Effect of T-activin on the production of immune interferon

A study was made of the effect of T-activin on the biosynthesis of immune gamma-interferon. It was shown that in 27% of patients with chronic nonspecific pulmonary diseases, production of gamma-interferon by lymphocytes was substantially reduced during exacerbation of inflammatory process in the lungs. It was discovered that T-activin was not an interferon inductor but enhanced its synthesis in patients with a low capacity of producing immune interferon even at small doses of interferon inductor. The preparation does not produce any effect on this process in normal subjects and in patients showing the normal level of gamma-interferon. Thus T-activin can be used for stimulation of interferonogenesis.     

Large scale production of human lymphoblastoid (Namalva) interferon

Summary Large scale production of human lymphoblastoid (Namalva) interferon (IF) is described. Cell propagation, in up to 50 1 culture volume, was carried out in a low cost medium by a semi-continuous cultivation method. IF was induced by Sendai virus, testing two induction methods. The yield of crude IF varied in the range of 12 – 100 × 10³ IF units.ml^-1. A weekly production output of 1 – 5 × 10⁸ units crude IF was obtained.     

Interleukin 2-mediated immune interferon (IFN-gamma) production by human T cells and T cell subsets

Human interleukin 2 (IL 2, or T cell growth factor), which was free of lectin and interferon activity (IFN), induced human peripheral T lymphocytes to produce immune IFN (IFN-gamma). In contrast, non-T cells and macrophages did not produce IFN-gamma in response to IL 2. IL 2 acted directly on unstimulated T cells to induce IFN-gamma production, and also acted in synergy with a suboptimal dose (2 micrograms/ml) of concanavalin A (Con A) to enhance IFN-gamma production. The IFN-gamma-inducing activity of partially purified IL 2 was absorbed along with the IL 2 activity by murine IL 2-dependent CT-6 cell line cells. This further supports the view that IFN-gamma-inducing activity is identical to IL 2. When T cells were separated further into helper/inducer T4+ and suppressor/cytotoxic T8+ subsets by negative selection with monoclonal antibody and complement, both T4+ and T8+-enriched cells produced significant levels of IFN-gamma in response to IL 2. Complete removal of macrophages from purified T lymphocyte populations by treatment of OKM1 plus complement consistently reduced IFN-gamma production in response to IL 2 to a limited degree; readdition of macrophages restored IFN-gamma production by both T cell subsets. This observation that IL 2 contributes to the production of IFN-gamma by human lymphocytes suggests that a cascade of lymphocyte-cell interactions participates in human immune responses.     

The human fibroblast and human immune interferon genes and their expression in homologous and heterologous cells

The genetic information coding for human fibroblast interferon (IFN-beta) has been cloned both as a DNA copy (cDNA) and as a genomic clone. Human IFN-beta is made as a precursor and consists of a signal sequence 21 amino acid residues long followed by the mature protein 166 amino acids long. A single site for glycosylation is present. The human IFN-beta gene does not contain introns. Transfection of monkey cells with a chimeric SV40 derivative containing the human IFN-beta cDNA clone under control of the late SV40 promoter leads to secretion of high levels of IFN-beta. When a genomic clone is used in the same vector, IFN-beta synthesis can be further enhanced up to 30-fold by treatment with poly(rI) . poly(rC); this shows that a cis-active control element is present in the clone. An efficient expression system in Escherichia coli was worked out based on a plasmid containing the promoter PL of bacteriophage lambda, which is regulated by a temperature-sensitive repressor. This promoter is followed by a segment derived from bacteriophage MS2 that contains the ribosome-binding site of the replicase gene. The latter, however, is replaced by the human IFN-beta gene. Upon induction, high levels (about 5 x 10(9) IU 1(-1)) of IFN-beta are synthesized by the bacteria; this corresponds to about 2% of the total bacterial protein. The human immune (type II) interferon (IFN-gamma) gene has similarly been cloned. Partly purified mRNA derived from human spleen cells that had been induced with staphylococcal enterotoxin A was used as starting material. A full-length cDNA clone was sequenced. The total cDNA sequence is about 1150 nucleotides long; it contains a single open reading frame coding for 166 amino acids, the first 20 of which constitute the transmembrane signal. There are two sites for glycosylation. The amino acid sequence is quite different from that of IFN-alpha or IFN-beta, although a few similarities can be noted. The untranslated 3'-terminal region is about 550 nucleotides long. The IFN-gamma gene was expressed in monkey cells, again by using the SV40-derived vector, and the secreted product was characterized as true human IFN-gamma. A genomic clone in the form of a bacteriophage lambda derivative was also obtained. The IFN-gamma gene extends over at least 5 kilobases and contains at least two introns.     

