Microtiter assay for interferon: microspectrophotometric quantitation of cytopathic effect.

This report describes an accurate, reproducible, and efficient microassay for human interferon, using a dye-uptake method to quantitate cytopathogenicity. The antiviral activity was measured by using a Gilford 300-N microsepctrophotometer, with the automatic programmer, sampler, and data lister. Two hundred interferon samples, each in a final volume of 1.0 ml, may be analyzed and recorded in 45 min. The reproducibility of a laboratory reference interferon and the human international reference B 69/19 on two different cell lines, using the model Q Oxford sampler, was found to be excellent, with the standard error of the log10 geometric mean of both references +/- 0.04 to 0.06.     

Radioactive human lymphoblastoid interferon. One-step purification, regulation of heterogeneous species production and its use for radioimmunoassay

Human lymphoblastoid interferon (alpha type), labeled with [3H]leucine added to virus-induced Namalwa cells, was purified quantitatively and in one step from the culture fluid by immune precipitation. The material showed, upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, only four radioactive bands with molecular weights ranging from 17000 to 21000, which coincided well with interferon activity. They coincided also with the four interferon protein bands in the electropherogram of unlabeled interferon purified by a different method. The purity of the labeled interferon was ascertained also by polyacrylamide gel electrophoresis in the absence of dodecyl sulfate. Pulse-labeling of interferon with [3H]leucine for 1 h at various times after induction indicated that the cells always synthesized and secreted the four interferon species in parallel during the interferon production period. Competitive radioimmunoassay for human interferon alpha was achieved by the use of purified radioactive interferon, anti-(interferon alpha) serum, and bacterial adsorbent. The immune precipitation of the labeled interferon was inhibited by unlabeled interferon alpha, and 100 international reference units of interferon alpha could be measured in this way.     

Interferon Production in Human Diploid Cell Strains Derived from Embryonic Lungs

Twenty-three strains of human diploid cells derived from embryonic lungs were tested for production of interferon by “superinduction.” Strain HAIN-55 produced a relatively high level of interferon. The optimal concentration of cycloheximide for superinduction was essentially equal to that reported with foreskin fibroblasts. On the other hand, actinomycin D at a concentration of 4 to 16 μg/ml enhanced the production of interferon more strikingly than at a concentration of 1 μg/ml, which was usually employed for superinduction in the foreskin fibroblasts. Inhibition of interferon production was observed when fetal bovine serum was added to the medium during treatment with metabolic inhibitors for superinduction. Minimal essential medium was superior to Eagle's basal medium as growth medium for interferon production, and serum added after removal of metabolic inhibitors could be replaced by bovine serum albumin. The yield of interferon produced under the best conditions in this study, with strain HAIN-55, was more than 10,000 reference units/ml.     

Determination of human leukocyte interferon by a microfluorimetric immunologic method]

The possibility of quantitative determination of human leucocyte interferon using FITC-labeled antiinterferon antibodies was studied. A highly specific antiinterferon immunoglobulin was obtained as a result of longterm immunization of a donkey with human leucocyte interferon followed by fractionation and immuno-absorbtion of immune plasma. This immunoglobulin was labeled with FITC and used for human leucocyte interferon assay in direct and indirect reactions of fluorescence immunoinhibition. The titres of different human leucocyte interferon preparations in this immunoassay were comparable with the titres of the same preparations detected by interferon inhibition of viral cytopathic effect.     

Synthesis of human fibroblast interferon by E. coli

A cDNA library was constructed using mRNA from human fibroblasts induced with poly(I):poly(C). A bacterial clone containing fibroblast interferon cDNA sequences was identified by hybridization to a cDNA probe synthesized using deoxyoligonucleotide primers which hybridize to fibroblast interferon mRNA specifically. Expression plasmids were constructed which permitted the synthesis in E. coli of 8 x 10(7) units of human fibroblast interferon per liter of culture. The bacterially produced fibroblast interferon is indistinguishable from authentic human fibroblast interferon by several criteria.     

