Interferons produced by the cells of newborn mice in the presence of sera from newborn and adult animals

Fibroblasts of newborn mice produced far less amount of interferon in the presence of sera from newborn animals than in the presence of sera from adult animals. The interferons obtained were purified by adsorption chromatography on porous glass and were analyzed by electrophoresis in polyacrylamide gel. It has been shown that antiviral activity of interferon preparations obtained in the presence of sera from newborn mice was associated with the fraction of 45 Kd. Addition into the growth medium of sera from adult animals led to the production by the same cells of interferon activity associated with 41 and 28 Kd fractions. It is assumed that the sera of newborn mice contained the components influencing the molecular content of interferon produced by the cells of newborn animals.     

Effect of vitamin E on transcription in isolated nuclei and rat liver chromatin in normal status and in E-hypovitaminosis]

The effect of alpha-tocopherol on the RNA-polymerase activity in isolated rat nuclei and chromatin from normal and E-deficient rats and the possible role of tocopherol-binding proteins in this process were studied. Some differences in the RNA-polymerase activities of the nuclei were found; however, in vitro added alpha-tocopherol had no effect on the level of the label incorporation into RNA. No effect of alpha-tocopherol on this process was observed after addition of cytosol either. Analysis of chromatins from normal and E-deficient rats revealed no differences in their RNA-polymerase activities. In vitro added alpha-tocopherol increased the RNA-polymerase activity of normal (but not of vitamin E-deficient) rats. Some differences in the RNA-polymerase activities were noted after addition to the incubation medium of the Triton X-100-solubilized nuclear fraction specifically binding alpha-tocopherol. This effect was enhanced in the presence of exogenous alpha-tocopherol. The susceptibility of chromatin from normal and E-deficient rats to DNAse I hydrolysis was also found to be different. It was concluded that vitamin E can influence the RNA-polymerase activity of the nuclei and chromatin as well as the chromatin structure and that alpha-tocopherol-binding proteins are necessary for the vitamin E effect on the RNA-polymerase activity to be manifested.     

Effect of glucocorticoid hormones on growth of human fibroblast cells and interferon production in a microcarrier culture system

Glucocorticoid hormones promoted the growth of fibroblast cells derived from human neonatal foreskins and prolonged their life span in a microcarrier culture system that used Eagle's minimum essential medium (MEM) supplemented with fetal calf serum (FCS). But, these hormones suppressed cell growth in conventional monolayer cultures. Precolostrum newborn calf serum (PNCS) was the only species that supported the serial propagation of fibroblast cells on microcarriers, possibly because of its high content of hydrocortisone (HC). Fibroblast cells grown on microcarriers in the presence of glucocorticoid hormones maintained their ability to produce interferon (IFN)-beta in a superinduction method with poly I: poly C and antimetabolites. These cells had more than 93% diploidy and no chromosomal aberration or translocation. Use of PNCS for the cultivation of human fibroblast cells has high potential for providing a microcarrier culture system for the mass production of human IFN-beta.     

Effect of brain cytosol proteins of embryonal and adult animals on RNA-polymerase activity of isolated brain nuclei]

Cytosol and its fractions obtained by the precipitation with ammonium sulphate and ion-exchange chromatography have been studied for their effect on the RNA-polymerase activity of isolated nuclei. We observed the discrepancies in the action of total cytosol of embryonal, newborn or adult animals on the label's incorporation in RNA. It was found that some fractions increased DNA-polymerase activity of isolated nuclei in cattle embryonal cytosol. The same fractions obtained from adult cytosol did not act in such a way. It was found that most fractions obtained from cytosol of adult brain inhibited the RNA-polymerase activity of brain nuclei.     

