The Experts below are selected from a list of 9 Experts worldwide ranked by ideXlab platform
Susan Solliday Rich - One of the best experts on this subject based on the ideXlab platform.
-
REGULATORY MECHANISMS IN CELL-MEDIATED IMMUNE RESPONSES: I. Regulation of Mixed Lymphocyte Reactions by Alloantigen-Activated Thymus-Derived Lymphocytes
Journal of Experimental Medicine, 2004Co-Authors: Susan Solliday RichAbstract:Functionally distinct subpopulations of thymus-derived lymphocytes (T cells) cooperate in the development of certain cell-mediated immune responses. Initially, Cantor, Tigelaar, and Asofsky (1-3) demonstrated that two distinct populations of T cells, termed effector and amplifier T cells, interacted synergistically in the development of graft-vs.-host (GVH) 1 reactions in the mouse. More recently, cooperative interaction between thymocytes and periph-eral T cells has been described for mixed lymphocyte reactions (MLR) (4) and for development of cytotoxic allograft responses in vitro (5). Evidence is now accumulating that a third T-cell subpopulation, suppressor T cells, may be important in the regulation of cell-mediated immune responses. Hardin, Chused, and Steinberg (6) described a population of splenic suppressor cells in young (6-wk old) (NZB/NZW)F1 mice which inhibited GVH activity of 25-wk old spleen cells when injected into newborn C3H mice. In addition, Zembala and Asherson (7) reported suppression of contact sensitivity to picryl chloride when T cells from mice unresponsive to picryl sulfonic acid were transferred to normal recipients. Suppression of cytotoxic killer cell generation by concanavalin A (Con A)-activated murine spleen cells has been reported by Peavy and Pierce (8). Folch and Waksman (9, 10) have observed enhanced responses to T-cell mitogens and alloantigens in rat spleen cell cultures depleted of glass-adherent T cells. We here report the effects of alloantigen activation on generation in vivo of a subpopulation of murine thymus-derived lymphocytes which regulate MLR. The data indicate that regulatory cells with suppressor and amplifier activities in MLR are anatomically segregated after alloantigen sensitization. The generation 1Abbreviations used in this paper: Con A, concanavalin A; FCS, fetal calf serum; GVH, graft-vs.-host; HBSS, Hanks' balanced salt solution; MLR, mixed lymphocyte reaction; PFC, plaque-forming cell(s); PHA, phytohemagglutinin. 1588 THE JOURNAL OF EXPERIMENTAL MEDICINE • VOLUME 140, 1974 SUSAN SOLLIDAY RICH AND ROBERT R. RICH 1589 in vivo and the kinetics and specificity of activated suppressor splenic lympho-cytes are characterized. These studies provide additional evidence to support a general concept of active, physiologic regulation of both cell-mediated and humoral immune responses by activated T cells. Materials and Methods Mice. BALB/c (H-2 ~) mice (Cell Biology Department, Baylor College of Medicine, Houston, Texas) and C57BL/6 (H-2 b) and A/Tex (H-2 ~) mice (Texas Inbred Mice Company, Houston) were used in these studies. Experiments were performed with 8-to 14-wk old male animals. MLR. Single cell suspensions of mouse spleen were prepared by gentle teasing and sedimentation of fragments and debris. Lymph node cell suspensions were similarly prepared from inguinal nodes or pooled cervical, brachial, axillary, and inguinal nodes. Responder, stimulator, and regulator cell populations were cultured in equal numbers, 1 × 10' cells of each, in a final vol of 0.2 ml in microculture plates (Linbro 1S-FB-96-TC, International Scientific Instruments, Cary, Ill.). Cultures were prepared in RPMI-1640 medium supplemented with 5% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, and