Binding of human interferon alpha to cells of different sensitivities: studies with internally radiolabeled interferon retaining full biological activity.

The characteristics of interferon binding to various cells with different interferon sensitivity were studied by using [3H]leucine-labeled, pure human interferon alpha from Namalwa cells. Scatchard analysis of the binding data on cells sensitive to interferon alpha (human FL and fibroblasts and bovine MDBK) indicated the presence of two kinds of binding sites with high and low affinities. The binding constants of the high-affinity sites in these cells were similar (4 X 10(10) to 11 X 10(10) M-1). Cells insensitive to human interferon alpha (human HEC-1 and mouse L cells) were shown to have only low-affinity sites, suggesting that high-affinity binding sites are indispensable for interferon sensitivity and represent interferon receptors. However, the number of sites in three human diploid fibroblast strains and one strain trisomic for chromosome 21 were not proportionally correlated to the interferon sensitivity of the cells. The high-affinity binding to human cells was completely inhibited by both nonradioactive human interferons alpha and beta in a similar manner, but binding to bovine MDBK cells, on which human interferon beta is practically inactive, was inhibited effectively only by interferon alpha and not by beta. These results suggest that the receptor for human interferon alpha is common to human interferon beta in human cells, whereas the receptor on bovine cells binds only human interferon alpha.     

Modulation of the human in vitro antibody response by human leukocyte interferon preparations

The ability of human leukocyte Interferon to modulate the plaque-forming-cell response of human peripheral blood leukocytes to horse red blood cells was examined. Human peripheral blood mononuclear cells were cultured in vitro with the addition of varying doses of human leukocyte interferon 24 hr prior to, simultaneously with, and 24 hr after sensitization of the cultures with horse red blood cells. Plaque-forming-cell responses were measured 5 days after sensitization with antigen using poly-L-lysine-coupled horse red blood cell monolayers. When human leukocyte interferon preparations were added 24 hr prior to sensitization with antigen, a significant enhancement of the plaque-forming-cell response was observed. When the interferon was added simultaneously with antigen, the plaque-forming-cell response was significantly suppressed. Therefore, human leukocyte interferon appears to have a time-dependent immunomodulatory activity. The kinetics of immunomodulation appear to be different from those of previously described mouse models.     

Genetic transfer of a functional human interferon alpha receptor into mouse cells: cloning and expression of its cDNA

A cDNA coding for the human interferon alpha receptor has been cloned using a gene transfer approach. This consists of transferring human DNA to mouse cells and selecting for cells sensitive to human interferon alpha. The transfected cells expressed the human interferon alpha receptor, and a 5 kb human DNA was isolated from a secondary transfectant. This DNA defects an mRNA present in human cells and was used to clone a 2.7 kb cDNA from a library constructed from human Daudi cells. The sequence of the cDNA is presented. It codes for a glycoprotein of 557 amino acids with an N-terminal hydrophobic region and a single transmembrane-spanning segment. Mouse cells expressing the cDNA become sensitive to the antiviral activity of and express binding sites for human interferon alpha, demonstrating that the cloned cDNA encodes a functional human interferon alpha receptor.     

Maximum likelihood methods for nonignorable missing responses and covariates in random effects models

This article analyzes quality of life (QOL) data from an Eastern Cooperative Oncology Group (ECOG) melanoma trial that compared treatment with ganglioside vaccination to treatment with high-dose interferon. The analysis of this data set is challenging due to several difficulties, namely, nonignorable missing longitudinal responses and baseline covariates. Hence, we propose a selection model for estimating parameters in the normal random effects model with nonignorable missing responses and covariates. Parameters are estimated via maximum likelihood using the Gibbs sampler and a Monte Carlo expectation maximization (EM) algorithm. Standard errors are calculated using the bootstrap. The method allows for nonmonotone patterns of missing data in both the response variable and the covariates. We model the missing data mechanism and the missing covariate distribution via a sequence of one-dimensional conditional distributions, allowing the missing covariates to be either categorical or continuous, as well as time-varying. We apply the proposed approach to the ECOG quality-of-life data and conduct a small simulation study evaluating the performance of the maximum likelihood estimates. Our results indicate that a patient treated with the vaccine has a higher QOL score on average at a given time point than a patient treated with high-dose interferon.     

