-1+ and Lyt-1-2-. Clone 9, as well as supernatant factor(s) derived from it, were able to enhance the primary cytotoxic responses of Db responder cells to alloantigens. Furthermore, clone 9 cells or its factor(s) were only active when added during the first 24 hr of a 5-day culture period. When a low stimulator cell dose (10(4) cells per 0.2 ml culture) was used, it was possible to demonstrate that clone 9 also required a source of irradiated allogeneic splenic accessory cells to exert its helper action. Under these conditions, clone 9 or its factor(s) could also synergize with IL 2-containing medium in mounting cytotoxic responses to alloantigens. Synergy between IL 2-containing medium and clone 9 or its factor(s) was observed only when Db responder cells were used. The helper activity in clone 9 supernatant was also specifically absorbed out by Con A-stimulated Db spleen cell blasts. Preincubation with clone 9 supernatant for 1 hr at room temperature also led to enhanced cytotoxic responses of Db responder cells to alloantigens. Clone 9 supernatant was also found to be devoid of detectable IL 2 activity. Thus, clone 9 or its helper factor(s) appear to exert its helper activity by an early interaction with Db cytotoxic T lymphocyte precursors (CTL-P).

Animals↗
PubMed1983

Cellular and genetic basis for suppression of cytotoxic T cell generation by haloaromatic hydrocarbons.

Generation of allospecific cytotoxic T cells in C57Bl/6 mice is significantly impaired following exposure to TCDD at doses as low as 4 ng/kg. T helper activity, as assessed by the ability to produce Interleukin 2, and frequency of CTL precursors appear unaffected in treated animals and thus the TCDD-induced suppressor cells we have described are primarily responsible for the reduction in the CTL response. Suppression of CTL generation in DBA/2 mice requires a 10--100-fold greater dose than in C57Bl/6, consistent with the observation that the Ah locus gene(s) of DBA/2 mice code for TCDD receptors with low binding affinity for TCDD. Other haloaromatic hydrocarbons (3,3'4,4'-tetrachlorobiphenyl and Aroclor 1254), capable of binding to the TCDD receptor, also suppress CTL generation, whereas the 2,2',4,4',6,6'-hexachlorobiphenyl molecule that lacks affinity for the TCDD receptor does not suppress CTL. The immunotoxic effects of TCDD in C57B/6 and DBA/2 mice occur at dose levels below those required to induce mixed-function oxidase enzymes in the liver. Suppression of CTL by TCDD is associated with increased susceptibility to lethal herpes virus type II infection. These data suggest that low levels of TCDD may interact with cytoplasmic receptors for TCDD in the thymus and induce biologically significant immunosuppression through activation of suppressor cells.

Animals↗
PubMed1982

The effects of cyclophosphamide on in vitro cytotoxic responses to a syngeneic tumour.

We have studied the effects of treating DBA/2 mice with high doses of cyclophosphamide upon their subsequent ability to generate cytotoxic cells in vitro against syngeneic tumour antigens or alloantigens. High doses of cyclophosphamide (100-200 mg/kg body weight) eliminated the response to both antigens. The addition of normal DBA/2 thymocytes into these cultures restored the response to allogeneic cells but not to tumour cells. The anti-tumour response could be restored by the addition of interleukin 2 to the cultures. Treatment with high doses of cyclophosphamide decreased the number of anti-tumour cytotoxic cell precursors in the spleen, but did not affect the capacity of bulk cultures of spleen cells to produce interleukin 2 when stimulated with the mitogen concanavalin A.

Animals↗
PubMed1982

Molecular cloning of a Cellulomonas fimi cellulose gene in Escherichia coli.

A sensitive and simple immunoassay was developed to screen Escherichia coli transformed with recombinant DNA plasmids carrying a cellulase gene. The assay was used to identify a recombinant DNA plasmid carrying at least one cellulase gene from Cellulomonas fimi. The enzyme present in extracts of E. coli carrying the plasmid was active in catalysing the hydrolysis of carboxymethylcellulose as indicated by the production of reducing sugars.

Cellulase↗
PubMed1981

Quantitation of helper factor activity. I. Utilization of the ELISA method to measure helper activity in the secondary antibody response.

We describe here a simple culture system for measuring the effects of regulatory cells or factors on the secondary antibody response which uses the enzyme-linked immunosorbent assay (ELISA) to detect antibody production. This technique offers several advantages over methods which measure hemolytic plaque-forming cells. Helper factors purified from the supernatant of in vitro primed helper cells were shown to specifically enhance antibody production from primed spleen cells by from 2- to 4-fold in this system.

Animals↗
PubMed1981

Antigen-specific helper factors present in the supernatant of concanavalin A-induced spleen cell cultures.

The supernatants from cultures of concanavalin A-induced spleen cells contained both antigen-specific and nonspecific (Interleukin 2) helper factors (Hf). The antigen-specific factor could be isolated from the supernatant by adsorption onto and elution from antigen-Sepharose immunoadsorbents. Specific Hf was produced in cultures of either immune or nonimmune spleen cells although in the latter case the quantity of Hf was significantly less. The specific Hf did not manifest the thymocyte stimulatory property of 112.

Animals↗
PubMed1981

Helper cells active in the generation of cytotoxicity to a syngeneic tumor.

We have examined a population of thymic lymphocytes that augment the generation in vitro of specific cytotoxic T cells against P815.X2, a syngeneic tumor in DBA/2 mice. These helper cells are evident in the thymus of DBA/2 mice 5 to 7 days after subcutaneous injection of live P815 cells. They are Thy-1 positive, resistant to gamma radiation, antigen specific, and appear to exert their influence most effectively when added at the beginning of the culture.

