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An improved bioassay for measuring neutralizing antibodies to interferons (IFN) is described. The assay is based upon an objective and precise quantification of the viral cytopathic effect. This effect is measured via the dehydrogenase-system in cells, and quantified spectrophotometrically. Virus-infected cells, in contrast to non-infected cells, possess low enzyme activity resulting in low OD signals. This fall in OD can be prevented by the addition of a small, but fixed amount of IFN before the addition of virus. Anti-IFN sera will neutralize the protective effect of IFN. This effect can be quantified by measurement of the reduction in the OD signals. Antibodies to recombinant IFN were found to cross-react with human leukocyte IFN although to a ten-fold lower degree. The assay requires no expensive reagents, it is performed in 96-well microtrays and the results can be measured in an ordinary ELISA scanner. The assay is highly reproducible, yielding inter- and intra-assay variability of less than 10%. The sensitivity is much higher than that reported previously for the CPE technique and that of ELISA techniques.

A biological method for precise quantification of interferons has been developed. The method is based upon the dehydrogenase system of the intact target cells, which will normally convert an artificial substrate, MTT, into formazan (blue), which, in turn, can be measured spectrophotometrically. This conversion is greatly reduced by cytocidal viruses in a dose-dependent manner. The protection of target cells by interferon against challenge virus is reflected in a diminished reduction in the production of formazan, thus giving a very precise method for quantification of interferon. The lowest level of detection is around 0.10 international units. The intra- and inter-assay variability appear to be below 10%. The assay, which makes no use of expensive ingredients, is performed in 96-well micro-trays and read in an inexpensive ELISA-scanner.

Normal human peripheral blood lymphocytes were incubated in the presence of different concentrations of interferon for various incubation periods. Subsequently, the amount of beta 2-Microglobulin and HLA-A, B and C surface antigens was estimated by means of quantitative immunofluorescence (flow cytofluorometry) and by a radioimmunoassay for beta 2-Microglobulin. It was found that the amounts of these MHC antigens increased in a dose and time-dependent way after interferon treatment. Furthermore, the influence of different temperatures on this IFN-induced increase in beta 2-Microglobulin was gradually enhanced after incubation at 37 degrees C to 39 degrees C incubation mostly suppressed the beta 2-Microglobulin increase observed at 39 degrees C. The total amount of membrane associated beta 2-Microglobulin was estimated by a radioimmunoassay. After interferon treatment a beta 2-Microglobulin increase was observed ranging from 39% to 122% in different experiments. It is shown that interferon is the substance responsible for the effects on lymphocytes reported here, because completely pure interferon-proteins had the same activity as has been shown for partially purified interferon preparations.

Human leucocyte interferon (HuLeIF) has now for the first time been purified by a series of techniques involving precipitation, gel filtration, and affinity chromatography with Cu-chelate, blue dextran, and antibody. The two major species of HuLeIF were identified as two clearly separable and stainable proteins representing 85% of the biological activity with molecular weights of 18,400 and 20,180 daltons. Three more subspecies of HuLeIF were demonstrated with molecular weights of 19,500, 20,900 and 22,130 daltons, representing 15% of the biological activity. Specific activity of pure interferon is 2 x 10(9) interferon units/mg protein. Recovery was about 50%, and the purification factor exceeded 350,000.

Human Leukocyte Interferon, Human Fibroblast Interferon and Namalva-interferon was purified on an antibody affinity colomn up to 2-20 X 10 IFU/mg protein with recoveries in the 80-120% range. The colomn was made by coupling highly absorped anti-leukocyte interferon immunoglobulins to Sepharose 4B. The absorption was performed by means of crude human leukocyte interferon covalently bound to Sepharose 4B.

Human fibroblast interferon preparations were completely stabilized to 100 degrees C by sodium dodecyl sulphate (SDS) in the presence of mercaptoethanol, but only a minor fraction of their activities were stabilized by SDS without mercaptoethanol. On the contarary, human leukocyte interferon preparations were completely stabilized to 100 degrees C by SDS in the absence of mercaptoethanol, but only a minor fraction of their activities were stabilized by SDS in the presence of mercaptoethanol. Furthermore, human fibroblast interferon preparations whose activities had been destroyed by boiling at 100 degrees C were completely reactivated by SDS under reducing conditions, but only a minor part of their activities were restored by SDS in the absence of reduction. On the contrary, human leukocyte interferon preparations whose activities had been destroyed by boiling at 100 degrees C were completely reactivated by SDS in the absence of reduction, but only a minor part of their activities were restored by SDS under reducing conditions. These data suggest that there are distinct molecular species of human interferons.

