The rapid purification of peritoneal exudate macrophages by ficoll (polysucrose) density gradient centrifugation

Macrophages can be obtained about 99 per cent pure and in a yield of about 50 per cent by ficoll (polysucrose) density gradient centrifugation of mouse peritoneal exudate cells. The macrophages appear in the lighter fractions. The lymphocytes appear in the denser fractions and can be further purified on a polystyrene bead, rayon wool column. The method also separates guinea-pig and rat peritoneal exudate cells.

Peritoneal exudate macrophages take up colloidal radioactive gold in vivo. However, macrophages differ in the ability to take up colloidal gold and the macrophages which phagocytose the most gold appear in the lightest fraction after density gradient centrifugation.

     

Opsonization of Staphylococcus aureus protects endothelial cells from damage by phagocytosing polymorphonuclear leukocytes.

When phagocytosis of Staphylococcus aureus by human polymorphonuclear leukocytes (PMN) takes place on the surface of cultured human endothelial cells, the endothelial monolayers are damaged by lysosomal enzymes that are released by the PMN. Because PMN can phagocytose opsonized as well as unopsonized staphylococci on an endothelial surface, we studied the role of bacterial opsonization in the damage caused to the endothelium. Phagocytosis of unopsonized S. aureus was accompanied by greater damage (expressed as the percentage of the endothelial cells detached from the culture plates) of the monolayers than was phagocytosis of opsonized S. aureus: 52 +/- 10% and 24 +/- 7%, respectively, after 30 min of phagocytosis and 73 +/- 5% and 50 +/- 6%, respectively, after 60 min of phagocytosis. When correlated to the amount of phagocytosis, this difference was even greater (uptake was 35 +/- 4% for unopsonized S. aureus and 56 +/- 5% for opsonized S. aureus after 30 min and 42 +/- 3% and 60 +/- 5%, respectively, after 60 min). Total release of lysozyme and myeloperoxidase and generation of superoxide anion were the same during phagocytosis of opsonized or unopsonized staphylococci. Adherence of PMN to the endothelial cells was greater during phagocytosis of unopsonized S. aureus: 42 +/- 4% verus 27 +/- 3% during phagocytosis of opsonized staphylococci. Possibly, increased adherence of the PMN resulted in a locally higher concentration of enzymes which induced more damage. We conclude that opsonization of bacteria not only improves bacterial uptake, but also protects bystander cells from damage by the phagocytosing PMN.     

Comparison of the effects of filtration leucapheresis and discontinuous flow centrifugation leucapheresis on granulocyte microbicidal function

In an investigation of the in vitro phagocytic and microbicidal function of granulocytes collected by filtration leucapheresis (FL) from 18 donors and by discontinuous flow centrifugation leucapheresis (DFC) from six donors, comparison was made with the function of granulocytes obtained from the same donors by venepuncture and density gradient centrifugation over Ficoll-Isopaque (FI). No significant impairment of the phagocytosis or killing of Candida guilliermondii by either FL- or DFC-granulocytes was observed. Although the ability of FL-granulocytes to phagocytose and kill Staphylococcus aureus did not differ significantly from the function of control FI-granulocytes, DFC-granulocytes were significantly less active.     

The effect of cytophilic IgG2 on phagocytosis by ovine polymorphonuclear leucocytes.

Non-lactating, multiparous ewes were immunized either by subcutaneous infection with live Staphylococcus aureus (seventeen ewes) or by intramuscular injections of a killed S. aureus-oil adjuvant vaccine (seventeen ewes). Polymorphs which were subsequently collected from the mammary glands of the animals were used in in vitro phagocytosis assays against Pseudomonas sp. or S. aureus. There was no difference between polymorphs from the two groups of ewes in their ability to phagocytose Pseudomonas organisms. Polymorphs from the infected ewes showed significant phagocytic superiority over cells from ewes given the killed vaccine when S. aureus was the target organism. This phagocytic superiority could be abrogated by removal of cytophilic immunoglobulin from polymorphs and restored by replacement of cytophilic immunoglobulin. It was shown by staining polymorphs with FITC-conjugated anti-immunoglobulin sera that cytophilic immunoglobulin on the surface of polymorphs belonged to the IgG2 class of immunoglobulins. When ''neutral'' polymorphs (from non-immunized ewes) were coated with IgG2 purified from the sera of infected ewes, they exhibited enhanced phagocytosis of staphylococci compared with ''neutral'' polymorphs carying IgG2 from the sera of ewes given the killed vaccine.     

