Human Fibroblast Interferon Research Articles

30] Production of human fibroblast interferon in the presence of the glycosylation inhibitor tunicamycin

Publisher Summary This chapter describes procedure that deals specifically with the use of tunicamycin in the production of unglycosylated interferon induced by polyinosinate-polycytidylate [poly(I) . poly(C)] in cultures of human foreskin fibroblasts. A similar procedure with 2-deoxy-o-glucose or o-glucosamine as inhibitor is reported. Production of interferon in FS-4 cells by the superinduction procedure has been described. Only the steps involving tunicamycin treatment of the cell culture are described. Tunicamycin is a complex molecule consisting of one residue of uridine and two residues of N-acetyl-o-glucosamine, with the amino hydrogen in one of these residues substituted by a long-chain fatty acid. Four major components is identified in tunicamycin preparations that differ in fatty acid chain length; the molecular weight of each component is determined to be 970, 984, 998, and 1012, respectively. A more recent study demonstrated that tunicamycin can be resolved into two major and eight minor components by high-performance liquid chromatography (HPLC).

18] Procedures for the measurement of interferon mRNA distribution in induced mouse cells

Publisher Summary This chapter presents the methodology used to extract IF mRNA, partially purify IF mRNA, and assay IF mRNA by translation in Xenopus laevis oocytes. It is shown that total cytoplasmic RNA from Newcastle disease virus (NDV)-induced L-929 cells can program the synthesis of interferon in Xenopus laevis oocytes. Moreover, if this RNA is further purified by sucrose-formamide gradient centrifugation, a linear dose-response relationship is obtained between the amount of RNA injected into the oocyte and the amount of interferon translated. A similar quantitative relationship has also been established for the oocyte and wheat germ systems programmed with human interferon mRNA. These observations have facilitated a study of the mechanism of superinduction of human fibroblast interferon production. For studying the mechanism of interferon priming on interferon production in NDV-induced L-929 cells, a advantage of this simple quantitative assay to study the kinetics and intracellular distribution of interferon mRNA (IF mRNA) is presented. Priming by interferon did not affect IF mRNA transcription or stability, but rather its rate qf translation in NDV-induced mouse L cells.

5] Amino acid analysis of protein bands on stained polyacrylamide gels

Amino acid analysis is carried out on proteins that are present as bands on stained polyacrylamide gels. After electrophoresis, the gel is stained with Coomassie Blue and destained extensively with 7% acetic acid-10% methanol in the usual manner. After hydrolysis, the hydrolyzate is removed from the gel residue, dried in vacuo, dissolved in the pH 2.2 diluent, and run on the fluorescamine amino acid analyzer. With this procedure, it is not necessary to extract the protein from the gel in order to carry out amino acid analysis. Extraction from the gel typically gives poor recovery of protein and introduces amino acid contamination. This procedure offers a unique opportunity to characterize a protein. In many instances gel electrophoresis is the simplest method for separating a protein from a mixture, and it is applicable at the low microgram level. In the studies on purified human fibroblast interferon, a minor contaminant of about 40,000 daltons was detected on gels. Amino acid analysis revealed it a dimer of interferon by its identity in composition with the 20,000-dalton band.

14] Induction and production of human interferon with a continuous line of modified fibroblasts

Publisher Summary The production of human fibroblast type interferon depends on the innovative induction procedures to increase production rather than on large-scale cell culture techniques as in the case of lymphoblastoid cell cultures. Interferon induction in transformed or modified human fibroblasts is described. It is important to note that, unlike the primary diploid fibroblast cultures, the modified human fibroblast cultures are capable of producing human interferon in response to a large number of substances structurally unrelated to the traditional inducer-type molecule like poly(I).poly(C). Various conditions are used to induce C-10 cell cultures optimally for interferon production on a large (100-200 flasks) scale including approximately 3 x l0 6 C-10 cells being seeded into each 175-cm 2 plastic culture flask; before inducing the C-10 cells, each culture is primed with 250 units/ml of partially purified human fibroblast interferon (specific activity of 1 x 10 6 units/rag protein) for 10-12 hr and the culture fluid is harvested at the end of 16 hour.

11] Translation of human fibroblast interferon mRNA in African fish ova (Tilapiamosaiabica peters oocytes)

Publisher Summary The induction of interferon in human fetal skin muscle diploid cell cultures by inducers or its superinduction by polyinosinic acid·polycytidylic acid [poly(I)·poly(C)] and cycloheximide results in the activation of the cellular interferon structural gene, leading to the transcription of interferon messenger RNA (mRNA). The total cell poly(A)-RNA, containing interferon mRNA, can be isolated from the induced fibroblasts and is found to direct the synthesis of human fibroblast interferon in Tilapiamosaiabica peters oocytes. These fish ova can be much more easily obtained than those from Xenopus laevis. The criteria by which the translation products in fish ova is found to contain human interferon are (1) marked antiviral activity in homologous cell culture being detected with the translation products from mRNA of induced fibroblasts, but not from that of non-induced cells or from the rRNA and tRNA of induced fibroblasts and (2) the antiviral activity being neutralized by antiserum against interferon prepared from human fibroblasts from which the interferon mRNA has been isolated.