A new quantitative air-lift sampler for collecting macroinvertebrates on stony bottoms in deep rivers

SUMMARY. 1. As three previous comparative studies of deep-water samplers for benthic macroinvertebrates in rivers highlighted the need for a quantitative sampler for use on stony substrata, a new air-lift sampler was developed. It can be operated from a small boat by two people, weighs 13.5–20.0 kg, depending on the length of riser used, and extends the maximum range of substrata that may be sampled quantitatively from 16–32 mm to 128—256 mm. The sampling area is isolated by forcing a collecting cylinder into the substratum, and rapid evacuation of the contents is assisted by a vibrator.
2. All the major specifications of the sampler were determined experimentally in a large tank using three sizes of substrata and plastic pellets to represent invertebrates. The sampler performed accurately to a depth of at least 8 cm on substrata ranging from gravel (2–4 mm) to large stones (32–36 mm long).
3. The performance of the sampler was compared with that of a Ponar grab and Pearson el at. air-lift sampler at two sites on a large river and also with a Naturalist's dredge and a diver-operated Hess-Waters sampler at one of the sites where there were large stones up to 280 mm long. In terms of both mean taxa per sample and mean numbers m⁻², samples taken using the new air-lift sampler provided estimates comparable to or belter than those obtained with the other samplers.     

A quantitative sampler for air-breathing aquatic insects

SUMMARY. A quantitative funnel-trap sampler for air-breathing aquatic insects is described. Performance tests with special reference to adult Glaenocorisa propinqua are presented. Abundance was calculated from pilot surveys with the funnel-trap sampler during 1976 and compared with results from a trial with the capture-recapture method. The influence of temperature on the frequency with which adult Glaenocorisa propinqua renews the bubble gilt was measured.     

Purification of human lymphoblastoid interferon by a simple procedure with high yields

Human Namalwa cell interferon, induced by Sendai virus and composed of a single species with molecular weight of 17,000, was purified to 4.5 X 10(8) international reference units/mg of protein by a combination of salt precipitation, ion exchange chromatography, metal chelate chromatography, hydrophobic chromatography, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. By immunization of a rabbit with this purified interferon and by extensive absorption with Namalwa cells and an impurity column, highly specific antibody was obtained. Namalwa cells, treated with 5-bromo-2'-deoxyuridine, produced 10-fold more interferon upon induction by Sendai virus. Interferon in this case consisted of heterogeneous species with molecular weight ranging from 15,000 to 24,000. These heterogeneous interferon molecules were purified to 7.6 X 10(8) international reference units/mg of protein by successive chromatography using immobilized highly specific rabbit anti-interferon antibody, Blue Sepharose, and immobilized goat anti-rabbit IgG antibody. The overall recovery of interferon activity was 72%, and the purity of the final preparation was ascertained by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.     

Fast Monitoring of Indoor Bioaerosol Concentrations with ATP Bioluminescence Assay Using an Electrostatic Rod-Type Sampler

