Pooled Human Serum Research Articles

Effects of the two varieties of Cryptococcus neoformans cells and culture filtrate antigens on neutrophil locomotion.

Cryptococcus neoformans var. gattii (serotype B and C) isolates have a relative predilection for immunocompetent hosts, and C. neoformans var. neoformans (serotype A and D) isolates have a relative predilection for immunocompromised hosts, suggesting that normal host resistance to the former may be relatively inefficient compared with that to the latter variety. In order to assess the possibility that normal cellular host defense is inadequate in protecting against C. neoformans var. gattii, we compared the two varieties of C. neoformans cells and their culture filtrate antigens (CneF) with respect to effects on neutrophil (polymorphonuclear leukocyte [PMN]) locomotion. In a 48-well modified Boyden chamber, the cells and CneF of C. neoformans var. neoformans (serotype A and D) isolates stimulated chemotaxis and chemokinesis of human PMN and activated a complement component(s) in pooled human serum to become a chemoattractant(s) for human PMN. In contrast, the cells and CneF of C. neoformans var. gattii (serotype B and C) isolates did not stimulate chemotaxis or chemokinesis in human PMN but rather inhibited chemokinesis and chemotactic responses of PMN to pooled human serum and formylmethionyl leucyl phenylalanine. Neither of the CneF from the C. neoformans var. gattii isolates was cytotoxic to PMN. Furthermore, with the mouse model, we found that CneF from C. neoformans var. neoformans caused migration of PMN into gelatin sponges implanted in naive and immunized mice, whereas CneF from C. neoformans var. gattii inhibited PMN migration into sponges. Our results, combined with findings of others showing reduced PMN infiltration in lungs of mice infected with C. neoformans var. gattii compared with PMN infiltration in lungs of mice infected with C. neoformans var. neoformans, indicate that the relative inadequacy of normal host resistance mechanisms to prevent infection with C. neoformans var. gattii results, in part, from inhibition of PMN migration to the site of the organism.

Behavior of frozen serum pools and lyophilized sera in an external quality-assessment scheme

To further improve analytical accuracy in clinical chemistry, proficiency testing needs amelioration in the quality of materials tested and in target value assignment. To obtain information on the actual state-of-the-art in the Lombardy region of Italy, and to examine the behavior of different types of control materials (fresh-frozen human sera and lyophilized materials), we developed the following experimental design. Two human serum pools and two lyophilized sera were distributed to 32 laboratories for determination of glucose, creatinine, cholesterol, sodium, potassium, and gamma-glutamyltransferase (gamma-GT). Each analyte was measured in triplicate on each of 3 days. Target values for the controls were obtained with Reference Methods. The results show a good intralaboratory precision for every component but some accuracy problems for glucose, creatinine, cholesterol, and gamma-GT. Lyophilized materials showed some commutability problems for glucose, electrolytes, and gamma-GT, mainly with dry chemistry technology.

Isotope dilution mass spectrometry as a candidate definitive method for determining total glycerides and triglycerides in serum

A new isotope dilution mass spectrometric method for total glycerides and triglycerides in human serum is described. Total glycerides are defined as the sum of tri-, di-, and monoglycerides plus free glycerol; triglycerides are defined as the pure triglyceride species. In both determinations, serum samples are supplemented by addition of [13C3]tripalmitin, processed, derivatized, and the abundance ratios of selected ions are determined. Bias is investigated by quantifying the analyte in the same samples under different chromatographic and ionization conditions. The analytes were determined in two human serum pools. The CV for a single measurement ranged from 0.35% to 0.72%, and the relative SEM ranged from 0.10% to 0.34%; there was no significant bias in the measurements. The combination of high precision and absence of significant bias in the results qualify this method for consideration as a Definitive Method as defined by the National Committee for Clinical Laboratory Standards.

