Ernesto Falcoff

125I-labelled human interferons alpha, beta and gamma: comparative receptor-binding data

Binding of 125I-labelled human recombinant DNA interferons (IFNs) alpha-2, beta and gamma was compared on various human lymphoid cells and embryonic fibroblasts. While binding constants were within an order of magnitude for all three interferons (10(-10) to 10(-9) M), no competition was observed between IFN-gamma on the one hand and IFN-alpha 2 and IFN-beta on the other. However, consistent with previous reports, IFN-alpha 2 and IFN-beta competed for presumably common receptors. Depending on the cell type, binding sites for IFN-gamma were expressed in different numbers compared to those for IFN-alpha 2 and IFN-beta. These direct comparative binding studies support the hypothesis that the receptor system for IFN-gamma is unrelated to the IFN-alpha/beta system.

Suppression of human interferon production by inhibitors of glycosylation

Abstract Treatment of diploid human foreskin cell cultures with either 2-deoxy- d -glucose or d -glucosamine resulted in dose-dependent inhibition of interferon production induced with polyinosinate-polycytidylate [poly(I) · poly(C)]. This inhibitory action was readily demonstrable even if the addition of the inhibitor was delayed until 1.5 hr after induction. The appearance of intracellular interferon activity was also inhibited, suggesting that synthesis of biologically active interferon, and not its secretion from the cell, was the main target of the inhibitory action. The inhibitory action of 2-deoxy- d -glucose on interferon production was completely reversible by d -mannose while the action of d -glucosamine was only slightly affected by the addition of mannose. Interferon produced in the presence of a partially inhibitory concentration of 2-deoxy- d -glucose was more heat-labile than control interferon. Residual active interferon made in the presence of d -glucosamine was less efficiently neutralized by anti-interferon antibody than control interferon. The results are compatible with the idea that the two inhibitors suppress the production of biologically active interferon by virtue of their interfering with the proper glycosylation of the molecule.

Production of antibodies against mouse immune T (type II) interferon and their neutralizing properties.

An antiserum to immune T-type mouse interferon was prepared in rabbits by repeated injections of interferon, induced by phytohaemagglutinin (PHA) in mouse spleen cells and purified to 1 X 10(5) units/mg protein. The antiserum neutralized mouse interferons synthesized in vitro in response to several T mitogens and Brucella organisms, and also the type II interferon present in the serum of BCG-sensitized mice after an injection of the specific antigen, tuberculin. All these interferons are thus antigenically related; they are also all unstable at pH 2. In contrast, the antiserum did not neutralize interferons induced by West Nile virus (WNV) and Newcastle disease virus (NDV) in vitro, or by lipopolysaccharide and Brucella organisms in vivo; all these interferons are stable at pH 2. It also failed to neutralize a non-glycosylated virus interferon prepared in the presence of tunicamycin.

Influence of preincubation on the development of the inhibition of protein synthesis in extracts from interferon-treated mouse L cells action on tRNA

Abstract Treatment of cells with interferon leads to an inhibition of cell-free protein synthesis in preincubated cell extracts. We show here that the observation of such a defect in the translation of both Mengo virus RNA and poly(U,C) depends critically upon the length of preincubation period. Control and interferon-treated cell extracts (Cont. S-10, Int. S-10) preincubated for up to 60 min have identical protein synthetic activity when assayed with either Mengo virus RNA or poly(U,C). Although preincubation of Cont. S-10 for more than 60 min leads to a gradual reduction in the protein synthetic capacity of the extract, the activity of Int. S-10 declines much more rapidly, and after 60 to 120 min of preincubation the translational activity of Int. S-10 is markedly inhibited (80–90%) compared to Cont. S-10. In extracts preincubated for times sufficient to allow the development of this impaired protein synthesis, addition of both total tRNA and a purified leucine tRNA restores synthesis by Int. S-10 to almost the same level as the corresponding controls. Addition of total tRNA, but not leucine tRNA, also overcomes the loss of activity observed in Cont. S-10 extracts which have been preincubated for more than 60 min. Addition of leucine tRNA to Int. S-10 during the preincubation step strongly reduces the ability of the tRNA to restore protein synthesis. Int. S-10 programmed with Mengo virus RNA synthesized the same polypeptides as Cont. S-10 when the preincubation period was 60 min or less. Although preincubation of Cont. S-10 for more than 60 min led to a decline in their activity, the pattern of polypeptides synthesized was not affected. However, preincubation of Int. S-10 for more than 60 min caused a decrease in the size of polypeptides as well as a reduction in their amount. Only peptides of MW 7000 to 26,000, were detected in such extracts. Addition of tRNA to the Int. S-10 restored the synthesis of larger polypeptides in a dose-dependent fashion and, with suitable amounts of total tRNA, the pattern of polypeptides synthesized was identical to that for control extracts. Our results indicate that preincubation of cell-free extracts leads to a decline in their capacity to utilize tRNA and that pretreatment of the cells with interferon strongly enhances this effect, at least with respect to leucine tRNA. The inability of extracts prepared from interferon-treated cells to utilize endogenous tRNA after prolonged preincubation (60–120 min) is most probably due to inactivation of the tRNA.

