Herman Friedman

Distribution of Antibody Plaque Forming Cells in Various Tissues of Several Strains of Mice Injected with Sheep Erythrocytes.

Summary Lymphoid cell suspensions from several strains of mice injected with a single inoculum of sheep erythrocytes were capable of forming localized zones of hemolysis (“antibody plaques”) in agar containing sheep erythrocytes and complement. The ability of lymphoid and non-lymphoid tissue from immunized NIH, C3H, C57, and AKR mice to form antibody plaques in agar were compared. Optimum time for plaque formation with the strains tested was 4 to 5 days following intravenous immunization. Cells from NIH mice resulted in the most consistent and highest levels of plaque formation. C3H and C57 mice yielded lymphoid cells which were less than half as efficient in plaque forming ability as compared to NIH cells. Lymphoid cell from AKR mice had the least plaque forming capability. Spleen cell suspensions formed the largest number of plaques following I.V. injection. Lymph node cell suspensions were somewhat less efficient. Suspensions of peripheral leukocytes (obtained from “buffy coats”) had low, but significant antibody plaque forming ability. Thymus and bone marrow cells had no significant plaque forming ability. Cell suspensions prepared from lungs, kidney, liver, brain, and skeletal muscle exhibited either low levels of plaque formation or were negative.

Suppression of the primary antibody plaque response of mice following infection with friend disease virus.

Summary Infection of mice with Friend Disease Virus prior to or simultaneous with immunization with sheep red blood cells resulted in a marked suppression of appearance of antibody forming cells, as determined by a localized hemolytic plaque technique in agar gel. Infection with virus had a marked suppressive effect on the number of 19S hemolysin forming cells in mouse spleen tissue at the peak of the expected immune response and during a period of two weeks or longer thereafter. Appearance of low efficiency 7S hemolysin forming cells late in the immune response, following a single injection of sheep red cells. Was also depressed in infected mice. Animals infected 3 days to a week or longer prior to immunization had the lowest number of detectable 19S or 7S antibody-forming cells. The number of plaque forming cells per million leukocytes tested was most suppressed in those mice which had obvious splenomegaly due to virus infection.

Leukemia Virus Suppression of Antibody-Forming Cells: Ultrastructure of Infected Spleens

Infection of adult BALB/c mice with Friend disease virus results in a leukemia-like disease characterized by erythropoietic changes and splenomegaly. A marked depression of formation of cellular and serum antibody occurs in infected animals. Electron-microscopic examination of the ultrastructure of spleen sections from infected mice with depressed immunity revealed that virus particles can be detected only in immature blastlike lymphoid cells and not in plasmocytes characteristic of the immune response in spleens of noninfected mice immunized with sheep erythrocytes.

ABSENCE OF ANTIBODY PLAQUE FORMING CELLS IN SPLEENS OF THYMECTOMIZED MICE IMMUNIZED WITH SHEEP ERYTHROCYTES.

Summary Surgical removal of the thymus of newborn NIH mice resulted in a marked suppression in subsequent ability to form circulating serum hemolysins to sheep erythrocytes. Spleen cell suspensions from young adult mice thymectomized at birth and immunized with sheep red blood cells were markedly less capable of forming hemolytic antibody plaques in agar gel than spleen cells from sham operated control mice. Although the number of antibody plaques formed by cells from thymectomized mice was markedly reduced, the size of individual plaques was generally similar to those formed by spleen cells from the control mice.

SPECIFIC RESTORATION OF AGGLUTININ FORMATION IN X‐IRRADIATED RABBITS BY NUCLEOPROTEIN FRACTIONS FROM IMMUNE DONORS*

Lymph-node cell-suspensions obtained from shigellaimmunized donor rabbits were fractionated into subcellular preparations, including cell-free homogenate, nuclei, mitochondria, desoxyribonucleoprotein and ribonucleoprotein- containing fractions. No serologically detectable shigella agglutinins or antigens could be observed with these preparations. Injection into nonimmunized recipient rabbits, either nontreated or whole-body-irradiated with 425 r 24 hr previously, resulted in appearance of antishigella agglutinins in the sera of the recipients. The time and magnitade of the agglutinin response was relatively similar to that observed following transfer of control whole cell suspensions. A majority of recipient rabbits receiving cell-free fractions had detectable shigella agglutinins within three to nine days, with peak titers at nine to twenty days. Possible mechanisms responsible for the appearance of these agglutinins were discussed. (auth)

Defect in Cellular Immunity of Leukemia Virus-Infected Mice Assessed by a Macrophage Migration-Inhibition Assay