Interlaboratory/intermethod differences in functional sensitivity of immunometric assays of thyrotropin (TSH) and impact on reliability of measurement of subnormal concentrations of TSH

Clinically relevant interassay precision profiles for thyrotropin (thyroid-stimulating hormone; TSH) were constructed with human serum pools measured over 4-8 weeks by six immunometric assays, in at least two different reagent lots. Functional sensitivities (the concentration at which the interassay CV is < or = 20%) were determined in four to eight clinical laboratories plus the respective manufacturer's laboratory. These studies revealed that the manufacturer's stated functional sensitivity limit is rarely duplicated in clinical practice. Loss of specificity (indicated by artifactually high values) was seen with some methods when used to measure certain unrefrigerated low-TSH sera. Measurement of TSH in four human serum pools (TSH < 0.05-0.25 mIU/L) by 16 different methods (each in at least eight UK or US laboratories) showed that some methods could not reliably distinguish subnormal from normal TSH values. Better pool rankings and fewer misclassifications of low-TSH sera as "normal" were seen with use of assays capable of "third-generation" functional sensitivity (0.01-0.02 mIU/L) than with assays with "second-generation" functional sensitivity (0.1-0.2 mIU/L). Because inter- and intramethod differences in functional sensitivity negatively impact the diagnostic accuracy and cost-effectiveness of a TSH-centered thyroid-testing strategy, laboratories should independently establish an assay's functional sensitivity by a clinically relevant protocol. Moreover, manufacturers should assess functional sensitivity more realistically and improve the robustness of assays to ensure that their performance potential is consistently met in clinical practice.

Lipopolysaccharide induces intracytoplasmic migration of the polymorphonuclear leukocyte CD11b/CD18 receptor.

We investigated the effects of lipopolysaccharide (LPS) on the subcellular location of the integrin receptor CD11b/CD18 (Mac-1). Cytoplast and subcellular fractionation experiments were performed to distinguish between receptor shedding and intracellular receptor transport as mechanisms involved for the effects of LPS on CD11b/CD18 expression. Cytoplast preparations demonstrated the same percentage of cell-associated receptors +/- LPS. Subcellular fractionation experiments demonstrated a shift from primarily plasma membrane fractions to azurophilic granules. Protein tyrosine kinase inhibition with genistein (50 microM) inhibited the LPS-induced translocation of CD11b/CD18 receptors to azurophilic granules. The effects of LPS (10 ng/mL) alone were reproduced with LPS (.1 ng/mL) plus heat-inactivated pooled normal human serum. Preincubation of PMN with anti-CD14 monoclonal antibodies prevented the effects of LPS+serum on the translocation of CD11b/CD18 receptors. These results demonstrate that LPS regulates CD11b/CD18 expression by inducing intracytoplasmic translocation of this receptor to azurophilic granules. This process involves activation of protein tyrosine kinase, and endosomal acidification contributes to the degradation of these receptors within azurophilic granules.

Immune pressure selects for Plasmodium falciparum parasites presenting distinct red blood cell surface antigens and inducing strain-specific protection in Saimiri sciureus monkeys.

The passive transfer of specific antibodies to a naive splenectomized Saimiri sciureus monkey infected with the Palo Alto FUP/SP strain of Plasmodium falciparum resulted in the emergence of parasites resistant to the transferred antibodies. Molecular typing indicated that the original and resistant parasites were isogenic. Saimiri monkeys primed with original parasites were fully susceptible to a challenge by the resistant ones, and vice versa. This absence of crossprotection indicates that strain-specific determinants would be the major targets of protective immunity developed in these monkeys. Phenotypic analysis showed that the surface of the infected red blood cells differed in both lines. Original parasites formed rosettes, autoagglutinated, presented characteristic knobs at the surface of the infected red blood cell, and did not agglutinate in the presence of a pool of human immune sera. In contrast, the resistant parasites did not form rosettes, did not spontaneously autoagglutinate, presented abnormal flattened knobs, and formed large aggregates in the presence of a pool of human immune sera. The presence of strain-specific determinants at the surface of the resistant parasites was confirmed by surface immunofluorescence and agglutination using homologous Saimiri serum. Neither the original nor the resistant parasites cytoadhered to an amelanotic melanoma cell line, suggesting that cytoadherence and agglutination can be dissociated. These results indicate that parasites that differ by the antigens exposed at the surface of the red blood cell induce strain-specific immunity. Furthermore they show that rosetting and nonrosetting parasites differ in their antigenic properties and do not crossprotect.