Interferon and cytotoxic factor (cytotoxin) released in the blood of mice infected with Mycobacterium bovis BCG. III. Interferon and cytotoxin induced by the specific antigen as compared with those induced by bacterial lipopolysaccharide

The time course of development and decline of the ability of BCG-infected mice to produce interferon in the serum in response to the intravenous infection of purified protein derivative of tuberculin (PPD) was very similar to that of their systemic hypersensitivity to PPD. A cytotoxic factor (cytotoxin) was produced in parallel with interferon in the serum of BCG-infected mice after stimulation with PPD. The duration of the period in which cytotoxin-production responsiveness to PPD was definitely detectable was much shorter than that for interferon-production responsiveness although the periods for the maximum production of interferon and cytotoxin coincided. The kinetics of release of interferon in the serum of BCG-infected mice after stimulation with PPD did not parallel that of release of cytotoxin. The four kinds of activities, interferons and cytotoxins induced by PPD and lipopolysaccharide (LPS) in the serum of BCG-infected mice, were compared for their stability to heating at 56 C and to treatment at pH 2. The kinetics of inactivation of these four activities differed significantly, when the serum was either heated at 56 C or treated at pH 2. Interferon produced in response to LPS could be neutralized by anti-L cell(NDV) interferon rabbit serum as easily as L cell (NDV) interferon, 16 times as much antiserum was required to neutralize the same amount of interferon in response to PPD, but cytotoxins induced by PPD and LPS were not neutralized at all by the antiserum. From these findings it is thought likely that interferons and cytotoxins induced by PPD and LPS in the serum of BCG-infected mice are different substances, although the antigenic relationship between cytotoxins induced by PPD and LPS remains unknown.     

Interferon γ in acute and subacute encephalitis

Intrathecal synthesis of interferon γ was shown in 14 out of 16 samples of cerebrospinal fluid collected in the first days of disease in adults, children, and newborn infants with herpes encephalitis. This synthesis was concomitant with that of interferon α and was switched off when the specific antibodies in the central nervous system increased. No endogenous interferon γ was detected in 11 serum samples or 13 samples of cerebrospinal fluid collected early in the course of the disease from patients with measles encephalitis and rubella encephalitis, or in serum and cerebrospinal fluid samples from seven patients with subacute sclerosing panencephalitis. In serum collected after the 10th day after the onset of neurological symptoms interferon γ was present at low concentrations in only three out of 11 serum specimens from patients with measles encephalitis or rubella encephalitis.Interferon γ was present in patients with acute herpes encephalitis and there was active virus replication, but it was not present in postinfectious encephalitis. Possibly the local production of specific antibodies masks the viral antigens and switches off the induction of interferons.     

Substitution of Milk for Serum in the Production of Human Leukocyte Interferon

The presence of serum in suspensions of Sendai-induced human leukocytes is necessary for the synthesis of significant amounts of interferon. Very little interferon is obtained from serum-free suspensions. Cow's milk or milk casein can substitute for serum in the production of high yields of human leukocyte interferon.     

Relationship between the Molecular Size of Poly I·Poly C and Its Biological Activity1

Seven polyinosinic·polycytidylic acid (poly I·poly C) preparations, ranging from 4.2 S to 21.2 S, prepared from various sizes of polyinosinate and polycytidylate, were examined for toxicity and interferon-inducing activity in mice. The increase in size of poly I·poly C was accompanied by increases both in the maximal amount of interferon produced and in the length of persistence of a high level of interferon in plasma. Toxicity of poly I·poly C was proportional to the molecular size within the range of 8 S to 16 S. The amount of interferon induced by 1/5 LD₅₀ of poly I·poly C depended on the size of the inducer, being increasingly lower with progressively smaller sizes. Next, activities of poly I·poly C in culture cells were examined. The resistance-inducing activity of poly I·poly C in primary chick embryo cells (CEC) increased with the size of the inducer (4.2 S to 11.6 S), whereas the activity in L cells was not so markedly dependent upon its molecular size as in CEC. In the presence of calf serum during induction of resistance the activity was lowered. The activities of preparations with small molecular sizes were affected by calf serum more markedly than those of large molecular sizes. The interferon-inducing activity in RK13 was not appreciably influenced by the size of poly I·poly C, especially in the presence of DEAE-dextran, while the activity in L cells was markedly dependent upon the size of the inducer. These results suggest that the influence of the molecular size of poly I·poly C upon the resistance-inducing and interferon-inducing activities varies among different kinds of cells, and alters in the presence of serum or DEAE-dextran.     