A culture-based colony counting method is the most widely used analytical technique for monitoring bioaerosols in both indoor and outdoor environments. However, this method requires several days for colony formation. In this study, our goal was fast monitoring (Sampling: 3 min, Detection: < 1 min) of indoor bioaerosol concentrations with ATP bioluminescence assay using a bioaerosol sampler. For this purpose, a novel hand-held electrostatic rod-type sampler (110 mm wide, 115 mm long, and 200 mm tall) was developed and used with a commercial luminometer, which employs the Adenosine triphosphate (ATP) bioluminescence method. The sampler consisted of a wire-rod type charger and a cylindrical collector, and was operated with an applied voltage of 4.5 kV and a sampling flow rate of 150.7 lpm. Its performance was tested using Staphylococcus epidermidis which was aerosolized with an atomizer. Bioaerosol concentrations were measured using ATP bioluminescence method with our sampler and compared with the culture-based method using Andersen cascade impactor under controlled laboratory conditions. Indoor bioaerosol concentrations were also measured using both methods in various indoor environments. A linear correlation was obtained between both methods in lab-tests and field-tests. Our proposed sampler with ATP bioluminescence method may be effective for fast monitoring of indoor bioaerosol concentrations.     

Detergent extraction of the human alpha-beta interferon receptor: a soluble form capable of binding interferon.

After examining a variety of detergents we find that receptors for human alpha interferon can be solubilized, in active form, from plasma membranes of lymphoid cells using the detergent CHAPS. The complexes formed, in solution, with interferon are stable enough to be separated chromatographically.     

Sensitivity of influenza a viruses to human interferons in human diploid cells

Abstract The sensitivity of influenza virus to the action of natural human interferon (IFN)-α+β and -γ, and to the action of highly purified recombinant HuIFN-αB, -αD, and -αF, has been investigated. A plaque assay for the fowl-plague strain of influenza A virus has been established using human embryonic foreskin (HEF) cells. The sensitivity of influenza virus to all IFNs tested in this assay was comparable to that shown by vesicular stomatitis virus (VSV) which was taken as the reference standard. The high sensitivity to IFN action found for the fowl-plague strain was confirmed for the WSN strain of human origin in a yield reduction assay.     

Inducible high level synthesis of mature human fibroblast interferon in Escherichia coli.

We have obtained high level synthesis in Escherichia coli of mature human fibroblast interferon using a plasmid vector that was designed to allow easy coupling of a DNA coding region to the initiator AUG of the replicase gene of the RNA phage MS2 cloned downstream of phage lambda's leftward promoter. The activity of the promoter can be regulated by temperature. Induced cells accumulated the interferon up to 4% of the total cellular protein. The biological activity of the product amounted to 4 X 10(9) international units per litre of culture. The synthesis of human fibroblast interferon was shown to drastically inhibit the growth rate of the bacterial host.     

Optimization of protective properties of interferons in human cells exposed to mutagens estimated by the DNA repair activity

Protective properties of human interferons against physical and chemical mutagens have been described earlier. This work was aimed at detecting an optimum of protective action of interferons in human fibroblasts using two criteria: the number of single-strand DNA breaks formed and the index of DNA repair synthesis. The protective ability of interferon was shown to be expressed starting after 4 h of cells' pretreatment and proceeding through 40 h in experiments with N-methyl-N-nitro-N-nitrosoquanidin. The phenomenon of stimulation of DNA repair synthesis in human cells pretreated with interferon proceeded even after replating cells during 8 h in the experiments with UV irradiation.     

Comparative study of airborne pollen counts located in different areas of the city of Córdoba (south-western Spain)

Airborne pollen counts are mainly determined using a volumetric suction sampler based on the impact principle, that is, a Hirst-type spore trap. As a consequence of their volumetric nature, samplers detect pollen from a wide area, and therefore, a single sampler is frequently used to acquire information on airborne pollen counts for the whole city. The main goal of the present study was to compare airborne pollen counts at two sites located at opposite ends (south-west vs. north-east) of the southern Spanish city of Córdoba, to assess the advantages and disadvantages of using more than one sampler in the city. Also, a comparative study was carried out using two samplers at the same site, in order to confirm the efficiency of the samplers. Results revealed that data from one volumetric sampler—located within a city of medium size with uniform topography and vegetation conditions—are sufficient to establish monitoring of the main airborne pollen types, the pollen seasons involved and the timing of peak counts. For clinical studies, however, data on pollen counts in specific areas of the city may be of value, since pollen intensity may vary from one district to another, mainly in the case of ornamental plants with a local distribution inside the city. Comparison of data obtained by the two samplers running at the same site indicated that potential inter-site differences could not be attributed to differences in sampler efficiency.     

