Pooled Human Serum Research Articles

A rapid fluorescent assay to distinguish attached from phagocytized yeast particles

In studies of phagocytosis and its consequences for cell activation, it is important to distinguish those particulate stimuli which are completely ingested and internalized from those which are only attached to phagocytic surfaces. Ingestion can be profoundly influenced by both the type of opsonins on the surface of the stimulus and the expression and activation of receptors on the phagocytes for these opsonins. We report a new fluorescent assay which facilitates rapid and reproducible quantitation of attached versus fully internalized live or dead yeast particles by phagocytes. The assay employs the fluorescent dye diaethanol (Uvitex 2B) which stains chitin on the cell wall of fungi and is excluded from live phagocytes. The diaethanol assay and a standard, previously published methylene blue dye exclusion assay yielded comparable results using human neutrophils or monocytes incubated with heat-killed, serum-opsonized Candida albicans. The diaethanol assay proved useful in distinguishing differences in effects of various opsonins, as human neutrophils selectively opsonized with either pooled human serum (PHS), IgG (heat-inactivated PHS) or complement (IgG-depleted PHS) completely internalized 69.5%, 91.3% and 52.5% of cell-associated zymosan particles respectively. Finally, the new assay permitted comparisons of differing macrophage populations, as resident murine peritoneal macrophages internalized only 10.6% of complement-opsonized, cell-associated zymosan particles compared with 41.7% when the macrophages were elicited with thioglycolate. The assay should prove useful to investigators studying both fungal phagocytosis and killing, as well as to those performing general studies of receptor expression, regulation and function.

Killing of Neisseria meningitidis by human neutrophils: implications for normal and complement-deficient individuals.

The contributions of complement-dependent phagocytosis and serum bactericidal activity (SBA) to the killing of 62 strains of meningococci were examined by using C8-depleted or pooled human serum (PHS). The complement-dependent nature of killing by neutrophils was confirmed by restoring survival to control values by using heated serum. Serogroups B and 29E, but not A, C, Y, and W135, were ingested and killed by neutrophils in C8-depleted PHS (PHS-C8Dep; 41.7% +/- 7.3% and 60.5% +/- 17.8% vs. greater than or equal to 100% survival, respectively, at 30 min). Group B meningococci were resistant to complement-mediated SBA, whereas group Y were susceptible. Deposition of C3 on serogroups B and Y was similar (28.5 +/- 2.9 vs. 23.5 +/- 2.7 C3 fluorescence units; P greater than .05); however, susceptibility to complement-dependent phagocytosis and complement-mediated SBA of serogroups B and Y did not correlate. We also examined meningococcal phagocytosis by using serum from a C8-deficient patient. In contrast to PHS-C8Dep, this serum supported rapid phagocytic killing of serogroups A, C, Y, and W135 meningococci. This finding suggests that vaccinating individuals deficient in late-complement components may shift the burden of host defense from SBA to phagocytosis.

Activation of the Human Complement Cascade by Bacterial Cell Walls, Peptidoglycans, Water‐Soluble Peptidoglycan Components, and Synthetic Muramylpeptides—Studies on Active Components and Structural Requirements

Cell walls isolated from 29 strains of 24 gram-positive bacterial species, whose peptidoglycans belong to the group A type of Schleifer and Kandler's classification, with one exception (Arthrobacter sp.), were shown to activate the complement cascade in pooled fresh human serum mainly through the alternative pathway and partly through the classical one. The complement-activating effect of cell walls (5 species) possessing group B type peptidoglycan, except those of Corynebacterium insidiosum, was weaker than that of the walls with group A type peptidoglycan. Preparations of peptidoglycan isolated from cell walls of Staphylococcus aureus, Streptococcus pyogenes, and Lactobacillus plantarum also activated the alternative pathway of the complement cascade, but less effectively than the respective parent cell walls. A water-soluble "polymer" of peptidoglycan subunits (SEPS), which was prepared from Staphylococcus epidermidis peptidoglycans by treatment with a cross-bridge degrading endopeptidase, retained most of the complement-activating ability of the parent cell walls. A peptidoglycan "monomer," SEPS-M, which was obtained by hydrolysis of the glycan chain of SEPS with endo-N-acetylmuramidase to disaccharide units did not activate complement. In conformity with this finding, neither synthetic N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) nor MDP-L-Lys-D-Ala activated the complement cascade. Among several lipophilic derivatives of MDP, 6-O-(3-hydroxy-3-docosylhexacosanoyl)-MDP-L-Lys-D-Ala (BH48-MDP-L-Lys-D-Ala) and 6-O-(2-tetradecylhexadecanoyl)-MDP (B30-MDP) were shown to activate complement through the alternative as well as the classical pathway and exclusively through the classical pathway, respectively. The finding that a D-isoasparagine analog of B30-MDP caused the same effect as the parent molecule strongly suggests that the activation of complement by B30-MDP is different from that caused by cell wall peptidoglycans and a water-soluble "polymer" of peptidoglycan subunits.