Properties of the rat liver DNA-dependent RNA-polymerase stored at -25 degrees C in the presence of glycerol or polyethyleneglycol-400

The effect of slow freezing (1-2 degrees C/min) was studied as applied to the template activity of DNA in the RNA-polymerase reaction. It was found out that the activity of each RNA-polymerase form decreases after 24-hour storage by freezing at -25 degrees C in the presence of glycol or polyethylene glycol-400. The study of the enzyme in the glycerol concentration gradient showed sedimentation constant changes.     

Interferon and cytotoxic factor (cytotoxin) released in the blood of mice infected with Mycobacterium bovis BCG. I. Enhanced production of interferon and appearance of cytotoxin stimulated by capsular polysaccharide of Klebsiella pneumoniae or bacterial lipopolysaccharide.

Interferon production stimulated by the active substance (neutral fraction) of the capsular polysaccharide of Klebsiella pneumoniae (neutral CPS-K) in BCG-infected mice was compared with that by bacterial lipopolysaccharide (LPS). Prior infection with BCG increased the responsiveness of mice to the lethal effect of neutral CPS-K as well as to that of LPS. Associated with this, BCG-infected mice showed a markedly enhanced ability to produce interferon after stimulation not only by LPS but also by neutral CPS-K. In addition, a cytotoxic factor (cytotoxin) was found to be released in the serum of BCG-infected mice after injection of these inducers. The kinetics of production of interferon and cytotoxin stimulated by neutral CPS-K were very similar to those stimulated by LPS. The time pattern of cytotoxin production was not in parallel with that of interferon production. Interferon reached a peak 2 hr and cytotoxin 3 hr after injection with these inducers. Interferon and cytotoxin produced by neutral CPS-K showed essentially the same stabilities to heating at 56 C and to treatment at pH 2 respectively as those produced by LPS. Interferon was inactivated by heating at 56 C more rapidly than cytotoxin. Cytotoxin was inactivated by treatment at pH 2 for 24 hr, whereas interferon activity was well preserved after this treatment. These results suggest that both activities are the result of different substances.     

Production of human immune interferon by recombinant mammalian cells cultivated on microcarriers

Recombinant Chinese hamster ovary (rCHO) cells were cultivated on microcarriers for the production of human immune (Gamma) interferon. The effect of basal medium, serum, and microcarrier concentration on interferon production was investigated. The specific interferon productivity in the post-confluent stage was similar to that in the growth stage. Control of the pH results in a significant improvement in the volumetric interferon production. The volumetric production rate of interferon by these rCHO cells did not decrease after one month of cultivation on microcarriers.     

Interferon Production in Human Diploid Cell Strains Derived from Embryonic Lungs

Twenty-three strains of human diploid cells derived from embryonic lungs were tested for production of interferon by “superinduction.” Strain HAIN-55 produced a relatively high level of interferon. The optimal concentration of cycloheximide for superinduction was essentially equal to that reported with foreskin fibroblasts. On the other hand, actinomycin D at a concentration of 4 to 16 μg/ml enhanced the production of interferon more strikingly than at a concentration of 1 μg/ml, which was usually employed for superinduction in the foreskin fibroblasts. Inhibition of interferon production was observed when fetal bovine serum was added to the medium during treatment with metabolic inhibitors for superinduction. Minimal essential medium was superior to Eagle's basal medium as growth medium for interferon production, and serum added after removal of metabolic inhibitors could be replaced by bovine serum albumin. The yield of interferon produced under the best conditions in this study, with strain HAIN-55, was more than 10,000 reference units/ml.     