Summary Cellular immunity in leukemic mice was studied using Friend leukemia virus and susceptible BALB/c mice. Injection of this virus into the test mice resulted in rapid development of splenomegaly and other symptoms of leukemia. There was a concomitant decrease in the ability of peritoneal cells from these animals to respond to PPD in vitro, as assessed by the macrophage migration-inhibition procedure. Sensitization of normal mice with mycobacteria in Freunds complete adjuvant resulted in rapid development of cellular immunity as assessed by the migration-inhibition assay. Failure to detect cellular immunity by this assay with peritoneal cells from leukemia virus-infected mice correlates with the marked decrease in humoral immunity, as well as cellular immunity previously assessed by skin graft rejection assays.

Comparison of Bacteriolysis, Passive Hemolysis, and Bacterial Adherence Colony Formation for Detecting Antibdy-Forming Cells∗

Summary Three procedures were compared in a study to determine the cytokinetics of antibody formation to the lipopolysaccharide antigen of Escherichia coli 0127:B8. A bacterial cytoadherence colony procedure, recently described for enumerating antibody-forming cells to Salmonella flagellin, was adapted to enumerate antibody-forming cells to E. coli. Antibody plaque procedures, using either viable E. coli or lipopolysaccharide-sensitized red cells as indicator targets, were also used. The bacterial cytoadherence procedure was more sensitive than the complement dependent plaque methods. Mice immunized with 50 μg of E. coli antigen had approximately 50,000 to 60,000 plaque-forming cells and over 130,000 colony-forming cells at the peak of the immune response 5 days after immunization. The first antibody forming cells were detected 24–48 hr after immunization. Normal mice had lower numbers of “background” antibody-forming cells. After a lag of 3 days serum antibody titers correlated with the appearance of antibody-forming cells.

Immunologic “Amnesia” of Antibody-Forming Cells after RES Blockade

Summary Cellular and humoral antibody formation was markedly inhibited in mice treated with colloidal carbon particles to blockade the reticuloendothelial system. Appearance of 19S IgM and 7S IgG hemolysin-forming cells was prevented when adult mice were treated with carbon prior to a single inoculation of sheep erythrocytes. Carbon treatment also reduced markedly the expected secondary immune response in mice primed several weeks previously with sheep red blood cells. Few antibody-forming cells, either IgG or IgM, and only low levels of serum hemolysins appeared in primed mice injected with carbon 1 to 4 days before a secondary immunization. Mice injected with carbon before the primary inoculation of antigen had normal 19S IgM antibody-plaque responses after a second injection of red blood cells 1 month later. However, there was a marked absence of 7S IgG antibody-forming cells. Also, there was little, if any, 2-mercaptoethenol-resistant antibody in the sera of these mice after booster immunization. The immune response of these animals was similar to that of a primary-type response of normal control mice injected only once with erythrocytes. Specific immunologic “amnesia,” as distinct from immunological tolerance, had developed as a consequence of RES blockade by carbon injection prior to initial immunization.

Migration of Mouse Lymphocytes in Vitro from Capillary Tube Cultures

Summary Cell suspensions from mouse lymphoid organs, including the spleen, deep and superficial lymph nodes, bone marrow, and thymus, and from induced peritoneal exudates, were compared as regards their ability to migrate from capillary tubes under conditions of tissue cultures. Cells from all lymphoid tissues migrated rapidly in vitro. Purified lymphocyte suspensions prepared by glass bead column or glass plate adsorption procedures containing few, if any, macrophages migrated just as well as unfractionated cell suspensions. The utilization of lymphocytes for migration procedures in vitro for various biologic, immunologic, and physiologic studies are discussed. We are grateful to Dr. Maurice Landy, National Institute of Allergy and Infectious Diseases, Bethesda, Md., for his helpful discussion and advice during the course of this study, as well as his excellent assistance in the preparation of this manuscript.

Direct Localization and Visualization of Hyaluronate Lyase Activity by Agar Gel Electrophoresis.

Summary A rapid agar slide method for localization and visualization of hyaluronate lyase (hyaluronidase) has been described. This procedure is based on enzymophoretic techniques developed for localization of mammalian enzymes following agar gel electro-phoresis. Differences in electrophoretic mobility between staphylococcal, streptococcal and bovine testicular hyaluronidase have been demonstrated. The use of enzymophoresis in agar for study of bacterial enzyme systems has been discussed as a valuable procedure for enzyme analysis during purification and characterization studies.