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E B Walker

Publications and source records attributed to E B Walker.

At least 37 records · Page 2Linked to original sources
PubMed1988

Characterization of a T-cell subset prevalent in immunoregulatory disorders in humans.

T cells from individuals with certain autoimmune diseases (rheumatoid arthritis, graft-versus-host disease, acquired immunodeficiency syndrome) express high levels of a cell surface sialoglycoprotein with a molecular weight of 140 kDa (gp140). Although a low frequency of gp 140+ T cells was detected in the blood of normal individuals, upon stimulation with autologous EBV-transformed B cells (AMLR), the frequency of expression of gp140 was increased threefold. To further characterize gp 140+ T cells, rosetting techniques with ox erythrocytes coated with monoclonal anti-gp 140 antibody were used to isolate T-cell subsets for phenotypic, cell cycle, and functional analysis. The majority of gp140+ T cells expressed cytotoxic/suppressor (CD8+) phenotype in both normal and AMLR-activated states. Unstimulated gp140+ T cells had significantly greater nucleic acid content, as measured by acridine orange and flow cytometry, than gp140- T cells. Surprisingly, the gp140+ T-cell subset had a less proliferative response in vitro to pokeweed or phytohemaglutinin mitogens. These results suggest that gp140+ T cells in normal individuals and in patients with autoimmune diseases may have been activated previously in vivo and that they are relatively resistant to reactivation in vitro.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1988

Hyperthermia in humans enhances interferon-gamma synthesis and alters the peripheral lymphocyte population.

Induction of hyperthermia (39 degrees C) in human volunteers by immersion in warm water (41-45 degrees C) rapidly alters the cell populations in the peripheral blood. In addition to granulocytosis, there is an alteration of the normal ratios among T-lymphocyte subsets. Following in vitro mitogen stimulation, lymphocytes from hyperthermic individuals produce as much as 10-fold more interferon-gamma (IFN-gamma) than cells withdrawn at basal core temperatures from the same individuals. A temperature threshold of 39 degrees C for this response suggests potential relevance to fever. No change was noted in the activity of the macrophage population. The possible involvement of interleukin-2 (IL-2) in this enhanced production is discussed. No changes were noted in the circulating levels of IFN-gamma.

Adult↗
PubMed1988

Functional and phenotypical studies of the Leu-4 (CD3)+, Leu-1 (CD5)- T lymphocyte.

A small T cell subpopulation expressing the phenotype Leu-5(CD2)+, Leu-4(CD3)+, Leu-1 (CD5)- can be found in peripheral blood and bone marrow of normal individuals. When these cells were sorted out by three colour immunofluorescence cell sorting and tested in limiting dilution assays, they were found to have lower frequencies of proliferating (9.0 +/- 5.6 times, n = 7) and of IL-2 producing cells (11.5 +/- 5.0 times n = 5), and a higher frequency of cytotoxic cells (3.1 +/- 2.6 times, n = 2) than T lymphocytes expressing the three markers. In peripheral blood lymphocytes, 1/3 of the CD3+, CD5- cells were positive for Leu-2a (CD8) while virtually all were negative for Leu-3a (CD4). Four colour flow cytometric analysis revealed a small subset of T cells positive for CD3 and negative for CD5, CD4 and CD8. Approximately 75% of the CD3+, CD5- cells were negative for Leu-7 and CD16 simultaneously. These results shed a light on the phenotype of T cells that escape killing by CD5 and complement in T cell depleted bone marrow and may explain why fewer residual T cells in the depleted marrow are detected by limiting dilution assays than by flow cytometric analysis.

Antigens, Differentiation↗
PubMed1987

The same cloned murine B lymphoma cell lines can be selectively induced to release either interleukin-1- or interleukin-2-like factor activity.

