F Dianzani

Immune interferon activates cells more slowly than does virus-induced interferon.

Summary The kinetics of activation of the antiviral state by virus induced interferon and by mitogen-induced immune interferon have been studied comparatively. It has been found that both human and murine virus-induced interferons are able to activate the antiviral state after a brief (minutes) contact with the cells. In contrast, several hours were required for both human and mouse immune interferons to induce a comparable level of antiviral resistance. Experiments measuring the binding of the two interferons to cells showed that there was no significant difference in the rate and degree of binding, suggesting that a different total association of interferon with cells could not account for the slower kinetics of activation by immune interferon. Additionally, the possibility that some contaminants present in the immune interferon preparation could nonspecifically interfere with the rapid induction phenomenon is not supported by the finding that the rapid kinetics of cell activation by virus-induced interferon was not modified by the presence of immune interferon. The interesting possibility which remains is that the two interferons may activate cells by different mechanisms. The authors are greatly indebted to Dr. G. Georgiades and Mr. M. Langford for the generous gifts of immune interferon and to Dr. S. Baron for helpful suggestions and criticism.

[51] Virus yield-reduction assay for interferon by titration of Sindbis virus hemagglutinin

Publisher Summary The basic steps in a hemagglutination (HA) reduction assay for interferon are (1) incubation of interferon (IF) dilutions on cultures, (2) challenge of the cultures with a high input multiplicity (MOI) of hemagglutinating virus, and (3) measurement of the reduction of hemagglutinin yield. The chapter presents the rationale for performing such an assay and a detailed description of the procedures. Some of the advantages of the HA reduction assay over the infectious virus yield-reduction assay are that HA titrations are simpler, more rapid, and less expensive than infectious virus titrations and hemagglutinin is usually more stable than infectivity, hence the time of collection of the samples after maximum virus production for HA titration is not as critical as it is for infectivity titrations. The major disadvantages usually encountered are (1) the preparation of reagents needed to obtain the proper pH for optimal HA activity, (2) inhibitors of HA activity that may be present in serum or culture fluids, and (3) the availability of male goose erythrocytes.

[21] Large-scale induction and production of human and mouse immune interferons

Publisher Summary Production of large quantities of immune interferon in cultures is greatly facilitated when a suitable inducer is available. Using the T cell mitogen staphylococcal enterotoxin A, a system for production of sufficient human and mouse immune interferon for partial purification, antibody production, and initiation of clinical trials in humans has been developed. There are two general methods for in vitro induction of immune interferon in lymphocyte cultures. One method involves the use of specific antigen usually added to cultures containing lymphocytes previously sensitized to the antigen. This method is generally impractical with poor yields of immune interferon. The second method involves the use of T cell mitogens. Three such mitogens, phytohemagglutinin P (PHA-P), concanavalin A (Con A), and staphylococcal enterotoxin A (SEA) are compared. An alternative way to produce high concentrations of human immune interferon is to treat lymphocyte cultures with the enzyme galactose oxidase. This method has not yet been adapted to large-scale production, but it shows strong potentiality for such use because of the high yields and the fast kinetics of production.

Inhibition of RNA synthesis in human lymphoid cells induces interferon production.

Abstract Human lymphoid cell cultures produced an interferon-like activity in the absence of inducers. Using actinomycin D, cycloheximide, and other metabolic inhibitors, we have shown that the cellular requirement for de novo RNA synthesis is not a requisite for interferon induction. We show here that the production of an interferon-like protein may be triggered in cultured lymphoid cells by substances capable of suppressing DNA-dependent RNA synthesis. Our data suggest that a rapidly turning over regulatory mechanism controls the production of an interferon-like molecule in lymphoid cells. In fact, the induction by inhibitors of RNA synthesis and the requirement for protein synthesis for production, but not for induction, suggests a preformed messenger RNA for an interferon-like molecule whose translation is normally prevented by a rapidly turning over mechanism which requires ongoing RNA synthesis. Experiments with neutralizing antibody for interferon suggest a new antigenic type of immune interferon or a slightly different molecule with lowered affinity for antibody. The very existence, however, of such a rapidly activated regulation of interferon production infers an important role in lymphocyte response and immune regulation.

Effect of Cell Density on Development of the Antiviral State in Interferon-Producing Cells: A Possible Model of in Vivo Conditions

Summary Tissue culture conditions which mimic the high cell densities n vivo were used to elucidate further the virus, cell, and interferon interactions. To help define some of the variables which govern the action of interferon in the same cells which produce the interferon, the technique of altering the effective interferon concentration by varying the extracellular volume was used. Specifically, mouse L cells were treated with a high-multiplicity Newcastle disease virus at 0° and then resuspended at 37° in two different volumes of prewarmed medium such that each had the same total number of cells but the concentration of cells in one culture was 5 × 106 and that in the other was 5 × 104. Under these conditions both cell suspensions produced the same total amount of interferon, but at interferon concentrations of 320 and 4 units/ ml, respectively. Antiviral resistance developed more rapidly and to higher levels in the more concentrated cells. It was calculated that at the high cell density (closer to that of solid tissue) the production of the antiviral protein occurred not later than 60-75 min after the induction by NDV and the production of interferon itself must have occurred even earlier. Also, the finding that interferon-producing cells developed different levels of antiviral activity in relation to the interferon concentration in the surrounding fluid, rather than to the same internal (total) amount of interferon produced, supports the view that interferon must be externalized to induce resistance. Finally the possibility was raised that secreted interferon might more efficiently induce resistance in the interferon-producing cell than in nonproducing cells.

Tissue Culture Models of In Vivo Interferon Production and Action

Interferon participation in recovery from viral infection is clearly documented by a series of in vitro observations. It has been shown in vitro that the inhibition of interferon production during infection often notably increases the amount of virus produced (Glasgow and Habel 1962). Cultures infected with viruses in conditions favoring good interferon production yield scarce quantities of virus and establishment of inapparent or abortive infections (Isaacs, 1963). The same effect can also be obtained by adding preformed interferon to the infected cultures.