Sensitive ELISA for quantitating the terminal membrane C5b-9 and fluid-phase SC5b-9 complex of human complement

Activation of the complement system to completion results either in the generation of a pore-forming, cytolytic C5b-9(m) complex, or of a cytolytically inactive, fluid-phase SC5b-9 complex. In this paper, we describe a sensitive and reliable, sandwich ELISA for C5b-9(m) and SC5b-9, which is based on the use of a monoclonal antibody to a neoantigen of C5b-9 in combination with affinity-purified, polyclonal rabbit antibodies. The ELISA has been calibrated with purified C5b-9(m) and SC5b-9, and can detect 3 ng/ml C5b-9(m) and 20 ng/ml SC5b-9. We show that maximal conversion of C5–C9 in pooled human serum by insulin or zymosan activation generates 220±40 μg/l SC5b-9. 65 of 100 normal human EDTA plasma samples analyzed in this study contained 100–600 ng/ml SC5b-9, corresponding to 0.04–0.24% of maximal conversion. Levels of circulating SC5b-9 in other donors were below the limit of detection. Incubation of serum at 37°C always led to spontaneous generation of SC5b-9; concentrations ranged from 490–4725 ng/ml after 60 min, 37°C, with a mean of 1848±1031 (SD) ng/ml amongst 25 donors studied. The terminal complement complex present in EDTA plasma was partially purified by PEG precipitation, DEAE-ion exchange chromatography and sucrose density gradient centrifugation, and was found to contain C8, C9 and S-protein as demonstrable by SDS-PAGE immunoblotting. Thus, the material most probably represented genuine SC5b-9. No significant age- or sex-dependent variations in SC5b-9 levels were noted. The present data call for a critical re-appraisal of several previously published methods for the determination of SC5b-9 levels in human plasma and serum.

Serum Factor Responsible for Thymidine Uptake by Lymphocytes Is Independent of Somatomedin-C

ABSTRACT Lymphocytes have specific receptors for somatomedin-C (Sm-C) and GH. To test whether these cells are sensitive to the growth-promoting properties of these growth factors, we studied serum (with low or normal Sm-C and GH levels) from children having various statural disorders by means of the lymphocyte proliferation assay. Peripheral lymphocytes from each subject were cultured in media containing either autologous serum (AS) or normal pooled human serum (HS). The mitogenic response (MR) was assessed by determining the amount of tritiated thymidine incorporated into cellular DNA. The MR was studied both in phytohemagglutinin-stimulated and in unstimulated lymphocytes. MR was expressed as thymidine uptake (CPM) in the presence of AS divided by CPM in the presence of HS. The MR in serum from (1) 27 control subjects was 1.01 ± 0.03 (SEM); (2) 14 children with short stature and normal serum GH-RIA and Sm-C levels was 0.98 ± 0.04; and (3) 12 children with GH deficiency showed two widely different responses. In four patients (subgroup 1), the MR was 0.05 ± 0.02 and in eight (subgroup 2) the MR was 1.11 ± 0.06. Eleven patients were studied after GH therapy (three from subgroup 1), and the MR was 1.12 ± 0.03. Neither Sm-C levels nor linear growth rate correlated well with the growth-promoting property of the serum when tested on lymphocytes. In seven additional patients with GHD, the MR when studied at varying concentrations of serum (0.1–5.0%), showed results identical to subgroup 2. Addition of purified Sm-C to cultures did not enhance the MR of serum from GHD patients prior to GH therapy. GH-dependent factors other than Sm-C may have a role in in vitro lymphocyte proliferation; in its current form, however, this bioassay is not sensitive enough to be useful in the diagnosis of children with GH deficiency.

