Discontinuous Bovine Serum Albumin Gradients Research Articles

Properties of density gradient-fractionated peripheral blood leukocytes from cattle infected with bovine leukemia virus

Discontinuous bovine serum albumin gradients were used to fractionate peripheral blood leukocytes from bovine leukemia virus (BLV)-free and BLV-infected cows. The release of infectious BLV and spontaneous incorporation of [3H]thymidine were not properties of density gradient-fractionated leukocytes from a BLV-free cow. When leukocytes from BLV-infected cattle were fractionated, B lymphocytes which spontaneously incorporated [3H]thymidine could be separated as a distinct subpopulation from B lymphocytes which replicated infectious BLV. Density gradient fractionation of leukocytes from a cow with lymphosarcoma is also reported. A fall in lymphocyte count at the time of tumor development is attributed to the loss of B lymphocytes which spontaneously incorporate [3H]thymidine.

Glucocorticoid receptors in corticosensitive and corticoresistant thymocyte subpopulations. I. characterization of glucocorticoid receptors and isolation of a corticoresistant subpopulation.

1. Separation of mouse thymocytes by centrifugation on a discontinuous bovine serum albumin gradient leads to the isolation of four subpopulations of cells. 2. The study of I13H]uridine incorporation in vitro by these subpopulation in the presence of steroid shows that one of them is corticoresistant. 3. However, the binding capacity of these subpopulations measured by incubation with [3H]dexamethasone is very similar. 4. It is therefore concluded that in mouse thymus, in contrast with lymphoma cells, corticoresistance may not be explained by a defect of cytoplasmic glucocorticoid receptors.

Stimulation of a subpopulation of hamster lymphoid cells by trypsin and chymotrypsin

Hamster lymph node and spleen cells can be stimulated to incorporate tritiated thymidine ([ 3H]TdR) in vitro under serum-free conditions by the proteases trypsin and chymotrypsin. Under similar conditions, thymocytes could be stimulated by concanavalin A (ConA) but not lipopolysaccharide (LPS) or the proteases. The subpopulation of cells responding to the proteases correlated with the cells responding to LPS on fractionation of spleen and lymph node cells on discontinuous bovine serum albumin (BSA) gradients or on nylon-wool columns. The stimulation induced by trypsin was completely blocked by soybean trypsin inhibitor (SBTI) while that induced by chymotrypsin was only partially blocked. The inhibition by SBTI of protease activation was not effective when added 24 h after initiation of stimulation. On the other hand, addition of clarified isologous serum to protease activated cultures after 24 h still lead to greater than 50% inhibition of [ 3H]TdR incorporation.

CELL INTERACTION IN TUMOR ALLOGRAFT IMMUNITY

The cytotoxic activity of lymphoid cells from allogeneic tumor-sensitized mice in three different strain combinations was tested at various intervals after i.p. sensitization. Peritoneal cells (PC) gave the highest cytotoxic index. The cytotoxic activity of the PC was due to both peritoneal lymphocytees (PL) and peritoneal macrophages (PM). Splenic macrophages, on the other hand, exerted an inhibitory effect as evidenced by increased cytotoxic activity of spleen treated with antimacrophages serum (AMS). Increased cytotoxic activity was also found in: (1) the nonadherent fraction of spleen after absorption on glass; (2) the effluent fraction of spleen after nylon wool column filtration; and (3) the medium density band after centrifugation of spleen on a discontinuous bovine serum albumin gradient. The results suggest the presence of an AMS-sensitive, glass- and nylon wool-adherent spleen cell which could have a suppressor role in allogeneic tumor-sensitized mice.

Detection of T-Precursor Cells in Human Bone Marrow and Foetal Liver

Human lymphoid cells that have been incubated with conditioned medium from confluent monolayers of human thymic epithelium (HTCM) show an increase in cells forming rosettes with sheep erythrocytes at 4 degrees C (E-rosettes). Those cells demonstrating this in vitro conversion have been interpreted to be T-cell precursors. Separation of human bone marrow cells on discontinuous bovine serum albumin (BSA) gradients, and on a single-step 23% BSA gradient showed enrichment of these T-precursor cells, not only in bone marrow, but also in human foetal liver cells. Bone marrow precursor cells from a patient with Wiskott-Aldrich syndrome (T-cell deficiency) showed a normal in vitro response to HTCM, but no response was seen in cells from a patient with severe combined immunodeficiency disease.

