Major histocompatibility complex diversity is positively associated with stream water temperatures in proximate populations of sockeye salmon.

Local adaptation to heterogeneous environments generates population diversity within species, significantly increasing ecosystem stability and flows of ecosystem services. However, few studies have isolated the specific mechanisms that create and maintain this diversity. Here, we examined the relationship between water temperature in streams used for spawning and genetic diversity at a gene involved in immune function [the major histocompatibility complex (MHC)] in 14 populations of sockeye salmon (Oncorhynchus nerka) sampled across the Wood River basin in south-western Alaska. The largest influence on MHC diversity was lake basin, but we also found a significant positive correlation between average water temperature and MHC diversity. This positive relationship between temperature and MHC diversity appears to have been produced by natural selection at very local scales rather than neutral processes, as no correlation was observed between temperature and genetic diversity at 90 neutral markers. Additionally, no significant relationship was observed between temperature variability and MHC diversity. Although lake basin was the largest driver of differences in MHC diversity, our results also demonstrate that fine-scale differences in water temperature may generate variable selection regimes in populations that spawn in habitats separated by as little as 1 km. Additionally, our results indicated that some populations may be reaching a maximum level of MHC diversity. We postulate that salmon from populations in warm streams may delay spawning until late summer to avoid thermal stress as well as the elevated levels of pathogen prevalence and virulence associated with warm temperatures earlier in the summer.

The incidence of antibody formation to OKT3 consequent to its use in organ transplantation.

Enzyme-linked immunosorbent assays were performed on 12,133 serum samples to determine the incidence of anti-OKT3 antibody formation among transplant recipients who had received OKT3 for rejection treatment or prophylaxis. High anti-OKT3 antibody titers (> or = 1:1000) were detected in 5.8% of samples drawn 2 to 8 weeks following initiation of OKT3 therapy. The frequency of high titers differed by organ (6.9%, 2.7%, and 5.3% for kidney, heart, and liver, respectively; P < 0.001) and by sampling times (P < 0.001). The highest frequency of positive titers was obtained in samples obtained between 2 and 4 weeks following the initiation of OKT3. For all transplant recipients and for kidney recipients alone, multivariate logistic regression showed that the risk of high anti-OKT3 titers varied significantly at 2 to 4 weeks and at 4 to 6 weeks (but not at 6 to 8 weeks) with age (the youngest patients had the highest incidence, with a steady decline after age 30; P < 0.05), course of therapy (lowest frequencies followed a first course of OKT3; P < 0.001), and transplant number (lowest frequencies followed a first transplant; P < 0.01). Analyses of a set of patients on whom immunosuppressive regimen information was available indicated that prophylactic or maintenance treatment with CsA was associated with a significantly lower frequency of high-titer anti-OKT3 antibodies than was therapy without CsA (P < 0.001). In conclusion, this series provides confirming evidence that high-titer anti-OKT3 antibodies, which are of concern whenever retreatment with OKT3 is contemplated, occur in a low percentage of patients and are associated with such factors as age, previous transplantation or courses of therapy with OKT3, and treatment with CsA.

Applications of electrospray mass spectrometry to erythropoietin N- and O-linked glycans.

The improved sensitivity and soft ionization characteristics of electrospray (ES) mass spectrometry (MS) has been applied to the glycan structures of recombinant erythropoietin (rEPO). The four glycopeptides (O-126, N-24, N-38, N-83) were mapped, isolated, deglycosylated, and the glycans profiled (without desialylation) by ES-MS as their methyl derivatives. The O-linked glycopeptide was also analyzed directly. In this fraction seven glycans were identified (two previously unreported) in addition to the unglycosylated peptide. The three N-linked fractions were divided with one fraction preceded by periodate oxidation and reduction to resolve isomeric structures, linkage, and branching patterns, and confirm the overall structural motif. All were methylated and profiled. Two biantennary, 8 triantennary, and 10 tetraantennary structures were identified with approximately 13% of each N-linked glycan possessing a single N-glycolylneuraminyl analog. The minimal energies of ionization, the absence of a matrix background, and enhanced sensitivity brings an improved technology for studying carbohydrate structural detail.