Role of human serum and cellular factors on the growth of peripheral blood CFU-gm.

The effects of fetal calf serum (FCS), autologous serum (AS) and pooled human serum (PS) on granulocyte-macrophage progenitors (CFU-gm) from normal human peripheral blood mononuclear non-adherent cells (MNAC) were studied to determine optimal growth conditions. PS provided fewer variations and better growth conditions than AS and FCS. Moreover, serum inhibitors affecting both autologous and heterologous CFU-gm were detected in a small group (10%) of donors. Since CFU-gm from different donors display a relatively uniform cell-cycle status and membrane phenotype, under optimal growth conditions the number of CFU-gm probably reflects the size of the circulating myeloid progenitor cell compartment.

Characteristics of neurite outgrowth from rat spinal ganglia: effects of serum and segmental level.

The outgrowth of neurites from spinal ganglia in tissue culture may be affected by many variables including medium constituents and the age of ganglia. If E15 rats were used, the rate of outgrowth from ganglion explants in response to nerve growth factor was independent of the cranial-caudal position from which the ganglia were derived. This contrasts with the cranial-to-caudal gradient present during embryonic development. The effect of serum on this outgrowth was also studied and ten of 17 lots of human placental serum, two of three lots of pooled human placental serum, and one of two lots of fetal calf serum significantly inhibited the outgrowth of neurites. This has relevance to the design of tissue culture studies and may be important in the interpretation of in vitro studies of disease mechanisms.

Ligand-receptor interactions in the phagocytosis of virulent Streptococcus pneumoniae by polymorphonuclear leukocytes.

We used polyclonal and monoclonal antibodies to neutrophil complement receptors CR1 and CR3 to assess the role of these receptors in the phagocytosis of virulent Streptococcus pneumoniae serotypes 3, 6A, and 14, which bear accessible C3 ligands covalently bound to the polysaccharide capsule. When the iC3b receptor (CR3) on normal polymorphonuclear leukocytes (PMNLs) was blocked by the monoclonal antibody OKM10, phagocytosis of pneumococcal serotypes 6A and 14 (which bear exclusively iC3b) was inhibited 50%-80% in pooled human serum and completely in nonimmune serum. Blockade of the PMNL C3b receptor (CR1) failed to inhibit phagocytosis for serotypes 6A and 14. For serotype 3, which bears C3b and C3d (as well as iC3b) on the capsule, CR3-mediated phagocytosis accounted for only 20% of the uptake; again, there was no evidence for CR1-mediated phagocytosis. The iC3b ligand elicited consistently greater release of superoxide, myeloperoxidase, and lactoferrin than did C3b. The iC3b/CR3 interaction is thus the primary trigger for phagocytosis of iC3b-bearing pneumococci and for stimulation of intracellular bactericidal processes.

Identification of non-cross-reacting antigens of Onchocerca volvulus with lymphatic filariasis serum pools.

Onchocerca volvulus proteins labelled with 125I were immunoprecipitated with onchocerciasis and lymphatic filariasis human serum pools in order to differentiate between cross-reacting and non-cross-reacting antigens. Analysis of the immunoprecipitates by two-dimensional gel electrophoresis revealed that all high molecular weight (Mr) O. volvulus antigens cross-reacted with the lymphatic filariasis serum pools. We observed, however, that at least 8 O. volvulus antigens were specifically immunoprecipitated only by the onchocerciasis serum pools, with Mr ranging from 20,000 up to 43,000 Daltons. These results suggest that the lower Mr O. volvulus antigens are more species specific than the other antigens. The significance of these findings for the immunodiagnosis of onchocerciasis is discussed.

The role of surface polysaccharide in determining the resistance of Serratia marcescens to serum killing.

