Douglas A. Weigent

[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.

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.

[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.

Interferon-induced transfer of viral resistance by human B and T lymphocytes

Enriched human B lymphocytes cocultivated with mouse L cells produced human leukocyte interferon (IFN-alpha) and shortly thereafter transferred antiviral activity to the recipient cells (99% inhibition of expected virus yield). In contrast, cocultivation of enriched T-cell populations with mouse L cells resulted in no IFN production or transfer of antiviral activity. In addition, both T and B lymphocytes pretreated with exogenous IFN or stimulated in vitro by mitogens could transfer antiviral activity to human WISH cells. The transfer of antiviral activity was not blocked by antibodies to IFN. The data indicate that both T and B cells can be recruited by IFN to transfer antiviral activity. Thus, once cells are recruited by IFN they can transfer antiviral activity in the absence of IFN and protect cells locally or distally from the site of infection.

Effects of 6-thiopurines on the transforming activity of Bacillus subtilis deoxyribonucleic acid

Abstract 6-Thioguanine (6-TG) and 6-mercaptopurine (6-MP) have been reported to produce cytotoxicity due to incorporation into DNA. Although some investigators have performed in vitro transcription studies with synthetic polymers containing 6-TG, the DNA isolated from a cell population exposed to thiopurines has not been evaluated for its biological activity. Bacillus subtilis UTH-8505 was grown in the presence of 6-TG, 6-MP, bromodeoxyuridine (BUdR), arabinosyladenine (AraA) and 2,6-diaminopurine. DNA isolated from these and control cultures was used to transform B. subtilis T 3 , a strain lacking tryptophan synthetase, to prototrophy. 6-MP exposure decreased the transforming activity 80 per cent; 6-TG, 20 per cent; BUdR, 50 per cent; and in single determinations AraA and 2,4-diaminopurine were without effect. 6-MP was also the most effective growth inhibitor of B. subtilis . Base composition and melting temperatures of the DNA from 6-MP, 6-TG and BUdR cultures were not altered. Although the methods employed did not permit unequivocal demonstration of 6-TG incorporation, a maximal estimate of one 6-TG molecule substituted for every 500 guanine residues in the DNA was inferred. Thus, extremely low levels of incorporation may alter DNA function, or the reduced transforming activity is produced by another mechanism.

Production and Function of Growth Hormone in the Immune System

An important and relatively new finding is the ability of cells of the immune system to produce and secrete neuroendocrine hormones (reviewed in 1). Currently, the list includes adrenocorticotropin (ACTH) and endorphins, thyrotropin, luteinizing hormone, and growth hormone (GH). In addition, the synthesis of corticotropin releasing hormone (2) and growth hormone releasing hormone (GHRH) (3) has also been documented. Further, functional receptors for the neuroendocrine hormones have also been observed in the immune system (4). Thus, the evidence taken together supports the existence of the same signal molecules and receptors that potentially mediate interactions both between and within the neuroendocrine and immune systems.

Lymphocytes and Hypothalamic Peptides

It has previously been shown that pituitary hormones and hypothalamic releasing hormones have direct effects upon cells of the immune system (1). Because cells of the immune system also produce pituitary hormones, we and others hypothesized that cells of the immune system might also produce hypothalamic releasing factors. In fact, there are now data to support the extrahypothalamic immune synthesis of corticotropin releasing hormone (CRH), growth hormone releasing hormone (GHRH), luteinizing hormone releasing hormone (LHRH), and somatostatin (SS) (1–3). Further, functional receptors for these same hypothalamic hormones have been identified on cells of the immune system (4). The results discussed in this chapter show that hypothalamic hormones produced in the immune system are structurally similar to, and are produced in some respects similarly to, their hypothalamic counterparts, whereas important differences also exist. The data taken together support the existence of hypothalamic signal molecules and their receptors in the immune system, and suggest they play important roles in lymphocyte function.