Animals↗
PubMed1981

Characteristics of an antigen-specific helper factor that enhances the in vitro generation of cytotoxic cells directed against a syngeneic tumor.

An antigen-specific helper factor (Hf) that enhances the in vitro generation of tumor-specific cytotoxic T cells (CL) has been isolated from the spleens of DBA/2 mice bearing the syngeneic mastocytoma, P815. This factor enhances the CL response to P815 but not to L1210 (a syngeneic T cell leukemia in DBA/2 mice), B10 minor antigens, or DBA/2 alloantigens. The factor binds specifically to P815 membrane immunoadsorbent columns and can be eluted with 2 M NaCl. P815 Hf did not resemble the lymphokine Il 2 because it did not enhance the proliferative or CL response of thymocytes nor would it support the growth of an IL 2-requiring line of T cells. This factor acted optimally if added late in the culture period in contrast to IL 2, which was more effective if added early. In limiting dilution experiments P815 Hf enhanced the level of CL activity generated but not the number of precursors triggered. IL 2 markedly amplified both of these parameters.

Animals↗
PubMed1979

Further characterization of thymic suppressor cells and a factor that suppress the generation of cells cytotoxic for a syngeneic tumor in DBA/2 mice.

Antigen-specific suppressor cells and suppressive extracts obtained from the thymuses of DBA/2 mice bearing small syngeneic P815 mastocytomas were compared for their immunogenetic properties and requirements. The assay for specific suppression involved the ability of either cells or extracts to inhibit the primary in vitro cytotoxic response of normal DBA/2 splenocytes to mitomycin-treated P815 cells. It was shown that pretreatment of suooressor cell populations with anti-Iad antiserum plus rabbit complement removed the suppressive activity. Similarly, absorption of the suppressor factor with anti-Iad antiserum removed the suppressive properties of the material. It was found that the suppressor cells, generated in DBA/2 tumor bearers, were capable of specifically suppressing the anti-P815 response of B6D2 F1 radiation chimeras possessing lymphoid cells of the H-2b or H-2t2 haplotype equally as well as they could suppress the response of H-2d-bearing effector cells. This indicates that the suppressor cells are not H-2 restricted with respect to K or D markers on the responder cells in this system.

Animals↗
PubMed1978

Effect of adult thymectomy on tumour immunity in mice.

The effect of adult thymectomy in DBA/2J mice on the in vitro response to syngeneic tumour cells was investigated. Spleen cells from adult mice which had been thymectomized 8 weeks previously demonstrated a severely impaired primary cytotoxic response to P815 tumour cells, whereas their cytotoxic responses to allogeneic cells (C57BL/6) and to non-H-2 antigens (BALB/c), and their ability to form a primary antibody response to sheep red blood cells was unimpaired. Suppressor T cells, specific for P815 cells, appeared early in the thymuses of animals inoculated with P815 cells (between 4 and 8 days after tumour-cell injection). No differences in tumour growth between animals thymectomized as adults and sham-operated controls were observed, and thymectomized tumour-bearing animals had levels of specific suppressor cells in their lymph nodes equivalent to the levels found in untreated controls. Severely thymocyte-deprived animals which had been thymectomized, irradiated and reconstituted with either marrow or spleen cells 8 weeks before tumour implantation succumbed more rapidly to metastatic tumour than did control animals.

Animals↗
PubMed1978

Characterization of a soluble factor that specifically suppresses the in vitro generation of cells cytotoxic for syngeneic tumor cells in mice.

A tumor-specific soluble factor found in extracts of thymocytes from mice bearing small P815 tumors has been demonstrated. This factor is capable of significantly suppressing the in vitro generation of syngeneic cells cytotoxic for P815 targets if it is added to culture vessels within the first 30 hr of culture. The suppressive factor eluted from Sephadex G-100 after hemoglobin and was estimated to have a m.w. in the range of 40 to 60,000. Preparative isoelectric focusing of thymic extracts established that the suppressive material has an isoelectric point in the range of pH 4.6 to 4.9. The suppressive activity of extracts could be removed by passage of the material through immunoadsorbent columns prepared from membrane proteins of P815 cells but not by analogous columns prepared by using L1210 membrane proteins. The suppressive material was not removed by its passage through immunoadsorbent columns containing anti-mouse immunoglobulin.

Animals↗
PubMed1977

Identity of a T-lymphocyte inhibitor with mouse immunoglobulin in the serum of tumour-bearing mice.

A component of the serum of tumour-bearing mice has been shown to be inhibitory to the immunological function of normal mouse T cells. This factor fractionates with monomeric immunoglobulin upon gel filtration. Studies were carried out utilizing goat antisera to the major immunoglobulin chains of mouse Ig (kappa, gamma, alpha and mu) mixed with the immunoglobulin-rich fraction of serum from normal mice and tumour bearers and passed through immunoadsorbent columns prepared with rabbit anti-goat immunoglobulin. Such studies showed that the inhibitory activity in tumour-bearing serum could be removed after treatment with anti-kappa, anti-gamma and anti-mu chain antisera but not by treatment with either anti-alpha chain or goat immunoglobulin. That the inhibitory activity of tumour-bearing immunoglobulin could be attributed to simple quantitative differences in the levels of IgM and IgG in the test samples was discounted by quantification of the amounts of immunoglobulins in normal and tumour-bearing sera.

Animals↗