Properties of DNA-dependent RNA-polymerase from spleens of mice infected with Rauscher leukemic virus]

The properties of RNA polymerases A, B and C isolated from the spleens of mice infected with Rauscher leukemic virus were studied. The solubilized RNA-polymerases A and B were purified 150--300-fold. The dynamic changes in the activities of all forms of RNA-polymerases at different stages of leukosis were studied. At the earliest steps of leukosis a 2-fold increase in the RNA-polymerase B activity followed by a 5-fold increase in the RNA-polymerase A activity was observed. At late stages of leukosis the activity of RNA-polymerase C also showed an increase. The properties of RNA-polymerases A and B from the spleens of virus-infected mice were compared to those of the controls. In leukemic tissues the specific activities of RNA-polymerases A and B were higher as compared to those of the enzymes isolated from the spleens of non-infected mice. However, no significant differences in the enzyme properties in normal and virus-infected animals were revealed. Dihydrorifampicine (200 mkg/ml) caused a 50% inhibition of RNA polymerase A in vitro but had no effect on the activities of RNA-polymerases B and C.     

Radioactive human lymphoblastoid interferon. One-step purification, regulation of heterogeneous species production and its use for radioimmunoassay

Human lymphoblastoid interferon (alpha type), labeled with [3H]leucine added to virus-induced Namalwa cells, was purified quantitatively and in one step from the culture fluid by immune precipitation. The material showed, upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, only four radioactive bands with molecular weights ranging from 17000 to 21000, which coincided well with interferon activity. They coincided also with the four interferon protein bands in the electropherogram of unlabeled interferon purified by a different method. The purity of the labeled interferon was ascertained also by polyacrylamide gel electrophoresis in the absence of dodecyl sulfate. Pulse-labeling of interferon with [3H]leucine for 1 h at various times after induction indicated that the cells always synthesized and secreted the four interferon species in parallel during the interferon production period. Competitive radioimmunoassay for human interferon alpha was achieved by the use of purified radioactive interferon, anti-(interferon alpha) serum, and bacterial adsorbent. The immune precipitation of the labeled interferon was inhibited by unlabeled interferon alpha, and 100 international reference units of interferon alpha could be measured in this way.     

Anomalous Effects of Heterologous Normal Serum on Interferon Production in Mice

WHILE studying the inhibitory effect of burro anti-mouse lymphocyte serum on the production of interferon in mice¹, we investigated whether rabbit anti-mouse lymphocyte serum (ALS) had similar activity. There was some inhibition of interferon production after intraperitoneal injection of polyinosinic/polycytidylic acid (poly IC) in NIH Swiss strain mice pretreated with three doses of potent rabbit anti-mouse lymphocyte serum. Animals treated with normal rabbit serum, however, showed a similar inhibition of interferon production (Table 1), although normal burro serum had no effect¹.     

Circulating Interferon Production in the Mouse : Origin and nature of cells involved and influence of animal genotype

A radiobiological study of circulating interferon production in the mouse was undertaken in the hope of elucidating the site(s) of circulating interferon production. After total body X-irradiation of the animals, different radiosensitivities of circulating interferon production were observed with different viral inducers. Myxovirus-induced circulating interferon production was especially radiosensitive. Moreover, a study of interferon production in syngeneic and xenogeneic radiochimeras demonstrated that cells producing NDV (Newcastle disease virus)-induced circulating interferon were derived from hematopoietic stem cells. In addition, treatment of mice with antilymphocyte serum significantly reduced NDV- and Sendai virus-induced circulating interferon, as opposed to other inducers. Taken together, these results strongly suggest that the lymphocyte is the major source of myxovirus-induced circulating interferon. A survey of interferon production in 12 inbred mouse strains, using NDV as inducer, revealed the existence of low and high producers. A Mendelian analysis carried out with low producing Balb/c and high producing C57BL indicated that the difference between low and high interferon producers was caused by a single, autosomal, codominant factor.     