The treatment of 10 patients having neoplastic disease with exogenous i.m. interferon therapy is described. The interferon given is partially purified interferon produced from human leukocytes. Sendai virus is used as interferon inductor. The patients reported in this paper have been on treatment for periods of 2-28 months. Apart from initial periods of fever, no side-effects have been recorded. Patients suffering from bladder papillomas have shown partial regression after a few months of therapy. The other cases treated are too few to warrant any conclusions, but the therapy does seem to have been beneficial.

Antibodies against partially purified human leucocyte interferon (PIF) were bound to Sepharose 4B and crude interferons applied on this affinity column were purified, up to 8 x 10(5) interferon units (IFU) per mg protein in one step. Antibodies against PIF were absorbed with immobilized crude human leucocyte interferon bound to Sepharose, whereby antibodies against impurities were predominantly removed. Extensively absorbed antisera were coupled to Sepharose and used for antibody affinity chromatography of crude interferon preparations. Leucocyte and fibroblast interferons were purified in one step with around 100% recovery, up to 1 x 10(8) IFU per mg protein, and Namalva interferon up to 2 x 10(7) IFU/mg. SDS electrophoresis of affinity-purified leucocyte interferon revealed that the interferon activity appeared in two bands (19,000 and 23,000 D).

Antibodies to crude leukocyte interferon (CIF) were bound to Sepharose 4B, and CIF was chromatographed. A relationship between recovered interferon and percentage binding of the antibodies to Sepharose 4B was observed. Highest recovery was obtained when only 50%-85% of th- antibodies were bound. When CIF was subjected to normal affinity chromatography, specific activity of 1-5 X 10(6) interferon units (IFU) (69/19B-units)/mg protein was obtained. When this purified preparation was further chromatographed on a column mainly directed against normal cellular proteins and then concentrated by normal affinity chromatography, a tenfold increase in 'direct' specific activity was obtained. It is suggested that the 'indirect' specific activity is about 2 X 10(8) IFU/mg protein.

Interferons produced in human peripheral leukocytes (LE) and foreskin fibroblast (FS-4) cells were subjected to affinity chromatography on Sepharose-bound globulins from rabbits immunized with these interferons. Anti-LE interferon sera neutralized both interferons, but titers against FS-4 interferon were consistently lower than those against LE interferon. Anti-FS-4 interferon sera neutralized only FS-4 but not LE interferon. Accordingly, affinity columns constructed with anti-FS-4 globulin excluded LE but not FS-4 interferon, whereas those prepared with anti-LE interferon globulin bound and eluted both LE and FS-4 interferons. Purification of native interferons of both types on anti-LE interferon-Sepharose ranged from 680- to 3,600-fold and recoveries from 72 to 126%. Specific activities of eluate pools varied from 4 to 30 times 10-6 reference (B, 69/19) units per milligram protien.

Polymorphonuclear leukocytes (PMN) from healthy volunteers were tested for ADCC activity against both erythrocyte and tumor targets with and without the addition of human leukocyte interferon (IFN). It was demonstrated that IFN within 30 to 60 min enhanced the reaction in a dose-dependent manner with minimal IFN doses ranging from 1 to 100 units. Formal proof that the augmenting agent was IFN was obtained by using pure IFN proteins in combination with both mock-IFN preparations, which showed no enhancing activity, and anti-IFN antisera, which inhibited the action of the completely purified IFN proteins. In the light of data demonstrating that the IFN effect was most pronounced when the IgG antibodies in the ADCC reaction were present in suboptimal amounts, it is hypothesized that IFN may play a special role in the early nonspecific immune response against non-self antigens.

Rabbit antisera prepared against interferon produced in human fibroblast cell cultures stimulated with poly(1).poly(C) neutralized the activity of interferon preparations produced in various human fibroblast cultures timulated either with poly(1)poly)C) or with viruses. However, these antisera showed no detectable neutralizing activity against interferon produced in cultures of human leukocytes. On the other hand, most rabbit antisera against the human leukocyte interferon were active in neutralizing both homologous interferon and fibroblast interferons. A preparation of antiserum against leukocyte interferon, active against both leukocyte and fibroblast interferons, was shown by affinity chromatography to have two distinct antibody populations, one of which was specific for the fibroblast interferon. We conclude that the heterologous neutralizing activity of sera from rabbits immunized with leukocyte interferon is liekly to be due to the presence of two antigenic species of interferon. The major antigenic species of leukocyte interferon preparations (designated "Le") is distinct from huamn fibroblast interferon. The minor species of leukocyte interferon ("F") is either identical with, or closely related to, interferon produced in human fibroblast cultures.

