Unusual apparently constitutive interferons and antagonists in human placental blood.

We have detected seemingly uninduced interferons (IFNs) in 29/37 human placental samples obtained during caesarian sections at different periods of pregnancy, mostly around the 37th week. The amounts were usually low and did not enable us to correlate our findings with any physiological or pathological conditions. Occasionally the presence of IFN was masked by a lectin-like antagonist. Therefore, in a number of cases, substantially higher amounts of IFN were found after purification by affinity chromatography using concanavalin A, Cibacron blue, or antiserum to IFN-alpha, each coupled to Sepharose. Analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed the presence of IFN-alpha and IFN-beta with molecular masses between 15 and 80 kilodaltons. Some of the high molecular weight components were neutralized either only by monospecific antiserum to IFN-alpha or, to the same extent, by antiserum to IFN-alpha or to IFN-beta, reminiscent of those previously reported after viral induction in the human amniotic membrane. We postulate that both IFNs and antagonist play a physiological role during fetal development.

Phosphorylation of recombinant interferon-gamma by kinases released from various cells.

Recombinant interferon-gamma (IFN-gamma) in contact with human embryonic fibroblasts or with a great variety of cells from different animal species was phosphorylated in the presence of [gamma-32P]ATP and magnesium ions by a protein kinase released in the culture medium. Using SDS-polyacrylamide gel electrophoresis, we found that both the monomeric (17 000 to 18 000) and dimeric (34 000 to 35 000) molecular weight forms of IFN-gamma became intensely radioactive. Serine, but not threonine or tyrosine, was phosphorylated. It is of interest that the kinase released from reputedly insensitive cells also phosphorylated IFN-gamma. The process did not noticeably degrade the antiviral functions of the molecule nor did it affect, at least in a detectable manner, its anti-proliferative effect on WISH or Daudi cells. Furthermore, the antigenic structure and its capacity to react with monoclonal antibodies were also unaltered. It is presently not known which biological function is regulated by the phosphorylating process.

Sarcolectin: an interferon antagonist extracted from hamster sarcomas and normal muscles. Isolation, characterization, and purification.

In the present work we show that sarcoma and normal hamster tissues contain a protein which agglutinates normal and transformed cells. The inhibition of agglutination by galacturonic acid and occasionally by fucose suggests a resemblance of this protein with vegetal lectins. When added 5 h after interferon, the crude semipurified and electrophoretically homogeneous preparations reduce within 20 h the antiviral state pre-established by interferon. These two biological tests have enabled us to monitor the subsequent purification steps. The isolation of the biologically active protein is greatly facilitated by its resistance to pepsin and nucleases, whereas its sensitivity to trypsin and Pronase suggests its proteinaceous character. Furthermore, the molecule is stable when heated 1-2 min at 100 degrees C in the presence or absence of sodium dodecyl sulfate. After pepsin treatment, Sephacryl G-200 gel filtration, and ion exchange chromatography on DEAE-cellulose, 25-40-fold purification can be obtained. When controlled by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a double band (DEAE-cellulose sample) or single band (octyl-agarose sample) is detected in the 65,000-dalton region and no other contaminator is present. The eluted protein retains full biological activity when assayed by the degradation of the interferon-induced antiviral protection in the cell or titrated by cell agglutination.

Unusual human interferons produced by virus-infected amniotic membranes.

Interferon (IFN) induced in the human amniotic membrane contains at least five different molecular species, as shown by analysis in NaDodSO4/polyacrylamide gels after heating and under reducing conditions. Three of the IFN components reported here--migrating at 26, 43, and 80 kilodaltons--are of unusual antigenic structure because they are neutralized to about the same extent by anti-IFN-alpha and anti-IFN-beta antibodies. The 15- to 17-kilodalton species belongs to the IFN-alpha group, while the 21- to 22-kilodalton species, the most frequently detected major peak, is IFN-beta. In addition to their unusual size and antigenic structure, these IFNs could play a role during embryonic development and in the immune tolerance of the mother with regard to the fetus.

[Production of human type I interferon by lymphocytes in contact with cells infected by herpesvirus and fixed with glutaraldehyde]. / Production d'interféron humain de type I par des lymphocytes au contact de cellules infectées par le virus herpès et fixées par le glutaraldéhyde.

Type I interferon was released from human lymphocytes cultured on cells infected with Herpesvirus and fixed with glutaraldehyde. This process of interferon induction did not involve histocompatibility antigens nor the Herpes immunity of the lymphocyte donors. Type I interferon can also be induced after interaction between lymphocytes and other intranuclear replicating virus infected cells.

Human leukocyte interferon: relationship between molecular structure and species specificity.

Human leukocyte interferon can be separated into two classes of subspecies by polynucleotide-agarose affinity chromatography; 30-40% of the molecular species have the polynucleotide-binding property and 60-70% lack affinity for the polynucleotide ligand. When analyzed on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the former class of interferon has a slower mobility corresponding to the migration of a polypeptide of 21,000 daltons, while the latter class has a faster mobility corresponding to a polypeptide of 13,500-15,000 daltons. By analogy to the behavior of other interferons and a class of nucleotidyl transferases on the polynucleotide-agarose chromatography, we suggest that the human leukocyte interferon having the polynucleotide-binding site is in a possibly "native" conformation and the loss of affinity for polynucleotide results from a degradative alteration of the native molecules. Moreover, the alteration of interferon is accompanied by an increase in heterospecific activity on bovine cells. It is suggested that the polypeptide domain responsible for species specificity may be closely related to the polynucleotide binding area. The modified interferon molecule, however, still conserves its antiviral activity. The simplicity and the high capacity of polynucleotide-agarose chromatography make this a powerful technique for the purification of interferon. The easy separation of these two classes of human leukocyte interferon makes the purification procedures more rational and will facilitate the preparation of both subspecies to a high degree of molecular homogeneity.

Effect of ammonium salts on the interferon-induced antiviral state in mouse L cells.

The addition of ammonium salts to cells treated with interferon prevents the development of the antiviral state and destroys it when already established. This treatment does not seem to act on the binding of interferon to the cells but blocks a further step of activation on the cell membrane. The anti-interferon effect of ammonium salts is reversible with a complete recovery of the antiviral state. It is postulated that these salts may stabilize the interferon-receptor complex and thus prevent the changes in configuration necessary for the establishment and maintenance of its biological functions.