Interferon stabilization and enhancement by rare earth salts.

The addition of salts of the rare earth elements (lanthanides) enhances by a factor of two or more the initial activity of human fibroblast, leukocyte and immune interferons as well as mouse L-cell interferon. Furthermore, the salts of the lanthanides (0.01 to 0.002 M) preserve the activity of human fibroblast interferon not only after 4 days of heating at 37 degrees C but also after exposure to severe shearing forces; human leukocyte interferon was stabilized to a lesser degree, albeit significantly.

64] Purification of human fibroblast interferon produced in the absence of serum by cibacron blue F3GA-agarose and high-performance liquid chromatography

This chapter describes the procedure that combines the use of affinity chromatography and high-performance liquid chromatography to purify human fibroblast interferon produced in the absence of serum. The final product has a specific activity of about 3 x 10 8 units per milligram of protein and is homogeneous by the criteria of sodium dodecyl sulfate (SDS)-gel electrophoresis, amino acid analysis, and amino acid sequencing. In addition, this procedure has the advantage that the pure interferon is left in a volatile buffer, and thus is easily concentrated or dried for further studies. Affinity chromatography provides a high purification factor, but yields a dilute solution of interferon in 50% ethylene glycol. The final product is then obtained in concentrated form by HPLC. The interferon may be recovered salt- and solvent- free by evaporation because only volatile eluents are used. As proof of homogeneity, a single band is obtained on SDS-polyacrylamide gel electrophoresis as well as a single NH2-terminal amino acid sequence.

32] Interferon induction by polynucleotides, modified polynucleotides, and polycarboxylates

Publisher Summary This chapter discusses the procedures for obtaining high interferon yields in primary rabbit kidney (PRK) and human fibroblasts (HF) cells, in response to (I) n . (C) n . The chapter discusses several procedures that are developed to enhance the interferon response of PRK and HF cell cultures to (I) n . (C) n . An appropriate interferon induction scheme has been applied routinely to assess the interferon-inducing potentials of double-stranded RNAs in HF cells and could be readily adjusted to obtain human fibroblast interferon in large quantities. Parenteral administration of (I) n . (C) n results in an appreciable interferon response in most animal species in which (I) n . (C) n has been tested: upon intravenous injection of (I) n . (C) n , peak serum interferon titers will be attained at 2 hours in rabbits, 2-4 hours in mice, 3-6 hours in cats, 8 hours in monkeys, and 12-24 hours in man. As regularly done with (I) n . (C) n , polycarboxylates could also be injected intravenously to elicit an interferon response, and, indeed, interferon has been detected in the serum of humans given single or repeated intravenous doses of 8-16 mg/kg of pyran copolymer.

Effect of human fibroblast interferon on uncoating and release of murine leukaemia virus in NIH/3T3 mouse cells.

The effect of human fibroblast interferon (IFN), a heterologous IFN, on the uncoating of murine leukaemia virus (MLV) was investigated in exogenously infected NIH/3T3 mouse cells. Virus uncoating was determined by following the disappearance of the penetrating virus particles from the cytoplasm of the infected cells. Intracellular virus particles were estimated by sedimenting them at high speed from the cytoplasmic fraction of the infected cells and assaying their reverse transcriptase activity. In untreated control cells, uncoating started immediately after penetration, but in cells treated with human IFN, uncoating was delayed for 2 to 3 h. This delay led to prolongation of the infectious cycle, with delayed release of progeny virus. The delay in release did not result from inhibition by IFN of the process of release, since in NIH/3T3 cells chronically infected with MLV, treatment with IFN had no effect on virus release.

The reactivation of human interferons by guanidine thiocyanate.

The addition of 1.5 M guanidine thiocyanate (GuSCN) reactivates inactive human leukocyte interferon. The biological activity of inactivated human fibroblast interferon can be only partially recovered with GuSCN if additional (thermal) energy is supplied.

In vitro inhibitory effect of interferon on colony formation of myeloma stem cells

The inhibitory effect of interferon on colony formation of myeloma stem cells in two layer plasma clot-soft agar cultures was studied. Human lymphoblast interferon inhibited in therapeutically attainable concentrations myeloma stem cell proliferation in 50% and human fibroblast interferon in 23% of the 14 myeloma patients in whom in vitro colony formation could be achieved. In interferon-sensitive patients the numbers of myeloma stem cell clusters and colonies were decreased to 34.4%–54.9% of control cultures. In addition, maturation of myeloma stem cells in differentiated plasma cells was reduced by interferon in most of these cases.

