G. John Stanton

The Interferons: Mechanisms of Action and Clinical Applications

The interferons (IFN) are one of the bodys natural defensive responses to such foreign components as microbes, tumors, and antigens. The IFN response begins with the production of the IFN proteins (alpha, beta, and gamma), which then induce the antiviral, antimicrobial, antitumor, and immunomodulatory actions of IFN. Recent advances have led to Food and Drug Administration approval of five clinical indications for IFN. Interferon alfa is approved for hairy-cell leukemia, condyloma acuminatum, Kaposis sarcoma in the acquired immunodeficiency syndrome, and non-A, non-B (type C) viral hepatitis. Interferon gamma has properties distinctive from those of IFNs alpha and beta and is approved as an immunomodulatory treatment for chronic granulomatous disease. Promising clinical results with IFNs have also been reported for basal cell carcinoma, chronic myelogenous leukemia, cutaneous squamous cell carcinoma, early human immunodeficiency virus infection, hepatitis B, and laryngeal papillomatosis. Future clinical uses of IFNs may emphasize combination therapy with other cytokines, chemotherapy, radiation, surgery, hyperthermia, or hormones.

[49] Virus plaque-reduction assay for interferon: Microplaque and regular macroplaque reduction assays

Publisher Summary This chapter describes procedures to perform microplaque and regular (macroplaque) plaque reduction Interferon (IF) assays. The plaque reduction IF assay is precise, sensitive, and reproducible, which can be used to assay all known types of IF. For optimum precision and reproducibility, the reduction in plaque numbers must be proportional to the IF dilution. Thus, it is important to prevent secondary plaque formation, which will result in an inaccurate end point determination. Various culture techniques and overlay media containing agar, agarose, methyl cellulose, carboxymethyl cellulose, starch, gelatin, and homologous antibody have been used for this purpose. Variations in IF titers with the plaque reduction assay are primarily dependent upon changes in sensitivity of the cells to IF and the virus. An internal reference standard should be established and titrated with each set of IF assays to monitor reproducibility and normalize IF titers. Some disadvantages associated with the plaque reduction assay are (a) the length of time required to develop and count plaques, (b) day-to- day variation in plaque numbers resulting from changes in cell sensitivity to IF and virus, and (c) subjective determination of what constitutes a plaque when certain viruses are used.

Role of interferon in streptococcal infection in the mouse

In previous studies, we have shown the rapid in vitro induction of IFN gamma from human T cells by highly purified peptic extracts of M proteins from Streptococcus pyogenes. The present report extends these in vitro studies and shows that a mixture of both alpha/beta and gamma IFN were present in spleen cell homogenates after in vivo treatment with M protein wild-type (M+) or mutant (M-) S. pyogenes strains. The levels of bacterial-induced IFN were found to be greater in M+ treated animals. Additional studies in vivo showed that pretreatment of mice with heat-killed M+ S. pyogenes organisms significantly protected mice to pneumococcal infection compared to similarly treated M- or control animals (P less than 0.001). Further, antibodies to mouse IFN alpha/beta and antibodies specific to a synthetic N-terminal peptide of mouse IFN gamma enhanced the death of animals due to pneumococcal infection and blocked the protection observed in animals previously treated with heat-killed M+ organisms. Most importantly, treatment of mice with either type of IFN alone enhanced the survival of mice to levels similar to that observed by treatment with M+ organisms (P less than 0.05). The results strongly suggest that IFN can play a crucial role, directly or indirectly, in controlling infection by Streptococcus pneumoniae and perhaps other streptococci.

Modulation of immune responses by anabolic androgenic steroids

Anabolic androgenic steroids (AS) have recently been placed on the Food and Drug Administrations (FDAs) list of controlled substances, because of the adverse effects seen in athletes taking accelerated dosages in attempts to enhance performance. Reported deleterious effects on abusers include sterility, gynecomastia in males, acne, balding, psychological changes, and increased risks of heart disease and liver neoplasia. Considering the roles of the immune and neuroendocrine systems and their interactions in many of these pathologies, it is important to determine the effects of these derivitized androgens on this connection. Little is known in this respect. We therefore determined the effects of anabolic steroids on certain immune responses and their effects on the extrapituitary production of corticotropin by lymphocytes. We present evidence that (1) both 17-beta and 17-alpha esterified AS, nandrolone decanoate and oxymethenelone, respectively, significantly inhibited production of antibody to sheep red blood cells in a murine abuse model; (2) the control androgens testosterone and dehydroepian-drosterone (DHEA) or sesame seed oil vehicle had no significant effects on antibody production; (3) nandrolone decanoate and oxymethenelone directly induced the production of the inflammatory cytokines IL-1 beta and TNF-alpha from human peripheral blood lymphocytes but had no effect on IL-2 or IL-10 production; (4) control androgens had no direct cytokine inducing effect; (5) nandrolone decanoate significantly inhibited IFN production in human WISH and murine L-929 cells; and (6) nandrolone decanoate significantly inhibited the production of corticotropin in human peripheral blood lymphocytes following viral infection. These data indicate that high doses of anabolic steroids can have significant effects on immune responses and extrapituitary production of corticotropin. Furthermore, the mouse model should provide an effective means by which to study other deleterious effects of anabolic steroid abuse in humans.

