Showing papers by "Walter and Eliza Hall Institute of Medical Research published in 1969"

An enzymic method for the trace iodination of immunoglobulins and other proteins.

Abstract: 1. A method is described for the trace iodination of immunoglobulins and other serum proteins by a system consisting of lactoperoxidase, hydrogen peroxide and iodide. 2. gammaG immunoglobulin that had been labelled to a specific radioactivity of 5muc/mug. by use of carrier-free [(125)I]iodide gave no evidence of denaturation when analysed by electrophoresis and density-gradient ultracentrifugation. 3. Tryptic hydrolysis and peptide ;mapping' of a completely characterized peptide radioiodinated by this method showed that the [(125)I]iodide was bound to tyrosyl residues. 4. Proteins differ in their susceptibility to iodination by this method. Human gammaG immunoglobulin, for example, is iodinated more than ten times as readily as is human alpha(2)-macroglobulin under the same conditions. 5. Lactoperoxidase catalyses the iodination of proteins much more readily than does horseradish peroxidase.

Thymus and Antigen‐Reactive Cells

Abstract: Neonatal thymectomy severely limits the ability of some rodents to engage in certain immune responses, particularly cell-mediated immunities such as delayed hypersensitivity and the homograft reaction (reviewed in Miller & Osoba 1967). Many antigens apparently elicit normal humoral antibody responses in thymectomized animals but some, such as heterologous erythrocytes and serum proteins, do not. To account for the immune defects following thymectomy, it was originally postulated that the thymus produced 'the originators of immunologically competent cells' which migrate to other sites (Miller 1961). The simplest relationship between the thymus and the cells taking part in immunity would be that, in response to antigenic stimulation, thymus-derived immunologically competent cells generate the effector cells which actually carry out the response. Recent experiments in our laboratory have attempted to examine this hypothesis in order to define more precisely the cellular basis of the immune defects in thymectomized mice. The purpose of this article is to summarize this work without reviewing the entire literature in the field.

Specific Inactivation of Antigen-reactive Cells with 125I-Labelled Antigen

Abstract: A QUESTION of current interest is whether the initial stages of an immune response—either the induction of antibody formation or of specific tolerance—may involve the reaction of an antigen with a specific lymphocyte. Naor and Sulitzeanu1 have demonstrated, both in vivo and in vitro, a reaction of 125I-labelled bovine serum albumin with a small proportion (about 1/5,000) of mouse spleen lymphocytes. We have since shown2 that both flagellin and polymerized flagellin (Salmonella adelaide) and haemocyanin (Jasus lalandii) labelled with 125I or 131I react in vitro with certain cells from spleens of rats and mice. Of the strongly reactive cells, almost all are mononuclear with a high nuclear/cytoplasmic ratio and 7–12 microns in diameter and, for any one antigen, comprise about 1/5,000 of the total cell population. These reactive cells adsorb between 4,000–40,000 molecules of labelled protein when allowed to react at 0° C with labelled protein (about 3 × 1012 molecules/ml.) in 10 per cent foetal calf serum. A similar proportion of such reactive cells was observed in cell suspensions from lymph nodes and thoracic duct lymph, while peritoneal exudate contained a higher proportion and thymus a lower proportion of these cells2. The reaction was not inhibited by concentrations of sodium azide which restricted the uptake of labelled antigen by macrophages but was inhibited, using mouse cells, by rabbit anti-mouse globulin serum. Spleen cells from germ-free and conventional mice reacted equally well with 131I-labelled flagellin2. We wished to know whether the ability of these cells to react with antigen was immunologically significant. Experiments were devised to distinguish between the following possibilities: that the reactive cells were (1) cells present because of a prior experience of the animal with a related antigen, (2) antigen-reactive cells3,4 which had not had prior antigenic experience but which were capable of contributing to a specific immune response such as formation and secretion of antibody, and (3) cells coated non-specifically with cytophilic antibody.

Studies on colony formation in vitro by mouse bone marrow cells. I. Continuous cluster formation and relation of clusters to colonies.