Phagocytosis and degradation of DNA-anti-DNA complexes by human phagocytes I. Assay conditions,quantitative aspects and differences between human blood monocytes and neutrophils

The uptake in vitro was studied of ³H-labeled DNA-anti-DNA complexes by neutrophils and monocytes from human blood. Complexes were prepared from ³H-labeled circular double-stranded (ds) DNA of bacteriophage PM₂ and anti-dsDNA-containing sera from patients with systemic lupus erythematosus. After phagocytosis, cells and medium were separated. The cells were treated with DNase to remove adherent and noningested complexes before the cell-associated radioactivity was counted. Thus, only complexes inside the cells were measured. The medium was analyzed for acid-precipitable radioactivity. In this way, we found that neutrophils only phagocytose the complexes, whereas monocytes phagocytose the complexes and degrade the antigen. In contrast, both types of phagocyte degraded the antigen in tetanus-antitetanus complexes. The degradation took place after phagocytosis, inside the cells. The difference in DNA degradation between neutrophils and monocytes correlated with the difference in acid DNase activity of the lysosomal fractions: monocytes contained DNase activity, neutrophils did not. With complexes made from DNA with ¹³¹I-labeled anti-DNA, we found that both cell types degraded the antibody. Uptake of complexes and degradation of antigen increased with incubation time and cell concentration and was saturable with respect to complex concentration. The processes were inhibited by 5 mM mono-iodoacetic acid or by low temperatures.     

Enhancement of infectivity by combination of two ribonucleic acid components from alfalfa mosaic virus

RNA was extracted from alfalfa mosaic virus 425. Infectivity was determined in density gradient fractions and in all possible combinations of two fractions. Certain combinations were remarkably infectious. This led to the purification through two cycles of sucrose gradient centrifugation of a 27 S and a 14 S RNA, from bottom component and top component a, respectively. The purified bottom component RNA had a low infectivity; when purified top component a RNA was added, infectivity increased considerably. Possible explanations for this effect are discussed.     

The use of protein A-sepharose affinity chromatography for separation and detection of specific IgM antibody in acquired rubella infection: A comparison with absorption by staphylococci containing protein A and density gradient ultracentrifugation

Protein A-Sepharose affinity chromatography has been investigated as a means of removing IgG from sera before testing for rubella-specific IgM. The method was compared with whole staphylococci containing protein A and with sucrose density gradient ultracentrifugation. Approximately 98% of IgG was removed by protein A-Sepharose absorption and 95% by whole staphylococcal absorption, but 50–60% of IgM was also removed. One hundred and two sera were tested — 49 had been shown to contain rubella-specific IgM by ultracentrifugation and 53 to be negative. Accepting 2-fold or greater reduction in antibody titre with 2-mercaptoethanol as the criterion for positivity after absorption, 4 of 49 (8%) sera were negative with protein A-Sepharose and 31% whith whole staphylococci. Of the 4 sera negative after protein A-Sepharose treatment three were obtained between 48 and 62 days after infection.It is essential to confirm positive results by demonstrating reduction of IgM antibody remaining after absorption by treatment with 2-mercaptoethanol. It is also essential that all post-absorption antibody be treated with 2-mercaptoethanol to eliminate false positives. Protein A-Sepharose affinity chromatography was more sensitive than absorption with whole staphylococci but less sensitive thn ultracentrifugation. The method has a place in screening convalescent sera collected less than 28 days after infection.     

Rat mast cell granules reacting with a mouse monoclonal antibody M6764 which recognizes the same epitope of a mouse monoclonal antibody HNK-1.

Rat mast cell granules and plasma membrane fractions were obtained by homogenization of highly purified rat mast cells and isolation in a Percoll gradient and a sucrose gradient, respectively. Immunostaining of rat mast cells, granules and plasma membrane fractions was performed with mouse monoclonal antibody M6764 which was produced against the crude membrane fractions of the neural tubes. Rat mast cells and granules were immunostained with the monoclonal antibody, but not the plasma membrane fractions. The granules fixed with glutaraldehyde-paraformaldehyde showed ring-like forms. Chloroform-methanol treatment did not effect the staining of rat mast cells and granules with the monoclonal antibody. Western blotting analysis of rat mast cells and granules with the monoclonal antibody showed broad protein bands ranging from 100 to 250 kD.     

