Kurt Paucker

Distinct Molecular Species of Human Interferons: requirements for Stabilization and Reactivation of Human Leukocyte and Fibroblast Interferons

Human fibroblast interferon preparations were completely stabilized to 100 degrees C by sodium dodecyl sulphate (SDS) in the presence of mercaptoethanol, but only a minor fraction of their activities were stabilized by SDS without mercaptoethanol. On the contarary, human leukocyte interferon preparations were completely stabilized to 100 degrees C by SDS in the absence of mercaptoethanol, but only a minor fraction of their activities were stabilized by SDS in the presence of mercaptoethanol. Furthermore, human fibroblast interferon preparations whose activities had been destroyed by boiling at 100 degrees C were completely reactivated by SDS under reducing conditions, but only a minor part of their activities were restored by SDS in the absence of reduction. On the contrary, human leukocyte interferon preparations whose activities had been destroyed by boiling at 100 degrees C were completely reactivated by SDS in the absence of reduction, but only a minor part of their activities were restored by SDS under reducing conditions. These data suggest that there are distinct molecular species of human interferons.

Interferon and Cell Division VII. Inhibitory Effect of Highly Purified Interferon Preparations on the Multiplication of Leukemia L 1210 Cells

Summary Highly purified mouse interferon preparations prepared by different techniques in different laboratories inhibited the mul–tiplication of mouse leukemia L 1210 cells. The dose–response curves of the antiviral activity of interferon and the anticell multiplication activity using a sensitive assay were parallel. Likewise, the kinetics of the appearance of the factor responsible for both activities and the curves of thermal inactiva–tion of this factor were also parallel. It seems reasonable to conclude that interferon itself can inhibit cell multiplication. I. G. is deeply indebted to Dr. Sidney Farber, Director of the Childrens Cancer Research Foundation, Boston, MA for his continued interest and support. K. P. acknowledges the aid of NSF Grant No. G. B.–26515. M. T. is the recipient of an EMBO fellowship.

Biological Properties of Human Leukocyte Interferon Components

Human leukocyte interferon, purified approximately 1000-fold by affinity chromatography on immobilized anti-interferon globulins and SDS-Sephadex filtration, was resolved into one major and one minor component by adsorption chromatography on hydroxylapatite and electrophoresis in polyacrylamide gels. These components were indistinguishable in their capacity to protect bovine, porcine and murine cells, and the antiviral activities of both were equally susceptible to reduction by beta-mercaptoethanol. They were neutralized to the same degree of rabbit anti-leukocyte interferon but were not neutralized by rabbit antifibroblast interferon serum. Mice immunized with either component developed antibodies to both but failed to form antibodies against human fibroblast interferon. Our present evidence indicates that the two components posses at most only minor structural and antigenic dissimilarities.

[74] Preparation and absorption of antiserum against mouse L cell interferon

Publisher Summary This chapter describes the production and absorption of sheep antiserum to live Newcastle disease virus (NDV) induced mouse L cell interferon. Antibody titers to known contaminants found in the interferon preparation are measured by a passive hemagglutination test as detailed. Three contaminants of NDV-induced mouse L cell interferon are chosen to monitor the success of the absorption procedure: (a) egg albumin, since the inducer virus is produced in embryonated eggs, (b) bovine serum albumin, present in the serum used to culture the mouse cells, and (c) L cell extract, a mixture of antigens from sonicated mouse L cells. The anti-mouse interferon serum is absorbed by the same procedure described for the absorption of anti-human leukocyte interferon serum. As in the case of the absorption of antiserum to human leukocyte interferon, the unbound material also had to be supplemented with egg albumin in order to remove a large portion of the antibodies to this contaminant. The level of antibodies to the test contaminant antigens that remained after absorption are reduced to background titers found in control serum taken prior to immunization.

Purification of acid ethanol-extracted human lymphoid interferons by Blue Sepharose chromatography☆

Abstract Human leukocyte and lymphoblastoid (Namalva) interferons were purified by a modified acid ethanol extraction procedure and chromatography of the dilute ethanolic interferon solution on Blue Sepharose. Both interferons were purified more than 1000-fold, with recoveries ranging from 35 to 40%.

Characterization of interferon-associated proteins.

