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Biomedical subjects

R E Morris

Publications and source records attributed to R E Morris.

At least 253 records · Page 14Linked to original sources
PubMed1982

Failure to demonstrate a role for line Ib tumor-associated surface antigen in the etiology of age-dependent polioencephalomyelitis.

In the preceding paper [Morris et al. (1982), Molec. Immun. 19, 973-982] we demonstrate an associative interaction between the line Ib tumor-associated surface antigen (Ib-TASA) and the Dk/Kk regions of the major histocompatibility complex, i.e. 'altered-self' antigen. We originally hypothesized that age-dependent polioencephalomyelitis (ADPE) occurred as the result of immune recognition of a 'self'-determinant on the 'altered-self' antigen. In this report we used the non-ionic detergent, NP-40, to solubilize Ib cell surface antigens. Although immunization of immunocompetent C58 mice with the soluble NP-40 Ib cell extract afforded protection to lethal tumor challenge, the extract failed to induce ADPE in immunosuppressed mice. Data presented here demonstrate that Ib-TASA is not involved in the etiology of ADPE. The evidence suggests that lactic dehydrogenase virus, which is a silent virus passaged with line Ib leukemia, is the causative agent of the paralytic disease.

Age Factors↗
PubMed1982

Cellular enzyme-linked immunospecific assay (CELISA). I. A new micromethod that detects antibodies to cell-surface antigens.

A novel, indirect immunoassay capable of detecting human anti-HLA antibody bound to lymphocytes has been developed. This cellular enzyme-linked immunospecific assay (CELISA) utilizes an antiglobulin covalently linked to alkaline phosphatase to quantitate the amount of anti-HLA antibody bound to cell-surface HLA antigens. During the CELISA, V-bottom polyvinyl microplates served as the receptacle in which as little as 5 microliters of sera and as few as 25,000 lymphocytes per well were incubated. We devised a rapid and simple technique to transfer the cells from the original V-bottom plate to a flat-bottom plate before adding the enzyme substrate. Using this strategy, background noise because of the nonspecific adsorption of the different protein immunoreactants to plastic was eliminated. This strategy, the identification of an optimal cell concentration and an optimal conjugate source and dilution, enabled us to detect the anti-HLA activity in sera diluted out 250-fold more than their maximum titer as determined by the microdroplet cytotoxicity test. Since this assay is capable of sensitively and objectively quantitating antibody bound to cell-surface antigens, it may be of value in the areas of transplantation, blood banking, autoimmune disease, tumor immunology, and the study of cell-surface differentiation and viral antigens.

Alkaline Phosphatase↗
PubMed1982

Essential role of calcium in cellular internalization of Pseudomonas toxin.

Pseudomonas exotoxin (PE) has been shown previously to enter mouse LM cells by receptor-mediated endocytosis, to block protein synthesis, and to cause cell death. The requirement for the divalent cation calcium in the binding and internalization of PE was examined. Biochemical studies showed that depletion of extracellular calcium with ethylene glycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid protected cells from the action of PE when the chelator was present during the internalization step. Extracellular calcium was not required for binding. We observed with immunoelectron microscopy that, in the cold, toxin bound to cell surfaces equally well in the presence or absence of chelator. In the presence of chelator, toxin was not cleared from the cell surface when cells were warmed to 37 degrees C. Replenishment of calcium (2 mM CaCl2), however, allowed normal rapid clearance of PE to occur. We suggest that internalization, but not binding, of PE by LM cells requires extracellular calcium.

ADP Ribose Transferases↗
PubMed1982

Combined use of immunoferritin and immunocolloidal gold for the simultaneous demonstration of two antigens by immuno-electron microscopy.

In this report we describe a double-label bridging technique for the simultaneous visualization of two cell surface antigens by immuno-electron microscopy. Mouse IgG-labeled horse spleen ferritin and rabbit IgG-labeled gold sols served as the electron dense marker-conjugates. The technique was found to discriminate reproducibly two cell surface antigens simultaneously in two separate murine systems, one an in vivo cell line and the other an in vitro cell line. The technique described in this report differs from other double-labeling procedures in two notable ways: (1) the electron dense markers are easily distinguished from one another, and (2) the primary and link antibodies are not modified.

Animals↗
PubMed1982

T cell subsets required for protection against age-dependent polioencephalomyelitis of C58 mice.

