Biomedical subjects
M Bennett
Publications and source records attributed to M Bennett.
Expression of three plant glutamine synthetase cDNA in Escherichia coli. Formation of catalytically active isoenzymes, and complementation of a glnA mutant.
Three cDNA clones encoding the closely related glutamine synthetase (GS) alpha, beta and gamma polypeptides of Phaseolus vulgaris (French bean) were recombinantly expressed in Escherichia coli. The GS expression plasmids correctly synthesised the recombinant alpha, beta and gamma polypeptides which then assembled into catalytically active homo-octameric isoenzymes. These isoenzymes behaved similarly to their native homologues on ion-exchange and gel-filtration chromatography. Furthermore, the alpha and gamma isoenzymes complemented a GS(glnA)-deficient mutant, thus demonstrating their physiological activity in E. coli. Differences were observed between the three recombinant GS plasmids in their quantitative expression of the GS polypeptides and their ability to complement the E. coli mutant. These differences were correlated to the degree of solubility of the polypeptide, which was observed to be dependent on the temperature of expression. The production of active GS isoenzymes in E. coli facilitates the isolation and characterisation of the individual P. vulgaris homo-octameric GS isoenzymes.
An absence of T cells in murine bone marrow allografts leads to an increased susceptibility to rejection by natural killer cells and T cells.
The mechanisms behind the increased incidence of marrow graft failure in recipients that receive allogeneic marrow depleted of T cells were studied. Recipient mice were lethally irradiated and challenged with bone marrow cells (BMC) from C.B-17 +/+ (+/+) donors. Radioisotope 125IUdR incorporation was assessed 5 to 7 days after transfer to determine the extent of engraftment. Some groups received BMC in which the T cells were removed by treatment with antibody and C. In addition, some groups received BMC from T cell-deficient C.B-17 scid/scid (SCID) mice to determine the postulated need for donor T cells in hematopoiesis and engraftment. In a model system that distinguishes between possible host NK cell and radioresistant T cell-mediated rejection of marrow allografts, it was determined that the absence of donor T cells in a marrow graft does not affect engraftment in syngeneic recipients. However, both host NK cell and radioresistant T cell rejection was markedly enhanced when SCID BMC or BMC from C.B-17 +/+ donors that had T cells removed by antibody and complement were infused into irradiated allogeneic recipients. Furthermore, the addition of alloreactive thymocytes as a source of T cells could abrogate this increased susceptibility of the BMC to host rejection mechanisms. As determined by histology and 59Fe uptake, the addition of thymocytes resulted in enhanced erythropoiesis. These results suggest that the increased incidence of marrow graft failure when BMC depleted of T cells are used is a result of active rejection by host effector cells and that the adverse effect of marrow T cell depletion can be reversed by the addition of thymocytes.
Natural killer cells in the thymus. Studies in mice with severe combined immune deficiency.
The relationship between NK cell and T cell progenitors was investigated by using mice with severe combined immune deficiency (scid). Scid mice are devoid of mature T and B cells because they cannot rearrange their Ig and TCR genes. However, they have normal splenic NK cells. Thymus of scid mice, although markedly hypocellular, contains cells that lyse YAC-1, an NK-sensitive tumor cell. By flow cytometry, two populations of cells were identified in the scid thymus. Eighty percent of the cells were Thy-1+, IL-2R(7D4)+, J11d+, CD3-, CD4-, CD8- whereas the remaining were IL-2R-, J11d-, CD3-, CD4-, and CD8-. By cell sorting, all NK activity was found in the latter population, which is phenotypically similar to splenic NK cells. To determine if the thymus contains a bipotential NK/T progenitor cell, J11d+, IL-2R+ cells were cultured and analyzed for the generation of NK cells in vitro. These cells were used because they resemble 15-day fetal and adult CD4- CD8- thymocytes that are capable of giving rise to mature T cells. Cultured J11d+ thymocytes acquired non-MHC-restricted cytotoxicity, but in contrast to mature NK cells, the resulting cells contained mRNA for the gamma, delta, and epsilon-chains of CD3. This suggests that J11d+ cells are early T cells that can acquire the ability to kill in a non-MHC-restricted manner, but which do not give rise to NK cells in vitro. The differentiative potential of scid thymocytes was also tested in vivo. Unlike bone marrow cells, scid thymocytes containing 80% J11d+ cells failed to give rise to NK cells when transferred into irradiated recipients. Together these results suggest that mature NK cells reside in the thymus of scid mice but are not derived from a common NK/T progenitor.
