Human Blood Fractions Research Articles

Arylsulfatase A activity and electrophoretic banding patterns from isolated human blood fractions.

Arylsulfatase A (ASA) activity was studied from five blood fractions, leukocytes plus platelets (LKPL), leukocytes (LK), neutrophils (N), lymphocytes (LYM), and platelets (PL). No significant difference was found among the mean ASA specific activity values for the fractions. Electrophoretic examination revealed four distinct activity bands for the LKPL and PL fractions, while the LK, N, and LYM fractions showed only a single, broad peak, which indicates that the four-band pattern is associated with enzyme obtained from platelets. The PL fraction gave a clearer, more distinct banding pattern than the other four fractions. This clearly resolved electrophoretic banding pattern of ASA from pure platelet preparations was demonstrated for variant human ASA as well. These findings may be significant to future work investigating the biochemical genetics of ASA.

Generation of a cloned NK cell line derived from the "null cell" fraction of human peripheral blood.

The present studies were designed to determine the conditions for generation of human NK clones. First the "null cell" (NC) fraction of human peripheral blood mononuclear cells, which contains the NK effectors, was purified using negative selection with anti-T3, anti-T8, anti-B1, and anti-Mo2 monoclonal antibodies. Subsequently, NC were cloned by limiting dilution using a combination of phytohemagglutinin (PHA) and lymphocyte-conditioned medium (LCM) as an initial stimulus. Colonies could be easily obtained with this procedure, but the maintenance of long-term growth of the cultures represented a major problem. A cloned cell line termed JT1 was generated and has been proliferating continuously in culture for more than 6 mo. The phenotype of JT1 cells was analyzed several times with a series of monoclonal antibodies. These cells did not express surface markers related to thymic (T3-, T4-, T8-, T11-), B cells (B1-, J5-), or myelomonocytic (Mo1-, Mo2-, MY7-) differentiation. In contrast, 95% of JT1 cells reacted with the anti-T10 monoclonal antibody. T9, Ia, and J2 antigens were also present on JT1 cells, but their expression appeared variable from one determination to another. This cloned cell line maintains a strong cytotoxicity against common NK targets, such as K562 and Molt 4 cell lines, and a moderate ADCC activity. In addition, after several months in culture, JT1 cells are still capable of being regulated by interferon, because this lymphokine rapidly enhances cytotoxicity against K562 cells.

Acrylonitrile solubility in human blood: Determination of the Ostwald partition coefficient

AbstractAn in vitro head space method was used to determine the Ostwald partition coefficient (OPC) of acrylonitrile in the following liquids: lactated Ringer's solution, fresh rat blood, fresh human blood, outdated human blood and cellular fractions of either fresh or outdated human blood. Fresh human blood cells have the strongest affinity for acrylonitrile (OPC >700). Fresh whole human blood has a lower OPC, approximately 500. Lactated Ringer's solution, outdated human blood and water had lower OPC values, approximately 200.

Quantification of erythroid and granulocytic precursor cells in plateletpheresis residues.

Mononuclear cell fractions of human blood and plateletpheresis residues were compared for their content of hemopoietic precursor cells. Erythroid burst-forming units (BFU-E) averaged 560 +- 130 per ml of blood and granulocyte--monocyte colony forming units (CFU-C) averaged 240 +- 90 per ml blood. Estimates based on a blood volume of 7% of body weight indicate that the total blood pools of BFU-E and CFU-C are about 3.5 x 10/sup 6/ and 1.5 x 10/sup 6/ cells respectively. Sequential studies were performed over 3 days following one plateletpheresis in 4 donors. CFU-C and BFU-E approximately doubled between 48 and 72 hours after a plateletpheresis. During this time there was no significant alteration in the percent of null, T or B lymphocytes in blood. Thus, plateletpheresis appears to lead to a mobilization of precursor cells, which results in a transient increase in their concentration in blood. Therefore, pheresis 48 to 72 hours after an initial short-term procedure could harvest much larger numbers of precursor cells. Moreover, such techniques would put blood precursor cell content of plateletpheresis residues within reach of the precursor cell content in the volume of human marrow used for transplantation.