A series of cloned murine B lymphoma cell lines including WEHI-5, WEHI-231, 2PK-3 and L10A/2J have been studied previously for their ability to present soluble protein antigen in a major histocompatibility complex (MHC) restricted fashion. These B cell lines have been shown to be effective accessory cells in the in vitro stimulation of antigen-specific, MHC-restricted, continuous T cell lines; and in the in vitro stimulation of antigen-specific, MHC-restricted T-T hybridoma cell lines. Using 2PK-3 and L10A/2J as examples of this group of B cell lymphomas we demonstrate in this study that these tumor cell lines constitutively release an interleukin-1 (IL-1) like factor activity as determined by the ability of the conditioned medium from these cultures to support the synergistic stimulation of thymocyte proliferation in the presence of concanavalin A (Con A). Conversely, these same constitutive supernatants will not stimulate the proliferation of IL-2 dependent cell lines such as CTLL-2 or HT-2. Stimulation of 2PK-3 and L10A/2J by lipopolysaccharide (LPS) results in the release of increased levels of the IL-1 like factor activity. By contrast, stimulation of the same cloned 2PK-3 and L10A/2J cell lines with the polyclonal activator Staph. aureus results in the release of a soluble factor activity which functionally acts like IL-2 since conditioned medium from S. aureus stimulated 2PK-3 and L10A/2J cultures will support a CTLL proliferation response as well as stimulate thymocyte proliferation. Thus, the same cloned B cell lines can be differentially stimulated to release lymphokine activity with either IL-1 or IL-2 like functional properties.

Animals↗
PubMed1987

Cellular control of in vitro progression of murine myeloid leukemia: progression accompanies acquisition of independence from growth factor and stromal cells.

The ability of bone marrow stroma cells of normal WCB6F1 (+/+) mice versus their congenic Sl/Sld stromal-defective littermates to support sustained proliferation and leukemic transformation of the growth factor-dependent myeloid cell line FDC-P1 was studied. Extensive proliferation of factor-dependent cells occurred on (+/+) normal long-term marrow culture stroma without the addition of growth factor, whereas factor-dependent cells dissipated from Sl/Sld stromal cultures after addition. The sustained proliferation that occurred on +/+ stromal layers later resulted in the appearance of factor-independent cell lines that were no longer dependent upon stroma. Factor-independent cell lines were cloned by limiting dilution and analyzed for expression of cell surface antigens to prove their origin from FDC-P1. Factor-independent cells, but not factor-dependent cells, formed tumors in syngeneic mice. These studies demonstrate a critical role for marrow stroma in the stepwise development of murine leukemia and are concordant with the previous data obtained in in vivo studies by McCool et al. that the splenic stroma of irradiated Sl/Sld mice do not support growth of Friend virus-induced preleukemic cell colonies. The present data demonstrate in a preleukemia model not induced by Friend virus complex that normal (+/+) stromal cells promote the in vitro proliferation of factor-dependent preleukemic cells and their subsequent transition to factor-independent leukemia cells, but Sl/Sld defective stroma do not efficiently promote this transition.

Animals↗
PubMed1986

GH3 pituitary adenoma cells can reverse thymic aging in rats.

Thymic size and T-cell function decrease with age, and it has not yet been possible to totally reverse this thymic atrophy and completely restore T-cell-dependent immune functions. In this study, GH3 pituitary adenoma cells, which secrete growth hormone and prolactin, were implanted subcutaneously into 16- and 22-month-old female Wistar-Furth rats and the rats were sacrificed approximately 2 months later. Only thymic remnants were detected in aged, non-implanted rats, but thymus glands were found in both the 18- and the 24-month-old rats that had been implanted with GH3 cells. Thymus glands from the GH3-implanted 18-month-old rats contained distinct cortical thymocytes and medullary epithelial cells. Depending on the concentration of phytohemagglutinin or concanavalin A, T-cell proliferative responses of splenocytes from these implanted rats were 2- to 5-fold greater than those of 18-month-old controls. At the optimal concentration of mitogen, proliferative responses to either lectin could be restored to those levels observed in splenocytes from 3-month-old Wistar-Furth females. Thymus glands from 24-month-old GH3-implanted rats contained more cortical thymocytes and fewer fat vacuoles than controls, but they were not totally reconstituted. No significant lectin-induced T-cell proliferative responses or IL-2 secretion were detected in 24-month-old control rats, but splenocytes from GH3-implanted rats showed augmented T-cell proliferative responses and increased synthesis of IL-2. Fluorescence-activated cell-sorter analysis of thymocytes revealed that 24-month-old rats implanted with GH3 cells had a higher proportion of lymphocytes with the Thy-1.1 and helper-T-cell phenotypes. These data show that it is possible to regenerate normal thymic tissue in situ and reverse the natural loss in cell-mediated immunity that occurs with aging.

Adenoma↗
PubMed1985

Characterization of subsets of bone marrow-derived macrophages by flow cytometry analysis.