C58 mice are rendered susceptible to age-dependent polioencephalomyelitis by the natural aging process or by treatment with immunosuppressive agents. The mechanism of immunologically-mediated resistance was investigated. Susceptibility to the disease results from the loss of protective T cells. Transfer of unfractionated, naive splenic T cells from young donors prevented disease induction in immunosuppressed mice. Spleen cells from aged mice did not render young animals susceptible to disease, nor were they protective. Lyt phenotype analysis of the protective cells showed that the Lyt-1, Lyt-1,2, and Lyt-2 T cell subsets were all required for the generation of a protective response. When young donor animals were presensitized with LDV, transfer of Lyt-2 cells alone was protective.

Aging↗
PubMed1981

Structural changes in lysosomes from cultured human fibroblasts in Duchenne's muscular dystrophy.

We have previously reported a decreased activity of the lysosomal enzyme dipeptidyl aminopeptidase-I (DAP-I) in cultured fibroblasts from patients with Duchenne's muscular dystrophy (DMD). Here we report that electron microscope examination of these cells reveals the presence of abundant lamellar bodies, a morphologic abnormalities commonly associated with impaired lysosomal function. Morphometric analysis of these cytoplasmic figures in dystrophic cells shows a sevenfold increase relative to normal controls (P less than 0.01). Analysis of lysosomal density profiles by density gradient centrifugation reveals similar patterns in normal and DMD cells. Treatment of lysosomes wit the nonionic detergent Triton X-100 causes an activation of DAP-I. This activation, attributable to structure-linked latency, is markedly diminished in DMD cells which show an optimal activation of only 180% compared to 255% for control fibroblasts (P less than 0.01). These data suggest an alteration in the properties of the lysosomal membrane in DMD fibroblasts. This suggestion is also supported by studies on the release of DAP-I from lysosomes by osmotic shock which show it to be a membrane-associated enzyme with membrane-binding characteristics intermediate between those of tightly bound beta-glucosidase and those of unbound N-acetylgalactosaminidase. The latency characteristics of these other lysosomal enzymes are not altered in the DMD cells, indicating that the effect is specific for DAP-I.

Cathepsins↗
PubMed1981

Quantitation of tumor antigen expression on the surface of malignant line Ib lymphocytes by immuno-electron microscopy.

Malignant line Ib lymphocytes obtained from strain C58/J mice moribund with the syngeneic, transplantable leukemia were examined by transmission electron microscopy. A large, multilobulated nucleus containing a prominent nucleolus was typically viewed. In addition to normal organelles, cytoplasmic elements included frequent intracisternal A-type virions. The cell membrane possessed pseudopodia, and occasionally C-type viruses were noted budding from the plasma membrane. On the basis of complement-mediated antibody cytotoxicity assays with the use of either antiserum against mouse IgM, antiserum against mouse IgG, or antiserum against Thy 1.2 antigen, Ib cells were characterized as T-cells. Similarly, leukemia lymphocytes were lysed by a highly specific rabbit anti-Ib tumor-associated surface antigen (TASA) in the presence of complement. The density and distribution of TASA on malignant lymphocytes were analyzed by immuno-electron microscopy with the use of a bridging technique in conjunction with antiserum against Ib TASA. The following observations were reported after the examination of more than 100 randomly selected Ib lymphocytes: a) The density of the TASA was low, b) the distribution of the TASA was random, and c) no evidence indicated cell cycle-dependent expression of the TASA.

Animals↗
PubMed1980

In situ fixation of cultured mouse peritoneal exudate cells: comparison of fixation methods.

Mouse peritoneal exudate cells grown in vitro on plastic petri dishes were fixed in situ with both glutaraldehyde and osmium tetroxide by a variety of contemporary methods. The goal of the investigation was to determine which method resulted in the best ultrastructural preservation. The parameters being tested included: (a) the method of fixation, i.e. either sequential or simultaneous; (b) the buffer vehicle for fixation, i.e. cocodylate, Mellonig's phosphate, Sorenson's phosphate, or s-collidine; and (c) the temperature of fixation. Results presented indicate that simultaneous fixation is far superior to sequential methods. Samples fixed sequentially at 4 degrees C consistently had better morphological preservation than samples fixed under similar conditions at 23 degrees C. With the exception of s-collidine, which was totally unacceptable for in vitro in situ fixation on plastic, comparable results were noted with different buffer vehicles.

Animals↗
PubMed1980

Receptor-mediated internalization of Pseudomonas toxin by mouse fibroblasts.