Tissue Factor Expression on Mesothelial Cells is Induced during in vitro Culture – Manipulation of Culture Conditions Creates Perspectives for Mesothelial Cells as a Source for Cell Seeding Procedures on Vascular Grafts

Lining the luminal surface of prosthetic small diameter bypasses with endothelial cells (EC) will lower its thrombogenicity. Unfortunately, human EC are only scarcely available. Mesothelial cells (MC) have antithrombotic properties in vivo and can be harvested in large numbers, from the omentum. Recent work demonstrated that the expression of tissue factor (TF) is induced in MC after isolation and culture. Different culture conditions were studied to suppress TF-expression. MC grown in pooled human serum (HS) are procoagulant (717 +/- 119 pM factor Xa/min.10(5) cells). Replacing HS for fetal calf serum, precoating the surface with extracellular matrix and the addition of the xanthine-oxidase inhibitor allopurinol, inhibited TF expression by 90% (p < 0.001). Allopurinol clearly reduced TF-mRNA levels. TF expression on cultured MC is an in-vitro effect due to culture conditions and the formation of oxygen free radicals. By reducing TF expression by 90%, we have established conditions in which MC are a good alternative for EC for seeding on synthetic grafts.

Impaired polymorphonuclear neutrophil function in end-stage renal failure and its correction by continuous ambulatory peritoneal dialysis.

The polymorphonuclear neutrophil (PMN) function of 22 patients with end-stage renal failure (ESRF) was studied immediately before and 3 months after starting continuous ambulatory peritoneal dialysis (CAPD) and compared with a control group of healthy normal volunteers. The PMN functions studied were phagocytosis and killing of Staphylococcus epidermidis and respiratory burst activity. The results show that in the presence of normal pooled human serum PMN from patients with ESRF before CAPD treatment phagocytose bacteria normally, but have impaired killing. Before treatment, the PMN from patients with ESRF also showed an increase in both unstimulated and stimulated superoxide anion production. Abnormal PMN function was corrected by CAPD treatment, suggesting the involvement of a dialyzable toxin.

Sulfated Polysaccharides Are Required for Collagen-Induced Vascular Tube Formation

We have previously shown that soluble type I collagen can induce vascular tube formation when it contacts the apical side of a confluent endothelial monolayer. In this study we have examined which soluble agent(s) are required for collagen-induced tube formation. Human neo-natal foreskin microvascular endothelial cells, maintained in basal medium, were preincubated with each test agent for 2 h prior to the addition of solubilised type I collagen (100 μg/ml). After 6 h, tube formation was quantitated using image analysis and expressed as the mean area of tube formation (mm2) per microscopic field of view. Collagen-induced tube formation did not occur in the presence of endothelial cell growth supplement, basic fibroblast growth factor, or normal pooled human serum. In contrast, the addition of heparin at 5 or 50 μg/ml caused extensive tube formation (0.22 ± 0.07 and 0.30 ± 0.12 mm2, respectively) whereas at 500 μg/ml little tube formation occurred (0.03 ± 0.02 mm2). Protamine sulfate, an antagonist of heparin, inhibited collagen-induced tube formation in a dose-dependent manner. Pentosan polysulfate, dextran sulfate, heparan sulfate, and chondroitin sulfate mimicked the action of heparin. Partially sulfated heparin (de-N-sulfated heparin) stimulated less tube formation compared to heparin (0.15 ± 0.06 mm2 at 50 μg/ml). The nonsulfated polysaccharides, xylan and dextran, had no effect on tube formation. In summary, sulfated polysaccharides are required for collagen-induced vascular tube formation in vitro. The sulfation of these molecules appears to be vital for collagen-induced tube formation.

Permeability of Ketorolac Acid and Its Ester Analogs (Prodrug) through Human Cadaver Skin