Antibody to staphylococcal enterotoxin A-induced human immune interferon (IFN gamma)

Antiserum to human gamma interferon (IFN gamma) was produced in rabbits immunized with partially purified (10(4.8) to 10(6.2) antiviral U/mg protein) staphylococcal enterotoxin A-induced IFN gamma. Staphylococcal enterotoxins, phytohemagglutinin M, concanavalin A, and pokeweed mitogen-induced antiviral activity in human leukocyte cultures was neutralized to undetectable levels by the antiserum. However, human leukocyte interferon (IFN alpha), human fibroblast interferon (IFN beta), and mouse interferons were not neutralized by the antiserum. After determining the antiserum was specific for IFN gamma and did not neutralize other known types of interferon, it was used with antibody to human IFN alpha to demonstrate the type(s) of interferon stimulated by some new inducers and antigens. Galactose oxidase- and calcium ionophore-induced interferons were neutralized to undetectable levels by the antiserum to IFN gamma. Interferon produced in leukocyte cultures from tuberculin-negative individuals stimulated with tuberculin-purified protein derivative or old tuberculin was IFN alpha, whereas interferon from tuberculin-positive individuals was a combination of alpha and gamma IFN. In addition, the antiserum neutralized the anticellular and natural killer cell enhancement activities of IFN gamma preparations. The specificity of this antiserum for IFN gamma indicates that it is an additional, powerful tool for identifying and classifying known and new interferons produced in vitro or in vivo and for investigating the role(s) of IFN gamma during the course of infectious, neoplastic, and autoimmune diseases.     

Effect of rabbit interferon on immune responses

Rabbit interferon was found to inhibit partially the proliferative response of rabbit lymph node cells to antigen. In contrast, neither the primary humoral immune response to sheep erythrocytes in vivo nor the secondary antibody response of lymph node fragments in vitro to diphtheria toxoid was significantly inhibited by interferon.It is suggested that the inhibition of lymphoid-cell proliferation by interferon is an expression of a more general tendency to inhibit mitotic activity.     

Effect of levamisole on interferon production by PHA-stimulated human leukocyte cultures

Mononuclear leukocytes of 10 normal blood donors were cultured in vitro and treated with phytohemagglutinin (PHA-P) and/or levamisole. Interferon-like activity was investigated in the supernatant fluids of the cultures, using VSV as challenge virus. In most of the cases the PHA-stimulated interferon-like activity was slightly but significantly enhanced by levamisole. The antiviral activity produced in the supernatant fluids was characterized as interferon since it was trypsin sensitive, species specific and inhibited by specific antiserum. This interferon has the characteristic sensitivity to PH2 of immune interferon.     

Participation of macrophages in enhanced in vitro immune interferon (IFN gamma) production with mouse spleen cells

IFN gamma production in cultures of spleen cells obtained from mice sensitized with TH69, a live Streptococcus faecalis preparation, was examined to determine how macrophages participate. It was demonstrated that sensitized spleen macrophages participated in enhanced IFN gamma production by T cells at an early stage (0-6 hr) of incubation, and that this production is mainly dependent on Ia-bearing macrophages In the reconstitution experiments where different combinations of spleen macrophages and T cells obtained from mice sensitized with TH69, OK-432, and BCG were used, T cells required that the identity between the sensitizing organisms in vivo and the stimulating organisms in vitro be the same for enhanced IFN gamma production while macrophages did not. Macrophage-mediated production of IFN gamma appears to be genetically restricted because IFN gamma was only produced in cultures where the H-2 region of macrophages and T cells matched. Further examination revealed that for macrophages to participate in enhanced IFN gamma production, first contact between cycloheximide-treated macrophages and T cells was required. Second, enhanced IFN gamma production occurred when culture supernatants of macrophages obtained from sensitized spleen cells were added to T cells. However, the addition of culture supernatant obtained from sensitized peritoneal macrophages resulted in inhibition of IFN gamma production. These results clearly showed the crucial role of macrophages in enhanced IFN gamma production by spleen T cells in vitro.     

Characterization of receptors for immune interferon in U937 cells with 32P-labeled human recombinant immune interferon

Recombinant human immune interferon (HuIFN-gamma) was labeled with [gamma-32P]ATP and cyclic-AMP-dependent protein kinase from bovine heart to a specific radioactivity of 11,000 Ci/mmol. At least two molecules of phosphate were incorporated per molecule of interferon. The binding of [32P]HuIFN-gamma to human U937 histiocytic lymphoma cells was time dependent, and displaceable by HuIFN-gamma but not by HuIFN-alpha A or HuIFN-beta. The specific binding was saturable with less than 10% nonspecific binding. The dissociation constant of [32P]HuIFN-gamma for U937 interferon receptors was calculated to be 1.5 X 10(-10) M with a total of 1,800 binding sites/cell. Dissociation of bound [32P]IFN-gamma at 24 degrees C exhibited two distinct rates. A fast dissociation with a specific rate constant of 0.141 min-1, and a slow dissociation with a specific rate constant of 0.0027 min-1. The Kd for [32P]HuIFN-gamma was calculated from kinetic constants to be 5.4 X 10(-10) M.     