Pseudomonas exotoxin (PE) was used as a probe to study the mechanism by which protein ligands are internalized by mammalian cells. Both biochemical and electron microscopic methods were used to look at the internalization of PE by mouse LM cell fibroblasts. Our data suggest that PE enters cells by receptor-mediated endocytosis, a process previously thought to be restricted to the entry of biologically significant molecules such as lysosomal enzymes and peptide hormones. Biochemical studies showed that methylamine (20 mM) and chloroquine (10 microM) protected LM cells from the action of PE. Full protection was observed if methylamine or chloroquine was added to the monolayers simultaneously with toxin or if they were added up to 10 min after toxin binding. Later addition of amine or chloroquine afforded partial protection to the monolayers. With immunoelectron microscopy we observed that in the cold toxin bound diffusely to the cell surface but was rapidly internalized when cells were warmed to 37 degrees C. In the presence of methylamine, chloroquine or ammonium chloride, internalization did not occur. We propose that PE enters mouse fibroblasts by receptor-mediated endocytosis and that chloroquine and methylamine, agents which are known to block this process, prevent expression of toxicity.

ADP Ribose Transferases↗
PubMed1979

Shigella infection of henle intestinal epithelial cells: role of the host cell.

The process of Henle 407 embryonic intestinal epithelial cell infection by Shigella flexneri 2a M42-43 was studied in an in vitro model system. The role of the Henle cell was assessed. It was established that entry of S. flexneri into cells was suppressed by reagents which inhibit uptake of particles by phagocytic cells. The compounds tested included cytochalasin B, dibutyryl-cyclic adenosine monophosphate, choleragen (Vibrio cholera enterotoxin), iodoacetate, and dinitrophenol. Cytochalasin B inhibited infection at concentrations of 1.0 mug/ml or greater. Dibutyryl-cyclic adenosine monophosphate at concentrations of 1 mM and choleragen at 0.1 mug/ml caused significant suppression of infection. Iodoacetate or dinitrophenol, at 0.1 mM concentrations, inhibited internalization of virulent shigellae, and a combination of these compounds inhibited infection at 0.01 mM concentrations. Preincubation of Henle cell monolayers with the combination of iodoacetate and dinitrophenol (0.05 mM) also inhibited infection markedly. The data suggest that infection of epithelial cells by S. flexneri in vitro is accomplished by an endocytic process induced by virulent bacteria. The process appears to be similar to uptake of particles by phagocytic cells. Ultrastructural analysis by transmission electron microscopy provided corroborative evidence of phagocytosis of shigellae by Henle cells in that intracellular bacteria were often observed within membrane-limiting vacuoles resembling phagosomes.

Bucladesine↗
PubMed1979

Chicken anti-rat lymphocyte globulin: dose response study and determination of strain-specific alloantibody.

Chicken anti-rat lymphocyte globulin (CARLG) is a new and uniquely specific immunosuppressant for which the mechanisms of immunosuppressive action are not yet known. This study defines its in vivo therapeutic dose range and in vitro allospecificity. A dose response study, using CARLG or normal chicken globulin (NCG), as the sole immunosuppressant, was conducted in Lewis (Ag-B1) recipients of Buffalo (Ag-B6) rat cardiac allografts. Groups of Lewis recipients were treated with 175 mg of NCG per kg or 30, 100, 175, or 300 mg of CARLG per kg for the first 8 post-transplant days. Doses of 175 and 300 mg of CARLG per kg resulted in significantly longer (P less than 0.01) prolongation of graft survival than doses of 30 and 100 mg of CARLG per kg, which were not significantly different (P less than 0.05) in their ability to prolong graft survival than a dose of 175 mg of NCG per kg. In vitro and in vivo absorption studies revealed that CARLG does not contain strain-specific alloantibody. When Lewis lymphoid cells were used to absorb, in vitro, a batch of CARLG produced with Brown Norway (Ag-B3) lymphoid cells as immunogen, all antibody activity toward Brown Norway cells as quantitated by a two-stage lymphocytotoxicity assay was completely removed. In another experiment, a preparation of CARLG, produced using Buffalo lymphoid cells as immunogen, underwent in vivo absorption as a result of i.v. injection into Lewis rats. Serum levels of CARLG-binding activity to Lewis or Buffalo lymphoid cells were quantitated in vitro at intervals after injection by an isotopic antiglobulin assay. Lewis rat tissues were able to remove completely serum CARLG-binding activity toward Buffalo antigens. A mechanism of action for CARLG derived from these observations is proposed and discussed.

Animals↗