Natural killer cells activated with interleukin 2 in vitro can be adoptively transferred and mediate hematopoietic histocompatibility-1 antigen-specific bone marrow rejection in vivo.
An adoptive transfer model was developed for examining natural killer (NK) cell-mediated bone marrow cell (BMC) graft rejection. Homogeneous populations of NK cells were obtained by culturing spleen cells from mice with severe combined immune deficiency in the presence of recombinant interleukin 2 for 7 days. These cells maintained a phenotypic (CD3-CD4-CD8-NK-2.1+ and asialo GM1+) and lytic pattern consistent with that of activated NK cells. The cells were infused into lethally irradiated recipients whose own NK cells were depleted by anti-NK-1.1 antibody treatment and could no longer reject marrow allografts. The transferred NK cells restored the ability of the hosts to reject BMC in a hematopoietic histocompatibility-1 (Hh-1) antigen-specific manner. Immunogenetic studies demonstrated that the rejection was a function of the donor, not host, NK cells. These studies demonstrate that NK cells can be cultured in vitro with recombinant interleukin 2 and are capable of mediating Hh-1-specific BMC rejection in vivo in the absence of other immune cell types.
Canine coronavirus infection in cats; a possible role in feline infectious peritonitis.
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Peroxisome proliferation and induction of peroxisomal enzymes in mouse and rat liver by dehydroepiandrosterone feeding.
Dehydroepiandrosterone (DHEA) treatment is effective in the prevention of various genetic and induced disorders of mice and rats. In studies designed to define some of the basic mechanisms that underline the beneficial chemopreventive effects exerted by the action of this steroid, we found that the liver undergoes profound changes that result in: (i) hepatomegaly; (ii) color change from pink to mahogany; (iii) proliferation of peroxisomes; (iv) increased cross-sectional area and volume density of peroxisomes; (v) increased or decreased number of mitochondria per cell; (vi) decreased mitochondrial cross-sectional area; (vii) marked induction of the peroxisomal bifunctional protein enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase; (viii) increased activities of enoyl-CoA hydratase and other peroxisomal enzymes assayed in this study, viz. catalase, carnitine acetyl-CoA transferase, carnitine octanoyl-CoA transferase, and urate oxidase; and (ix) increased activity of mitochondrial carnitine palmitoyl-CoA transferase. In addition, feeding DHEA to mice resulted in increased plasma cholesterol levels in two strains of mice evaluated in this study, and either slightly decreased or markedly increased plasma triglyceride levels, depending on the strain. Whether liver peroxisome proliferation, induced by DHEA feeding to mice and rats, plays a role in the chemopreventive effects elicited by this steroid remains to be established.
Children's understanding of the mitigating function of disclaimers.
Despite earlier work indicating young children's competence in the use and understanding of retrospective "facework strategies" such as apologies, justifications, and excuses, it was hypothesized that an understanding of the disclaimer, a prospective strategy, would be relatively late appearing. An experiment is reported in which 144 British children between the ages of 5 and 11 years were presented with stories concerning an actor who had brought about certain negative consequences, having either previously issued, or not, a disclaimer. Children were asked to indicate and justify whether they felt the actor should be punished. Broadly consistent with the hypothesis guiding the present study, significant reductions in punishment recommendations following the provision of a disclaimer were found only among the 11-year-old subjects. However, from subjects' justifications for the decision not to punish, it was noted that nearly 50% of the 8-year-olds and 25% of the 5-year-olds provided evidence of understanding the mitigating function of disclaimers.
Resistance to Mycoplasma pulmonis mediated by activated natural killer cells.
Infection of C57BL/6J mice with Mycoplasma pulmonis (MP) enhanced NK cell function 3-7 days later, as detected by in vitro and in vivo assays. Moreover, spleen and lung cells of acutely infected C57BL/6J mice inhibited MP growth in vitro. The effectors were eliminated by treatment with anti-NK antibody in vivo and anti-asialo GM1 serum or anti-3A4 antibody plus complement in vitro. Clearance of viable and radiolabeled MP from the lungs was also enhanced in acutely infected mice. Acutely infected mice with severe combined immunodeficiency (SCID) eliminated viable MP faster than did uninfected mice. Antibodies to interferon-gamma (IFN-gamma) impaired clearance of MP from the lungs of SCID mice and decreased their survival times. Activated NK cells can function in resistance to early stages of infection with MP. NK cells directly inhibit MP with secrete IFN-gamma, which may activate macrophages or inhibit the growth of MP or both.