The in vitro skin permeabilities of ketorolac acid (KA), a potent nonsteroidal analgesic, and its two ester analogs as prodrug through human cadaver skin were investigated. The two esters of KA, namely, the ethyl ester (KEE) and [(N,N/‐dimethylamino)carbonyl]methyl ester (KDAE), were selected. The melting temperature of the two esters was significantly lower than that of ketorolac free acid. The partition coefficients (Ko/w) were 600, 3541, and 124 for KA, KEE, and KDAE, respectively. The enzymatic hydrolysis of KEE and KDAE by human pooled serum at 37 °C was investigated. The esters were hydrolyzed to KA by the serum esterases; the metabolic rate constants were 0.0418 and 0.0148 min‐1for KDAE and KEE, respectively. The serum half‐life of KDAE was about 3 times shorter than KEE. When split‐thickness cadaver skin was incubated with ester solution at 32 °C, the enzymatic hydrolysis of these esters was observed. The metabolic rate in the skin, however, was significantly lower than in the human pooled serum. The skin permeations of KA, KEE, and KDAE through heat‐separated epidermis from propylene glycol (PG), PG/glyceryl monocaprylate (GMC) (9:1), and PG/Azone (19: 1) vehicle mixtures were evaluated using modified Franz flow‐through diffusion cells. The skin fluxes of KA, KEE, and KDAE from PG/GMC (9:1) were 50 ± 10, 15 ± 4, and 57 ± 6 μg/cm2/h, respectively. KA was detected in the receiver compartment, albeit to a lesser extent. In conclusion, KDAE appeared to be a better ester prodrug than KEE because it exhibited relatively higher skin flux and faster enzymatic hydrolysis by human serum to liberate the parent drug.

Sequential Determination of Triglycerides and Free Fatty Acids in Biological Fluids by Use of a Continuous Pretreatment Module Coupled to a Gas Chromatograph

A continuous system coupled to a gas-liquid chromatograph was used for the sequential determination of triglycerides and free fatty acids in serum and urine. The module provides compositional information and hence more detailed information on lipid metabolism changes in patients suffering metabolic disorders. Lipids in biological samples are manually extracted in methanol- n-hexane and introduced into the flow system; free fatty acids are then separated by retention on an ion-exchange resin and triglycerides (not retained) are transesterified with acetyl chloride in methanol. The resulting methyl esters are continuously injected into the gas chromatograph and determined by using a flame ionization detector. In a second step, retained free fatty acids are eluted and derivatized (also with acetyl chloride in methanol) and subsequently determined similarly as the triglycerides. The proposed method was applied to the determination of triglycerides in a lipid control serum; free fatty acids were determined in a human pool serum by the proposed method and compared with the volumetric method used in clinical practice. The results obtained in both instances showed good agreement between the results provided for triglycerides and free fatty acids. The proposed method was also applied to urine samples; a parallel recovery study was also made in order to asses the performance of the method.

The susceptibility of S-layer-positive and S-layer-negative Aeromonas strains to complement-mediated lysis.

Forty strains of Aeromonas hydrophila and Aeromonas veronii recovered from invasive and non-invasive infections were tested for their susceptibility to complement-mediated lysis by 65% pooled human serum (PHS). Based upon the results of this assay, two major populations could be defined. The first group (n = 20) consisted of serogroup O:11 strains, all of which possessed a paracrystalline surface layer (S layer); all of these strains were refractory to the bactericidal activity of 65% PHS with the exception of A. hydrophila strain AH-121, which was composed of mixed subpopulations of serum-susceptible and serum-resistant clones. A second collection of isolates (n = 20), all of which were S-layer-negative, contained a subgroup of strains (n = 7) that were highly susceptible to complement-mediated lysis, showing a greater than 100-fold reduction of viable progeny within 30 min of exposure to 65% PHS. Serum-resistant strains from both groups could not be lysed by exposure of bacterial cells to polyclonal somatic or whole cell antisera or to 30 micrograms ml-1 of polymyxin B nonapeptide prior to challenge with 65% PHS. Analysis of selected serum-resistant and serum-susceptible strains from both groups showed that all isolates activated the complement pathway and most bound C3b to the cell surface, indicating that the inability of complement to lyse serum-resistant strains was related to a defect in the terminal portions of the complement pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of altretamine in human plasma with high-performance liquid chromatography.

A fast, simple, and sensitive isocratic HPLC method has been developed and validated for the determination of the anticancer drug altretamine in human plasma. Spiked serum samples and clinical plasma samples are extracted with acetonitrile at 4 degrees C and the precipitate removed by filtration. The plasma sample volume required (ca. 0.2 ml) is small and the total analysis time is less than 15 min per sample (including batch-wise pre-treatment). Recovery of altretamine is 99 to 106% for pooled human serum spiked with altretamine in the range 200 ng/ml to 10 mg/ml. In this concentration range, the R.S.D. varies from 1 to 8%. The limit of quantitation is ca. 150 ng/ml for an R.S.D. of 10%. The intra-day R.S.D. for human samples spiked at 5 mg/ml varied between 1.7 and 4%; the inter-day R.S.D. at this concentration was ca. 3%. A preliminary study with one patient receiving 260 mg/m2 by mouth indicated that the peak altretamine concentration was significantly lower after a standard breakfast than in the fasting state.