Effect of different substrates and their preliminary treatment on cyclodextrin production

Summary Various kinds of substrates were tested for cyclodextrin production with cyclodextrin glucanotransferase (CGTase) from Bacillus megaterium. The enzyme formed cyclodextrin from different kinds of starch, dextrins, amylose, and amylopectin. However, the highest degree of conversion was obtained from starch. Corn starch appeared to be the best substrate – the cyclodextrin yield was 50.9%. The effect of molecular mass and preliminary treatment of starch with α-amylase on the CD yield was investigated. It was proved that CGTase preferred native starch with high molecular mass and low dextrose equivalent. The preliminary treatment with α-amylase occurred to be inefficient and unnecessary since it did not lead to an increase in the CD yield. Some of the substrates were treated with pullulanase. The effect of debranching was highest in the case of corn starch: the cyclodextrin yield increased by 10%.     

Effect of mouse genotype on interferon production

Upon induction with Newcastle disease virus, peritoneal macrophages derived from C57BL/6 mice produced ten times as much interferon as macrophages derived from BALB/c mice. This suggested that the alleles of theIf-1 locus are expressed in vitro by these cells. Further evidence for this was obtained by studying interferon production by peritoneal macrophages derived from seven recombinant inbred and one congenic line: in each case there was complete correlation between in vivo and in vitro phenotype: macrophages fromIf-1^l mice were low producers in vitro, and macrophages fromIf-1 ^h mice were high producers in vitro.     

Messenger RNA of mouse immune interferon (MuIFN-gamma)

A lysate of $\text{Staphylococcus aureus}$ ($\text{S. aureus}$) induced high-titered (10^3.6 ～ 10^4.6 units/ml) mouse immune interferon (MuIFN-γ) in spleen cell cultures. mRNA was extracted from such cells, purified by oligo(dT) cellulose chromatography and sucrose gradient centrifugation, and injected in $\text{Xenopus laevis}$ oocytes. Fractions containing RNA of 15 to 16 S gave rise to IFN activity, that was characterized as IFN-γ by virtue of its acid lability, neutralization by antiserum to partially purified MuIFN-γ, and lack of neutralization by antiserum to NDV-induced L-cell IFN.     

Interleukin 2 regulates immune interferon (IFN gamma) production by normal and suppressor cell cultures

A Lyt-2+ lymphocyte is responsible for immune interferon (IFN gamma) production in mitogen-stimulated mouse spleen cell cultures. A Lyt-1+ helper cell is required for the induction of IFN gamma. Interleukin 2 (IL 2) can specifically replace the Lyt 1 cell requirement. IL 2 will also abrogate suppressor cell inhibition of IFN gamma production. IFN gamma production appears to be regulated by a dynamic interaction between helper cells (source of IL 2), suppressor cells (absorption of IL 2?) and IFN gammna-producer cells.     

Mouse immune interferon enhances fibronectin production of elicited macrophages

By using mouse immune interferon (IFN-gamma) either produced by recombinant DNA technology or partially purified from a T cell lymphoma, L12-R4, we conclusively demonstrated that mouse IFN-gamma enhances fibronectin synthesis and secretion of proteose-peptone-elicited macrophages, but not of normal macrophages. The enhancing activity of both IFN-gamma preparations was acid-labile and was neutralized by a rabbit antiserum made against native IFN-gamma. In addition, IFN-gamma induced the disappearance, as revealed by immunofluorescence studies, of the characteristic fibronectin streaks from the cell surface of elicited macrophages. Taking into account the opsonizing activity of fibronectin, these findings offer some explanations for the IFN-gamma-mediated increase of macrophage phagocytosis and tumor cell killing.     

Crystallization and preliminary X-ray diffraction studies of bovine recombinant immune interferon

Reproducible conditions have been established for the crystallization of recombinant bovine immune interferon. Two cystalline forms of this protein were obtained. A tetragonal form, space group P422, with unit cell dimensions a = b = 59.0 A and c = 125.7 A and an orthorhombic form, space group P2(1)2(1)2(1), with unit cell dimensions a = 42.80 A, b = 79.90 A and c = 85.64 A were obtained under similar crystallization conditions. The orthorhombic form diffracts to 2.6 A resolution, contains a single interferon dimer in the asymmetric unit of structure and is suitable for X-ray diffraction analysis.