DNA polymerase, thymidine kinase and DNA synthesis in erythropoletic mouse spleen cells separated on bovine serum albumin gradients

A single dose of erythropoietin stimulates DNA synthesis in the spleen of the polycythemic mouse with the maximum effect occurring 48 h after the hormone is administered. The increase in DNA synthesis is accompanied by morphologic evidence of increased erythropoiesis and by increases in the activities per cell of both thymidine kinase and cytoplasmic high molecular weight DNA polymerase-α. The activity of low molecular weight DNA polymerase-β does not change significantly. Spleen cells from mice which had received either erythropoietin or saline 48 h previously were separated into 7 density classes on discontinuous bovine serum albumin gradients. Following the administration of erythropoietin, thymidine incorporation and thymidine kinase activity showed the greatest relative increases per nucleated cell in layers 3, 4 and 5 of the gradient. DNA polymerase-α showed the greatest increase in cells of the denser layers 5, 6 and 7. Each layer contained normoblasts and lymphocytes. The less well differentiated erythroid elements constituted a larger proportion of cells in layers of lower density. Increases in the rates of thymidine incorporation were better correlated with increases in thymidine kinase activity than with increases in DNA polymerase activities. Measurement of iron incorporation into heme confirm the morphological impression that the cell type responsible for increased thymidine incorporation and increased DNA polymerase-α activity is the young normblast.

Null cells in peripheral blood of normals and systemic lupus erythematosus

Enriched null cell populations were obtained by fractionating blood mononuclear cells on discontinuous bovine serum albumin gradients. Null cells from 22 normals were compared to those from 14 patients with systemic lupus erythematosus who had various degrees of disease activity and to 8 patients with other autoimmune phenomena. Null cell layer lacked T cells (E rosette forming cells), cells with surface membrane immunoglobulin, cells capable of latex particles phagocytosis or cells capable of binding to IgG-coated human erythrocytes (EA rosette assay). Cells carrying receptors for complement were present. Cells of the null cell layer failed to respond in vitro to mitogens and allogeneic cells but were capable of Ig synthesis upon incubation with pokeweed mitogen. On transmission electron microscopy, null cells had large nucleus, narrow cytoplasm, and short surface processes. Null cells of SLE patients, when compared to normals, were increased in numbers ( p < 0.01), and had the capacity to bind to native DNA ( p < 0.01). Binding of cells to DNA could not be blocked by prior treatment of cells with anti-Ig antisera. There was a positive correlation between disease activity, null cell numbers, and DNA binding to serum or to the null cells. Defective maturation and/or differentiation of the null cells of SLE into mature cells with expansion of cell clones reactive to native DNA could be important in the pathogenesis of SLE. This could be due to the lack of maturation factors and/or rapid emigration of the precursor null cells from the stem cell compartment to the circulating pool.

Thymopoietin-induced acquisition of responsiveness to T cell mitogens

A population of murine spleen cells, enriched by flotation in discontinuous bovine serum albumin gradients, was induced to differentiate in vitro by incubation with the purified thymic polypeptide hormone thymopoietin. These cells, normally unresponsive to both T and B cell mitogens, acquired the capacity to respond to the T cell mitogens concanavalin A (Con A) and phytohemagglutinin (PHA) but remained unresponsive to the B cell mitogen lipopolysaccharide from Escherichia coli. The acquisition of responsiveness to mitogens was not impaired by treatment with anti-Thy-1 serum + complement before induction but was prevented by this treatment after induction; thus the cells acquiring the functional capacity to respond to T cell mitogens had also been induced to express the T cell alloantigen Thy-1. Like the expression of T cell alloantigens, the capacity to respond to Con A developed rapidly and reached its maximum within 6 hr. Responses to Con A always greatly exceeded those to PHA. Our data suggest that committed precursor cells, which we believe to be prothymocytes, are induced by thymopoietin to differentiate to cells with an antigenic phenotype and mitogen responsiveness similar to cortical thymocytes.

The intercellular content of the three transcobalamins at various stages of normal and leukaemic myleoid cell development.