Tumor necrosis factor alpha stimulates killing of Mycobacterium tuberculosis by human neutrophils

The ability of human neutrophils to aid in defense against pulmonary infection with Mycobacterium tuberculosis is controversial. In this study, we have shown that neutrophils respond to and phagocytose M. tuberculosis in human lesions. Neutrophils from healthy individuals were able to kill significant fractions of an inoculum of M. tuberculosis within 1 h of phagocytosis, and this ability was enhanced by tumor necrosis factor alpha but not by gamma interferon. The mycobactericidal mechanism was nonoxidative, as inhibitors of reactive oxygen or reactive nitrogen intermediates did not interfere with killing. However, the mycobactericidal mechanism was associated with increased exposure of intracellular M. tuberculosis to neutrophil defensins. In vitro, human neutrophil peptides 1 to 3 were not able to kill the bacilli even at much higher levels. These studies support the concept that human neutrophils are directly involved in defense against infection with M. tuberculosis.     

Association between phage DNA injection and generation of the protonmotive force in Staphylococcus aureus

Suppression of protonmotive force generators in staphylococci lead to a loss of phage infection efficiency. KCN inhibited phage infection by 49.5-53.5%; DCCD by 51.0-61.4%; CCCP by 59.2-68.8%. Suppression tock place at the stage of phage DNA transport. Valinomycin in concentration of 0.5 microM evoked dissipation of the membrane potential, nigericin caused a reduction of the gradient pH in the staphylococci membrane at a concentration of 12.0 microM. Individually, these antibiotics did not have an essential influence on the efficiency of phage infection but when combined, a maximum inhibition of phage infection (64.5%) occurred at the stage of introducing phage DNA into the bacterial cell implicating the participation of the membrane potential and pH gradient in the transportation of phage nucleic acid to the staphylococcal cell.     

Effects of staphylococcal products on locomotion and chemotaxis of human blood neutrophils and monocytes.

The effects of staphylococcal products as chemo-attractants for human blood neutrophils and monocytes and as inhibitors of locomotion of these cells were studied with bacterial cells, culture filtrates and isoelectrically focused fractions from culture filtrates of nine strains of Staphylococcus aureus. Little direct chemotactic activity of staphylococcal products for neutrophils was observed, although a chloroform-soluble extract of the whole organisms contained such activity. The major chemotactic effect of staphylococci for neutrophils was indirect, i.e., generated when the organisms or their products were incubated with plasma, perhaps due to activation of complement. In contrast, direct chemotactic activity for monocytes was found in a large number of staphylococcal fractions. Staphylococci also produced inhibitors of locomotion of both neutrophils and monocytes. Isoelectric focusing showed more fractions inhibitory for neutrophils than for monocytes. Some of the inhibitors could be identified. Staphylococcal alpha-toxin inhibited migration of both neutrophils and monocytes. Sphingomyelinase C (beta toxin) inhibited migration of monocytes but not of neutrophils. Leucocidin-rich strains were strongly active as inhibitors of neutrophil locomotion but less so as inhibitors of monocyte locomotion.     

The in vitro immune response to sheep erythrocytes by fractionated spleen cells: biological and immunological differences

Normal spleen cells were fractionated using equilibrium density gradient centrifugation. Three fractions were taken and the ability of those fractions to produce antibody-forming cells (PFC) in vitro was examined. The three fractions were distinct from each other in susceptibility to anti-θ serum, nutritional requirements, requirements for radiation-resistant cells, antigen dose optima, kinetics and participation in cell cycle. The results are interpreted as showing a differentiation series with the immunocompetent cells moving from the dense to the less dense region of the gradient following antigen stimulation. The correlation between steps in the rate of production of PFC's by unfractionated spleen cells and the contribution of each fraction to the total response are discussed.     

Identification and characterization of the immunoprecipitating medium- and low-density tryptic fragments of bovine nasal cartilage proteoglycan