penicillin-streptomycin mixture (50 U each per ml) (Grand Island Biological Company, Grand Island, N. Y.), and incubated at 37°C in a humidified atmosphere of 5% CO2 in air. To block appreciable DNA replication by either regulator or stimulator cells, these populations were treated before addition to MLR with mitomycin C (Sigma Chemical Company, St. Louis, Mo.), 50 ~g/ml for 30 rain at 37°C and washed three times with Hanks' balanced salt solution (HBSS). Regulator cells, syngeneic to the responding population, were obtained from mice injected with syngeneic or allogeneic spleen cells. Spleen cell suspensions in various doses were injected intravenously, intraperitoneally, or into the hind foot pads. The usual protocol for suppressor cell generation, illustrated in Fig. 1, utilized 2 × l0 T cells injected into the hind foot pads. 4 days after injection, single cell suspensions of spleen or lymph node were prepared, treated with mitomycin C and added to MLR. DNA synthesis in MLR was assayed by adding 1.0 ~Ci of tritiated thymidine ([3H]TdR, spec act 2.0 Ci/mM, New England Nuclear Corp., Boston, Mass.) to cultures for the final 18 h of a 72-h incubation period. Exceptions to this protocol for certain experiments are subsequently detailed. Cultures were harvested by aspiration of cells onto glass-fiber filters with a saline wash utilizing a multiple automated sample harvester (MASH-II, MicroBiological Associates, Inc., Bethesda, Md.). Dry filters were placed in a scintillation cocktail containing toluene, Permaflour, and Soluene (Packard Instrument Co., Inc., Downers Grove, Ill.) and radioactivity was measured in a liquid scintillation spectrometer (Mark II, Nuclear-Chicago, Des Plaines, Ill.). Data are expressed as mean counts per minute (cpm) of three to six replicate cultures with the standard error of the mean. The stimulation index (E/C) was calculated by dividing cpm from cultures containing stimulating cells allogeneic to the responder population by cpm from cultures containing syngeneic stimulating cells. To standardize results and permit evaluation of regulator cell effects in replicate experiments with differing positive and negative control responses, a ratio was calculated of the stimulation index of cultures incubated with activated regulatory cells to the stimulation index of cultures incubated with nonaetivated cells. Each E/C value was first adjusted to express the increment of stimulation over 1.0 (i.e., 1.0 equals no stimulation). The following formula was used: [(E/C) of MLRwith activated regulators] 1 [(E/C) of MLR with nonactivated regulators] -1 x 100 = % control MLR response.
Marc E. Lippman - One of the best experts on this subject based on the ideXlab platform.
-
Myelogenous Leukemia Lines Glucocorticoid Sensitivity and Receptors in Cells of Human
2020Co-Authors: H. Phillip Koeffler, David W. Golde, Marc E. LippmanAbstract:VeteransAdministrationHospital,Sepulveda,California91343(H.P.K.,D.W.G.j;andMedicineBranch,NIH.Bethesda,Maryland20014(M.E.L.]cells are ASD-chboroacetate estenase positive, and the lineretains the same karyotypic abnormality that was present in thefresh leukemia cells of the patient. The HL-60 cell line wasderived from a patient with promyebocyticleukemia. These cellsare predominantly at the pnomyebocytestage of developmentand stain positive for peroxidase and ASD-chboroacetate esterase. The cells retain the karyotype of the fresh beukemiccells and are abbeto produce tumors in nude mice. The KG-iand HL-60 cells form myeboidcolonies in soft-gel culture andare not infected with Epstein-Barr virus.The cell lines were maintained in T-flasks (Lux ScientificCorp.) with a medium(Flow Laboratories), 2O%fetal calf serum(Grand Island