Human leucocyte interferon (HuLeIF) was purified by a series of techniques involving precipitation, gel filtration, Cu-chelate-, blue dextran- and antibody-affinity chromatography. The two major species of HuLeIF were identified in SDS-PAGE as two clearly separable and stainable proteins representing 85% of the biological activity. Three more species of HuLeIF representing 15% of the biological activity were also demonstrated. The specific activity of pure interferon proteins was approx. 10(9) IFU/mg protein. Recovery was about 50% and the purification factor exceeded 350000. All five species of HuLeIF had definite anticellular activities when tested with Daudi cells (inhibition of thymidine uptake).

Purified human lymphocyte interferon (PIF) was added to mixed lymphocyte cultures; DNA synthesis was measured and killer-cell generation was determined. At certain concentrations, PIF ingibited proliferation, but at the same time increased the killer efficiency of the resultant culture cells. It is suggested that lymphokines, apart from their normal mediator function, may play a role as regulators of the generating immune response.

Mononuclear cells from the blood of healthy normal humans were kept in cultures under nonstimulating conditions for 16 hr in the presence or absence of human interferon. The relative quantities of HLA antigens and beta(2)-microglobulin on the cultured cells were determined by quantitative immunofluorescence (fluorescence-activated cell sorter) and by the capacity of cells to absorb out cytotoxic antibodies against the relevant antigens. Interferons of different origin and purities enhanced the expression of HLA antigens and beta(2)-microglobulins, whereas membrane immunoglobulins and antigens recognized by antiserum raised against human brain and T cells were the same on interferon-treated and control cells. Similar interferon effects were observed on an Epstein-Barrvirus-negative Burkitt lymphoma cell line. The enhanced expression of histocompatibility antigen subsequent to intereferon treatment was observed on B- and T-enriched lymphocyte populations and was found to be dose dependent with the optimum with "physiological" concentrations of interferon. Pretreatment of lymphocytes with interferon for 2 hr was found to be as effective as having interferon present during the total culture period. The interferon-induced enhancement of antigen expression on cells was dependent on active protein synthesis.

Mixed lymphocyte cultures (MLC) showed decreased thymidine incorporation when interferon (IF) had been added to the culture medium. The cell-mediated lympholysis (CML) potential generated in mixed lymphocyte cultures grown in the presence of IF was substantially augmented at a certain concentration range. Highly purified leucocyte IF also had this effect, whereas mock preparations did not. The CML-augmenting property was found in the antiviral fraction after separation of semipurified leucocyte IF (by the method of Dahl & Degree). The possibility that the effects were due to a shift in kinetics after culturing in the presence of IF has been excluded. The killer-enhancing properties of IF did not seem to be due to an enhanced expression of histocompatibility antigens, the selective inhibition of CML suppressor cells, or a change in the specificity of recognized target cells. The results of secondary in vitro MLC-CML experiments, in which primary MLC had been carried out in the presence or absence of IF, indicated that the compound acts (in vitro) through preferential selection of cells that are less sensitive to inhibition by IF and more likely to kill.

Background: CD3(+) CD56(+) natural killer T (NKT)-like cells are a subset of T cells characterized by expression of NK receptors and potent antitumour activity. It has also been suggested that they have a role in autoimmune disease, and levels of NKT-like cells are elevated in patients with coronary disease.Objectives: To investigate whether high levels of CD3(+) CD56(+) NKT-like cells are associated with an increased incidence of cardiovascular disease and a lower incidence of cancer.Methods: This was a prospective study including 700 subjects participating in the baseline investigation of the Malmö Diet and Cancer study between 1991 and 1994. Leucocytes obtained at the baseline investigation and stored at -140 °C were thawed and CD3(+) CD56(+) cells analysed by flow cytometry. The incidence rates of cancer and coronary events during a mean follow-up of 15 years were determined through national registers.Results: Subjects in the lowest tertile of interferon (IFN)-γ-expressing CD4(+) CD56(+) cells were found to have an increased risk of incidence of coronary events (log-rank test: P < 0.05). This association remained significant after controlling for age, sex, smoking, body mass index, hypertension, diabetes and the Th1/Th2 and Th1/Treg cell ratios in a Cox proportional hazards regression model (hazard ratio 1.98, 95% confidence interval 1.24-3.16), but not when the LDL/HDL ratio was included in the model. There were no associations between CD3(+) CD56(+) NKT-like cells and incident cancer.Conclusions: The present results could not confirm the hypothesis that low levels of CD3(+) CD56(+) NKT-like cells are associated with a higher incidence of cancer and a lower incidence of cardiovascular disease. However, we found that low levels of IFN-γ-expressing CD3(+) CD4(+) CD56(+) NKT-like cells were associated with an increased incidence of coronary events and that this association may be dependent on lipoproteins. [ABSTRACT FROM AUTHOR]