Quantitative mapping of the N-linked sialyloligosaccharides of recombinant erythropoietin: combination of direct high-performance anion-exchange chromatography and 2-aminopyridine derivatization.

A rapid quantitative analysis of the sialylated N-linked oligosaccharides of recombinant erythropoietin (EPO) expressed in Chinese hamster ovary (CHO) cells has been developed. The procedure utilizes a glycoamidase (glycopeptidase F) to release all of the N-linked oligosaccharides from the native glycoprotein, followed by direct chromatographic analysis using high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection. The eight sialyloligosaccharides isolated from HPAEC were characterized by derivatizing with 2-aminopyridine followed by two-dimensional HPLC mapping of the pyridylaminated asialooligosaccharides (Tomiya et al., 1988, Anal. Biochem. 171, 73-90). Seven kinds of complex-type asialooligosaccharides were identified ranging from a biantennary structure to N-acetyllactosamine-extended tetraantennary structure. Approximately 3% of the terminal galactose residues of the oligosaccharides released from EPO were not sialylated whereas 97% contained an alpha(2-->3)-linked sialic acid. Quantitative oligosaccharide mapping of four different lots of EPO from CHO cells was performed to quantify the molar balance and distribution of the N-linked oligosaccharides. The sialyloligosaccharides were distributed with approximately 5% disialylated (single type), 20% trisialylated (six types), and 75% tetrasialylated (four types) oligosaccharides with an average molar recovery of 85% starting from 750 pmol of EPO.

Separation of carbohydrate-mediated microheterogeneity of recombinant human erythropoietin by free solution capillary electrophoresis. Effects of pH, buffer type and organic additives.

Free solution capillary electrophoresis has been investigated as an alternative to isoelectric focusing for the separation of the glycoforms of recombinant human erythropoietin (r-HuEPO), a primary regulator of erythropoiesis. A systematic approach was used to study the effect of pH, buffer type and organic modifiers on the resolution of the microheterogeneity of erythropoietin. The main factors for improving the resolution were the regulation of the electroosmotic flow of the running buffer and the reduction of solute-wall interaction. The best resolution of the glycoforms of r-HuEPO was obtained with a mixed buffer pH 4.0 (100 mM acetate-phosphate, 10 h preequilibration time).

Monoclonal-antibody-based enzyme-linked immunosorbent assay for Trichomonas vaginalis.

Trichomonas vaginalis is estimated to infect 4 million women per year in the United States. The diagnosis of trichomoniasis is predominantly achieved by direct microscopic examination of vaginal exudates. This subjective diagnostic procedure is reported to be 75% sensitive under ideal circumstances. We have developed an enzyme-linked immunosorbent assay (ELISA) for the detection of T. vaginalis directly from vaginal exudates. The ELISA employs a monoclonal antibody specific for a 65-kilodalton surface polypeptide of T. vaginalis as the capture antibody in a sandwich format. A polyclonal rabbit anti-T. vaginalis antibody labeled with horseradish peroxidase serves as the probe. An evaluation of vaginal specimens from women attending clinics revealed a sensitivity and specificity of the ELISA of 89 and 97%, respectively, versus the culture technique. These results indicate the usefulness of this ELISA as an alternative to microscopic and culture methods for the detection of T. vaginalis in vaginal exudates.

Regulation by interferon gamma of function in the acute monocytic leukemia cell line, THP-1.

THP-1 is an acute monocytic leukemia cell line which acquires phenotypic and functional monocytoid-like features following incubation with mezerein. The current study concerned the modulation of these features by rIFN gamma. rIFN gamma induces the time-dependent enhancement of HLA-DR expression in the presence or absence of mezerein but has no effect on the expression of Leu-M1, Leu-M2, or Leu-M3 antigens. CSF-1 production following mezerein activation was reduced by incubation in the presence of 10(3) and 10(4) units/ml rIFN gamma. This was confirmed through both biological assays with mouse bone marrow cells and an indirect ELISA. In contrast, the concentration of growth inhibitory activity in conditioned medium was increased by rIFN gamma. A small but significant increase in IL-1 beta concentration in conditioned medium was detected using a sensitive double-antibody ELISA and a radioimmunoassay. The results infer that the functional characteristics of this leukemia cell line are modulated by rIFN gamma in a manner qualitatively similar to that reported for IFN gamma treated normal monocytes.