Hemopoietic histocompatibility (Hh-1) regulatory and structural genes of the f haplotype map to H-2.
Natural resistance to bone marrow stem cell (BMC) grafts in lethally irradiated mice is a consequence of natural killer cell recognition and elimination of BMC that express hemopoietic histocompatibility (Hh) antigens inherited noncodominantly. The major Hh genetic region, Hh-1, maps to H-2. The phenotype of the BMC was determined by grafting BMC into panels of irradiated mice, and measuring splenic incorporation of the radioactive specific DNA precursor, 5-iodo-2'-deoxyuridine [125I] (IUdR) 5 days after cell transfer. Our previous analysis of Hh-1 antigen expression on BMC of intra-H-2 recombinant inbred strain mice indicated that Hh-1 regulatory genes (Hh-1r) map in the H-2S/H-2D interval. Nine recombinants described here shared the following: (1) the DL region was donated by H-2f that was associated with expression of determinant 2, shared with H-2d/Hh-1d, and (2) had crossovers in the S/D or Ea/S intervals or within Eb, and (3) had BMC that were Hh-1 null. F1 hybrids of crosses between mice of these recombinant strains with H-2d strain mice had BMC that expressed determinant 2, suggesting that a structural gene had been lost by the crossover event. Crosses with H-2b strain mice produced mice whose BMC were Hh-1 null, indicating that the H-2f/Hh-1f regulatory genes were still intact. We suggest that Hh-1r maps to the S/D interval of all haplotypes so far studied, even H-2f. The structural gene for the f haplotype is centromeric of Eb and may be Kf, based on recent data supporting the role of class I antigens in Hh antigen expression.
Combined inheritance of epithelial and erythrocyte receptors for Haemophilus influenzae.
Haemophilus influenzae type b expressing fimbriae showed no adherence to buccal epithelial cells and no agglutination of erythrocytes from three AnWj-negative siblings in one family. Hemagglutination of erythrocytes from 13 AnWj-positive members of the same family and from 24 controls was normal, and H. influenzae adhered well to buccal epithelial cells from them. These data indicate that the expression of epithelial and erythrocyte receptors for H. influenzae is inherited concomitantly. Combined with previous data (L. van Alphen, J. Poole, L. Geelen, and H. Zanen, Infect. Immun. 55:2355-2358, 1987), the results show that the receptor molecules on the surfaces of the epithelial cell and the erythrocyte are different but that the binding sites for the fimbriae of H. influenzae are similar.
Biological evaluation of Mycoplasma pulmonis temperature-sensitive mutants for use as possible rodent vaccines.
Temperature-sensitive mutants (TSMs) of Mycoplasma pulmonis were produced by treating the wild-type strain with N-methyl-N'-nitro-N-nitrosoguanidine. Three TSMs were selected at 38 degrees C, as a restrictive temperature, and at 34 degrees C, as a permissive temperature. Two TSMs, UTCMI and UTCMII, were proven to be nonpathogenic but immunogenic. In addition, they did not induce pneumonia, tracheitis, or tympanitis but did induce mild rhinitis. They were stable after 10 passages in vitro and in vivo. They elicited excellent antibody production and cell-mediated immunity in vaccinated rats. They also were not mitogenic to rat lymphocytes. Rats immunized intranasally with these TSMs were significantly protected against challenge with wild-type organisms. These mutants were morphologically and serologically indistinguishable from the wild-type organisms. The growth characteristics and antibiotic sensitivities were similar to those of wild-type organisms, except that they grew only at 34 degrees C. In contrast to wild-type organisms, they did not bind to or lyse sheep erythrocytes. Thus, these TSMs may qualify as a vaccine to prevent M. pulmonis infection in rats.
Mechanisms of chemoprevention by dietary dehydroisoandrosterone. Inhibition of lymphopoiesis.