The fragments responsible for the immunodiffusion reactivity of middle- and low-density fractions of trypsin-digested bovine nasal cartilage proteoglycan have been identified and obtained in relatively homogeneous fractions. Glycosaminoglycan-bearing tryptic fragments were isolated from 4 M guanidinium chloride extracts of cartilage by ion-exchange chromatography and fractionated by dissociative equilibrium density gradient ultracentrifugation at a starting density of 1.50. Fragments in the middle fractions of the density gradient were digested with chondroitinase ABC and subfractionated by Sepharose 6B column chromatography. Middle-density subfractions contained fragments which were chemically and immunologically identical to those in high-density fragment subfractions of similar elution from Sepharose 6B. The middle-density subfractions contained two additional immunoprecipitating fragments. One, with alanine as N-terminal amino acid, was isolated by virtue of its retention by a column of concanavalin A-Sepharose 4B and its resistance to digestion with keratanase; the second was concentrated in a subfraction whose elution from concanavalin A-Sepharose 4B was retarded. The gradient fraction of lowest density contained fragments with the properties of the major tryptic fragments of the hyaluronic acid-binding segment of the proteoglycan monomer and the link proteins. These were recovered as a complex in the void volume upon Sepharose gel chromatography in saline-buffer and were resolved into relatively homogeneous fractions by column chromotography on CL-Sepharose 6B in 4 M guanidinium chloride. In all, tryptic digests of cartilage proteoglycan contain at least seven different immunoprecipitating fragments, some of which may not have been correctly identified previously.     

Morphological and biochemical evidence that scrapie-associated fibrils are derived from aggregated amyloid-like filaments.

The membrane fraction from scrapie infected mouse brains was dissolved in saturated urea, centrifuged on a 10 to 50% glycerol gradient at 35,000 rpm for 24 h, and fractionated from the bottom of the tube into 11 fractions. PrP was detected throughout the gradient. However, the relative PrP concentrations of fractions 4 and 8 were the highest. The relative PrP concentration versus protein concentration of fractions 1 to 4 was higher than that of the other fractions. Scrapie infectivity also was detected in all fractions. Fractions 2, 3, 4, 7, and 8 produced the shortest incubation periods. Positively stained filamentous aggregates with sizes varying from about 40 x 60 nm to more than 4 microns were observed in fractions 2 and 4 by negative staining. These resembled amyloid filaments. Congo red-stained aggregates showed birefringence under polarized light. Aggregation of the filamentous aggregates was observed by incubation with anti-mouse SAF serum. Fine fibrils 10-18 nm in width were partially dissociated from the aggregates by brief exposure to the detergent Sarkosyl. These facts suggest that SAF are not products of self-assembly from subunit structures liberated from membranes by exposure to detergent, but exist as aggregates of amyloid-like filaments from which SAF are dissociated by detergent extraction.     

Subcellular fractionation of hypothalamus and pituitary stalk of the bull. Morphologic study and serotonin-norepinephrine distribution

Summary Hypothalamus and pituitary stalk of the bull were fractionated in a sucrose density gradient according to the Hebb and Whittaker (1958) procedure. The crude mitochondrial-synaptosomal fraction was layered on a discontinuous sucrose density gradient (0.6; 0.8; 1; 1.2 M) and, after 63,000 g (average) centrifugation for 2 hours, 4 bands and one pellet were obtained. The fraction layered on 0.8 M sucrose contained mainly myelin and some rare synaptosomes. The fractions layered on 0.8 M, 1.0 M, and 1.2 M sucrose were very rich in synaptosomes. Different populations of the latter were present: one type contained only clear synaptic vesicles; the second type, in addition to clear synaptic vesicles, had a population of vesicles with homogeneous dense core (about 1000 Å diameter). There was no significant difference between the light fractions of hypothalamus and of pituitary stalk. However, in the heavier fractions derived from the pituitary stalk a considerable number of large profiles containing dense core granules of 2000 Å diameter were present.In the primary fractions norepinephrine is recovered mainly in the crude synaptosomal fraction, while serotonin is present in higher concentration in the supernatant. In the density gradient serotonin and norepinephrine distribute principally in the three synaptosome-rich fractions, serotonin being more concentrated in the lightest synaptosomal fraction, while norepinephrine is mainly recovered in the heaviest fraction. The differences in the amine distribution in the fractions derived from hypothalamus and pituitary stalk are described and discussed in relation to the different morphology of those fractions.Supported in part by a CNR research grant.Presented in part at the 2nd International Meeting of the International Society for Neurochemistry, Milano 1969.     

Separation of proximal tubule cells from suspensions of rat kidney cells in density gradients of Ficoll in tissue culture medium.