The transformation of adult but not newborn human lymphocytes by Epstein Barr virus and phytohemagglutinin is inhibited by interferon: the early suppression by T cells of Epstein Barr infection is mediated by interferon

In a previous study, it was demonstrated that T cells from adult individuals were able to suppress the transformation of B cells after infection by EBV. In this paper, it is shown that this suppression is mediated by interferon. Thus, the suppression is abrogated by anti-interferon serum and mimicked by human leukocyte interferon. Furthermore, the interferon is released in response to the virus-infected B cell, not the virus alone. The relevance of these results to previous clinical evidence, indicating a role for interferon in recovery from EBV infection, is discussed. Interferon will also suppress the transformation of adult B lymphocytes by the mitogen phytohemagglutinin (PHA). However, interferon at concentrations 2 to 3 orders of magnitude higher was unable to suppress the transformation of neonatal B lymphocytes by either EBV or PHA. These experiments suggest that EBV and PHA induced transformation share a common interferon sensitive step. Lastly, the resistance of newborn lymphocytes to the protective effect of interferon may be an important consideration in the application of interferon as an antiviral or anti-tumor agent.     

鱼类培养细胞干扰素的诱导

对紫外线灭活的草鱼出血病病毒（ＧＣＨＶ）－Ｆ９株,在几种鲤科鱼类培养细胞中诱导产生干扰素的能力及影响干扰素产量的各因素进行了研究。纯化的ＧＣＨＶ在紫外线照射５分丧失感染性,但获得了在ＣＡＢ等５种鲤科鱼类培养细胞中高铲诱导产生干扰素的能力。干扰素的诱导需要病毒高复数感染细胞。干扰素主要在诱导后１４小时时内产生。培养上清中的新生牛血清对干扰素的产量有抑制作用。而在ＰＨ６．２－７．８范围内对干扰素产量无     

Separation of mitogen-induced suppressor and helper cell activities during inhibition of interferon production by cyclic AMP.

The effect of exogenous cyclic AMP on mitogen-induced suppression and enhancement of the in vitro plaque-forming cell (PFC) response and on mitogen induction of immune interferon (also called type II) in cultures was examined. Mitogen induction of immune interferon was quantitatively associated with mitogen-induced suppressor activity, and cyclic AMP blocked both the suppressor activity and the production of immune interferon in mouse (C57B1/6) spleen cell cultures. The evidence is as follows: (a) The concentrations of dibutyryl cyclic AMP that blocked T-cell mitogen (staphylococcal enterotoxin A) suppressor activity were the same as those that blocked mitogen induction of immune interferon. (b) The blocking action of dibutyryl cAMP on both the suppressor and interferon effects of mitogen was a function of the time of dibutyryl cAMP addition to cultures relative to mitogen addition. (c) A dramatic immunoenhancing effect of mitogen occurred in the presence of dibutyryl cAMP under conditions that blocked production of immune interferon. Specifically, mitogen-induced helper cell function is dramatically enhanced in the presence of dibutyryl cyclic AMP, if the mitogen is added to cultures 24 to 48 hr after SRBC and dibutyryl cyclic AMP. Dibutyryl cyclic GMP did not affect the mitogen- or cyclic AMP-induced effects under the conditions of our test system. Under the conditions described here, then, cyclic AMP appears to selectively block suppressor cell activity while allowing or aiding mitogen-induced helper cell activity. It is possible that the immune response is a reflection of the ratio of helper to suppressor activities in the system.     

Interference of a human leukocyte interferon preparation with stimulatory activities in leukocyte-conditioned medium for human hematopoietic stem cells

Medium conditioned by leukocytes in the presence of phytohemagglutinin (PHA-LCM) promotes the growth of multilineage hemopoietic progenitors derived from human bone marrow. However, PHA-LCM prepared in the presence of a human leukocyte interferon preparation does not support mixed colony formation. Crude PHA-LCM preparations were characterized by gel filtration, affinity chromatography, and gel electrophoresis. The elution profile on Sephacryl S-300 of PHA-LCM prepared without interferon showed a distinct peak that stimulated the growth of pluripotent stem cells (CFU-gemm) and committed precursors (CFU-c, BFU-e). Gel filtration of PHA-LCM, prepared with 1000 U/ml of interferon, revealed a change in the elution profile. The eluted material demonstrated no growth-promoting activities. We conclude that the abolished stimulatory activity of PHA-LCM, prepared with human leukocyte interferon, might be due to a reduced production of stimulatory molecules, suggesting that interferon interferes with the molecular events required for colony formation of committed and noncommitted hemopoietic progenitors.