Heterogeneity of poly(I) x poly(C)-induced human fibroblast interferon mRNA species.

Three classes of human interferons (IFNs) have been defined on the basis of their immunological properties: the 'Le' or 'alpha' IFN, mainly derived from leukocyte or lymphoblastoid cells; the 'F' or 'beta' IFN, mainly derived from fibroblast cultures; and the 'T', 'immune' or 'gamma' IFN, mainly derived from mitogen- or antigen-stimulated lymphoid cells. Whereas several individual species of Le IFN have been purified to homogeneity, it is generally considered that F IFN represents a single protein. Thus current efforts to clone human fibroblast IFN mRNA sequences are based on the observation that F IFN mRNA sediments in sucrose gradients as a single RNA species of size corresponding to 12-14 S (refs 7-10). We show here, using gel electrohporesis of mRNA, that two populations of translationally active human fibroblast IFN mRNA molecules exist--an abundant '14 S' species and a scarce '11 S' species. Microinjection of either species of mRNA into Xenopus oocytes leads to the synthesis of biologically active F-type human IFN. These data agree with and complement recent RNA hybridization studies of Weissenbach et al.

Le INTERFERON PRODUCTION BY HUMAN FIBROBLASTS

Abstract Human leukocyte ( Le ) and fibroblast ( F ) interferons differ in antigenic properties, physicochemical characteristics, and activity on cells of heterologous species. Previous work from this laboratory showed that two human foreskin fibroblast strains can produce either F interferon alone or both F and Le interferons, depending on the nature of the inducer. The present study demonstrates that both the cell type and the inducer determine the proportion of Le interferon produced by human fibroblast strains. Of eight strains tested, the human GM-258 strain, which is trisomic for chromosome 21, produced the highest absolute and relative amounts of Le interferon after Newcastle disease virus (NDV) inoculation. However, there was no correlation between Le interferon production and number of copies of chromosome 21 in other cell strains examined. Although both NDV and vesicular stomatitis virus (VSV) induced a substantial proportion of Le interferon in GM-258 cells, little or no Le interferon was induced in the same cells by polyinosinic-polycytidylic acid (poly(I)·poly(C) under five different conditions. In GM-258 cells induced with NDV, it was possible to partially manipulate the relative proportion of F and Le interferons and the time at which they were made by altering the conditions of induction. The percentage of Le interferon was increased by decreasing the multiplicity of infection or irradiating the virus with ultraviolet light. Kinetics of F and Le interferon production after different modes of NDV inoculation paralleled each other closely. F and Le interferon production was inhibited to similar degrees in the presence of the glycosylation inhibitors 2-deoxy- d -glucose and d -glucosamine. The bovine EBTr cell strain was used for the selective assay of Le interferon. These cells were as sensitive to Le interferon as human diploid fibroblasts. In contrast, less than 0.1% of human F interferon activity crossed the species line in EBTr cells. Le and F interferons produced by NDV-induced GM-258 cells were physically separated by affinity chromatography on immobilized anti- Le and anti- F globulins. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the Le interferon component produced in GM-258 cells formed two separate peaks (with molecular weights of approximately 18,000 and 25,000), closely resembling a control Le interferon preparation. In contrast, the F interferon moiety made in GM-258 cells formed a single peak on SDS-PAGE, as did control F interferon (molecular weight approximately 26,000).

NH2-terminal amino acid sequence of human fibroblast interferon.

The purification of human fibroblast interferon by chromatography on Blue Sepharose and high-performance liquid chromatography is described. The amino acid composition and a partial sequence of the homogeneous protein are reported. The NH2 terminus was determined to be NH2-Met1-Ser-Tyr-Asn-Leu-Leu-Gly-Phe-Leu-Gln-Arg-Ser-Ser-Asn-Phe-Gln-X-Gln-Lys.

Spontaneous production of human interferon.

