Human leukocyte interferon (HuLeIF) preparations contain distinct molecular forms of interferon exhibiting significant heterogeneities in sizes when analyzed by electrophoresis in sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gels, migrating in two broad bands of activity with peaks at about 21,000 and 15,000 daltons. HuLeIFs Even as the important roles played by interferons in processes of natural recovery from viral infections are becoming increasingly evident (1, 2), it also becomes apparent that production potentials will drastically restrict the interferons' availability for the clinic. Several laboratories have, therefore, felt it important to attempt complete purification of interferons with the goal of determining their essential components for possible chemical synthesis.However, many laboratories involved in attempting purification of human leukocyte interferons (HuLeIFs) to homogeneity have found their efforts thwarted by the realization that there are, in fact, distinct molecular forms of interferons in HuLeIF preparations, differing in size (3-6) and charge (7)(8)(9). These findings have prompted considerable speculation on the nature of these heterogeneities. Some workers have claimed that HuLeIFs are glycoproteins and that their size and charge heterogeneities can both be reduced by glycolytic enzymes (7). However, other investigators have reported that the charge heterogeneities are unaffected by treatment with glycosidases (9), and, on the basis of failure of HuLeIFs to bind to lectins, some authors have maintained that HuLeIF is not a glycoprotein (10).Analysis of the broad distribution of interferon activities within the two constant peaks of activity isolated by sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gel electrophoresis of HuLeIF preparations (3,4) This reagent was stored at 40 in a foil-wrapped bottle, and has given identical results over a period of several weeks.Interferon samples were diluted with equal volumes of periodate buffer and were kept at 40 for the indicated times, when 0.1 ml aliquots were removed and diluted 1:10 with 50% (vol/vol) ethylene glycol solution to stop the reaction. Samples were then divided into two equal parts, one for NaDcdSO4/ polyacrylamide gel electrophoresis, and one for isoelectric focusing. Samples for NaDodSO4/polyacrylamide gels were dialyzed against 0.01 M sodium phosphate buffer, pH 7.1. Samples for isoelectric focusing were dialyzed against 0.03 M NH4HCO3 buffer, pH 7.6, lyophilized, and resuspended in 0.2 ml of focusing sample buffer which contained 9.5 M urea (Schwarz/Mann, Ultrapure), 2% (wt/vol) Nonidet P40 (NP-40; Shell Imperial), and 2% Ampholine (vol/vol) (LKB; 4:1 mixture of pH 5-7 and pH 3-10).Isoelectric Focusing. A modification of the isoelectric focusing procedure of O'Farrell (13) was used to determine the various components of HuLeIF preparations. Briefly, these gels consisted of 4% acrylamide, 9 M urea, 2% Nonidet 40, and 2% Ampholine mix. Cylindrical gels (0.25 cm X 24 cm) were used. An aliquot of 0.02 ml of in...

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Human mononuclear leukocyte populations separated into T-, B-, and monocyte-enriched fractions, were evaluated for their abilities to produce interferons when induced with either virus, double-stranded RNA or mitogens. The main producer of alpha-interferon was the B-cell enriched subpopulation, but these cells did not produce gamma-interferon. In contrast, the T-cell enriched population produced either alpha or gamma interferons, depending upon the type of inducer used. The monocyte-enriched population was also able to produce either alpha or gamma interferon. The presence of monocytes in T-cell enriched populations enhanced the levels of alpha interferon production, and removal of monocytes from T-cell enriched populations diminished levels of alpha interferon produced. Even though each of the mononuclear populations (B-, T-, and monocytes) were able to produce alpha-interferon in response to poly rI.polyrC, it was found, by using a single-cell production assay, that only about 0.1% of the total mononuclear population actually produced alpha-interferon when so induced. Similarly, using the single-cell production assay, it was demonstrated that gamma-interferon was produced by only a small proportion of induced T-cells (possibly on T-cell subpopulation), as only about 1 T-cell per 1,000 responded to PHA by production of gamma-interferon.

SUMMARYThe molecular heterogeneity of acid-stable (Type I) mouse interferons induced in C2~3 cells by Newcastle disease virus was analysed by SDS-polyacrylamide gel electrophoresis under non-reducing and reducing conditions, and the profiles of antiviral activities obtained were characterized biologically in mouse cells and in heterologous (guinea-pig) cells. Two bands of activity, A and B, were consistently present in all interferon preparations tested: under reducing conditions, the activity in all fractions of band A (with a peak of activity at about 380oo daltons) was uniformly increased, while that of band B (with a peak at about 22ooo daltons) was uniformly diminished. All the active fraction in band A had only slight activity (less than IO 9/00 of homologous titres) on guinea-pig cells, whereas all those in band B were significantly more active on guinea-pig cells than on homologous L cells. Thus, mouse type I interferon preparations contain two molecular populations of interferons that can be distinguished physically (by size), biochemically (by the effect of reduction on reactivation from SDS) and biologically (by activity in heterologous cells).

Human white blood cells produced interferon (IFN) in response to Staphylococcus enterotoxin B (SEB) in vitro. The majority of this IFN was classified as IFN-gamma by virtue of its acid instability, non-neutralization by antisera recognizing alpha or beta IFNs and species-specificity for human cells. The IFN activity appeared after 3-6 hr and reached maximum levels between day 2 and 3. The SEB-induced IFN was partially purified to about 10(5.5) units/mg protein by chromatography on controlled pore glass beads (CPG) and Sephadex G-100 columns. While the CPG column purification step increased the specific activity 50 to 100-fold, two peaks of IFN activity were eluted from the Sephadex G-100 column. The first activity peak containing the majority of the loaded IFN appeared to be active only on human cells and was not neutralized by anti-HuIFN-alpha and had an apparent molecular weight of about 44,000 daltons, the second peak (about 5% of the loaded IFN) showed antiviral activity on both human and bovine cells, was at least partially neutralizing by anti-HuIFN-alpha and had an estimated molecular weight of 21,000 daltons. The specific activity in the first IFN peak was about 30-fold increased by the gel filtration chromatography with a recovery of approximately 60-80%.