Role of rabbit lysozyme in in vitro serum and plasma serum bactericidal reactions against Bacillus subtilis.

The antibacterial activity of purified rabbit lysozyme was kinetically investigated at concentrations comparable to those in normal rabbit serum and plasma serum. The bactericidal capability, lysozyme content, and electrophoretic composition of "purified beta-lysin," fractionated from normal rabbit serum, were also examined. In contrast to the extensive antibacterial activity of dilute normal rabbit serum observed in vitro, rabbit lysozyme was only weakly bactericidal for Bacillus subtilis. Inhibition of lysozyme enzymatic and bactericidal activities in normal rabbit serum by antilysozyme immunoglobulin G slightly reduced the initial rate of killing. The addition of neutralizing antibody or histamine (another lysozyme inhibitor) to partially purified bactericidal serum fractions had no effect on killing kinetics. Increasing the ionic strength of reaction mixtures containing normal serum or partially purified bactericidal fractions to levels which completely inhibited lysozyme activity resulted in stimulation of their respective killing kinetics. The addition of inhibitors to normal rabbit plasma serum completely eliminated its bactericidal activity. With regard to the killing of B. subtilis by rabbit and human blood fractions, these analyses clearly demonstrated that (i) although lysozyme is not a significant antibacterial component of normal rabbit serum, it represents the principal factor in normal rabbit plasma serum; (ii) different primary bactericidal mechanisms which are not detectable by singlepoint analyses operate in the sera of different species; and (iii) purified beta-lysin isolated from normal rabbit serum by the classical procedure is a heterogenous mixture of components.

Demonstration of Fcγ Receptors on Human Basophil Granulocytes

Abstract Fcγ receptors were detected on human basophil granulocytes. The mononuclear cell fraction of human peripheral blood was incubated with heat-aggregated human IgG (HGG) followed by 125I-anti-HGG. Autoradiography of the cells showed that the majority of basophil granulocytes gave a significant number of grains. Basophils were not labeled by preincubation of the same cells with monomeric HGG followed by 125I-anti-HGG. However, the binding of aggregated HGG to basophils was inhibited by the presence of a high concentration of monomeric HGG or its Fc fragment but not by the Fab fragment. Evidence was obtained that Fcγ receptors are distinct from IgE receptors on the same cells: i) Saturation of basophils with IgE did not affect the binding of aggregated HGG to the cells. ii) Preincubation with and the presence of aggregated HGG failed to affect the binding of 125I-IgE to basophils, or to block passive sensitization of the cells with IgE antibodies. iii) The Fcγ receptors did not co-cap with IgE receptors. Aggregated HGG failed to induce histamine release from basophils even in the presence of D2O. It was also found that the presence of aggregated HGG on basophils did not modulate IgE-mediated histamine release from the cells.

936 RAPID FLUOROMETRIC DETERMINATION OF BILIRUBIN LEVELS AND THE RESERVE BINDING CAPACITY IN THE BLOOD OF NEONATES

Binding of (unconjugated) bilirubin (BR) to human blood fractions has been studied using spectrofluorometry, and the results obtained were used to interpret the titration curve of BR in Whole blood as determined by front face fluorometry. It is found that BR fluorescence from whole blood reflects only that BR tightly bound to albumin the normal BR transport protein. A significant amount of BR may be bound to erythrocytes as well as to plasma proteins other than albumin, and is essentially non-fluore scent. Detergents can extract BR from all blood sites. Since BR is fluorescent in detergent micelles, total blood BR can be assayed. A significant difference between albumin-bound BR and total blood BR reflects a saturation of the albumin binding or a reduced albumin binding affinity. The reserve albumin binding capacity for BR can be obtained from the incremental fluorescence measured upon adding a saturating quantity of BR to the specimen. All three parameters (albumin-bound BR, total blood BR, and reserve albumin capacity) can be measured simply and within a few minutes on less than 100ul blood by the use of a dedicated front face fluorometer (hematofluorometer), facitating effective management of neonatal jaundice. Prototypes of the BR-hematofluorometer in two intensive care nurseries have yielded results in excellent agreement with those obtained by present, more laborious methods, which are impractical for routine use.

