Faculty Bibliography

Attempts were made to demonstrate human alpha interferon production in virally induced human-rodent cell hybrids. In all hybrids investigated human beta-interferon production was detected when the relevant human chromosomes were retained, but only low levels of human alpha interferon could ever be detected even when human chromosome 9, which contains at least eight alpha-interferon genes, was present in the hybrid. The detection of human alpha interferon was made possible only by the use of specific antisera to human alpha and beta interferon and the sensitive virus yield-inhibition assay. Evidence is presented for independent regulatory mechanisms governing the expression of human alpha and beta interferons in human-rodent hybrids

The diterpene ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and several structurally related compounds were tested for their ability to stimulate interferon (IFN) production in primary cultures of human leukocytes. In cultures of Ficoll-Hypaque-purified mononuclear cells, TPA treatment alone induced only low levels of IFN, but TPA pretreatment of cells caused significant enhancement of IFN yields produced with phytohemagglutinin or several other T cell mitogens. In cultures of unprocessed cells derived from plateletpheresis residues or buffy coats, TPA treatment alone induced high levels of IFN and costimulation with TPA and phytohemagglutinin produced some further enhancement of IFN production. Phorbol 12,13-dibutyrate was comparable to TPA in its ability to enhance phytohemagglutinin-induced IFN production. Several other phorbol ester analogs were also active, but maximal stimulation occurred only at higher drug concentrations. Mezerein, a structurally related diterpene ester, was at least as active as TPA in stimulating IFN production in either Ficoll-Hypaque-purified or unprocessed cells. IFN produced after stimulation with TPA or mezerein, singly or in combination with phytohemagglutinin, had several properties characteristic of IFN-gamma, e.g., it was largely inactivated by dialysis at pH 2, or after exposure to sodium dodecyl sulfate, whereas it was not neutralized by antibody to IFN-alpha and IFN-beta. The stimulatory effect of diterpene esters has proved helpful in producing IFN-gamma for physicochemical analysis and other studies

gamma (immune) interferon (IFN-gamma) was induced in cultures of fresh human lymphocytes by combined treatment with a phorbol ester (12-O-tetradecanoylphorbol 13-acetate, TPA) and the T cell mitogen phytohemagglutinin (PHA). Compared to the IFN-gamma yields obtained with PHA induction alone, the inclusion of TPA caused a significant enhancement of IFN-gamma production. Poly(A)-containing mRNA was isolated from mononuclear cells induced with TPA and PHA. Injected into Xenopus laevis oocytes, this mRNA preparation gave rise to IFN activity with characteristic properties of human IFN-gamma. Sucrose density gradient centrifugation analysis showed that IFN-gamma mRNA sedimented at 15 S, suggesting that it contains a total of about 1400 nucleotides

Human gamma (immune) interferon (IFN-gamma) was produced in lymphocyte cultures stimulated with a phorbol ester (12-O-tetradecanoylphorbol 13-acetate) and purified phytohemagglutinin. Physicochemical analysis showed that human IFN-gamma is a glycoprotein with an isoelectric point around 8.6 and an apparent molecular weight of 58,000 +/- 3000. A purification process for IFN-gamma was developed consisting of sequential chromatographic separations on controlled-pore glass, concanavalin A-Sepharose, and Bio-Gel P-200. This purification process resulted in an increase in specific activity from about 10(4) (crude culture fluid) to an estimated 10(7) units per mg of protein with a cumulative recovery of about 40% of the IFN activity

Human leukocyte interferon has been trapped in the aqueous interstices of multilamellar liposomes (phosphatidylcholine, dicetyl phosphate, and cholesterol [7:2:1]). Such liposomes trapped [3H]inulin (aqueous space marker) and interferon to the extent of 0.22 +/- 0.01 mg (n = 8) and 350 +/- 54 U (n = 4) per mumol of liquid, respectively, as judged by molecular sieve chromatography. Interferon trapped within liposomes was resistant to tryptic digestion under conditions which completely inactivated free interferon. Studies in which interferon was added to preformed liposomes excluded the possibility that interferon bound nonspecifically to the outer layer of the multilamellar liposomes. When interferon was added to the aqueous medium in which liposomes of various net surface charges were permitted to form, trapping of interferon varied directly with the interlamellar aqueous compartments of the liposomes. The demonstration that stable liposomes can entrap interferon suggests that these may constitute suitable vectors for the delivery of interferon to cells