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W E Stewart

Publications and source records attributed to W E Stewart.

At least 37 records · Page 2Linked to original sources
PubMed1981

Size characteristics of human leucocyte interferon under reducing and non-reducing conditions.

Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was used to characterize human leucocyte interferon (HuIFN-alpha) under reducing and non-reducing conditions. Under non-reducing conditions HuIFN-alpha possesses two size forms, but under reducing conditions (r-HuIFN-alpha) only one is observed. The apparent molecular weight of this one form varies with the concentration of 2-mercaptoethanol used. When r-HuIFN-alpha is permitted to reoxidize the bimodal configuration of HuIFN-alpha is restored. The size heterogeneity of native HuIFN-alpha can be eliminated by mild treatment with NaIO4 [HuIFN-alpha/IO4; Stewart II, Lin, Wiranowska-Stewart & Cantell (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 4200-4204]. The size of the HuIFN-alpha/IO4 increases after treatment with 2-mercaptoethanol (r-HuIFN-alpha/IO4) and the apparent molecular weight of this component also varies with the concentration of 2-mercaptoethanol used. In the case of r-HuIFN-alpha the single peak observed apparently originates from both the higher- and lower-molecular-weight components.

Electrophoresis, Polyacrylamide Gel↗
PubMed1981

Heterogeneity of interferon mRNA species from Sendai virus-induced human lymphoblastoid (Namalva) cells and Newcastle disease virus-induced murine fibroblastoid (L) cells.

Cytoplasmic polyadenylated RNA preparations obtained from Sendai-induced human lymphoblastoid (Namalva) cells and from Newcastle disease virus (NDV)-induced murine (L) cells were denatured in 10-12.5 mM CH3HgOH and then electrophoresed in 2% agarose tube gels containing 10 mM CH3HgOH, the RNA eluted from gel slices and translationally active interferon mRNA species located using the Xenopus oocyte assay. The interferons synthesized were characterized as alpha or beta types based on neutralization tests using specific antisera against human or murine interferon-alpha and interferon-beta. At least two species of mRNA for human interferon-alpha and two for human interferon-beta were detected in RNA from Sendai-induced Namalva cells. These are (approximate mRNA length in parentheses) alpha (1.3 kb), alpha (1.9 kb), beta (1.1 kb) and beta (1.9 kb). Two populations of murine interferon mRNA of lengths approximately 1.4 kb and 3 kb were detected in mRNA preparations from NDV-induced L cells by electrophoresis. However, since the translation products of each of these two populations of mRNA consist of both murine interferon-alpha and murine interferon-beta it is likely that both the 1.4 kb and 3 kb populations contain at least one species each of murine interferon-alpha and murine interferon-beta mRNA.

Animals↗
PubMed1981

Target cell specificity of two species of human interferon-alpha produced in Escherichia coli and of hybrid molecules derived from them.

Plasmids containing cDNAs for human interferon (IfN) alpha-1, IFN alpha-2, and several hybrids of the two cDNAs, all joined identically to an Escherichia coli lac promoter fragment gave rise, in E. coli, to fused interferons (fIFNs) that had very different target-cell specificities. fIFN alpha-1 had a lower specific activity on human WISH cells than on bovine MDBK cells, while fIFN alpha-2 showed the opposite behavior. fIFN hybrids with the NH2-proximal half of fIFN alpha-2 behaved qualitatively like fIFN alpha-1, and those with the NH2-proximal half of fIFN alpha-2, behaved like fIFN alpha-2. On mouse L929 cells, fIFN alpha-2 was almost inactive, while fIFN alpha-1 showed relatively high activity. In this case, the fIFN hybrids with the COOH-proximal half of IFN alpha-1 showed activity on mouse cells, while the reciprocal hybrid did not. In many cases, the activity spectrum of the hybrids was very different from that of either parent. We propose that the IFN molecule has either two binding sites or two regions constituting the binding site, one in the COOH- and the other in the NH2-proximal half. The experimental findings can be accounted for if the fits of the two sites to their receptor counterparts on different cell lines are independent of one another.

Animals↗
PubMed1981

Determination of human leukocyte populations involved in production of interferons alpha and gamma.

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.

B-Lymphocytes↗
PubMed1981

Isolation of a Biologically active fragment of human alpha interferon.

When treated with pepsin under limited digestion conditions, human leukocyte-derived alpha interferon (HulFN-alpha) is partially inactivated. While the initial HulFN-alpha preparation is about equally active on human and on bovine cells, the residual antiviral activity of HulFN-alpha after pepsin treatment is significantly more active on bovine cells than on human cells. Furthermore, pepsin-treated HulFN-alpha, when assayed on bovine cells, yields an active production which is smaller and antigenically distinguishable from the native forms of HulFN-alpha.

Animals↗
PubMed1981

Binding and capture of human interferon-alpha by reverse evaporation vesicles, multilamellar vesicles, and small unilamellar vesicles.

Liposomes of various physical and chemical compositions were prepared and their ability to externally bind and internally capture human interferon alpha (HuIFN-alpha) was determined. HuIFN-alpha was bound by preformed liposomes composed of either dipalmitoyl phosphatidyl choline alone or dipalmitoyl phosphatidic acid (calcium salt), and cholesterol, with or without a phosphatidyl choline component. HuIFN-alpha could also be internally captured within liposomes both of compositions that bound or did not bind interferon. The interferon could be associated with liposomes in at least three manners: bound to the outside surface of the liposome, associated partially within the liposomal membranes, or completely internalized either within the aqueous compartments of the liposome or completely buried within the liposomal bilayer membrane. HuIFN-alpha was stably-associated with reverse-evaporation vesicles, multilamellar vesicles, and small unilamellar vesicles for 30 days at 4 degrees C. Incubation of the multilamellar vesicles at 37 degrees C caused an initial decrease in the amount of externally bound interferon, and incubation with mouse serum caused a further dissociation of the externally-bound interferon. Depending on liposome composition, incubation at 37 degrees C either had little effect on stability of internalized interferon or caused leakage of internalized interferon.