Abstract: Colony formation in vitro by mouse bone marrow cells following stimulation by human urine was analysed over a 7-day incubation period. There was a linear increase with time in the number of cell aggregates (clusters) developing in such plates. Early in the incubation period all clusters were granulocytic although later macrophage clusters developed. Although most fully developed colonies were composed of macrophages, mapping and transfer studies showed that at least half of these had initially arisen early in the incubation period as granulocytic clusters.

Immunological tolerance in vitro: kinetic studies at the cellular level.

Abstract: When normal mouse spleen, cells in suspension are cultured in vitro in the presence of polymer from S. adelaide flagellin, an immune response can be obtained as measured at the level of single antibody-forming cells. Cultures were stimulated with different doses of antigen, ranging from 0.2 ng to 3 µg/ml of tissue culture fluid and it was found that the peak number of approximately 500 antibody-forming cells per 106 harvested cells by day 4 was antigen dose dependent, 2–20 ng/ml being the optimal concentration. When more than 1 µg/ml of polymer from S. adelaide together with either 20 ng/ml of polymer from S. waycross or with 4 x 106 sheep erythrocytes were placed in the system, unresponsiveness to S. adelaide, but immunity to the other antigens occurred simultaneously. Cells made immunologically tolerant in vitro to S. adelaide H antigens were transferred into syngeneic lethally irradiated recipients and challenged with the same antigen. The adoptive immune capacity in these mice, as measured at the level of the immunologically competent cell was reduced by 80–90% as compared with relevant controls. Attempts to induce low zone tolerance in vitro were without success. To study the kinetics of tolerance induction in vitro, cells were cultured with tolerogenic doses of antigen for various periods of time, washed, and subsequently cultured with immunogenic doses of antigen for 4 days. It was found, that immunological tolerance may be induced to a significant degree in vitro within a period of 15 min. Similar results were obtained when spleen cells were exposed for various lengths of time to tolerogenic doses of antigen but at a temperature of 4°C instead of 37°C. The results are taken as suggestive evidence that the initial step in tolerance induction is related to the direct interaction between the surface of immune competent cells and antigen molecules.

Antigen-reactive cells in normal, immunized, and tolerant mice.

Abstract: The numbers of antigen-reactive cells (ARC) responding to a purified protein, the polymer of S. adelaide flagellin, have been assayed in cell populations derived from several lymphoid tissues of mice. The assay, which employs the cell transfer into lethally irradiated mice, indicates that there is a response of ARC in bone marrow in the absence of thymus cells. This suggests that the immune response to this protein antigen is not thymus dependent. The presence of relatively large numbers of ARC in Peyer's patches argues for their direct participation in the immune response in the adult mouse. The kinetics of ARC and antibody-forming cells in the early primary response employing the transfer system is described. The numbers of ARC declined during the first 2 days of the immune response, but by day 6 had increased to about five times the number in unprimed spleen cells. The rise is believed to be a result of the primary injection of antigen and therefore may be described as memory; however, these experiments have not been able to further elucidate any specific qualities of the "memory cell." Tolerance induction in C(57)BL/Brad mice produced by repeated injections of a cyanogen bromide digest of the antigen is described. The ARC or its precursor is shown to be the site of the lesion of tolerance by direct investigation.

THE SEPARATION OF DIFFERENT CELL CLASSES FROM LYMPHOID ORGANS : III. The Purification of Erythroid Cells by pH-Induced Density Changes