Biological Co.), and penicillin and streptomycin(Grand Island Biological Co.). The cloning studies were performed with cells in the logarithmic phase of growth (2 to 4days after reseeding in fresh media). Five thousand KG-i cellsand 2000 HL-60 cells were plated in 0.3% agan-containingCSA3as previously described (20). The CSA source was conditioned media from a line of T-Iymphocytes established froma patient with hairy-cell leukemia (15). The same lot of fetalcalf serum and conditioned medium was used for all expeniments. The KG-i cells have a marked response to CSA. In thepresence of optimal CSA concentrations, the cloning efficiencywas 3%, representing a 32-fold increase in colony numberover plates not containing CSA. The HL-60 cells had a cloningefficiency of 7.2% in cultures without CSA, and there was a2.2-fold increase in colony number when optimal concentrations of CSA were added. The CSA dose-response curves forboth KG-i and HL-60 cells consistently showed maximal cobony formation with 5% conditioned medium in the plate. Asuboptimal CSA concentration (i %) was used for the steroidstudies.Dexamethasone and other steroids were added directly tothe culture dishes. Four to 5 replicate culture plates wereincubated in a humidified atmosphere of 5% CO2in air for i 4days. Colonies with a minimum of 50 cells were enumeratedwith an inverted microscope. The steroids, dexamethasone,progesterone, i i -deoxycortisone, i 7$-estradiob, testosteronepropionate, fluoxymesterone (fluoxy i i $, 17f1-dihydroxy-9a-fbuoro-i 7a-methyb-4-androstane-3-one), 5-a-dihydrotestostenone, 5-fl-dihydrotestosterone, androsterone, and etiochobanolone (Sigma Chemical Co., St. Louis, Mo.) were dissolved inethanol and diluted to appropriate concentrations with tissueculture medium. Control cultures contained equivalent concentrations of ethanol. Each drug was tested in 3 or more expeniments.Studies of [3H]dexamethasone binding, competition studies,and Scatchard analysis were performed with whole cells aspreviously described (29, 34). The binding studies were done
George Miller - One of the best experts on this subject based on the ideXlab platform.
-
Tissue culture contamination with nontuberculous mycobacteria.
Microbiology and Immunology, 2013Co-Authors: Donald Coope, Alexander Von Graevenitz, Josefino Corrales, George MillerAbstract:Contamination of cultured eukaryotic cells with unwanted viruses, mycoplasmas, protozoa, fungi, and bacteria is a major problem for those who use suchcells in biologic and biochemical research (2). Among the many microorganismswhich have been isolated as cell culture contaminants, nontuberculous mycobacteria have not been described previously. We recently encountered these organisms in various lymphoblastoid cell lines such as CEM and RAJI as well as indiploid human fibroblasts and monkey kidney lines such as VERO. The presenceof contaminants had been suspected because of low virus yields.Gram stains of culture fluids bathing the tissue cultures, such as RPMI 1640or Eagle's basal medium with 10% bovine serum (Grand Island Biological Co.,Madison, Wis.) showed pleomorphic gram-positiverods resembling corynebacteria.There was, however, no gross alteration of the tissue culture and cells and no turbidity of the medium. Subcultures to media which would normally supportgrowth of corynebacteria, i.e., aerobic Tryptic Soy Agar (Difco Laboratories, Detroit, Mich.) with 5% sheep blood agar, Columbia Broth (Difco), Brain HeartInfusion (Difco), and Sabouraud Dextose Agar (Difco), did not reveal growth.Light growth consisting of slight flocculation near the top of the tube was occasionally seen in Fluid Thioglycollate Medium (Difco) after two weeks' incubation at