Intracellular location of newly synthesized interferon in human FS-4 cells

Abstract The distribution of interferon was examined in fractions of human FS-4 diploid fibroblasts induced with polyinosinate-polycytidylate [poly(I) · poly(C)]. In order to synchronize interferon production in the cultures, cells were first induced with poly(I) · poly(C) in the presence of the protein synthesis inhibitor, cycloheximide. After 4 hr, protein synthesis was allowed to resume by removing cycloheximide. Cells were harvested for the analysis of intracellular interferon at 20 min after the removal of cycloheximide, i.e., before substantial quantities of newly synthesized interferon could reach the extracellular environment. Much of the intracellular interferon present in the postnuclear supernatant (PNS) was associated with a membrane fraction that formed a single peak in sucrose density gradients. Treatment with pancreatic ribonuclease which abolished free polysomes did not result in a marked alteration of the distribution of interferon activity in the sucrose gradient, whereas treatment of the PNS with deoxycholate (0.5%) resulted in the release of all interferon activity from the membrane. Unlike free interferon, membrane-associated interferon was resistant to treatment with trypsin-chymotrypsin. Treatment with puromycin in high salt, which caused release on membrane-bound ribosomes, failed to remove interferon from the membrane fraction. These observations suggest that the membrane-associated interferon was contained inside membraneous vesicles. In addition to the interferon found in membrane-bound form, some interferon activity in the PNS of induced cells was present in free form. However, available evidence strongly suggests that this “free” interferon is an artifact due to the release of interferon from membraneous vesicles during the cell homogenization procedure. It is concluded that probably all interferon in this cell system is synthesized on membrane-bound polysomes, discharged into the lumina of the rough endoplasmic reticulum, and further processed during passage through various intracellular membrane compartments.

[78] General procedures for purification of mouse immune interferon

Publisher Summary This chapter presents three techniques based on different chromatographic systems that make it possible to obtain preparations of partially purified interferon. Specific activity is comparable for all three chromatographic methods, but the contaminants accompanying the antiviral activity are different. Simultaneous assay of type II interferon samples purified by these three techniques helps to minimize the possible role of contaminants and allow more reliable examination of this interferons biological and biochemical properties. The relatively large amounts of type II interferon prepared by the method made it possible to prepare a rabbit anti-type II mouse interferon serum. This antiserum neutralized several T mitogen induced interferons as well as the type II interferon that is induced in BCG-sensitized mice by injection of the specific antigen, tuberculin. Furthermore, the anti-type II interferon serum did not neutralize any of the type I interferons tested (virus-induced, lipopolysaccharide-induced, and such). These results indicate that mitogen- and antigen-induced type II interferons are antigenically related, and confirm the earlier observations about the different antigenic properties of type I and type II interferons.

LEPROMATOUS LEPROSY TREATED WITH RECOMBINANT INTERFERON GAMMA: CUTANEOUS HISTOLOGIC CHANGES

We report on the histologic changes occurring in single cutaneous lesions, from six active lepromatous patients, 1 week following the administration of three daily intradermal injections, 35 jag each, of recombinant interferon gamma (nFN‐γ). Except for a strong induration at the injection site, nFN‐γ produced no adverse systemic reactions and was able to promote a remarkable influx of T‐lymphocytes, mononuclear phagocytes with large nuclei, nonvacuolated cytoplasm, and reduced lysozyme reactivity. Furthermore, despite no clear‐cut reduction of mycobacterial dermal burden, bacilli showed a clear increase in the granular appearance. Present findings provide a basis for further elucidation of nFN‐γ as an additional tool for leprosy treatment.

[62] Assay of effect of interferon on vitro primary plaque-forming response to sheep red blood cells

Publisher Summary This chapter presents the assay of effect of interferon on in vitro primary plaque-forming response to sheep red blood cells (SBRC). Various antigens can be used to induce specific primary in vitro antibody production. They are of three classes—namely, (a) particular antigens, such as SRBC (the most commonly used) and burro red blood cells, (b) carrier-hapten complexes, in which a small molecule (hapten) is coupled to a carrier protein. Under these circumstances, the antibodies produced are directed against the hapten. In this class of antigens, the most commonly used are dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH) or dinitrophenyl-human y-globulin (DNPHGG), and (c) antigens, usually haptens, coupled to a B cell mitogen. These antigens directly stimulate B cells and the response does not require T cell help. DNP-aminoethyldextran (DNP-AE-dextran) or TNPlipopolysaccharide (TNP-LPS) are such antigens. The technique describes a typical stimulation of murine spleen cells by SRBC. A batch of SRBC should be selected for its good stimulating capacity and the same sheep used throughout the study. The measure of the interferon-induced suppression of the in vitro plaque-forming cell (PFC) response can easily been done in the highly reproducible culture system described if interferon is added at the beginning of the culture. It is useful to use partially purified interferon because especially when immune interferons are tested, cell culture supernatants may contain factors influencing the in vitro antibody response, such as amplifying the helper factors. The reproducibility of the technique is also dependent on the use of selected batches of Fetal calf serum (FCS) and SRBC.

CHARACTERIZATION OF IMMUNE INTERFERON INDUCED IN NUDE MICE SPLEEN CELLS

Publisher Summary This chapter describes the characterization of immune interferon induced in nude mice spleen cells. Interferons are antiviral proteins synthesized by many kinds of somatic cells in response to viral infection. Specific antigens and T and B lymphocytes stimulants are also able to induce the production of interferon but only in immunocompetent cells. This immune induced interferon, which appears in the course of many immunological events at the same time as other lymphokines, may play a part in regulating the immuno response. In an experiment described in the chapter, it was found that when the total population of spleen cells from nude mice was fractionated on a column of Degalan beads coated with anti-mouse immunoglobulins that removed the Ig + cells, the excluded cell population constituting a fraction enriched in theta-positive cells failed to produce a significant amount of interferon when incubated with PHA. The interferon titer was enhanced when Degalan-excluded cells were added to the total spleen cell population from nude or Balb/C mice. The enhanced phosphorylation of the 67000 MW protein indicates that pretreatment with PHA-interferon induces in the cells a protein kinase system similar to that induced by viral interferon.