Abstract: 1. Mammalian erythrocytes swell as the pH of the isotonic suspending medium is lowered, as a direct consequence of the specialized permeability properties of the erythrocyte membrane. Lymphocytes and granulocytes from a variety of sources did not exhibit this property. 2. The behaviour of mouse bone marrow erythroid cells at various stages of differentiation was studied by using a change in buoyant density with pH as an index of swelling. The ability to swell with a pH drop was acquired while the cell was still nucleated. All non-nucleated cells showed swelling. Most small erythroblasts shared this property, whereas most large erythroblasts did not. 3. The density shift with pH was used to provide a purification scheme specific for erythroid cells. The bone marrow cells were first centrifuged to equilibrium in an isotonic albumin density gradient at neutral pH. Regions of the gradient containing the erythroid cells were collected, and the cells were recovered and redistributed in an albumin gradient at acid pH. The erythroid cells showed a specific density shift which removed them from contaminants. Preparations containing 90–97% erythroblasts were obtained by this technique. 4. Differentiation within the erythroid series was accompanied by a general increase in cell buoyant density at neutral pH. This density increase may have been a discontinuous process, since erythroid cells appeared to form a number of density peaks. 5. The pH shift technique, in association with established density distribution and sedimentation velocity procedures, provides a range of cell separation techniques for biological or biochemical studies of erythroid cell differentiation in the complex cell mixtures in bone marrow or spleen.

The effect of bleeding on the number of in vitro colony-forming cells in the bone marrow.

Abstract: Summary: Bleeding 0.3 ml. daily for 3 days reduced the total number of in vitro colony-forming cells in the bone marrow of C57Bl and BALB/c mice to less than 50 per cent of that in control mice, but caused a significant increase of erythropoietic cells in the bone marrow. Transfusion of packed red cells combined with bleeding prevented this fall in in vitro colony-forming cells. Bled mice developed spleen enlargement with some increase in the total number of in vitro colony-forming cells in the spleen but this did not compensate for the reduced number of such cells in the bone marrow. Splenectomy did not affect this response of in vitro colony-forming cells in the bone marrow. Cortisone reduced the bone marrow content of in vitro colony-forming cells. Serum levels of colony-stimulating factor were not elevated in response to bleeding. The results suggest that in vitro colony-forming cells are not erythropoietic cells but may share a common ancestor with erythropoietic cells.

Cortisone Action on Serum Colony-Stimulating Factor and Bone Marrow in Vitro Colony-Forming Cells

Abstract: SummaryCortisone acetate in adult C57BL mice, in doses of 0.25-2.0 mg, caused an acute fall in serum levels of colony-stimulating factor and a slower fall in the bone marrow content of in vitro colony-forming cells reaching minimum values 3 days after injection. Doses of cortisone as low as 10 μg inhibited colony formation in vitro by bone marrow cells.

Cleavage of bacterial flagellin with cyanogen bromide. Antigenic properties of the protein fragments

Abstract: 1. Four polypeptide fragments, obtained by cyanogen bromide treatment of the protein flagellin from Salmonella adelaide, were tested for their antigenic activity by using them as inhibitors in three different assays: bacterial immobilization, haemagglutination of sensitized erythrocytes and quantitative micro precipitation. Immunodiffusion studies were also performed on the protein fragments. 2. Cleavage of the flagellin molecule in this way gave no detectable loss of antigenic determinants. Fragment A (mol.wt. 18000), the largest of the polypeptides, contained all the antigenic specificities present on flagellin that were recognized by the antisera used. In one test, fragment B (mol.wt. 12000) also contained antigenic activity to an extent not easily explainable by contamination with fragment A. Fragments C (mol.wt. 5500) and D (mol.wt. 4500) appeared to be antigenically inactive.

The Effects of Whole Body Irradiation on Serum Colony Stimulating Factor and in Vitro Colony-Forming Cells in the Bone Marrow

Abstract: Summary. Using in vitro cultures of mouse bone marrow cells, levels of colony stimulating factor were assayed in serum from mice following whole body irradiation. No significant differences from control levels were noted in the period 1–32 days following 50, 150, 250 or 450 rads. A dose of 250 rads caused a sharp fall in the level of colony-forming cells in the bone marrow followed by regeneration between 6 and 16 days following irradiation. Six to 8 hours following whole body irradiation, blood polymorphs and serum colony stimulating factor levels were consistently elevated. Splenectomy did not affect this response. The results suggest that serum colony stimulating factor is unlikely to be the major regulator determining regeneration of in vitro colony-forming cells in the bone marrow following irradiation.

