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Biomedical subjects

H Lewis

Publications and source records attributed to H Lewis.

At least 181 records · Page 10Linked to original sources
PubMed1972

Quantitative features of a sandwich radioimmunolabeling technique for lymphocyte surface receptors.

The present study was designed to devise and characterize an indirect or sandwich radioimmunolabeling technique for the study of lymphocyte surface receptors of immunoglobulin nature. Mouse lymphocytes from various sources were treated by the method of Shortman et al. to remove debris and damaged cells. This was an important preliminary step, as without it, little meaning could be attached to bulk scintillation counting of labeled cell suspensions, in view of the marked tendency of dead or damaged cells to adsorb protein nonspecifically. Next, cells were reacted at 0 degrees C for 30 min with graded dilutions of unlabeled rabbit antisera against defined mouse Ig chains. After washing, the cells were reacted with a sheep anti-rabbit globulin reagent labeled with (125)I, again at graded concentrations. After further washing, lymphocyte labeling was quantitated by both bulk scintillation counting and radioautography. Conditions were defined in which nonthymus-derived cells (B cells) but not thymus-derived cells (T cells) could be labeled. Most B cells displayed kappa- and micro-chains on their surface, but some also displayed alpha- and gamma(2)-chains, though in smaller amounts. When the concentration of both the first and the second reagents were raised considerably, conditions were defined under which virtually all T cells could be labeled by polyvalent antiglobulin sera, anti-kappa sera, or, with more difficulty, by anti-micro sera. A large series of control experiments confirmed the serologic specificity of this labeling. It was shown that under equivalent conditions, B cells bind 100-400 times more antiglobulin than do T cells. The theoretical implications of the results are briefly discussed. It is argued that the sandwich approach offers certain technical advantages over direct labeling procedures for further analyses of T cell receptors and for studies of receptor metabolism.

Animals↗
PubMed1970

In vitro stimulation of antibody formation by peritoneal cells. I. Plaque technique of high sensitivity enabling access to the cells.

An improved method for the short-term culture of mouse peritoneal cells in a medium containing carboxymethylcellulose (CMC), sheep erythrocytes (SRBC), and guinea pig complement is described. It involves preparation of microcultures, of thickness 12-15 micro and volume 3.6 microl, under paraffin oil. With such cultures, peritoneal cells from normal, unimmunized young male CBA mice give about 3000 hemolytic plaques per million cells cultured, this figure being attained within 24 hr. The plaque detection method is about four times as sensitive as the Jerne technique. A method is described whereby such plaque-forming cells (PFC) can be transferred, by micromanipulation, to fresh monolayer cultures containing SRBC, CMC, and complement. In this fashion, the secretory capacity and susceptibility to inhibitors of peritoneal PFC can be tested in detail. Using this technique, evidence is presented that the hemolytic substance responsible for plaque formation is actually secreted by the cell at the center of the plaque, and is not a complement component but probably an antibody. Studies on the time of plaque appearance after cell transfer, and the subsequent growth rate of the zone of hemolysis, have been performed. They speak against the idea that the PFC is either a reservoir of cytophilic antibody or a "background" PFC. Rather they suggest that active antibody secretion is induced in the cell at some defined time point in culture. Detailed kinetics of the rate of appearance of plaques in peritoneal cell cultures revealed an exponential phase lasting from about 3 to about 13 hr with a doubling time of 2 hr. The reasons for this are not known. A greatly heightened reactivity was shown in peritoneal cells of mice that had been pregnant several times. Cultures of such cells showed more rapid plaque appearance and a peak activity about 20 times higher than with cells from young male mice. Cultures in which 1 cell in 10 formed a plaque were not infrequent. A series of experiments on germ-free mice showed reactivity similar to that of conventional mice from the same strain and source. The significance of the findings for cellular immunology are discussed.

Animals↗
PubMed1970

In vitro stimulation of antibody formation by peritoneal cells. II. Cell interactions and effects of immunochemical or metabolic inhibitors.

Peritoneal cells (PC) from normal, unimmunized mice were placed in ultra-thin monolayer cultures containing carboxymethylcellulose (CMC), sheep red blood cells (SRBC), and complement, and tested for the appearance of plaques of lysis. The behavior of PC from young male mice and from female mice that had given birth to several litters (retired breeder mice) was studied. It was found that cells from spleen, mesenteric lymph node, thymus, bone marrow, thoracic duct lymph, or Peyer's patches could not form plaques in the CMC microcultures. Also, various combinations of these cells did not lead to plaque formation. When cells from any of these sources were mixed with PC, there was either no effect or an actual inhibition of plaque formation, the plaque counts being lower than would have been expected from the number of PC present in the mixture. Optimal plaque formation by peritoneal cells was found to be dependent on an optimal cell concentration, this optimum being around 5 x 10(6)/ml for young male mice and 0.5 x 10(6)/ml for retired breeders. Inhibition of plaque formation was found with either supra- or suboptimal cell concentrations. The inhibition by excess cell concentration may have been a simple nutritional or nonspecific overcrowding effect, as it could also be induced by an addition of an excess of spleen or lymph node cells. The failure of more dilute PC preparations to give adequate numbers of plaques appeared to be more specific, as plaque numbers could not be restored to normal by addition of spleen cells. The suggestion was that some cell to cell interaction between PC was involved. This dependence on cell concentration was not seen with immunized spleen PFC. Plaque appearance could be specifically and reversibly suppressed by placing PC in a medium containing rabbit anti-mouse IgM serum. Anti-IgG serum had no such effect. These experiments strengthened our view, expressed in the accompanying paper, that plaque formation was due to the formation of IgM, hemolytic antibody to SRBC by the PC. Metabolic inhibitors were incorporated into monolayer cultures and had different effects with the different types of PFC used. In the case of spleen cells from mice actively immunized against SRBC 4 days before killing, actinomycin D had no effect on plaque counts and puromycin reduced plaque numbers by a factor of 2. In the case of PC from young male mice, actinomycin D in concentrations above 0.01 microg/ml caused reductions down to < 2% of control values in plaque counts, and puromycin (10 microg/ml) had a similar effect. The PC from retired breeder mice occupied an intermediate position between the two cases just discussed. A compartment of cells, equal to about one-fifth of the total normal PFC compartment, was identified as resistant to high concentrations of either actinomycin D or puromycin, being similar in these respects to PFC from spleens of intentionally preimmunized mice. The mitotic poison, Colcemid, did not affect plaque counts in any situation tested. The theoretical implications of these results are briefly discussed.

Animals↗