Identification of antigens of pathogenic free-living amoebae by protein immunoblotting with rabbit immune and human sera.

Prominent antigens of pathogenic and nonpathogenic free-living amoebae were identified by using polyclonal rabbit immune sera in immunoblot assays. The intent was to determine if prominent epitopes identified with rabbit immune sera could also be recognized by human sera. With rabbit sera, the development of immunoreactive bands was restricted to molecular masses of greater than 18.5 kDa for Naegleria, Hartmannella, and Vahlkampfia antigens. Two or more broad bands of less than 18.5 kDa were prominent features in three different Acanthamoeba species. Few cross-reactive antibodies could be detected between representative species of the three different subgroups of Acanthamoeba. Naegleria antigen was likewise serologically distinct, as were Hartmannella and Vahlkampfia antigens. The relative lack of cross-reacting antibodies between the pathogenic amoebae suggested that i would be desirable to use a panel of amoebic antigens to represent the range of serologically distinct antigens for assessing reactive antibodies in human sera. In pooled human sera (10 serum specimens per pool), the appearance of minimally reactive bands ranging from 32.5 to 106 kDa was a common feature of all six antigens. A prominent band of less than 18.5 kDa was identified in the Acanthamoeba culbertsoni antigen lane in 2 of the 10 human serum specimen pools. When sera from each of the two groups were tested individually by immunoblotting, the reaction with A. culbertsoni antigen could be associated with one individual. By using a panel of amoebic antigens, this method could prove useful in recognizing undiagnosed amoebic infections by revealing specific reactive antibodies.

Immune response to Moraxella catarrhalis in children with otitis media: opsonophagocytosis with antigen-coated latex beads.

Opsonic antibody activity against Moraxella catarrhalis was determined in sera from children with otitis media. The antibody was determined with a new assay utilizing outer membrane antigen-coated latex beads. Antigen-coated beads opsonized in heat-inactivated pooled human serum phagocytosed 47.5 +/- 36.1 beads per 100 neutrophils compared to 15.6 +/- 10.2 beads per 100 neutrophils opsonized in hypogammaglobulinemic serum (p < .025). Antigen-coated beads opsonized in homologous sera from 11 children with M. catarrhalis otitis media demonstrated increased opsonic activity in convalescent sera (34.6 +/- 27.1) compared to acute sera (15.5 +/- 6.7; p < .05). These data suggest that infection with M catarrhalis is associated with the development of opsonic antibody.

Complement and Antibody in Neutrophil-Mediated Killing of Type V Group B Streptococcus

Serious infections caused by type V group B streptococci (GBS) are increasing. The requirements for antibody, complement, and neutrophil receptors in the killing of 12 clinical type V GBS isolates were investigated. When tested at concentrations of 33%, 5% and 1%, a human serum pool promoted neutrophil-mediated killing of the 12 isolates at a mean of 83% +/- 9%, 77% +/- 17%, and 14% +/- 28%, respectively. Addition of heated immune rabbit serum to the 5% or 1% pool increased killing significantly (97% +/- 2% or 80% +/- 15%, respectively, P < .01). With hypogammaglobulinemic serum, complement-mediated killing ranged from 74% +/- 2% for a strain designated resistant to 98% +/- 1% for a strain designated sensitive. Neutrophil-mediated killing was not altered by use of human sera deficient in C4 or C7 but was reduced significantly with C3-deficient serum (P < .05). Maximal inhibition of neutrophil-mediated killing was observed by monoclonal antibody blockade of complement receptor (CR) 3 alone or in combination with CR1 or Fc receptor III. Thus, C3 is required and specific antibody promotes neutrophil-mediated killing of type V GBS. Neutrophil CR3 and either CR1 or Fc receptor III optimize phagocytosis. A number of host responses function in concert to effect optimal neutrophil-mediated killing of clinical isolates of type V GBS.