Cleavage of bacterial flagellin with cyanogen bromide. Chemical and physical properties of the protein fragments.

Abstract: 1. Flagellin, isolated from the flagella of Salmonella adelaide, was shown by various criteria to be a pure protein. It had a molecular weight of about 40000 and contained three methionine, six tyrosine, 11 arginine and 25 lysine residues/mol., of which 11 of the lysine residues were present as -N-methyl-lysine. 2. After treatment of flagellin with cyanogen bromide in formic acid, four main fragments (A, B, C and D) were obtained, with as many as six minor components that represented partial degradation products. The major fragments were estimated by amino acid analysis to have molecular weights of about 18000 for fragment A, 12000 for fragment B, 5500 for fragment C and 4500 for fragment D. Fragments A, B and D, but not fragment C, were recovered pure by gel chromatography as monitored by polyacrylamide-gel electrophoresis. 3. A complex between fragments C and D was also isolated (mol.wt. 10000) after limited oxidation of flagellin by chloramine-t before digestion by cyanogen bromide. After oxidation essentially only two fragments were released from flagellin by cyanogen bromide: the C,D'comp≤xandapre∑edC,D′comp≤xandapre∑edAB' fragment. 4. The sum of the amino acid analyses of fragments A and B and the `C,D' complex gave residue values that agreed well with the amino acid composition of native flagellin. 5. Fragments A and D contained tyrosine, and ten of the 11 -N-methyl-lysine residues of the molecule were in fragment A. Reaction with [125I]iodide at small extents of substitution showed that, in flagellin, the tyrosine residue of fragment D was more readily substituted than those of fragment A. By contrast, in polymerized flagellin, the tyrosine residues of fragment A were more readily substituted. 6. Treatment of flagellin with carboxypeptidases A and B revealed the C-terminal sequence -Leu-Leu-Leu-Arg. Arginine and leucine were released by carboxypeptidase from the `C,D' complex but not from fragment D, indicating that fragment C was C-terminal. 7. On the basis of the results from amino acid analysis, carboxypeptidase digestion, N-terminal analysis, iodination studies and polyacrylamide-gel electrophoresis, the sequence of fragments in flagellin was considered to be B–A–D–C; in the polymer, fragment A was exposed. It is suggested that methylation of the lysine residues occurred in the organism after flagellin had polymerized.

Density distribution analysis of antigen-sensitive cells in the rat.

Abstract: Analysis of the cell populations capable of initiating a response to sheep and horse erythrocyte antigens has been carried out by means of equilibrium density gradient centrifugation. The results indicate that there are at least six distinguishable AS cell populations for sheep erythrocytes, but only three for horse erythrocytes in the spleen of the Lewis rat. Evidence is presented for the existence of metabolic, physiological, and immunological differences among these populations. It is suggested that at least one population of AS cells responds only to the specific antigen and at least one other population is sensitive to stimulation by a broad range of antigens. It is assumed that the difference between these two AS cells results from a process of differentiation of AS cells primed into DNA synthesis by antigen stimulation.

A new method for the enumeration of antigen-reactive cells responsive to a purified protein antigen

Abstract: A new technique for the enumeration of antigen-reactive cells (ARC) responsive to the polymer antigen of S. adelaide flagellin (POL) is described for two strains of mice. Foci have been shown to be antibody dependent, may be mimicked by IgM as well as IgG antibodies, and contain specific antibody-forming cells (AFC). The use of POL offers a system unencumbered by relatively high numbers of background foci which, when present, appear to be basically different from those found using the SRBC antigen. The response of 1 antigen-reactive cell (ARC) focus/1 x 106 CBAT6T6 mouse spleen cells is linearly related to the injected number between 1 x 106–3 x 106 donor spleen cells and since 5% of injected cells remain in the spleen, there are an estimated 2400 ARC/spleen. The number of ARC foci does not increase significantly after the 5th postantigen day, and by the 8th day the AFC progeny of ARC have reached the maximum mean of 280 AFC/ARC focus. In response to increasing antigen concentrations, an initial rise in the number of AFC as well as ARC is observed, resulting in a relatively constant AFC/ARC ratio. This suggests that the number of ARC stimulated determines the total number of AFC produced under these conditions rather than a variable mitotic rate of the ARC offspring. The main significance of this technique is that it will allow a study of the kinetics of the ARC in the primary and secondary immune response as well as in immunological tolerance.