Summary L cells in which interferon had been induced by u.v. irradiated Newcastle disease virus were exposed for varying periods of time to [3H]-l-methionine, [3H]- or [14C]-d-glucosamine, [3H]-protein hydrolysate or [14C]-l-fucose. Interferon was subsequently purified by SE-Sephadex chromatography and polyacrylamide gel electrophoresis to contain in excess of 1 × 106 units/mg. of protein. Electropherograms disclosed that methionine, amino acid mixture and, to a lesser degree, glucosamine, were incorporated into proteins associated with the dominant interferon fraction. No incorporation of fucose was noted. Methionine and glucosamine labels were absent from the major interferon band in control preparations from non-induced cells, co-electrophoresed in acrylamide gel with non-labelled chromatographed marker interferon.

Biological properties of human interferon fractions obtained by Blue Sepharose chromatography

SummaryHuman interferons of lymphoid and nonlymphoid origins were segregated into several fractions by virtue of their elution properties from Blue Sepharose. According to desorption of the main portion of antiviral activity, three major classes could be discerned: 1. Leukocyte interferons eluting predominantly in 0.5m NaCl buffer, 2. Lymphoblastoid interferons eluting mostly in 1m NaCl buffer, and 3. Cell culture-derived fibroblast and bladder carcinoma interferons requiring ethylene glycol in addition to 1m NaCl for elution. Desorption behavior from Blue Sepharose did not necessarily correlate with the presence of specific antigenic markers and no consistent segregation of Le and F antigens in individual fractions was observed. All fractions exhibited comparable activity in heterologous sheep and homologous human cells. Therefore, no distinctive biological features could be associated with multiple interferon species isolated by Blue Sepharose chromatography.

Molecular Species of Interferon Induced in Mouse L Cells by Newcastle Disease Virus and Polyriboinosinic-polyribocytidylic Acid

Interferons were stimulated in mouse L cells by Newcastle disease virus (NDV) or by polyriboinosinic-polyribocytidylic acid poly(rI).poly(rC). These were fractionated by sequential affinity chromatography on bovine plasma albumin (BPA)-Sepharose and on omega-carboxypentyl (CH)-Sepharose. Based on their interaction with CH-Sepharose, interferon induced by NDV was resolved into three major bands of activity (L/NDV-1,2,3) and poly(rI).poly(rC)-interferon into two (L/rI:rC-1,2). These interferon components were purified to a specific activity of 3 X 10(7) to 4 X 10(7) units/mg protein by antibody affinity chromatography and examined by electrophoresis in SDS-polyacrylamide gels. A total of five molecular species was thus identified for NDV-induced interferon and three for poly(rI).poly(rC) induced interferon, as summarized in Table 1. We conclude from our observations that mouse interferons can be produced by L cells in multiple forms with specific physiochemical properties and in proportions determined by the type of agent employed for induction.

Affinity chromatography of primary human amnion interferon

SummaryThe chromatographic behavior of human amniotic interferon on various affinity chromatography ligands was studied. Most of this interferon bound strongly to bovine plasma albumin-agarose, cibacron blue F3 GA-agarose, concanavalin A-agarose and L-tryptophyl-L-tyrosine-θ-carboxyl-pentyl-agarose. After binding most of the interferon activity was eluted only with 50 percent ethylene glycol, showing the high hydrophobicity of this interferon. Smaller quantities could be recovered after phosphate-buffered saline elution or with increased salt concentration. On BPA-θ-carboxy-pentyl-agarose and θ-aminohexyl-agarose, the majority of the biological activity was found in the break-through fraction (eluted with phosphate buffered saline) while some interferon was displaced with high salt or ethylene glycol. Increasing the salt concentration and lowering the pH was necessary to elute interferon from zinc chelate-agarose.These patterns indicate that human amniotic interferon is similar to human fibroblast (β) interferon but different from human leukocyte (α) interferon. However, the heterogeneity displayed by amniotic interferon on bovine plasma albumin-agarose requires further investigation.

Blocking of interferon production by chromatographically purified L cell interferon.

Summary Pretreatment of L cells with mouse interferon diminishes their responsiveness to interferon induction (blocking effect). The blocking property of interferon preparations is liberated simultaneously with interferon from stimulated cultures. Both activities are reduced to the same degree by partially inhibitory concentrations of actinomycin D. Chromatographic purification of interferon (specific act, 1–3×106 units/mg protein), fails to dissociate a separate blocking component from interferon. Nonpurified and chromatographed interferon preparations display the same blocking potency in relation to interferon content. Blocking can be partially or completely reversed by increasing the multiplicity of infectious NDV. Under the same conditions, reversal of blocking does not occur with excessive inducer concentrations of NDVuv and poly I:C. The authors are grateful for the competent assistance of Marilyn J. Mundy.