Binding of human fibroblast interferon to concanavalin A-agarose. Involvement of carbohydrate recognition and hydrophobic interaction.

Human fibroblast interferon binds to a concanavalin A-agarose (Con A-Sepharose) equilibrated with methyl alpha-D-mannopyranoside, or levan; in contrast, it is only partially retarded on a similar column equilibrated with ethylene glycol. Interferon does not bind, however, to a lectin column equilibrated with both methyl alpha-D-mannopyranoside and ethylene glycol. Thus, a hydrophobic interaction between fibroblast interferon and the immobilized lectin seems to account for a large portion of the binding forces involved. Other hydrophobic solutes, such as dioxane, 1, 2-propanediol, and tetraethylammonium chloride, were found equally or more efficient than ethylene glycol in displacing interferon from the lectin column. The elution pattern of interferon from a concanavalin A-agarose (Con A-Sepharose) column, at a constant ehtylene glycol concentration and with an increasing mannoside concentration, reveals the existence of four distinct interferon components. The selective adsorption to and elution from a concanavalin A-agarose (Con A-Sepharose) column resulted in about a 3000-fold purification of human fibroblast interferon and complete recovery of activity. The specific activity of the partially purified interferon preparation is about 5 X 10(7) units per mg of protein. The chromatographic behavior of human leukocyte interferon is remarkable in that it does not bind to concanavalin A-agarose at all indicating the absence of carbohydrate moieties recognizable by the lectin, or if present, their masked status. When concanavalin A was coupled to an agarose matrix (cyanogen bromide activated) at pH 8.0 and 6.0 human fibroblast interferon bound to both lectin-agarose adsorbents and could be recovered with methyl alpha-D-mannopyranoside. Concanavalin A, immobilized directly on agarose matrix at pH 8.0 and 6.0, thus displays only carbohydrate recognition toward interferon. By contrast, unless a hydrophobic solute was included in the solvent containing methyl mannoside, human fibroblast interferon could not be recovered from concanavalin A-agarose coupled at pH 9.0. When concanavalin A was immobilized via molecular arms, in tetrameric as well as dimeric forms, the binding of interferon again occurred exclusively through carbohydrate recognition. Thus, the hydrophobic interaction can be eliminated by appropriate immobilization of the lectin, and then adsorbed glycoproteins, as exemplified here by interferon, can be recovered readily with methyl mannoside alone.     

Novel Approach to Activity Evaluation for Release-Active Forms of Anti-Interferon-Gamma Antibodies Based on Enzyme-Linked Immunoassay

Selection of a suitable assay to measure the activity of drug agents based on release-active forms of anti-interferon-gamma antibodies (RA forms of Abs) is an important step forward in the investigation of such agents. In this study, the enzyme-linked immunosorbent assay was utilized to examine the effect of RA forms of Abs specific for human interferon gamma on the interaction between monoclonal anti-interferon gamma antibodies and recombinant human interferon gamma. The experimental data and the results obtained by using relevant mathematical analysis showed that such RA forms of Abs are able to modulate the monoclonal antibody interaction with both soluble and immobilized (to the assay plate well) interferon gamma. These data demonstrated the importance of using relatively low concentrations of both soluble and plate-immobilized interferon gamma to detect the effects of RA forms of Abs to interferon gamma on the binding of monoclonal antibodies to interferon gamma. It has been suggested that the observed influence of RA forms of Abs on ‘antibody-antigen’ interaction could be used to detect and analyze the activity of drugs containing RA forms of Abs.