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A Lifetime with Interferon
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A culture of P. funiculosum isolated on Guam proved capable of elaborating a substance which exerted a favorable therapeutic effect against swine influenza virus infections in white mice. The culture was extremely variable and irregular in its production of the antiviral substance, and during maintenance in the laboratory for several years gradually lost this property. Efforts to restore it were unsuccessful.
Subsequently it was found that the mold elaborated a substance, now designated helenine, which is therapeutically effective against Columbia SK encephalomyelitis virus infections in mice. Helenine appears to differ from the substance earlier procured from the mold, which was active against swine influenza virus infections in mice. It is frequently present in greater or lesser amount in the fluid portions of stationary cultures of P. funiculosum but is more regularly obtained and in larger amount, from the cellular components of the pellicles. When liberated from these latter by mechanical bruising and fracturing, it goes into solution in the culture fluids. It is precipitable from aqueous solution by 50 per cent acetone.
Infected mice injected with helenine in amounts less than the amount which produces a maximal therapeutic effect exhibit a dosage response. Increasing the dose above the optimum fails to increase the therapeutic effect.
Helenine exerts its maximum effect when given within the first 10 hours after viral infection but its influence is apparent even when treatment is delayed for up to 24 hours. It is not effective against massive amounts of virus and gives the best therapeutic results when used in the treatment of animals infected with from 10 to 1000 fatal doses of virus. Treatment of infected mice with helenine delays the entrance of virus into their brains for from 24 to 48 hours.
The mechanism by which helenine exerts its therapeutic effect against SK virus is not known but the findings presented suggest either that it causes an inhibition or interruption of multiplication of the virus, slowing down the whole process of infection and spread to the central nervous system, or that in some way it interferes temporarily with the neuroinvasiveness of the virus.
The period and rate of liberation of influenza virus from entodermal cells of the allantois have been studied by deembryonating eggs within a few minutes after infection, exchanging the medium thereafter at hourly intervals and assaying the virus concentration in the harvests thus obtained (differential growth curves). If the inoculum was sufficiently large, presumably all available cells immediately became infected and only 1 infectious cycle was expected to occur. If the inoculum was small, so that only a fraction of the cells adsorbed virus, the infectious process was held to 1 cycle by continuous exposure of the remaining susceptible cells to RDE. In either case, the results obtained indicate that once cells have been infected they produce and liberate virus at nearly constant rates for periods of 30 hours or longer before the yields decrease rapidly. Evidence has been presented which strongly suggests that such prolonged periods of liberation are observed not only in deembryonated eggs but also in the intact chick embryo.
Attempts have been made in the discussion to reconcile these findings with previous estimates of the liberation period and to integrate them with histologic observations and electron micrographs of thin sections of infected allantoic membranes having a bearing on the mode of liberation.
The usefulness of the deembryonation technic has been analyzed as a tool in the study of various problems in the growth cycle of influenza virus in the entodermal cells of the allantoic of chick embryos. Various improvements in the deembryonation technic have been described. The method readily permits repeated sampling of the medium at various stages after infection (cumulative growth curves) or frequent exchanges of the medium (differential growth curve). However, the yield of infectious virus or of hemagglutinins is less than that observed in the intact chick embryo. The difference observed is greater than can be accounted for by the reduction in the available host cells and is assumed, therefore, to be due in part to interruption of blood and nutrient supply to the cells. This handicap can be overcome by the combined in ovo-deembryonation technic, in which deembryonation is performed at any desired time after infection of the intact chick embryo, and the medium is collected and analyzed after 1 to 3 hours of further incubation. The value of the technic is demonstrated by the fact that liberation of virus from infected cells can be detected earlier than in the intact egg. Furthermore, it continues at a nearly constant rate for many hours, thus proving to be erroneous previous inference which had been based upon in ovo experiments. The technic also permits readily the addition and subsequent removal of substances that might interfere with viral propagation. As an example a study was made of the effect of the receptor-destroying enzyme of V. cholerae (RDE) when added to the medium of eggs infected prior to deembryonation. By carefully grading the dose of virus and using an appropriate amount of RDE, one-step growth curves were obtained indicating that those cells not directly invaded by the seed virus were subsequently protected against infection by action of the enzyme. The smaller the amount of virus the less RDE was required in order to note a protective effect. With a decrease in the period of exposure to RDE regeneration of cell receptors became increasingly more apparent in that correspondingly greater amounts of virus were produced and liberated late in the incubation periods. These results confirmed and extended those reported by Stone. More extensive applications of these technics will be reported in subsequent papers of this series.
An analysis has been made of factors contributing to the von Magnus phenomenon; i.e., the emergence of increasing quantities of non-infectious hemagglutinins (NIHA) in successive passages in the allantois of chick embryos of undiluted allantoic fluids infected with influenza virus.
Using the PR8 strain, the von Magnus phenomenon was pronounced when the serial seeds were obtained under conditions which permitted extensive inactivation of infectious virus during individual passages. Correspondingly, it was reduced but not abolished when precautions were taken to avoid accumulation of inactivated virus in the inocula. Thus, inactivated virus may be taken as a contributing factor.
Preparations of infectious virus obtained under conditions largely excluding the presence of inactivated virus were capable of yielding some NIHA on passage as long as sufficient amounts were injected to permit each host cell to adsorb several infectious virus particles. However, the fact remains that more NIHA was found in the harvests when the inocula contained a large proportion of non-infectious virus material.
Following injection of various types of seeds NIHA appeared in the allantoic fluids as soon as liberation of virus became detectable. This time relationship and the rates of release of non-infectious virus components seemed to exclude that the NIHA obtained consisted entirely of infectious virus which had been inactivated during incubation in ovo. It was apparent rather that NIHA other than that due to heat-inactivated virus was released.