A study of the three serum transcobalamins (TC) in myeloproliferative disorders and acute leukaemia has suggested a granulocyte origin for the three binders; the later stages of this cell line might be releasing 'TC I' and 'TC III' while 'TC II' which is increased in sera from patients with acute myelogenous leukaemia, probably originates at least in part from myeloblasts. The intracellular content of each TC has been studied in normal and leukaemic bone marrow cells after separation on a discontinuous bovine serum albumin gradient. Myeloblasts contain a TC II-like protein almost exclusively but promyelocytes contain a TC I-like protein which increases as the cells mature whilst 'TC II' decreases. A TC III-like binder parallels 'TC I' but appears later and reaches a maximum in the polymorphonuclear cells. In various types of leukaemia, abnormal TC distributions are related either to marrow replacement by the blast cells or to a disorder of the maturation process in the leukaemic cell line.

Isolation on discontinuous gradients of bovine albumin of a subpopulation of human lymphocytes exhibiting precursor characteristics.

Purified subpopulations of lymphocytes were obtained from discontinuous bovine serum albumin (BSA) gradients and by rosetting sheep erythrocytes (E) or EAC1-3. Cells from layers 1 to 3 of the BSA gradient which failed to rosette with E were highly reactive with EAC1-3 and with anti-human IgM. Demonstrating a high spontaneous rate of DNA synthesis and primitive morphology, these cells responded to pokeweed mitogen, concanavalin A and alloantigens, but not to phytohemagglutinin or soluble antigens. Constituting from 0.5% to 3.0% of all lymphocytes from each of the tissues studied, these lymphocytes appear to represent an earlier stage in human lymphocyte development distinct from both T and B cells.

Brief Communication: Biochemical Properties of Simian Virus 40-Transformed 3T3 Cell Mitochondria2

Mitrochondria isolated from simian virus 40-transformed 3T3 and nontransformed 3T3 cells were compared by various biochemical criteria. Transformed and nontransformed cell mitochondria had identical densities in linear sucrose and discontinuous bovine serum albumin gradients. The activities of several mitochondria-specific enzymes including cytochrome oxidase, adenylate kinase, nicotinamide adenine dinucleotide (NADH)-cytochrome c reductase, and NADH oxidase were similar in both cell types. However, the activity of the mitochondrial outer membrane enzyme, monoamine oxidase. In the virus-transformed cell mitochondria was reduced to 50% of that in nontransformed cell mitochondria.

Functional and morphological heterogeneity among rabbit peritoneal macrophages

Peritoneal exudate cells obtained from rabbits injected intraperitoneally with mineral oil were separated into several subpopulations by equilibrium density centrifugation on continuous or discontinuous bovine serum albumin gradients. On discontinuous gradients of 5, 8, 11, 15, 20, and 30% bovine serum albumin and, with appropriate attention to technical details, highly reproducible separation of the cells into 5 subpopulations was attained. Cells in bands B, C, and D, which accumulated at the 8 11% , 11 15% , and 15 20% interfaces, respectively, were mainly macrophages, and these cells were tested for endocytic activity for several antigens or particles (ferritin, T2 phage, E. coli, latex beads), and for the ability to produce immunogenic RNA after in vitro incubation with T2 phage. Band D cells showed high endocytic activity but failed to produce immunogenic RNA, whereas band B (15% of recovered cells) cells displayed low endocytic activity but produced immunogenic RNA that gave rise to both 7S and 19S antibodies when incubated with spleen cell cultures. Phase and electron microscopy studies revealed the presence of two morphological cell types in bands B and C. In addition to the typical macrophage, there was a second cell type with a centrally located round or oval nucleus, disperse chromatin, prominent nucleoli, and light staining cytoplasm containing polysomes, dilated lakes of granulated endoplasmic reticulum, densely staining mitochondria, and lacking a ruffled membrane. This glass-adherent cell, which neither meets the morphological criteria for macrophages or lymphocyte, will be referred to as cell “A”. Partial separation of typical macrophages from “A” cells was achieved by recentrifugation of band B cells on a discontinuous gradient of 8, 9, 11, and 30% bovine serum albumin. Band B I ( 8% 9% ), enriched in “A” cells (40%), yielded immunogenic RNA which gave rise to 19S antibody whereas band B II ( 9% 11% ), consisting of about 10% “A”, was responsible for 7S antibody formation.

Relationship between killer and rosette-forming cells reactive to H-2 antigens.