Binding of insecticides to human serum proteins

This investigation concerns the binding of DDT, dieldrin, carbaryl, and parathion to human serum proteins. Fresh human serum and commerically prepared human blood fractions were used to investigate the binding characteristics of the insecticides. Binding was demonstrated to be of a low affinity, hydrophobic nature with the albumin and lipoprotein fractions exhibiting the highest affinity for the insecticides. The binding of DDT and dieldrin to the lipoproteins was inferred to be partially protective in acute poisoning because of the relative stability of the protein-pesticide complex. All the protein fractions examined can clearly play some role in the transport of these insecticides.

Epstein-Barr virus (EBV) receptors, complement receptors, and EBV infectibility of different lymphocyte fractions of human peripheral blood: II. Epstein-Barr virus studies

In B-cell fractions isolated from human peripheral blood, the frequency of surface immunoglobulin-positive and of complement receptor-positive cells showed a good correlation with the frequency of EBV-binding cells, as detected by membrane fluorescence or by a quantitative bioassay for infectious virus in the absorbed supernatant fluid. There was a close relationship between all three parameters mentioned, the frequency of EBNA-positive cells 2 or 3 days after the infection, and the stimulation of cellular DNA synthesis. So-called O-cell fractions remaining after the removal of nylon adherent and E-rosetting cells contained a certain frequency of complement receptor-positive cells and absorbed EBV to a limited extent, but did not respond to EBV infection with EBNA induction or stimulation of DNA synthesis. None of the T-cell fractions absorbed EBV to a detectable extent. This includes the T ea+ fraction that contained a certain proportion of complement receptor-positive cells. It is concluded that the previously demonstrated relationship between EBV receptors and complement receptors on B-lymphoblastoid lines also holds for peripheral B lymphocytes. In these cells, virus absorption is followed by an intracellular infectious process, signaled by the appearance of EBNA and cellular DNA synthesis. O cells carry complement receptors and absorb EBV to a certain extent, but do not respond with EBNA synthesis or DNA stimulation, presumably due to intracellular restrictions. T cells do not bind EBV, and the complement receptors present on some cells of the T ea+ fraction do not function as EBV receptors.

Epstein-Barr virus (EBV) receptors, complement receptors, and EBV infectibility of different lymphocyte fractions of human peripheral blood: I. Complement receptor distribution and complement binding by separated lymphocyte subpopulations

Human peripheral lymphocytes were fractionated into a variety of B-, T-, and O-cell fractions and were characterized with regard to several surface receptors. There was a strong correlation between the frequency of EAC receptor-positive cells and the percentage of complement membrane fluorescence (CMF)-stained cells following exposure to fresh human serum and subsequent staining with an anti-C3 conjugate. CMF staining did not diminish in C4-deficient or hypogammaglobulinemic serum, or in the presence of EDTA or EGTA-Mg 2+, but was completely negative with C3-depleted normal human serum. In all likelihood, the staining is therefore due to the direct binding of C3 to preformed receptors on the lymphocyte surface. In addition to the surface Ig-positive B-cell fractions, C3 receptors were also detected on part of the O-cell population and on a proportion of the Fc receptor-positive T cells.

Enhancement by Irradiated T Cells of Human Plasma Cell Production: Dissection of Helper and Suppressor Functions in Vitro

Abstract The addition of irradiated T-enriched lymphocytes to B cell-enriched fractions of human peripheral blood or to unseparated mononuclear cells stimulated the differentiation of plasmacytoid cells in culture with pokeweek mitogen beyond the synergy obtained by the addition of unirradiated T cells. This stimulation was observed in both the proportion and absolute number of plasmacytoid cells recovered from the cultures, and in the amount of IgM detected in culture supernatants by a hemagglutination inhibition assay. Irradiation-induced enhancement was observed with normal and hypogammaglobulinemic T cells, but not with monoclonal T cells from two patients. Inactivation of T cells by heating or by repeated freezing and thawing did not produce the same effects as did irradiation. These data suggest that cell-mediated suppressor function in man is selectively radiosensitive, while helper activity is not. Irradiation may be a useful method for the functional isolation of helper cells and for the manipulation of the balance between suppressor and helper cell activities.