Correlations between the infectivities and hemagglutinating capacities of over 50 standard and undiluted passage seeds and the compositions of the harvests derived therefrom on passage without dilution indicated that the corresponding activities in the yields did not depend entirely upon the relative concentrations of infectious virus and non-infectious hemagglutinins in the inocula but that apparently different forms of NIHA were obtained in successive undiluted passages.
Helenine exerts a therapeutic effect against Semliki Forest virus infections of mice. Cures, that is to say the survival of treated animals, were more frequently observed in Semliki Forest virus infections than they were in SK virus infections. It is believed that this difference in end-result probably represented only a quantitative difference in the therapeutic effect of helenine against these two viruses and not a qualitative difference in its mechanism of therapeutic action. The findings reported in this paper with regard to the treatment of Semliki Forest virus infections with helenine parallel very closely those described in an accompanying paper which deals with the action of helenine on SK. virus infections.
The usefulness of the deembryonation technic has been analyzed as a tool in the study of various problems in the growth cycle of influenza virus in the entodermal cells of the allantoic of chick embryos.
Various improvements in the deembryonation technic have been described.
The method readily permits repeated sampling of the medium at various stages after infection (cumulative growth curves) or frequent exchanges of the medium (differential growth curve). However, the yield of infectious virus or of hemagglutinins is less than that observed in the intact chick embryo. The difference observed is greater than can be accounted for by the reduction in the available host cells and is assumed, therefore, to be due in part to interruption of blood and nutrient supply to the cells. This handicap can be overcome by the combined in ovo-deembryonation technic, in which deembryonation is performed at any desired time after infection of the intact chick embryo, and the medium is collected and analyzed after 1 to 3 hours of further incubation.
The value of the technic is demonstrated by the fact that liberation of virus from infected cells can be detected earlier than in the intact egg. Furthermore, it continues at a nearly constant rate for many hours, thus proving to be erroneous previous inference which had been based upon in ovo experiments.
The technic also permits readily the addition and subsequent removal of substances that might interfere with viral propagation. As an example a study was made of the effect of the receptor-destroying enzyme of V. cholerae (RDE) when added to the medium of eggs infected prior to deembryonation. By carefully grading the dose of virus and using an appropriate amount of RDE, one-step growth curves were obtained indicating that those cells not directly invaded by the seed virus were subsequently protected against infection by action of the enzyme. The smaller the amount of virus the less RDE was required in order to note a protective effect. With a decrease in the period of exposure to RDE regeneration of cell receptors became increasingly more apparent in that correspondingly greater amounts of virus were produced and liberated late in the incubation periods. These results confirmed and extended those reported by Stone.
More extensive applications of these technics will be reported in subsequent papers of this series.
When neutral red is added to the liquid medium in tissue cultures, more dye is taken up by healthy cells than by damaged cells. This forms the basis of a method for assessing the relative extent of cell damage in different cultures, the amounts of bound dye being determined directly by inspection of the stained cell sheets, or indirectly by estimating extracted dye colorimetrically. This procedure can be used to measure the effects of a cytocidal virus and can also be applied to the assay of interferons. Here it provides a precise and reproducible method which is unusually sensitive for the assay of mouse interferon, and has also been used to assay rat, chick, rabbit and human interferons.
Excerpt
Human proteins required for medical purposes, e.g., insulin, interferons, tissue plasminogen activator, and growth hormone, can now often be produced by recombinant DNA procedures. They can also be obtained by extraction from the appropriate human organ (e.g., growth hormone from pituitaries) or by stimulating human cells grown in culture to make the protein of interest. We pioneered production by the latter approach because we needed to make enough human interferon for large-scale clinical trials; we continue to use this route today, because we think it has certain advantages. To understand our reasons, some points from the history of interferons must be considered.
Discovery and Development of Interferons
Fifty years ago, Hoskins (1935) and Magrassi (1935) independently discovered the interference phenomenon: A virus replicating in an animal's organ, e.g., the brain, prevented that organ during the next few days from superinfection with any of a number of serologically unrelated viruses. Although...
During a study of the interference produced by heat-inactivated influenza virus with the growth of live virus in fragments of chick chorio-allantoic membrane it was found that following incubation of heated virus with membrane a new factor was released. This factor, recognized by its ability to induce interference in fresh pieces of chorio-allantoic membrane, was called interferon. Following a lag phase interferon was first detected in the membranes after 3 h incubation and thereafter it was released into the surrounding fluid.
CERTAIN influenza A viruses multiply readily in bovine kidney cells1. Calf kidney cells infected with the influenza A strain WS produce virus continuously for about 2 days and then cease to do so. Few cells degenerate. After a few more days influenza virus begins to appear again (Fig. 1). Periods in which the amount of virus formed rises and falls may alternate in this way for up to three months. Cultures, infected 6-12 days previously, which were yielding no detectable influenza virus hæmagglutinins, were found to be resistant to superinfection with Sendai virus. No hæmagglutinin was produced and no degeneration occurred.
It is a well-known fact that infection of a bacterium, plant or animal with one virus frequently prevents or partially inhibits simultaneous propagation of another viral agent in the same host. Such interference phenomena, which have been called also “sparing effect”, “cell blockade”, or “Schienenimmunität”, have aroused great interest on account of their theoretical, and possibly practical implications. As a result, the literature on this subject has been increasing rapidly in recent years. It may be profitable, therefore, to take stock at this time and to attempt formulation of general principles which have evolved in studies of the various phenomena. It will be apparent that the present knowledge is still very fragmentary, so much so that the plural in the title of this review is being employed advisedly because of the fact that it is not known whether the various instances of interference described are all based upon related mechanisms, or whether several entirely different reactions are involved in the various observations.
Hairy cell leukaemia. In-duction of remission with alpha interferon
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