Immunological Tolerance in Organ Transplantation Fair Prospect or Fanciful Folly

Abstract: A formidable array of practical problems must be solved at laboratory level before tolerance induction in organ transplantation can be considered a realistic clinical possibility. I believe it is too early for even tentative human experimentation in this field. However, the work of the last decade has shown unequivocally that even adult animals can readily be rendered tolerant of even exceedingly powerful antigens. The principle of tolerance has such great specificity and such compelling elegance when compared with present-day aids to organ transplantation that intensive effort must go into harnessing it to clinical use. It will certainly enter the clinical homograft scene within a decade, and xenografts are inconceivable in its absence.

Interaction Between Two Distinct Cell Lineages in an Immune Response

Abstract: In 1961, an immunological role for the thymus was revealed by experiments which indicated that thymectomy, at birth in mice, caused a severe depletion of lymphocytes in peripheral blood, lymph nodes, and spleen and a marked deficiency in the capacity to reject foreign skin grafts [1]. Since then numerous experiments have indicated that the thymus must perform a similar function in many species (reviewed by Miller and Osoba [2]). In spite of extensive research on the thymus in the last decade, there is much controversy regarding the fate and function of the thymus lymphocytes — the predominant cell type in thymus tissue. Recent work in our laboratory suggests that thymus lymphocytes are capable of recognizing and interacting with antigen by giving rise not to antibody-forming cells, but to a progeny of recirculating small lymphocytes through the intermediary of large pyroninophilic cells. As this work was performed using one antigenic system in mice, no generalization can yet be made with respect to other systems.

The expanding family of human immunoglobulins.

Abstract: The immunoglobulins comprise ail molecules having antibody activity as well as certain pathological proteins. These proteins can be divided into classes on the basis of many physiochemical properties, with antigenic delineation being particularly useful. Five major classes are presently recognized, IgM, IgA, JgG, IgD, IgE, together with four sub-classes of IgG and two of IgA. By the use of a sensitive radioimmunoassay, the IgM class is also observed to consist of at least two sub-classes. Recent evidence has shown that many of the pathological immunoglobulins also possess antibody activity, particularly of an autoantibody specificity. Our studies in laboratory mice have also suggested that susceptibility to plasma cell tumour induction is only observed in mice known to carry genes predisposing for autoimmunity. The hypothesis is therefore entertained that plasma cell tumours (multiple myeloma, Waldenstrom macroglobulinaemia) are very frequently, if not exclusively, malignancies of autoantibody-producing cells.

The role of blood cells in immunity

Abstract: Various types of nucleated cells in the blood play an essential role in immunity: progenitor or stem cells with potentialities for populating lymphoreticular tissues; immunologically competent cells that initiate certain immune reactions ; immunologically activated cells that actually mediate these reactions ; precursors of macrophages involved in phagocytosis and resistance mechanisms, and a variety of cells which can deal with antigens or antigenantibody complexes or which take part in various inflammatory responses associated with some immune reactions.

Enzyme-treated bovine arterial heterografts. I. Preparation and physical properties.

Abstract: In an attempt to render bovine carotid arteries suitable for use as arterial replacement grafts in the human, the vessels have been modified by a technique of preparation which removes the cellular tissue and elastin from the wall of the graft to leave an almost pure collagen tube. Electron microscopy has revealed that the collagen fibrils remain intact after preparation. Tensiometric studies performed in vitro on vessels prepared by this technique have shown that they retain most of their original strength.