The ingestion of dehydroisoandrosterone (DHA), a naturally occurring steroid, inhibits the development of autoimmunity, neoplasias, and other disorders of rodents. Potential mechanisms of action include (1) the induction of peroxisomal proliferation and (2) the conversion of DHA to androgens. We evaluated the immune system of mice fed DHA. Dietary DHA had no significant effect on antibody responses, cutaneous sensitivity reactions, natural killer activity, or graft-versus-host reactions. However, a decrease in lymphoid organ cellularity and an absence of splenic germinal centers were observed. We assessed progenitor cell activity in irradiated mice by evaluating the repopulation of marrow and lymphoid organs. Dehydroisoandrosterone feeding resulted in an inhibition of lymphopoiesis but not myelopoiesis. Clofibrate, another peroxisomal proliferator, failed to inhibit lymphocyte repopulation after irradiation. Androgen-non-responsive Tfm/Y mice were as susceptible as control mice to the inhibitory effects of DHA on lymphopoiesis. Thus DHA itself may act on lymphoid progenitor cells and/or the microenvironment.
Identification of a subset of murine natural killer cells that mediates rejection of Hh-1d but not Hh-1b bone marrow grafts.
NK cells demonstrate many immune functions both in vitro and in vivo, including the lysis of tumor or virus-infected cells and the rejection of bone marrow allografts. However it remains unclear whether or not all NK cells can mediate these various functions or if NK cells exist in functionally distinct subsets. We have developed a new NK-specific mAb, SW5E6, which binds to approximately 50% of murine NK cells. The 5E6 antigen identifies a distinct and stable subset of NK cells and is expressed on about one-half of fresh or rIL-2-activated murine NK cells. Both 5E6+ and 5E6- NK cells are capable of lysing YAC-1 tumor cells in vitro and in vivo. By treating animals with SW5E6, we demonstrate that the 5E6+ subset is necessary for the rejection of H-2d/Hh-1d but not H-2b/Hh-1b bone marrow cells. Thus NK cells exist as functionally separable subsets in vivo.
Murine natural killer cells express functional Fc gamma receptor II encoded by the Fc gamma R alpha gene.
We report evidence that murine NK cells express a functional Fc gamma RII encoded by the Fc gamma RII alpha gene. Several lines of indirect evidence indicate that freshly obtained NK cells from mice of several strains bear a functional Fc gamma RII: (a) anti-Fc gamma RII antibody 2.4G2 detects a small but significant proportion of sIg- cells and a small proportion of the 2.4G2+ cells are included in the Thy-1+ population; (b) sIg- lymphocytes contain 2.4G2+ and Fc gamma R-bearing cells in similar proportions; (c) binding of particulate immune complexes by sIg- lymphocytes is completely inhibited by 2.4G2; (d) 2.4G2+ cells mediate greater than 50% of the spontaneous cytotoxicity in sIg- splenic lymphocytes. Direct evidence for the presence of Fc gamma RII on murine NK cells is provided by the results of two-color immunofluorescence studies performed on splenic lymphocytes from C57BL/6 mice showing coexpression of NK-1.1 and 2.4G2. Studies of in vitro propagated homogeneous NK cell populations confirm that murine NK cells express only Fc gamma RII and that this Fc gamma R is functional, as shown in experiments of inhibition of ADCC by the anti-Fc gamma RII antibody 2.4G2. The results of studies at the molecular level show that an Fc gamma RII alpha transcript identical to that expressed in macrophages is the only molecule encoding Fc gamma RII in murine NK cells.
Effect of the venodilated state on sympathetic-induced venoconstriction in normal subjects.
The interaction between venoconstriction induced by application of ice to the forehead and nitroglycerin-induced venodilation was examined in 19 healthy male volunteers, ages 25 +/- 5 years (mean +/- standard deviation). Venous tone was determined by the equilibration technique. Mercury-in-silastic plethysmography was used to measure changes in forearm volume before and after ice application during control conditions, and before and after ice application in the venodilated state (nitroglycerin spray, 0.8 mg). Venous tone and arterial pressure increased significantly after the application of ice to the forehead in both the control and venodilated states, indicating that ice increased sympathetic tone. Nitroglycerin increased venous volume by 0.28 cc/100 cc arm. The venoconstrictive effect of ice after nitroglycerin (a decrease in venous volume of 0.53 cc/100 cc arm) was quantitatively similar to the venoconstrictive effect of ice during control conditions (a decrease in venous volume of 0.54 cc/100 cc arm). These results suggest that sympathetic-induced venoconstriction is not attenuated in the venodilated state.