Two O14:H12 strains of Serratia marcescens with different sensitivities to killing by normal pooled human serum were investigated. Complement binding, studied by measuring hydrophobicity and using rocket immunoelectrophoresis with anti-human C3, showed the sensitive cells (S1220) rapidly bound and fixed complement whereas the resistant cells (4444-60) bound less C3b. The strains had identical membrane protein composition. Crossed immunoelectrophoresis suggested that in S1220 cells the polysaccharide material including LPS was less antigenic and present in smaller amounts than in 4444-60 cells. This was confirmed by examining extracted polysaccharide material chemically and by SDS-PAGE. The resistant strain had 33% more phenol-extractable polysaccharide material than the sensitive strain, possibly comprising LPS with longer O antigen chain lengths, or a microcapsule of O antigen polysaccharide. Extra polysaccharide material on the surface of the resistant strain prevents complement components binding and reaching the hydrophobic membrane where lytic lesions occur.

Aqueous and serum-based materials compared for use as simulated calibrators for three ionized calcium analyzers.

To determine if bias between different ionized calcium analyzers could be decreased, we analyzed 10 control fluids during a study in which ionized calcium was measured in more than 150 serum and whole-blood samples. After calibrating three ionized calcium analyzers (Radiometer ICA 1, Nova 8, and AVL 980) with the manufacturers' respective calibrators, we used the between-instrument differences of the control fluids to simulate recalibration of the analyzers during each analytical run. A filtered human serum pool containing ionized calcium at 1 mmol/L concentration, with CO2 removed and having no added buffer, was the only material that consistently decreased between-analyzer bias of both serum and whole blood. Another human serum pool containing about 1.3 mmol of ionized calcium and about 10 mmol of bicarbonate per liter was even better at minimizing analyzer biases for serum samples, but was not as effective for whole-blood samples. Some additives used to buffer pH apparently adversely affected both the accuracy and precision of some, but not other, calcium ion electrodes. We conclude that if a reference material is developed for calibration of ionized calcium analyzers, it should be tested on several analyzers for use with both serum and whole blood, and it should be at least as effective as a human serum material, such as that used here.

Chemiluminescent response of polymorphonuclear leukocytes to Streptococcus pneumoniae and Haemophilus influenzae in suspension and adhered to glass.

We measured the luminol- and lucigenin-enhanced chemiluminescent response of human polymorphonuclear leukocytes (PMN) stimulated by various strains of Streptococcus pneumoniae and Haemophilus influenzae. In the absence of opsonin, phagocytosis of either bacterial species elicited good PMN response when the bacteria were adhered to a surface but minimal PMN response when they were in suspension. When 10% pooled human serum was used as a source of opsonin, a moderate to excellent chemiluminescent PMN response was elicited during phagocytosis of opsonized bacteria both in suspension and adhered to surface. We conclude that opsonin significantly enhances PMN chemiluminescence when a suspension-type assay is used and that opsonin-independent mechanisms play a significant role in the chemiluminescent response of PMN during phagocytosis of adherent bacteria.

Use of pooled human serum in the standardization process of the serum fructosamine determination for the estimation of glycosylated serum proteins

Measuring of serum fructosamine (FMN) concentration is a simple and reliable method for the estimation of glycosylated serum proteins [l]. Other methods are more time consuming [2,3] or require special equipment [4,5]. While we have found the method of Johnson et al [l] to be of value in screening for diabetes mellitus, but we also have had difficulty in standardization of serum FMN determinations: the use of different batches of albumin produced different changes in absorbance at the same concentration of l-deoxy,l-morpholinofructose (DMF). To overcome this difficulty we have used pooled human serum instead of human albumin for dilution of DMF for FMN determination in serum.

Two major human allergenic sites on ragweed pollen allergen antigen E identified by using monoclonal antibodies.