Normal C3H bone marrow cells were grown 7 days in medium containing L cell-derived colony stimulating factor-1 (CSF-1). During the first 4 days of culture, erythroid and granulocytic cells decreased while macrophages increased exponentially with a doubling time of about 31 hr. Only 0.3% of all cells in the initial bone marrow suspension formed discrete colonies of mononuclear phagocytes, but by day 6 60% of the nonadherent cells were capable of forming macrophage colonies, representing a 200-fold enrichment of the original progenitor population. Using flow cytometry, mononuclear phagocytes obtained after 4 days of culture were separated into two distinct phenotypes based on their autofluorescence. Nonadherent cells were a discrete population of small cells exhibiting low autofluorescence, and the adherent cells were a broad heterogeneous population of large cells exhibiting high autofluorescence. A panel of currently available rat monoclonal antibodies (MABs) against murine hematopoietic cells were used to determine whether unique subsets of macrophages could be resolved. The MABs RA 31B6 and H-11 stained virtually all the nonadherent cells but not adherent cells. The MABs E-2 and 11-4.1 (anti-H-2Kk) stained almost all the adherent cells and demonstrated no significant staining of nonadherent cells. Nearly all the nonadherent and adherent cells were stained by the MABs DNL 4.4 and MAC-1. Additionally, the data suggest that the epitopes for MAC-2 and MAC-3 and gamma 2a Fc receptors develop late in nonadherent progenitor cells as they mature into adherent macrophages.

Animals↗
PubMed1984

Murine gamma interferon activates the release of a macrophage-derived Ia-inducing factor that transfers Ia inductive capacity.

In this report we demonstrate that when the murine macrophage tumor cell line P- 388D1 is incubated for 48-72 h with either concanavalin A-stimulated rat spleen cell supernatant or cloned murine immune interferon (IFN-gamma), the cultured cells release a cell-free factor activity that in turn induces the cell surface expression of Ia antigen on the murine monocyte cell line WEHI-3. This IFN-gamma-stimulated, Ia-inducing activity cannot be blocked with an anti-IFN-gamma heteroantiserum that does block the induction of Ia expression on WEHI-3 by both cloned murine IFN-gamma and rat Con A supernatant. The Ia-inducing factor ( IaIF ) generated from P- 388D1 after stimulation by IFN-gamma does not demonstrate any antiviral activity. The P- 388D1 -derived IaIF is not shed plasma membrane Ia glycoprotein molecules, as demonstrated by the inability of the active component to bind specifically to an anti-I-Ad affinity column or to a protein A column after the active supernatant is first treated with an excess of anti-I-E/Cd,k monoclonal antibody.

Animals↗
PubMed1983

Photoreactivity of lysergic acid diethylamide and its possible utility as a photoaffinity labeling reagent.

Aqueous solutions of lysergic acid diethylamide (LSD) are extremely sensitive to light in the near-ultraviolet region of the spectrum. This rather efficient photoreaction yields a variety of products which have very low affinity for LSD-binding sites on plasma membranes from Fasciola hepatica. Since this photoreaction may be elicited by normal white fluorescent lighting in the laboratory, it represents a potential source of error in determining the binding affinity of LSD. Utilizing this photoreactivity advantageously, [3H]LSD was used to photolabel membrane proteins. Covalent binding of [3H]LSD was shown to be a function of the duration of illumination and was inhibited by 5-hydroxytryptamine and nonradioactive LSD. Sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis of [3H]LSD labeled membranes from F. hepatica showed two proteins which were selectively labeled by the photoreactive [3H]LSD. This method of direct photolabeling with non-derivatized [3H]LSD may allow identification of LSD-binding proteins in a variety of systems.

Affinity Labels↗
PubMed1982

Concomitant induction of the cell surface expression of Ia determinants and accessory cell function by a murine macrophage tumor cell line.

This study demonstrates that an uncharacterized soluble factor produced in concanavalin A-induced rat spleen cell suspensions has the capacity to induce the increased expression of cell surface H-2K and H-2D molecules and the expression of I-region gene products on murine monocyte-macrophage lineage tumors that are not Ia positive in the absence of the factor. In parallel with induction of serologically defined Ia specificities, Ia-induced WEHI-3 macrophage tumor cells are capable of providing accessory cell function in stimulating IL-2 production by T-T hybridomas that are activated in a major histocompatibility complex-restricted, antigen-dependent fashion. The uninduced Ia-negative WEHI-3 tumor cells do not trigger a comparable response in this assay system.

Animals↗
PubMed1982

Characterization and functional properties of tumor cell lines in accessory cell replacement assays.