Cell Membrane IgD: Demonstration of IgD on Human Lymphocytes by Enzyme-Catalyzed Iodination and Comparison with Cell Surface Ig of Mouse, Guinea Pig, and Rabbit

A substantial fraction of human cord blood and peripheral blood lymphocytes have recently been shown to bear IgD. Although IgD has not been identified in mice, it has been suggested that it is also a major surface immunoglobulin of murine lymphocytes. Thus, lactoperoxidase-catalyzed iodination of surface immunoglobulin of mouse spleen and lymph node cells reveals the existence of an IgH chain differing from mu, gamma, and alpha-chain both antigenically and by mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This new H chain class has been previously proposed to be the mouse homologue of delta-chain. In this paper, we analyzed human, mouse, guinea pig, and rabbit lymphoid cell membrane Ig by lactoperoxidase-catalyzed iodination, extraction with non-ionic detergent precipitation with a variety of specific anti-Ig sera, and electrophoresis of dissolved reduced precipitates on sodium dodecyl sulfate-polyacrylamide gels. Our studies confirm the previous reports of a new mouse cell membrane H chain with a mobility more rapid than that of mu-chain. However, we fail to detect a molecule with this electrophoretic mobility on the surface of guinea pig or rabbit lymph node and spleen cells. Moreover, neither anti-kappa nor anti-delta antibody precipitates a molecule with an H chain of this mobility from labeled extracts of human cord blood or peripheral blood lymphocytes. Cell surface delta was identified on both human cord blood and peripheral blood lymphocytes, but it proved to have mobility similar to human and mouse mu-chain. This result indicates either that mouse delta-chain has an electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels which differs appreciably from that of human membrane delta-chain or that the newly described mouse H chain is not the homologue of human delta-chain.

Epstein-Barr virus interactions with human lymphocyte subpopulations: virus adsorption, kinetics of expression of Epstein-Barr virus-associated nuclear antigen, and lymphocyte transformation

In order to further understand Epstein-Barr virus (EBV)-lymphocyte interactions, we investigated a chain of events including: (i) EBV binding to human lymphocyte subpopulations; (ii) the earliest appearance of EBV-determined nuclear antigen (EBNA) in the lymphocytes after EBV infection; and (iii) establishment of continuous lymphoblastoid cell lines (LCL) by infecting with EBV different types of lymphocyte preparations from the same as well as from different donors. By using direct membrane immunofluorescence assay, we found that only a small fraction of human peripheral blood and cord blood lymphocytes (CBL), and possibly less than 31% of the T cell-depleted lymphocyte population, carry receptors for P3HR-1 strain of EBV. The number of cells carrying receptors for EBV did not vary considerably among different blood lymphocyte populations from several normal donors. EBV adsorption on lymphocyte subpopulations showed that purified thymus-dependent (T) cells and thymocytes did not adsorb EBV, in contrast to T cell-depleted lymphocyte populations and lymphoid cells from fetal liver and spleen. In CBL infected with EBV strain B95-8, EBNA was detected by anti-complement immunofluorescence as early as 18 h after infection. This indicates that EBNA is the earliest detectable EBV-determined intracellular antigen to appear after infection and before or during lymphocyte transformation by EBV. Transformation was observed only in lymphocyte cultures containing detectable thymus-independent B cells but not in cultures of purified T cells. With one exception (es-b-1), all the EBV-transformed LCL from different origins carried surface-bound immunoglobulins (a B cell marker). These included also the 10 LCL obtained by infecting cultures of adherent cells from different donors. With regard to its surface markers, ES-B-1 appeared to be an exceptional EBV genome-carrying line, and it also lacked the ability to form spontaneous rosettes with sheep erythrocytes (a T cell marker). Therefore, it is possible that ES-B-1 was derived from an atypical B cell or B cell precursor or from a so-called "null cell" transformed by EBV.