Optimum conditions were selected for testing of direct and indirect H‐2 reactive rosette‐forming cells (RFC) with the use of sheep or mouse erythrocytes coated with soluble H‐2 antigens. Rosette formation with mouse erythrocytes is shown to be an immunologically specific reaction and to be inhibited by the pretreatment of cells with the corresponding soluble H‐2 antigen preparation. Rate of killer cell and RFC formation is different after primary and secondary immunization with an allogeneic tumor, Unlike killer cells. RFCs are inactivated without complement by rabbit antibodies against mouse γ‐globulin (MGG). In contrast to killer cells, which are eliminated in the presence of complement by anti‐θ antibodies but not by anti‐plasma cell antigen (PC.1) and anti‐MGG antibodies, direct RFCs are not inactivated by anti‐θ but are eliminated partiallv by anti‐MGG and anti‐PC1. The RFCs are shown to be concentrated in the low‐density fractions of the discontinuous bovine serum albumin gradient, whereas cytotoxic lymphocytes are distributed less closely, being concentrated in the intermediate fraction, which does not differ morphologically from the initial cell suspension. Killer and rosette‐forming cells reactive to the same H‐2 antigens appear to be two non‐overlapping populations of T and B cells, respectively, and no T‐B cooperation is required for destruction of target cells.

Selective hybrid cell formation by human lymphoid cells.

AbstractSubpopulations of human lymphoid cells are capable of spontaneously fusing with fibroblasts of human or murine origin to form human‐human or human‐mouse hybrid cells. These cells were present in thymus, spleen, and bone marrow. After fractionation on discontinuous bovine serum albumin gradients, the cells were found in the less dense layers of the gradient. Cells of fetal origin, except for bone marrow, fused spontaneously at a higher rate than those of adult origin. The highest rate of fusion was found with adult bone marrow cells. These lymphoid cells appear to be “thymus‐derived” cells.

Thymus cell subpopulations separated on discontinuous BSA gradients: Antigenic properties and circulation capacity

Subpopulations of thymus cells were separated on discontinuous bovine serum albumin (BSA) gradients. The cells in each fraction were tested for their sensitivity to the cytotoxic effect of normal guinea pig serum (GPS), and of antisera against the TL, θ, and H-2 alloantigens. The more buoyant fractions of strain A thymus cells were found to possess a high sensitivity to H-2 antibodies, but a low sensitivity to GPS, and to TL and θ-antisera. In contrast, high density A thymus cells were highly sensitive to the cytotoxic effect of GPS, TL-antibody and θ-antibody, and resistant to the cytotoxic effect of H-2 antibodies. Among subpopulations of C57BL thymus cells, GPS showed a marked different activity on buoyant and dense fractions. Subpopulations of thymus cells varied in their capacity to migrate to lymph nodes of isogeneic recipients. 51Cr-labeled cells of one of the more buoyant subpopulations showed the highest capacity of localizing in lymph nodes, whereas the densest cell subpopulation had the lowest capacity of migrating to lymph nodes.

Studies on the isolation of lymphocytes active in cell-mediated immune responses: I. Demonstration of an active population of thymus-derived cells in mouse bone marrow

Mouse bone marrow was fractionated on discontinuous bovine serum albumin gradients and ten subpopulations of cells were recovered. A graft-versus-host (GVH) assay was used to assess the cell-bound, immune potential of these groups of cells. Dose response studies showed that unfractionated marrow cells would produce a significant splenic enlargement in test animals, but was markedly inferior to spleen or lymph node lymphocytes. The virulence of the marrow cells could be brought to a par with other peripheral lymphoid cells by the fractionation procedure which yielded at least two functional cell types. Fractions 1–5, the larger less dense cells, were inert at all dose ranges and comprised 80% of the total cell number. Fractions 6–10 were equal to cells from spleen and lymph nodes in their GVH activity. The dose response studies help to rule out this activity as being a function of peripatetic lymphocytes. The mouse bone marrow, therefore, was concluded to be on the route of recirculation of, and a domicile of, small long-lived thymus-derived lymphocytes.