Evaluation of Different Medium Supplements for In vitro Cultivation of Brugia malayi Third-Stage Larvae

Growth and development of Brugia malayi (Nematoda: Filarioidea) third-stage larvae (L3) were compared in 5 medium supplements. The basic culture medium (NI) consisted of a 1:1 (v/v) mixture of NCTC-135 and Iscove's modified Dulbecco's medium, an antibiotic/antimycotic mixture, and 1 of the following 5 supplements: 25 mg/ml bovine albumin fraction-V (BAF), 10% fetal bovine serum (FBS), 10% commercially obtained human serum (CHS), 10-15% pooled human serum from hospital patients (PHS), and 10-15% human serum from a single individual (SHS). Cultures were maintained at 37 C in an atmosphere of 5% CO2 in air. NI-BAF and NI-CHS did not support molting of L3 to fourth-stage larvae (L4), whereas NI-FBS, NI-PHS, and NI-SHS did support molting of L3 to L4 but only the larvae in NI-SHS attempted the fourth molt. Growth and development of in vitro larvae in NI-PHS and NI-SHS were comparable to that observed in jirds for the first 28 days, after which the in vitro larvae lagged behind in vivo larvae. Optimal growth and development may be dependent on certain as yet unidentified components of specific human serum.

Lack of evidence for increased adherent growth in broth or human serum of clinically significant coagulase-negative staphylococci

The relevance of adherent growth of coagulase-negative staphylococci (CNS) in vitro to their clinical significance is controversial. We have investigated the accumulation on polymer surfaces of clinically significant CNS strains, isolated from blood cultures and intravascular catheter tips, compared with controls. The adherent growth of significant CNS isolates in a microtitre tray assay was not greater than controls, and this finding was repeated when different combinations of target polymer surface, culture medium and incubation atmosphere type were investigated. The surface accumulation of CNS cultured in pooled human serum was poor compared with culture in broth. Whole cell and surface protein profiles of serum grown Staphylococcus epidermidis differed markedly from broth cultured cells. We recommend the use of human body fluids, such as serum, as useful culture media to assess whether potential virulence determinants are likely to be expressed in vivo. The microtitre tray assay of adherent growth is not helpful in determining the virulence of individual CNS isolates.

Antigenicity of the oligosaccharide moiety of the Japanese cedar (Cryptomeria japonica) pollen allergen, Cry jI.

Cry jI, a major allergenic glycoprotein of Cryptomeria japonica (Japanese cedar, Sugi), is the most common pollen allergen in Japan. Carbohydrate analysis and lectin staining indicated that Cry jI possesses the fucose/xylose-containing N-linked oligosaccharide which previously has been found in some plant glycoproteins. Rabbit polyclonal anti-Cry jI IgG antibodies were found to be highly cross-reactive with two other plant glycoproteins which have the same type of oligosaccharides, and the cross-reactivities were completely abolished on chemical deglycosylation of the glycoproteins. Enzyme-linked immunosorbent assay inhibition showed that the majority (up to 70%) of the anti-Cry jI rabbit IgG antibodies recognized the oligosaccharide moiety of Cry jI. The carbohydrate epitopes were found to be only partially involved in the binding of specific IgE antibodies from a pool of human patient sera.

Serum-hydroxyapatite interaction in vitro

In a project to develop hydroxyapatites for bone replacement, biological and synthetic types were prepared at 600°C, ground to 300–600μm and immersed in a pooled human serum for periods up to 1 month at 4°C to assess material interaction. It was found that the levels of calcium in the serum were reduced at 6h immersion, followed by an increase to reach maximum at 48h and then stability up to 1 month. Phosphorus levels showed the opposite behaviour. Both apatites showed similar trends, although higher values were recorded for the synthetic type, suggesting higher activity. Infrared spectral analysis complemented the biochemical values, where the optical densities (O.D.) of phosphate groups were reduced, reflecting the increased phosphorus in serum and denoting leaching. Also O.D. values of both CO 3 2− and OH − groups were reduced at 10h, then returned to original levels. Scanning electron microscopy revealed a spongy appearance parallel with reduced O.D. and higher levels of serum Ca 2+. At longer periods (48h) the concentric needles of hydroxyapatite are clearly shown to be deposited on biological apatite. Differences in responses were attributed to their original crystalline structure assessed by X-ray diffraction analysis, as well as pore analysis using a mercury porosimeter.