Colony-Stimulating Factors in Cultures of Human Peripheral Blood Cells<xref ref-type="fn" rid="fn2"><sup>2</sup></xref>

Conditioned media containing colony-stimulating factors (CSF) were prepared in culture from unfractionated leukocytes and a lymphocyte-rich fraction of human peripheral blood. The mitogen phytohemagglutinin increased CSF production in low concentrations (106 cellsjml) of lymphocytes in the presence of homologous human plasma. In a semisolid agar medium system, the leukocyte-conditioned medium stimulated growth of granulocytic cell clones both from mouse and from human marrow cells; the lymphocyte-conditioned medium stimulated those from human marrow cells but had little or no activity with mouse marrow cells. This low colony-stimulating effect on mouse marrow cells was not due to the presence of inhibitors. The findings indicated that there were at least two types of CSF with respect to species specificity, that the lymphocyte-rich fraction contained CSF-producing cells, and that the factor(s) relatively specific for human cells could be separated from less specific factors.

Granulocyte colonies derived from lymphocyte fractions of normal human peripheral blood.

Granulocyte colonies can be grown from leukocyte-rich plasma obtained from normal human peripheral blood. Purification of lymphocytes by the Ficoll-Hypaque gradient method and subsequent removal of monocytes achieved a 10-fold increase in the number of colonies formed, suggesting that the colony-forming cell is nonadherent, nonphagocytic, and morphologically difficult to distinguish from a lymphocyte. Ultrastructural analysis of intact colonies by a new method developed for this purpose showed orderly differentiation and maturation. Only undifferentiated cells were seen in colonies on day 5, promyelocytes with developing primary lysosomes were present on day 9, whereas myelocytes and metamyelocytes were most prevalent on days 15 and 21, respectively. The studies suggest where further concentration of precursor cells may be attempted, and where further functional and histochemical analysis of colonies on the ultrastructural level will be possible.

Rate of Digestion of Three Human Blood Fractions in Aedes aegypti (Diptera: Culicidae)1

Commercially available purified albumin, alpha globulin, and gamma globulin fractions of human blood were used as antigens (10% solution in distilled H2O) to produce fraction-specific antisera in rabbits. The immunization schedule consisted of eight 1-cc injections administered on alternate days. The initial injection was intravenous; subsequent ones were subcutaneous. Rabbits were trial bled 8–10 days after the final injection. Antisera with interfacial-test titers in excess of 64,000 (in 3-mm ID tubes) were obtained for each of the 3 fractions.

Reduction of thymine by leukocytes.

The maximal activity of the dihydrothymine dehydrogenase of fractions of human blood was found in leukocytes, especially in the supernatant after centrifugation of homogenates at 100,000 × g in the course of isolation of the cell components. The dihydrothymine dehydrogenase from human and pig leukocytes was purified tenfold with a yield of activity of about 60%. Gel filtration with Sephadex G-200 showed several peaks of enzymatic activity. A quantitative method for the estimation of thymine and dihydrothymine by means of thin layer chromatography and subsequent liquid scintillation counting is described. This method may have application in the study of the genetically determined β-aminoisobutyric acid excretion in man.

Vitamin distribution in human plasma proteins.

APART from vitamin B12, very little is known about the relation of vitamins and plasma proteins1–3. By coupling the availability of pure human blood fractions with sensitive, specific microbial and chemical vitamin assays, the amount of vitamin in various fractions of human plasma was determined. Such information is needed for charting transport mechanisms for the various vitamins or, conversely, assigning transport functions to the various blood proteins.

Instrumental radioactivation analysis of selenium in biological materials

A correction procedure has been established for the instrumental radioactivation analysis of selenium in dialyzed biological materials, by means of the 19-sec isotope Se 77m. Based on the oxygen content of samples, the procedure has enabled a more accurate and sensitive determination than previous methods utilizing Se 77m for analysis of selenium in such a matrix. The amounts of selenium bound to protein in various fractions of human blood and other tissues were investigated using the technique.

Viremia: Isolation of measles virus from the washed leukocytic fraction of human blood

Viremia: Isolation of measles virus from the washed leukocytic fraction of human blood