Cholesterol↗
PubMed1981

Human gamma interferon production by leukocytes induced with monoclonal antibodies recognizing T cells.

The murine monoclonal antibody OKT3 (IgG2), which recognizes the majority of human E-rosette-forming cells, was found to induce interferon (IFN) production in human peripheral lymphocytes in vitro. This IFN was classified as IFN-gamma by virtue of its acid stability, nonneutralization by antisera recognizing human IFN-alpha and beta, and non-cross-reactivity on bovine kidney cells. OKT3 elicited considerable IFN-gamma yields at a concentration of 1 ng/ml, at which only very low mitogenesis of the lymphocytes measured by 3H-thymidine incorporation was seen. The IFN-gamma activity appeared beginning 3 to 6 hr after stimulation and reached its maximum after 36 hr. Anti-human Lyt3, anti-human Lyt2, or anti-human Lyt1 monoclonal antibodies, recognizing all or the majority of the E-rosette-forming cells, did not elicit IFN-gamma production. The OKT4 monoclonal antibody, which defines a subpopulation of T cells containing cells with helper activity, induced marginal levels of IFN-gamma. In contrast, OKT8 monoclonal antibody (defining cytotoxic/suppressor cells) as well as monoclonal or heterologous antibodies recognizing histocompatibility antigens or surface immunoglobulin failed to induce IFN-gamma production. Heterologous anti-thymocyte globulin or anti-lymphocytic serum were effective IFN-gamma inducers. Cell separation studies of the leukocytes demonstrated that E-rosette-forming cells are responsible for production of IFN-gamma induced by OKT3. Treatment of the leukocyte suspension with OKt3 monoclonal antibody plus complement abolished IFN-gamma production induced by this antibody. These results demonstrate that the OKT3 antibody recognizing an antigen present on the majority of the E-rosette-forming cells alone is sufficient to induce IFN-gamma production. OKT3-positive cells are apparently the producers of this IFN-gamma.

Antibodies↗
PubMed1980

Comparisons of several biological and physicochemical properties of human leukocyte interferons produced my human leukocytes and by E. coli.

Human interferon (IFN) prepared from virus-induced human leukocyte suspensions (leukocyte-derived interferon) was compared to the IFN extracted from Escherichia coli harboring a human interferon-alpha cDNA hybrid plasmid (Hif-SN35-AH-L6). E coli-derived IFN was 20 to 50 times more active than leukocyte-derived IFN on heterologous bovine, feline, murine and guinea pig cells, relative to the activity on human cells. After partial purification by affinity chromatography on an anti-human lymphoblastoid IFN antibody column, the IFN was analyzed by SDS-polyacrylamide gel electrophoresis. While leukocyte-derived IFN gave a heterogeneous pattern with major peaks of activity of 24000 and 19000 daltons, E. coli-derived IFN gave a heterogeneous peak of activity at about 17-18000 daltons. The leading edge of leukocyte-derived IFN in SDS-polyacrylamide gels was significantly more active on bovine cells than on human cells and coincided in mobility with E. coli-derived IFN, which was also much more active on bone than on human cells. After reduction with mercaptoethanol in SDS, the E. coli-derived IFN lost no activity, whereas the leukocyte-derived IFN lost about 90% of its activity. After reduction, E. coli-derived IFN migrated in SDS-polyacrylamide gels as a single peak at 24000 daltons, as did the residual activity of reduced leukocyte-derived interferon. Out data suggest that the interferon produced by the E. coli harboring the clone Hif-SN35-AH-L6 is analogous in size and cross-species activity to one of the molecular species of leukocyte-derived interferon.

Chromatography, Affinity↗
PubMed1980

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.

Cell Division↗
PubMed1980

Production and initial characterization of guinea pig interferon.

Guinea pig cell cultures produced extremely low levels of interferon when induced with Newcastle disease virus (NDV), or double-stranded RNA, poly rI.poly rC. Priming guinea pig cells with mouse interferon or guinea pig serum interferon did not significantly enhance interferon production. However, intracardial injection of 10(9) plaque forming units of NDV into guinea pigs lead to interferon production to levels over 6000 units per ml of serum 6 hr after the inducer was administered. The antiviral agent in the serum had the characteristics of interferon. Guinea pig interferon showed low levels of activity on mouse and bovine cells, and no detectable activity on human or rabbit cells.

Animals↗
PubMed1980

Skin reactions to interferon inoculations are reduced but not abolished by purification.

The skin reactions to partially-purified fibroblast-derived (HulFN-beta) and leucocyte-derived (HulFN-alpha) interferons and to HulFN-alpha of increasing purity were studied in normal volunteers. Reactions consisted of an immediate flare followed by a dense well-circumscribed erythema maximal at 4-8 hours. HulFN-beta caused significantly larger reactions than HulFN-alpha of approximately the same purity. Immediate flare reactions occurred even to the most pure preparation tested but the late reactions were significantly reduced. Experiments suggested that the early reactions to impure HulFN-alpha were in part mediated by local histamine release and late reactions in part by local prostaglandin synthesis. This suggests that interferon itself may not be the cause of general symptoms during a virus illness.

Chromatography, Gel↗