This study reports the initial phenotypic and functional characterization of a series of cloned, murine, myelomonocytic tumors and their parent cell line WEHI-3, and a group of murine B lymphoma tumors. The tumor cell lines of the myelomonocytic lineage demonstrated the ability to reconstitute a macrophage-depleted, primary in vitro anti-SRBC PFC response, but only marginally enhanced an accessory cell-depleted, ova-primed, lymphocyte proliferation in vitro response. The B lymphoma tumors displayed exactly the reverse functional profile, being highly efficient in reconstitution of the proliferation response, but not supporting the SRBC PFC response. Detailed analysis of the cell surface phenotype of the various B lymphoma tumors used in this study show they display cell surface markers characteristic of normal B lymphocytes but are heterogeneous in their various stages of differential arrest. Future work will concentrate on the orchestration of Ia-mediated and soluble factor-mediated (IL 1) modalities of antigen-triggering of lymphocytes by these B lymphoma and myelomonocytic tumor cell lines.

Animals↗
PubMed1981

The pH dependence of photosensory responses in Stentor coeruleus and model system.

1. Live Stentor coeruleus exhibits a substantially red-shifted fluorescence maximum, corresponding to the anionic species of the photoreceptor chromophore. No change was observed in either the absorption or fluorescence excitation spectrum, indicating an efficient deprotonation of the photoreceptor pigment upon excitation by light. 2 Changes in external pH exhibit a dramatic effect on the pulmonary response of Stentor. Phototaxis is specifically inhibited at pH less than 6, with loss of photosensory perception which is restored when the pH is returned to pH greater than 6. 3. Fluorescence changes of 9-aminoacridine in suspensions of live Stentor indicate the generation of a pH gradient upon irradiation with light. Both pH gradient and phototaxis were inhibited by the addition of nigericin and p-tri-fluoromethoxy carbonyl cyanide phenylhydrazone (FCCP). 4. Incorporation of the Stentor photoreceptor protein in to artificial liposomes demonstrates the ability of the system to generate pH gradients across model membranes as monitored by the quenching of 9-aminoacridine fluorescence. The effect of external pH on net proton movement in the model system is strikingly similar to the pH dependent of the liver Stentor, thus lending support for transient proton flux being an important mode of light signal processing for photosensory transduction.

Animals↗
PubMed1981

Proton release from Stentor photoreceptors in the excited states.

Steady-state and picosecond pulse excitations of the photophobic-phototactic receptors isolated from Stentor coeruleus produced anionic species predominantly in the excited singlet state, although neutral photoreceptors in the ground state were exclusively excited. The same photoreceptor in vivo also emits fluorescence from the excited state of its anionic species, with an excitation spectrum identical to the absorption spectrum of the neutral species in the ground state. The excited state dissociation of protons from the photoreceptor chromophore (stentorin; hypericin covalently linked to protein) efficiently occurs in less than 10 ps. A possible role of the transient-proton release from the photoreceptor, in the signal transduction photoresponse of Stentor, is briefly discussed.

Animals↗
PubMed1979

Spectroscopic characterization of the Stentor photoreceptor.

1. On the basis of chromatographic and spectroscopic (absorption, fluorescence and its polarization, fluorescence lifetime, circular dichroism) characterization of the Stentor photoreceptor (stentorin) for photophobic response, the photoreceptor chromophore released from mild acid hydrolysis has been identified as hypericin. 2. The native chromophore is apparently linked to a protein (65 K) containing Lys and several hydrophobic residues, which is soluble in acetone and n-pentane. The peptide-linked stentorin (I) chromophore exhibits circular dichroism in the visible region due to the induced optical activity provided by the peptide. 3. The sodium dodecyl sulfate polyacrylamide gel electrophoresis of a 38% fraction of the sucrose density centrifugation has resolved stentorin II proteins having molecular weights of 13 000, 16 000, 65 000 and 130 000. These proteins, as well as the acetone-soluble peptide, have been spectroscopically characterized with particular emphasis on their primary photoreactivity as the photophobic receptor of Stentor coeruleus. 4. Irradiation of whole living Stentor in dilute buffer solutions induces a decrease in the pH of the medium. A strong dependence upon pH in the fluorescence spectra of both synthetic and native chromophores is also evident, showing a significant drop in the pKa of one or more hydroxyl groups in the excited state. A mechanism for the photophobic response, based on this lowering of the pKa as the primary photoprocess, has been discussed.

Animals↗