Development and use of a radioimmunoassay for human interleukin-1 beta.

Interleukin-1 (IL-1) mediates immunological, physiological, and metabolic changes associated with inflammation and host defense systems. Measurement of IL-1 activity can vary with the bioassay employed and substances have been described in several bioassay systems that either inhibit or mimic IL-1 activity. We now report a sensitive, rapid (24 hour), and specific radioimmunoassay for human IL-1 beta. Using 125I-labeled IL-1 beta and polyclonal rabbit antisera raised against recombinant human IL-1 beta, a competitive inhibition assay is described which detects 250 pg/ml of recombinant human IL-1 beta and 500 pg/ml of pI 7 human monocyte IL-1. The assay does not detect human IL-1 alpha, human interleukin-2, human tumor necrosis factor-alpha or human interferon-gamma. Nearly 100% of IL-1 beta added to human serum or urine can be quantitatively recovered. Substances such as fetal calf serum, phytohemagglutinin, opsonized Staphylococcus albus or E. coli endotoxin do not affect the assay. Using this assay, IL-1 beta was measured in both the intracellular and extracellular compartments of stimulated human blood mononuclear cells. These results were unaffected by the presence of substances that interfere with bioassays for IL-1 such as indomethacin or BW 755C, a lipoxygenase inhibitor. These studies establish the usefulness of quantitating immunoreactive human IL-1 beta produced by human blood cells and that which may be present in human body fluids in the presence of other cytokines.

A fluorescence immunoassay for soluble antigens employing flow cytometric detection.

A "sandwich" fluorescence immunoassay is described which does not require the physical separation of bound from free label. Antibody coated microspheres, sample and fluorescent antibody are reacted together as in a conventional 'sandwich' immunoassay except that separation and washing steps are omitted. After the reaction is completed, the suspension is introduced directly into a flow cytometer equipped with a laser light source and both fluorescent and scattered light detection capabilities. By gating fluorescence light accumulation on scattered light pulses, particles associated fluorescence may be selectively measured. The system was evaluated in a model immunoassay for human immunoglobulin (hIgG), employing anti-hIgG coated microspheres (1--5 micrometer and 40--50 micrometer polyacrylamide beads and 30--40 micrometer dextran beads), fluorescein-labeled rabbit anti-hIgG and a Spectrum III flow cytometer. Sensitivities of 10 ng/ml and intra-assay precisions of 2--10% were achieved in a serum matrix. The approach potentially provides a general nonseparation immunoassay format for quantitatively measuring both small and large molecular weight soluble antigens, as well as cell surface antigens.

Improved radioimmunoassay technique for measuring serum thymopoietin.

An improved radioimmunoassay technique for measuring serum thymopoietin has been developed which obviates the spurious displacements previously obtained with serum samples, and increases sensitivity to 20 pg. Key improvements involved further purification of labeled thymopoietin, sequential incubations to enhance sensitivity and the use of charcoal-treated serum blanks to render controls and unknown samples comparable to the standard curve.

Graphical determination of specific activity, binding constants, and antibody-site concentrations for radioimmunoassays, with application to thymopoietin.

A graphical procedure for determining the specific activity of radiolabeled ligands has been developed for use with radioimmunoassays. Although with this procedure we utilize the same experimental information required for displacement analysis, we are also able to determine both the specific activity and the binding constants of the labeled and unlabeled materials without assuming that these constants are equal; the concentration of antibody-binding sites can also be calculated. Thus, this graphical technique permits calculation of additional information without additional experimentation. We applied this procedure to the labeled materials used in a thymopoietin assay, testing two different preparations of radiolabeled material, and saw negligible differences between the two. The specific activity determined from the displacement analysis correlated well with that calculated by the graphical procedure.