Recombinant gamma interferon enhances natural killer cell activity similar to natural gama interferon

Abstract The enhancement of human natural killing activity by recombinant human gamma interferon (IFNγ) and natural human IFNγ were similar over a wide concentration range. Enhancement of natural killing activity by both interferons was neutralizable by antibody to natural IFNγ, as well as by antibody to a synthetic peptide representing the first 20 N-terminal amino acids of IFNγ provide conclusive evidence that IFNγ is responsible for the enhanced natural killing activity seen in IFNγ preparations.

[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.

Modulation of 4HNE-mediated signaling by proline-rich peptides from ovine colostrum

In previous studies we showed that colostrinin (CLN), a complex of proline-rich polypeptides derived from ovine colostrum, induces mitogenic stimulation, as well as a variety of cytokines in human peripheral blood leukocytes, and possesses antioxidant activity in pheochromocytoma (PC12) cells. In this study we investigated the effects of CLN on 4-hydroxynonenal (4HNE)-mediated adduct formation, generation of reactive oxygen species (ROS), glutathione (GSH) metabolism, and the modification of signal transduction cascade that leads to activation of c-Jun N-terminal kinase (JNK) in PC12 cells. Here we demonstrate that CLN (1) reduced the abundance of 4HNE-protein adducts, as shown by fluorescent microscopy and Western blot analysis; (2) reduced intracellular levels of ROS, as shown by a decrease in 2′,7′-dichlorodihydro-fluorescein-mediated fluorescence; (3) inhibited 4HNE-mediated GSH depletion, as determined fluorimetrically; and (4) inhibited 4HNE-induced activation of JNKs. Together, these findings suggest that CLN appears to down-regulate 4HNE-mediated lipid peroxidation and its product-induced signaling that otherwise may lead to pathological changes at the cellular and organ level. These findings also suggest further that CLN could be useful in the treatment of diseases such as Alzheimer’s, as well as those in which ROS are implicated in pathogenesis.

Colostrinin-driven neurite outgrowth requires p53 activation in PC12 cells.

Summary1. Colostrinin (CLN) induces maturation and differentiation of murine thymocytes, promotes proliferation of peripheral blood leukocytes, induces immunomodulator cytokines, and ameliorates oxidative stress-mediated activation of c-Jun NH2-terminal kinases.2. Here we report that upon treatment with CLN, medullary pheochromocytoma (PC12) cells ceased to proliferate and extend neurites.3. The arrest of CLN-treated PC12 cells in the G1 phase of the cell cycle was due to an increase in the phosphorylation of p53 at serine15 (p53ser15) and expression of p21WAF1. PC12 cells treated with inhibitory oligonucleotides to p53 lacked p53ser15 and p21WAF1 expression, and did not show morphological changes after CLN exposure. Transfection with inhibitory oligonucleotides to p21WAF1 had no effect on p53 activation; however, cells failed to arrest or extend neurites. An oligonucleotide inhibiting luciferase expression had no effect on CLN-mediated p53 activation, p21WAF1 expression, growth arrest, or neurite outgrowth.4. We conclude that CLN induces delicate cassettes of signaling pathways common to cell proliferation and differentiation, and mediates activities that are similar to those of hormones and neurotrophins, leading to neurite outgrowth.

Enhanced in vivo sensitivity to interferon with in vitro resistant B16 tumor cells in mice

Mouse B16 melanoma cells rapidly develop resistance to the antiproliferative effects of interferon α (IFNα) and interferon β (IFNβ) when they are exposed to the interferons in vitro. This resistance was characterized to be non-genetic and dose-dependent, and does not alter other IFN-induced effects such as antiviral effects and elevation of 2′,5′-oligoadenylate synthetase activity in IFN-treated cells. The study of these IFN-resistant cells has been extended to an in vivo tumor model. Resistance, if it occurred in vivo, did not adversely affect the survival of IFN-treated mice. Further, IFN-treated mice inoculated with B16 cells that were resistant in vitro (B16αres cells) survived significantly longer than IFN-treated mice inoculated with B16 cells that were sensitive in vitro. The IFN-treated B16αres-inoculated mice had a significantly higher cure rate as well. The prolonged survival of the mice bearing B16αres cell tumors did not seem to be caused by the slower growth rate of the B16αres cells, since experiments performed with a tenfold higher B16αres cell inoculum and a tenfold lower B16 cell inoculum did not show any change in the survival pattern. It is clear that in vitro resistant B16αres cells are more sensitive to antitumor effects induced by IFN in vivo than in vitro sensitive B16 cells.

[50] Virus yield-reduction assay for interferon by titration of infectious virus

Publisher Summary This chapter presents the temporal changes in antiviral activity that occur within a cell following interferon (IF) treatment and the ultimate effect of these changes on the virus growth cycle, the yield-reduction assays are the method of choice. The critical advantage is that yield-reduction assays are based on the inhibition of virus replication during a single growth cycle, which is achieved either by the use of a high-input multiplicity of challenge virus or by harvesting the progeny virus after one growth cycle. This assay consists of three sequential parts: (1) the incubation of IF dilutions with sensitive cell cultures, (2) the challenge of IF-treated cells with a high multiplicity of virus, and (3) measurement of the degree of inhibition of viral yield. This chapter presents the rationale for the relative advantages of this assay and procedures for performing the assay. Some of the advantages of the yield-reduction assay are that (1) the effect of endogenous IF induced in the system is minimal because the input multiplicity (MOI) of the challenge virus is high enough to infect approximately 100% of the cells [5-10 plaque-forming units (PFU) per cell], (2) the effects of the decay of antiviral activity during the time the challenge virus is replicating in the cells is minimal, and (3) high accuracy can be achieved by titrating progeny virus in a cell type that allows plaquing of the virus.