Murine monoclonal antibodies specific for antigen E (AgE), the major allergen isolated from short ragweed pollen, have been produced and characterized. These monoclonal antibodies, when coupled to Sepharose and used as immunoadsorbents, specifically bound AgE when a crude pollen extract was passed through the column. Three antigenic sites (A, B, and C) on AgE were identified by using five of these monoclonal antibodies in both inhibition and double-bind solid-phase ELISA. These three antigenic sites appear to be nonoverlapping and nonrepeated, that is, present only once on each AgE molecule. Site C on AgE could readily be bound by the monoclonal antibody specific for that site, but only when AgE was in solution or "presented" by an anti-site A or anti-site B antibody. Site C appears to be only marginally available for binding when AgE is directly adsorbed to polyvinyl chloride microtiter wells. The majority of monoclonal antibodies isolated after immunization of BALB/c mice were specific for site A on AgE. In addition, the binding to AgE of pooled BALB/c polyclonal, hyperimmune antisera against AgE was blocked approximately 80% by a monoclonal antibody directed against site A, but was only blocked approximately 20% by an anti-site B monoclonal antibody. This suggests that site A on AgE is the predominant antigenic site in the BALB/c immune response and that site B represents a less dominant site. The binding of IgE in pooled human serum from ragweed-allergic individuals is blocked approximately 50% by a monoclonal antibody directed to site A on AgE and also approximately 50% by a monoclonal antibody directed against site B. A series of individual human short ragweed allergic antisera also showed significant, although varied, inhibition of IgE binding to AgE by both anti-site A and anti-site B monoclonal antibodies. Simultaneous addition of anti-site A and anti-site B was somewhat additive and inhibited up to 80% of the binding of human IgE specific for AgE. The conclusion from these data is that site A and site B defined by two murine monoclonal antibodies represent two very major allergenic sites in the human response to this molecule.

Effects of cyclosporine on human endothelial cell cultures.

Reports of vascular changes in renal biopsies of transplant patients treated with cyclosporine prompted review of our own renal biopsies and examination of human endothelial cell cultures exposed to cyclosporine in vitro. Endothelial cells were isolated from human umbilical cords by collagenase digestion and cultured in Medium 199 with Earle's salts plus 20% pooled human serum in the absence of antibiotics. Cultures exposed to cyclosporine (0, 0.4, 1.0, 5.0, 10.0 micrograms/ml) for 0, 3, 7, 10, and 14 days were subsequently fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer. Vascular thrombosis was seen in renal biopsies of cyclosporine- and azathioprine-treated patients but the incidence was the same in both groups. No change in the morphology of endothelial cell cultures was observed until 7 days when an increase in size and number of cytoplasmic inclusions became apparent in both control and cyclosporine-treated cultures. By electron microscopy, these inclusions were identified as secondary lysosomes. Their number and size increased with the length of time in culture but did not appear to correlate with the concentration of cyclosporine in the medium. No other morphologic change was identified. It is concluded that the appearance of increased numbers of secondary lysosomes in human endothelial cell cultures is a function of culture age as opposed to cyclosporine exposure. Furthermore, the data indicate that small vessel thrombosis is not specific to treatment with cyclosporine.

The human immune response against major allergens from house dust mite, Dermatophagoides pteronyssinus. II. Idiotypic cross-reactions of allergen-specific antibodies.

Antisera were prepared in rabbits against the idiotypic (Id) determinants of antiallergen antibodies. These antibodies were isolated from the plasma of 10 unrelated patients by immunoabsorption. Three major allergens isolated from the house dust mite, Dermatophagoides pteronyssinus (DPT), were used. The anti-Id antisera were rendered specific by successive absorption on insolubilized allergens, IgG and IgM from a pool of human sera and Ig from the donor after removal of anti-DPT antibodies by absorption. Anti-Id antibodies raised essentially against IgG antibodies were found to inhibit the agglutinating activity of both IgG and IgM anti-DPT antibodies toward allergen-coated latex. These anti-Id antibodies were also able to inhibit partly the binding of radiolabeled allergen to IgE anti-DPT antibodies. Matching the anti-Id antisera with the various individual anti-DPT antibodies revealed an Id cross-reactivity of about 71% for IgG, 61% for IgM and 62% for IgE. A second and predominant type of anti-Id antibodies recognized bystander idiotopes as the allergen did not inhibit Id-anti-Id reaction. These idiotopes were apparently more specific to individuals (private) as the anti-DPT antibodies of a given patient did not agglutinate latex particles coated with anti-Id antibodies prepared against Id of another patient.

