Biomedical subjects
Publications and source records attributed to D L Nelson.
During a routine oral examination of a 19 year-old Black male, a bony impaction of his mandibular left third molar fused to an inverted supernumerary tooth with a cystic cavity was found. Histologic examination confirmed the presence of a dentigerous cyst.
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Studies were undertaken to investigate the role of anti-target cell antibodies in determining whether lymphocytes can mediate antibody-dependent cellular cytotoxicity (ADCC) in vitro. Trinitrophenyl (TNP) modified Chang liver cells and human erythrocytes were employed as target cells and were coated with xenogeneic and allogeneic antibodies against TNP and natural cell surface antigens. Two cytotoxic effector cell populations were used: human peripheral blood mononuclear cells (PBMC) containing both lymphocytes and monocytes, and monocyte-depleted peripheral blood lymphocytes (PBL). With Chang targets, both PBMC and PBL mediated ADCC with xenogeneic anti-Chang and xenogeneic anti-TNP sera. With human erythrocyte targets, PBMC but not PBL mediated ADCC with human anti-blood group B serum, while both PBMC and PBL mediated ADCC with xenogeneic anti-TNP sera and also with a human anti-CD serum. These results demonstrate that the source of anti-target cell antibodies employed in ADCC reactions may determine whether or not lymphocytes are capable of mediating cytotoxicity.
The virus specificity of human in vitro cytotoxic T cell responses to influenza virus was studied with the use of peripheral blood mononuclear leukocytes from normal adult volunteers. Previous natural exposure of these donors to a variety of type A influenza viruses was documented by HI antibody titers. Cells sensitized in vitro with A/HK or A/PR8 were cytotoxic for autologous target cells infected with A/HK, A/PR8, or A/JAP 305 type A influenza viruses, but not for B/HK-infected or uninfected cells. B/HK-sensitized effector cells lysed target cells infected with B/HK but not targets infected with type A viruses. A/HK- and A/PR8-immune effector populations were shown to recognize cross-reactive antigens on A/HK- and A/PR8-infected target cells by cold target competition. Influenza-immune effector cells were cytotoxic for virus-infected autologous targets but much less so for virus-infected allogeneic targets. This self-restriction suggested that the cytotoxicity was largely T cell-mediated and was confirmed by cell separation analysis. Thus, the human secondary cytotoxic T cell response in vitro to influenza viruses is predominantly directed against cross-reactive determinants on cells infected with serologically distinct type A influenza viruses.
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Norepinephrine-containing storage particles in rat heart have been disrupted to determine their stability and to quantitate the amount of dopamine beta-hydroxylase (DBH) that exists in a soluble, membrane enclosed pool. Vesicles were relatively resistant to lysis by hypotonic shock or by up to five cycles of freezing and thawing. These procedures have been shown to release catecholamines from adrenal chromaffin granules readily. However, rat heart vesicles were sensitive to temperature and they could be lysed by mechanical disruption using a sonicator or a Polytron homogenizer. Increasing the force or duration of the disruption resulted in an increased release of norepinephrine and DBH into a supernatant fraction after high-speed centrifugation. Extrapolation of the data from such experiments gave an estimate for membrane-enclosed DBH of 37% of the total amount of enzyme activity. This estimate of the proportion of membrane-enclosed enzyme is considerably higher than those previously reported for membrane-enclosed DBH in sympathetic neurons.
Populations of lymphoid cells were obtained from the gastrointestinal tract of guinea pigs and compared with splenic cells for effector cell activity in assays of spontaneous cell-mediated cytotoxicity (SCMC), mitogen-induced cellular cytotoxicity (MICC), and antibody-dependent cellular cytotoxicity (ADCC). Lymphoid cells were isolated from mesenteric lymph nodes (MLN), Peyer's patches (PP), and the small intestinal mucosa. Mucosal lymphoid cells were separated into a population of cells containing intraepithelial lymphocytes (IEL), and a second population of cells containing intraepithelial lymphocytes (IEL), and a second population of cells containing lamina propria (LP) lymphocytes by a combination of physical and enzymatic methods. All populations of lymphoid cells from the gastrointestinal tract were capable of mediating MICC. Relative to spleen cells, IEL possessed more MICC effector cell activity whereas cells from MLN, PP, and LP possessed less activity. In contrast, SCMC and ADCC effector cell activities were present only in the IEL population and these cytotoxic activities were comparable to that of splenic cells. These results demonstrate that mucosal lymphoid cells possess cytotoxic effector cell activities that may contribute to local mucosal immunologic reactivity.
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Rat parotid acinar cells dispersed by a combination of enzymatic treatments remain sensitive to adrenergic and cholinergic agonists. Previous studies have implicated Ca2+ in both adrenergic and cholinergic responses. This paper describes the effects of adrenergic and cholinergic stimulation upon 45Ca2+ fluxes in isolated parotid acinar cells. Suspensions of dispersed cells took up 45Ca2+ from the medium. The net rate of isotope influx was increased by the adrenergic agonists epinephrine, norepinephrine, isoproterenol, and phenylephrine, and by the cholinergic agonists acetylcholine and carbamylcholine. In 1 mM Ca2+, epinephrine was capable of increasing the 45Ca2+ influx in 40 min to three times that of resting cells. Isoproterenol, a beta-adrenergic agonist, was only half as effective as epinephrine in stimulating maximal calcium uptake although it was equally effective in stimulating maximal amylase release in the same cells. Experiments with the alpha-adrenergic antagonist phentolamine, the beta-adrenergic antagonist propranolol, and the cholinergic antagonist atropine confirmed that alpha- and beta-adrenergic and cholinergic stimulation each had a direct stimulatory effect on 45Ca2+ uptake. N6,O2'-Dibutyryl adenosine 3':5'-monophosphate also caused some stimulation of net calcium uptake. Direct measurement of Ca2+ efflux indicated that the increased calcium uptake in the presence of epinephrine was not the indirect result of a decrease in efflux. The rates of both basal and epinephrine-stimulated calcium uptake increased with increasing calcium concentration in the medium. Epinephrine had little effect on the rate of calcium uptake at 0.15 mM Ca2+. Although the energy poison NaCN had little effect on the basal rate of calcium uptake, the stimulable component of calcium uptake was inhibited by NaCN at all calcium concentrations tested (0.2 to 4.1 mM).