Rat kidneys were disaggregated with 0.25% trypsin. Cell were separated by velocity sedimentation in a previously described isokinetic gradient, by isopycnic sedimentation, and by velocity sedimentation followed by isopycnic sedimetation. In some fractions from the isokinetic gradient, 71.8+/-2.4+ of the nucleated cells contained histochemically demonstrable alkaline phosphatase (HDAP); in semithin sections, 62.7+/-2.3% of these cells had brush borders. The correspondence between fractions enriched for cells with HDAP and fractions enriched for brush border suggested that HDAP might be a suitable marker for rat proximal tubule cells. These cell constituted 46.5+/-2.6% of the nucleated cells in the starting sample suspension of kidney cells, and 81.9+/-2.7% of nucleated cells in the purified fractions from the gradients. More than 98% of nucleated cells in these fractions excluded typan blue. Following isopycnin centrifugation, the purest fractions contained 87.3+/-1.5% nucleated cells with HDAP, 9.6+/-2.5% nucleated cells iwithout HDAP, and 3.1+/-2.5% red blood cells. These proximal tubule cells had densities of 1.036 to 1.052 g/ml. With rate-zonal separation followed by isopycnic separation, the purest gradient fraction contained 93.0+/-1.9% nucleated cell with HDAP, 6.0+/-2.3% nucleated cells with HDAP, and 1.0+/-0.9% red blood cells. These proximal tubule cells sedimented a density of 1.041 g/ml. More than 98% of these cells excluded trypan blue.     

Density gradient separation of chicken thymus cells

Chicken thymus cells were separated into six fractions by a discontinuous albumin gradient centrifugation. When 10 or 16-week old cell donors were used, thymus cells responding with antibody formation to Brucella abortus were clearly separated from those active in graft-vs.-host assays. The former were found in low and the latter in high density fractions. The results suggest that B and T cells in chicken thymus may be preferentially concentrated into separate fractions according to density.     

HBV颗粒的纯化及生物学活性鉴定

目的优化纯化HBV患者血清中HBV颗粒的技术方法。方法在蔗糖介质中,分别以100 000 g和70 000 g进行不连续蔗糖梯度(60%,45%,35%,25%,15%)离心5 h,离心后,分段吸取6份收集液,PCR荧光定量检测各组分HBV滴度;收集前5份收集液进行超滤,去蔗糖,PCR荧光定量检测HBV超滤液;电镜观察70 000g纯化后的HBV超微形态;70000 g纯化后的HBV颗粒感染树鼩肝细胞,检验70 000 g纯化后的HBV感染活性。结果100 000 g离心造成大量HBV在离心过程中损失,收获率只有21%,70 000g离心不仅降低了离心中的病毒损失,而且可有效对HBV和血清进行分离,收获率达到78%以上,较100 000g还提高3.7倍多。电镜可观察到纯化后病毒液中有大量的病毒颗粒;纯化后的HBV颗粒感染活性良好,可有效感染原代树鼩肝细胞。结论70 000 g不连续蔗糖梯度离心可有效分离纯化HBV病毒,较100 000 g收获率明显提高,纯化后的HBV不但具有典型的HBV特征,还具有良好的生物学感染活性。     

Induction of active immunity with membrane fractions from Haemophilus influenzae type b

Using Escherichia coli strain E-1 as a model, we developed procedures for the preparation of outer- and inner-membrane-enriched fractions as structural units. These procedures could be used to prepare relatively pure inner and outer membrane fractions as determined by succinate dehydrogenase activity, ketodeoxyoctonate levels, and polyacrylamide gradient gel electrophoresis. The use of these procedures to fractionate membrane components from Haemophilus influenzae type b strains H-2 and H-E led to good separation of outer- and inner-membrane-enriched fractions as determined by succinate dehydrogenase and ketodeoxyoctonate levels but incomplete separation as determined by polyacrylamide gradient gel electrophoresis. Although there were differences between the electrophoresis profiles of outer membrane fractions of strains H-2 and H-E, immunization with outer membrane of either strain led to the induction of a high degree of immunoprotection against challenge with the H-2 strain. Protection could also be elicited with inner membrane preparations, but such protection may be due to contamination with outer membrane. Extracted membrane protein induced levels of protection that were comparable to those induced by whole membrane fractions.     

Characterization of rat spleen cell populations. II. Activation by antigens and allogeneic cells.

Spleen cells from BN rats were separated in a discontinuous density gradient. Cells from different fractions were shown to be functionally diverse. Light cells were highly responsive in MLR, generated little killer activity and enhanced syngeneic recipient's IgM and IgG production in response to SRBC. Dense cells were unable to respond in MLR and suppress Ab response. Maximum killer activity was recovered from medium density fractions which were probably mixtures of helper and suppressor cells.