Non-Organ Specific Thermostable Tissue Antigens

Thermostable ethanol insoluble antigens (BE antigens) were identified that occur in all human tissues and human dispersed cells, but which are absent from normal human serum. In contradistinction to previously described organ-specific BE antigens, these antigens were referred to as non-organ-specific tissue antigens (NOST). Antisera obtained by immunization of rabbits with BE preparations of human organs had been selected for being devoid of any organ specificity and had been absorbed by BE preparations of pooled human serum. Such antisera could be used as reagents for detection of NOST BE antigens in pathological human sera. Inhibition of enzyme immunoassay proved to be a convenient procedure for these studies. Inhibition of 35% or more was only exceptionally noted in studying sera of normal subjects 20-40 years old, but inhibition exceeding 35% (positive results) was noted in 48% of sera from subjects at the age of 70 years or more. A high incidence of positive results was also encountered in sera of patients with end-stage renal disease (30%), renal graft recipients (18%), and in patients with lymphoma or leukemia (44%), but interestingly enough, no positive tests were noted in patients with lepromatous leprosy.

Evaluation of a mouse monoclonal antibody for the determination of serum theophylline by fluorescence polarization immunoassay.

Theophylline concentrations in spiked human serum and serum specimens obtained from patients with normal and impaired renal function were measured by fluorescence polarization immunoassay (FPIA) using a mouse monoclonal antitheophylline antibody. The interday coefficients of variation of the assay after 14 days were 4.2, 3.3, and 2.4% at serum theophylline concentrations in pooled human serum of 7.1, 12.2 and 26.9 mg/L. Theophylline concentrations determined by FPIA and high performance liquid chromatography (HPLC) were used to generate the following linear regression equations relating the corresponding theophylline concentrations measured by each method in serum specimens from 50 patients with normal renal function (FPIA = 1.00 HPLC + 1.01, r = 0.98) and 50 patients with end-stage renal disease (FPIA = 1.04 HPLC + 0.08, r = 0.99). In contrast to previous studies performed with a polyclonal antitheophylline antibody, the precision, accuracy, and specificity of the FPIA were adequate for analysis of theophylline in serum specimens obtained from patients with end-stage renal disease.

Effects of soil infection potentiating factors on neutrophils in vitro

Although the ability of soil silicate fractions to potentiate infection is well recognized, the precise mechanisms by which they do so remain unexplained. This study was carried out to investigate the effects of montmorillonite clay, the most potent of these soil infection potentiators, on human neutrophils, erythrocytes, and serum complement in vitro. Using phase microscopy, rapid neutrophil lysis was observed when cells were exposed to untreated clay. After lysis, the cytoplasmic marker enzyme lactate dehydrogenase rapidly adsorbed to the surface of the clay. Both enzyme surface adsorption and cell lysis could be blocked, however, by pretreatment of the clay with human albumin. Likewise, neutrophil chemiluminescence could be stimulated by untreated clay, but not by clay pretreated with 5 percent albumin or 10 percent pooled human serum. Maximal chemiluminescence was stimulated by clay pretreated with 0.1 percent albumin, probably because the partially protective albumin coating delayed cell lysis. Compared with the effect on neutrophils, clay lysis of erythrocytes was incomplete. When zymosan-activated serum samples were exposed to clay, complement activity as measured by neutrophil chemotaxis was suppressed in a dose-dependent fashion. We conclude that montmorillonite clay may potentiate infection by a direct cytotoxic effect on the neutrophil, making it unavailable for bacterial phagocytosis, by local reduction in bacterial opsonization due to depletion of activated complement, and by the release of toxic tissue substances, such as lysosomal enzymes and oxygen free radicals, from leukocytes which may damage host tissue and thus create an environment favorable for bacterial survival.

A note on the increased permeability of opsonized luminous bacteria

Barak, M., Merzbach D. & Ulitzur, S. 1985. A note on the increased permeability of opsonized luminous bacteria. Journal of Applied Bacteriology59, 57–59.Opsonization of a dark variant of the luminous bacterium Photobacterium leiog‐nathi by pooled human serum caused an increase in the permeability of the organism to actinomycin D, as judged by the inhibition of the proflavin‐induced synthesis of its luminescence system.