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The in vitro cytotoxic function and target cell specificity of peripheral blood lymphocytes from selected patients with primary biliary cirrhosis and hepatitis B surface antigen-negative chronic hepatitis were investigated using 51Cr-labeled human Chang and EL-4 mouse sarcoma cell targets in assays of spontaneous cell-mediated cytotoxicity (SCMC) and mitogen-induced cellular cytotoxicity (MICC). In addition, antibody-dependent cellular cytotoxicity (ADCC) against Chang cells was assessed. At an effector-to-target cell ration of 100:1, the mean SCMC against Chang cells was much less in patients with primary biliary cirrhosis than that in either the controls (P less than 0.001) or the patients with chronic hepatitis (P less than 0.005) whereas the value for patients with chronic hepatitis did not differ significantly from that of the controls. The mean SCMC against EL-4 mouse sarcoma cells was also less in patients with primary biliary cirrhosis than in controls (P less than 0.005) whereas the value for chronic hepatitis was not significantly different from that of the controls or patients with primary biliary cirrhosis. In contrast, MICC against both targets and ADCC against Chang cells were similar for each group. Comparison of SCMC and MICC against both target cells, measured simultaneously, showed similar cytotoxic potenital against both target cells for each group. Effector cells capable of mediating cytotoxicity in each assay were defined by testing the cytotoxic function of lymphocyte subpopulations isolated from two representative patients with each disease using techniques of immunoabsorbent affinity chromatography and Fc receptor binding to antigen-antibody complexes. In both primary biliary cirrhosis and chronic hepatitis SCMC and ADCC were mediated by a subpopulation of lymphocytes which lack surface immunoglobulin (sIg-) and bear Fc receptors (Fc+). In contrast, MICC was mediated by sIg- cells which lack Fc receptors. Lymphocytes bearing sIg- were not cytotoxic in any assay. These results establish a difference in cytotoxic function in primary biliary cirrhosis and chronic hepatitis by defining the presence of a defect in spontaneous cytotoxic function of sIg-, Fc+ lymphocytes against Chang cells in primary biliary cirrhosis.
The maintenance of adrenergic function has been investigated in organ cultures of adult rat superior cervical ganglia. Tyrosine hydroxylase and monoamine oxidase activities decreased gradually through 72 hours of culture. Dopamine beta-hydroxylase activity gradually increased in the cultured ganglia to 150% of that seen in fresh ganglia after 14 hours in culture. The level of norepinephrine increased in the cultured ganglia to a maximum of 225% of that seen in control ganglia. The increases in both dopamine beta-hydroxylase activity and in norepinephrine levels required protein synthesis but they were not dependent on the presence of nerve growth factor. The accumulation of norepinephrine in the cultured ganglia could not be explained by an increased rate of amine synthesis. Turnover measurements suggest that the accumulation can be explained by a decreased rate of amine metabolism in the cultured ganglia. A comparison of the distribution of amine storage vesicles showed that the number of heavy vesicles was greater in cultured than in fresh ganglia. The results suggest that sympathetic ganglia develop an increased capacity for amine storage in culture and that this leads to an increase in intracellular levels of norepinephrine.
Human peripheral blood lymphocyte subpopulations were analyzed for the presence of B cell alloantigens with a microcytotoxicity assay. B cell alloantigens were found exclusively on sIg-positive lymphocytes and were not present on sIg-negative, Fc receptor-bearing lymphocytes or sIg-negative, Fc receptor-negative T lymphocytes.
Surface immunoglobulin (sIg)-positive and sIg-negative subpopulations of macrophage-depleted murine splenic lymphocytes were obtained by Sephadex anti-Fab immunoabsorbent fractionation. These lymphocyte subpopulations were analyzed for the presence of Thy 1 and Ia alloantigens and also for Fc receptors by fluorescence microscopy. Concurrently, these lymphocyte subpopulations were studied for effector cell activity in antibody-dependent cellular cytotoxicity (ADCC). Effector cells mediating ADCC were contained in the sIg-negative lymphocyte subpopulation and sIg-positive lymphocytes did not mediate cytotoxicity. The majority of sIg-positive lymphocytes were found to bear Ia antigens and Fc receptors, and these cell surface structures were associated in that treatment of these cells with anti-Ia sera inhibited binding of complexed immunoglobulin to Fc receptors. In contrast, most sIg-negative, Thy 1-negative lymphocytes lacked Ia Antigens, and the Fc receptors detected on such cells were not blocked by anti-Ia sera. In addition, a small subpopulation of sIg-negative, Ia antigen-positive, Fc receptor-positive lymphocytes was found. Elimination of this subpopulation of Ia antigen-positive cells from sIg-negative lymphocytes, by treatment with anti-Ia serum and complement, did not diminish ADCC effector cell activity in the resultant cell population when compared with untreated sIg-negative lymphocytes. Thus, in murine spleen, nonphagocytic mononuclear cells that lack both sIg and Ia antigens were shown to mediate ADCC.
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