Studies on the isolation of lymphocytes active in cell-mediated immune responses: II. Identification and recovery of an immunocompetent subpopulation of mouse thymocytes

Suspensions of mouse thymocytes were separated into subpopulations of cells by centrifugation on discontinuous bovine serum albumin (BSA) gradients. Nine bands or fractions of cells localized at the interfaces of the ten different concentrations of albumin used. The fractions were numbered from 1 through 9, beginning with least dense harvested at the 17/19% interface and thereafter counted sequentially to the most dense. Fraction 5 comprised at least 50% of the cell population while fractions 1–4 constituted approximately 20% of which 15–18% were in fraction 4. Each fraction was then evaluated individually to assess its reactivity. Five parameters of responsiveness were used: 1. Incorporation of tritiated thymidine in tissue culture and in vivo; 2. Response to stimulation with the mitogen phytohemagglutinin ; 3. Homing or migratory potential to peripheral lymph nodes; 4. Ability to provoke a significant graft-versus-host reaction; and 5. Sensitivity to heterologous antilymphocyte serum. Fractions 2, 3 and 4 each possessed at least one of these attributes of thymus derived small lymphocytes, although others were lacking. Fraction 5 was inert in all systems tested. The striking depletion of bands 2–4 by ALS was reversible by the injection of marrow precursor cells obtained by differential centrifugation of syngenic marrow. Marrow cells from fractions 6–10 of marrow gradients did not replenish the depleted thymus. In whole organ perfusion culture fractions 2–4 were found to egress from the gland while the bulk of cells remained behind. It was concluded that there is a small active immunocompetent population of thymocytes within the thymus which undergoes sequential maturation and/or modulation, so that some surface changes may be necessary for circulation but not for competence.

Potentiation of the T-lymphocyte response to mitogens. I. The responding cell.

Human and mouse lymphoid cells, stimulated by phytohemagglutinin (PHA) or lipopolysaccharide W (LPS), release supernatant factor(s) which are mitogenic for mouse thymocytes and which potentiate their responses to PHA or concanavalin A (Con A), The term LAF (lymphocyte-activating factor) is proposed for this activity. LAF not only enhances the mitotic responses of the less dense thymus subpopulations (A, B, and C) separable on discontinuous bovine serum albumin (BSA) gradients but also gives substantial responses in the otherwise inert cells of the denser fractions D and P. LAF does not exert a potentiating stimulatory effect on the responses of unfractionated mouse spleen cells, but does act synergistically with PHA on nonadherent spleen cells and on spleen cells of mice of several strains 5 days after irradiation and injection of thymocytes. Similarly LAF, which has no visible effect on unfractionated human peripheral blood cells, strongly potentiates the PHA response of column-purified lymphocytes, when these are cultured at low concentration. We conclude that LAF stimulates both central and peripheral T lymphocytes and enhances their responses to other stimulants.

Size and Density Analysis of Rodent Lymphoid Cells

Summary Rat and mouse lymphoid cells have been fractionated on discontinuous bovine serum albumin gradients and analyzed for size with the Coulter counter. The thymocyte is smaller and denser than the small lymphocyte of thoracic duct lymph. If the major fraction of thoracic duct lymphocytes is derived from small thymocytes, then thymus cells must enlarge and become less dense either before or after their release into the circulation. Hemolysin-forming cells of the spleen are much less dense than either small lymphoid cell, but there is no apparent difference between 7S and 19S hemolysin-formers at any time during the 1st month after antigenic stimulation.

Specific Isolation of B Lymphocytes by Means of Their Receptor for Antigen-Antibody-Complement (Ag-Ab-C) Complexes

Abstract A population of lymphocytes (CRL) from many mammalian species including man has a receptor for Ag-Ab-C complexes. CRL interact with sheep red blood cells (E) coated with antibody (A) and complement (C) forming rosettes (Bianco, C. and Nussenzweig, V., J. Exp. Med., 132: 702, 1970). In the mouse, CRL are part of the population of B lymphocytes (Dukor, P., Bianco, C. and Nussenzweig, V., in preparation). The interaction between CRL and EAC appears to be mediated by a receptor for modified C3 on the lymphocyte membrane. Papain fragments of rabbit γG anti-mouse C3 competitively inhibit and revert the binding of EAC to CRL. Based on this finding, a method has been devised to purify CRL, which consists of: 1) incubation of EAC with lymphocytes for rosette formation; 2) separation of rosettes by sedimentation in a linear bovine serum albumin (BSA) gradient (The purified clusters contain less than 5% free leukocyte contamination); 3) treatment of the clusters with papain fragments of anti-C3 antibodies in order to dissociate EAC from CRL; 4) separation of CRL from EAC by ultracentrifugation in a discontinuous BSA gradient.