Several established lines of human lymphoblastoid cells were evaluated for abilities to produce interferons. Some cell lines were able to produce interferon when induced with either Newcastle disease virus or Sendai virus, whereas others failed to produce detectable interferon when so induced. However, several cell lines were able to spontaneously produce interferon without induction. Spontaneously produced interferon was liberated by cells only during logarithmic growth phase, reaching levels ranging from about 10 reference units/ml of growth medium for some cell lines to 1000 reference units/ml for others. The interferons produced by induced lymphoblastoid cells and the spontaneously produced interferons were all characterized as type I human leukocyte interferon by high levels of cross-species antiviral activities on bovine cells and by neutralizations by antiserum to human leukocyte interferon but not by antiserum to human fibroblast interferon. However, analysis by electrophoresis in sodium dodecyl sulfate/polyacrylamide gels revealed that spontaneously produced interferon was less size heterogeneous than human leukocyte interferon, migrating as a single band of activity with a peak at 20,000 daltons, whereas human leukocyte interferon contained peaks of major activity at 23,000 and 18,000 daltons and virus-induced Namalva lymphoblastoid cell interferon migrated predominantly as the 18,000-dalton form. Also, although neither virus-induced primary leukocyte interferon nor any of the virus-induced lymphoblastoid cell interferons were neutralized by antiserum to mouse interferon, all of the spontaneously produced interferons were neutralized by antiserum to mouse interferons. These data suggest significant structural similarities between the active cores of certain interferons from phylogenetically diverse animal species.

Human fibroblast interferon in tears of patients with picornavirus epidemic conjunctivitis.

We report the levels of coxsackievirus type A24 (CA24) and the levels and type of interferon produced early during naturally acquired picornavirus epidemic conjunctivitis. Virus levels ranging from 10(1.8) to 10(5.8) 50% tissue culture infective doses per ml were detected in 29 of 37 acute (collected 1 to 4 days after onset of conjunctivitis) tear samples. Interferon (10(1.5) to 10(3.3) U/ml) was detected in 12 of 29 tear samples collected on day 1, in 2 of 6 tear samples collected on day 2, and in 1 tear sample collected on day 3 after onset of conjunctivitis. The interferon activity in pooled tear samples was completely neutralized by antiserum against human fibroblast interferon, stable at pH 2.0, and active against different viruses. In addition, the interferon activity in tears, like human fibroblast and leukocyte interferons produced in vitro, protected human and rabbit, but not mouse, cells. This is the first report of production and identification of the antigenic type of interferon induced by an enterovirus during natural infection. The early appearance of fibroblast interferon suggests that it may be an important host defense at the local site of implantation against this and possibly other enterovirus infections.

Augmentation of human monocyte-mediated cytolysis by interferon

Human monocytes, separated by either plastic adherence or adherence to microexudatecoated surfaces, from the peripheral blood of most normal donors were shown to have significant cytolytic activity against TU5, a mouse SV40-transformed target cell. Spontaneous cytolysis ranged from 0 to 32% at a 40:1 effector:target (E:T) ratio. Augmentation of cytolysis was usually seen when human fibroblast interferon (IF) (10 3–10 4 units/ml) was cultured with the effector and target cells for the duration of the assay. The mean increase in percentage cytolysis at 40:1 and 20:1 E:T ratios was greater with monocytes obtained by a microexudate method (24.1 and 22.4%) than with monocytes obtained by a plastic adherence method (16.0 and 8.1%). Only a slight augmentation of cytotoxicity was observed when the effector cells were pretreated with IF for 1-hr. The increased levels of cytotoxicity observed when IF was present during the assay did not appear to be due to the toxic effects of IF on the target cells or to a stable increase in the susceptibility of the target cells to lysis.

Expression of human fibroblast interferon gene in Escherichia coli.

The human fibroblast interferon gene was inserted in a thermoinducible expression plasmid under control of the phage lambda PL promoter. The primary translation products predicted on the basis of the plasmid constructions were hybrid proteins starting with beta-lactamase or phage MS2 polymerase information followed by the total preinterferon. On induction, antiviral activity, whose physico-chemical, immunological and biological characteristics closely corresponded to those of authentic human fibroblast interferon, was synthesized. Processing to a size compatible with mature but unglycosylated authentic product was observed.

Human interferon enhances gunaylate cyclase activity

Partially purified human leukocyte interferons, partially purified human lymphoblastoid interferon, and human fibroblast interferon enhanced rat liver, kidney, and splenic guanylate cyclase {E.C.4.6.1.2.} activity 2–4 fold at 5 μIU concentration. Dose-response relationships revealed that the human leukocyte interferons enhanced splenic guanylate cyclase activity at concentrations as low as 0.01 μIU while a concentration of 1 μIU for partially purified human lymphoblastoid interferon and 10 μIU concentration for human fibroblast interferon were necessary to see any effect on guanylate cyclase activity.

Antagonism in action between mouse or human interferon and platelet growth factor.

Human leukocyte and mouse fibroblast interferons abolished the mitogenic effect of human or bovine platelet growth factor (PGF) on human or mouse cells. Conversely, the antiviral and anticellular activity of both forms of interferon was inhibited by PGF in the homologous and in certain heterologous cell systems.