H. Phillip Koeffler - One of the best experts on this subject based on the ideXlab platform.
-
Myelogenous Leukemia Lines Glucocorticoid Sensitivity and Receptors in Cells of Human
2020Co-Authors: H. Phillip Koeffler, David W. Golde, Marc E. LippmanAbstract:VeteransAdministrationHospital,Sepulveda,California91343(H.P.K.,D.W.G.j;andMedicineBranch,NIH.Bethesda,Maryland20014(M.E.L.]cells are ASD-chboroacetate estenase positive, and the lineretains the same karyotypic abnormality that was present in thefresh leukemia cells of the patient. The HL-60 cell line wasderived from a patient with promyebocyticleukemia. These cellsare predominantly at the pnomyebocytestage of developmentand stain positive for peroxidase and ASD-chboroacetate esterase. The cells retain the karyotype of the fresh beukemiccells and are abbeto produce tumors in nude mice. The KG-iand HL-60 cells form myeboidcolonies in soft-gel culture andare not infected with Epstein-Barr virus.The cell lines were maintained in T-flasks (Lux ScientificCorp.) with a medium(Flow Laboratories), 2O%fetal calf serum(Grand Island Biological Co.), and penicillin and streptomycin(Grand Island Biological Co.). The cloning studies were performed with cells in the logarithmic phase of growth (2 to 4days after reseeding in fresh media). Five thousand KG-i cellsand 2000 HL-60 cells were plated in 0.3% agan-containingCSA3as previously described (20). The CSA source was conditioned media from a line of T-Iymphocytes established froma patient with hairy-cell leukemia (15). The same lot of fetalcalf serum and conditioned medium was used for all expeniments. The KG-i cells have a marked response to CSA. In thepresence of optimal CSA concentrations, the cloning efficiencywas 3%, representing a 32-fold increase in colony numberover plates not containing CSA. The HL-60 cells had a cloningefficiency of 7.2% in cultures without CSA, and there was a2.2-fold increase in colony number when optimal concentrations of CSA were added. The CSA dose-response curves forboth KG-i and HL-60 cells consistently showed maximal cobony formation with 5% conditioned medium in the plate. Asuboptimal CSA concentration (i %) was used for the steroidstudies.Dexamethasone and other steroids were added directly tothe culture dishes. Four to 5 replicate culture plates wereincubated in a humidified atmosphere of 5% CO2in air for i 4days. Colonies with a minimum of 50 cells were enumeratedwith an inverted microscope. The steroids, dexamethasone,progesterone, i i -deoxycortisone, i 7$-estradiob, testosteronepropionate, fluoxymesterone (fluoxy i i $, 17f1-dihydroxy-9a-fbuoro-i 7a-methyb-4-androstane-3-one), 5-a-dihydrotestostenone, 5-fl-dihydrotestosterone, androsterone, and etiochobanolone (Sigma Chemical Co., St. Louis, Mo.) were dissolved inethanol and diluted to appropriate concentrations with tissueculture medium. Control cultures contained equivalent concentrations of ethanol. Each drug was tested in 3 or more expeniments.Studies of [3H]dexamethasone binding, competition studies,and Scatchard analysis were performed with whole cells aspreviously described (29, 34). The binding studies were done
Donald Coope - One of the best experts on this subject based on the ideXlab platform.
-
Tissue culture contamination with nontuberculous mycobacteria.
Microbiology and Immunology, 2013Co-Authors: Donald Coope, Alexander Von Graevenitz, Josefino Corrales, George MillerAbstract:Contamination of cultured eukaryotic cells with unwanted viruses, mycoplasmas, protozoa, fungi, and bacteria is a major problem for those who use suchcells in biologic and biochemical research (2). Among the many microorganismswhich have been isolated as cell culture contaminants, nontuberculous mycobacteria have not been described previously. We recently encountered these organisms in various lymphoblastoid cell lines such as CEM and RAJI as well as indiploid human fibroblasts and monkey kidney lines such as VERO. The presenceof contaminants had been suspected because of low virus yields.Gram stains of culture fluids bathing the tissue cultures, such as RPMI 1640or Eagle's basal medium with 10% bovine serum (Grand Island Biological Co.,Madison, Wis.) showed pleomorphic gram-positiverods resembling corynebacteria.There was, however, no gross alteration of the tissue culture and cells and no turbidity of the medium. Subcultures to media which would normally supportgrowth of corynebacteria, i.e., aerobic Tryptic Soy Agar (Difco Laboratories, Detroit, Mich.) with 5% sheep blood agar, Columbia Broth (Difco), Brain HeartInfusion (Difco), and Sabouraud Dextose Agar (Difco), did not reveal growth.Light growth consisting of slight flocculation near the top of the tube was occasionally seen in Fluid Thioglycollate Medium (Difco) after two weeks' incubation at
