Interferon production by the preimplantation sheep embryo.

Ovine trophoblast protein-1 (oTP-1), the major product secreted by the trophectoderm of the sheep conceptus between days 13 and 21 of pregnancy, is considered to mediate maternal recognition of pregnancy by maintaining the function of the corpus luteum. Its amino acid sequence has 40-55% identity with various mammalian interferons-alpha (IFN-alpha), and it has been shown to have antiviral activity. The present results confirm that oTP-1, which at days 15-17 of pregnancy is produced by a single embryo at more than 100 micrograms (greater than 1 million antiviral units) per day, is a functional IFN. A preparation of purified oTP-1 was made. Its amino-terminal sequence suggested that it consisted of a single homogeneous protein, so that its antiviral activity probably was not due to a contaminant. In a cytopathic effect inhibition assay with GBK-2 bovine cells challenged with vesicular stomatitis, its specific activity was 1.3 X 10(7) end point units/mg protein. It also protected GBK-2 cells against four other viruses, and A549 human cells against encephalomyocarditis virus. The antiviral activity was neutralized by an antiserum to human leukocyte IFN. Like human IFN-alpha, oTP-1 at concentrations as low as 10(-9) M inhibited the growth of GBK cells in culture and suppressed mitogen-stimulated incorporation of [3H]thymidine into ovine lymphocytes. Possible roles for oTP-1, functioning as an IFN-alpha during early pregnancy, are discussed.

UCLA conference. Interferon: immunobiology and clinical significance.

Interferons are proteins elaborated by infected cells that protect noninfected cells from viral infection. These proteins produce a temporary "antiviral state" by altering nucleotide metabolism and cytoplasmic enzyme induction. Interferons appear early after viral infection locally and systematically to limit spread of viral infection; they also affect cell differentiation, growth, surface, antigen expression, morphologic findings, and immunoregulation. Several human disorders have diminished interferon production. Newborns have normal interferon alpha but deficient interferon gamma production. Infants with congenital infections may also have defects in interferon production. Immunosuppressed patients receiving transplants (marrow, heart, of kidney) have diminished interferon production, particularly immediately after transplant. Deficiencies of interferon have also been noted in Down's syndrome, cellular immunodeficiencies, uremia, malnutrition, and hematopoietic malignancy. Leukocyte interferon has been of therapeutic value in herpes zoster infections, in patients with cancer, and in patients with hepatitis B infection. Interferon has not been proved to help children with congenital cytomegalovirus or rubella. Interferon can shrink lymphoid tumors, particularly non-Hodgkin's lymphoma.

Psoralen inactivation of influenza and herpes simplex viruses and of virus-infected cells.

Psoralen compounds covalently bind to nucleic acids when irradiated with long-wavelength ultraviolet light. This treatment can destroy the infectivity of deoxyribonucleic acid and ribonucleic acid viruses. Two psoralen compounds, 4'-hydroxymethyltrioxsalen and 4'-aminomethyltrioxsalen, were used with long-wavelength ultraviolet light to inactivate cell-free herpes simplex and influenza viruses and to render virus-infected cells noninfectious. This method of inactivation was compared with germicidal (short-wavelength) ultraviolet light irradiation. The antigenicity of the treated, virus-infected, antigen-bearing cells was examined by immunofluorescence and radioimmunoassay and by measuring the capacity of the herpes simplex virus-infected cells to stimulate virus-specific lymphocyte proliferation. The infectivity of the virus-infected cells could be totally eliminated without altering their viral antigenicity. The use of psoralen plus long-wavelength ultraviolet light is well suited to the preparation of noninfectious virus antigens and virus antigen-bearing cells for immunological assays.

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.

Combined antiviral effects of interferon, adenine, arabinoside, hypoxanthine arabinoside, and adenine arabinoside-5'-monophosphate in human fibroblast cultures.

Adenine arabinoside and human interferon are currently being evaluated in clinical trials against herpes- and poxvirus infections. Interferon production is also a normal antiviral response. It is therefore important to examine the combined actions of interferon and antiviral arabinosides for possible synergy or antagonism. We have examined the antiviral activities of human fibroblast interferon, adenine arabinoside, hypoxanthine arabinoside, and adenine arabinoside 5'-monophosphate individually, using plaque inhibition of vaccinia and herpes simplex type 2 viruses in human skin fibroblast cultures. By combining doses of interferon and arabinosides that, acting alone, give intermediate degrees of plaque inhibition, we were able to compare the combined antiviral activity with that calculated from the activity of each inhibitor alone, assuming that the activities are statistically independent. Our results show that the plaque-inhibitory activities of interferon and the arabinosides tested are statistically independent. The results also show that the arabinosides do not destabilize the antiviral state previously induced by interferon, and that interferon pretreatment does not interfere with subsequent arabinoside action in infected cells. We have also found that arabinosides do not affect the induction of interferon synthesis by either Newcastle disease virus or double-stranded ribonucleic acid, and are not themselves interferon inducers.

Relative quantitative assay of the biological activity of interferon messenger ribonucleic acid.

RNA extracted from cells previously stimulated to synthesize the antiviral protein, interferon, causes species-specific interferon synthesis when added to heterospecific cell cultures. Our results confirm the report of De Maeyer-Geugnard, De Maeyer & Montagnier (1972). We have used this observation to obtain a relative quantitative assay for the interferon messenger RNA activity. At appropriate RNA concentrations, the yield of interferon is proportional to the concentration of RNA adsorbed to recipient cell cultures.