Search

The alpha-interferons (IFN-alpha) belong to a family of polypeptides comprising several subtypes. Using recombinant DNA technology, it has been possible to create IFN hybrids that provide novel combinations of the amino acid residues from the parental protein sequences. They have been used to study structure-activity relationships of IFN-alpha and interactions with the IFN-alpha receptor, and to create analogs of natural IFNs with novel properties for potential therapeutic application. The biological data obtained with these hybrids are now evaluated in terms of the published structural and homology models of IFN-beta and -alpha.

MxA protein is interferon inducible, and its role as an antiviral mediator is being studied in various viral diseases. Several cytokines, including type 1 interferons (alpha and beta), interleukins 2 and 12, and granulocyte, macrophage, and granulocyte-macrophage colony-stimulating factors, were tested for their ability to induce human MxA protein synthesis in peripheral blood mononuclear cells from 15 chronic hepatitis B virus-infected patients and 6 healthy subjects as controls. Constitutive MxA expression was scarce in patients and controls but increased significantly in response to type I interferons. MxA responsiveness to interferon alpha was diminished significantly in chronic hepatitis B patients, compared with healthy donors (P0.05); this effect was more marked in patients with high viremia levels. Interleukins 2 and 12, and none of the colony-stimulating factors tested, induced low, but detectable, MxA protein levels. These results indicate that chronic infection by hepatitis B virus may impair activation of the immune cells and their capacity to respond to type 1 interferons.

Serum neopterin (Np), beta 2-microglobulin (beta 2-M), and 2',5'-adenylate (2',5'A) levels and intracellular 2',5'A and human Mx (Hu-Mx) protein synthesis were measured in 20-24 chronic myeloid leukemia patients before and during 1 year of IFN-alpha treatment and in a further 8-9 patients before and at the end of the first and second treatment weeks only. Univariate analysis showed that IFN-alpha increased Np and 2',5'A serum levels and intracellular concentrations of 2',5'A and Hu-Mx significantly from the end of the first week to month 12 of therapy. The biologic marker profiles were similar in cytogenetic responders and nonresponders, as well as in patients treated with IFN-alpha early (12 months from diagnosis) or late (after12 months standard chemotherapy). Further, there were no differences in the short-term (first 14 days) or long-term (during 12 month therapy) induction of the biologic markers irrespective of whether IFN-alpha 2a or IFN-alpha 2b was given. Because multivariate analysis revealed no significant interactions between cytogenetic response, time to treatment, and type of IFN-alpha used, increments in intracellular 2',5'A and Hu-Mx protein were similar at all study times for all factor combinations tested. Np levels varied significantly only during the first 14 therapy days; changes in serum 2',5'A were never statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

The human MxA protein is a new specific marker for type I interferon activity both in vitro and in vivo. In the study presented here, this interferon-induced marker, as well as the 2',5'-oligoadenylate synthetases, was measured in circulating mononuclear cells from 21 patients with acute hepatitis A, 20 patients with acute hepatitis B and 14 patients with acute hepatitis C for determination of the activation of the interferon system in these viral diseases. In acute hepatitis A a strong expression (10 of 10 patients) of the MxA protein and the 2',5'-oligoadenylate synthetase activity in peripheral-blood mononuclear cells was observed during the first 2 wk after onset of clinical symptoms. In this period the MxA protein concentrations reached levels similar to those measured in patients treated with up to 5 x 10(6) IU interferon-alpha three times a week. Beyond wk 3, in eight of eight patients with hepatitis A no increased MxA protein levels were found. In contrast, peripheral-blood mononuclear cells from patients with acute hepatitis B contained either no measurable MxA protein or only slightly higher levels of the MxA protein, as did those of most patients (12 of 14) with acute hepatitis C. The MxA protein levels of both hepatitis B and C patients were significantly lower (p0.05) than those found in hepatitis A patients. Furthermore, sera from 6 of 10 patients with hepatitis A, but none of 10 patients with acute hepatitis B and C, contained measurable MxA protein. This serum MxA protein may originate from interferon-exposed and subsequently damaged liver cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The clinical tolerance and biological properties of 6 x 10(6) IU of Chinese hamster glycosylated recombinant interferon-beta (rHuIFN-beta) and natural IFN-beta (Frone) given i.v. were compared in 12 healthy volunteers in a randomized cross-over, double-blind trial. All subjects received a single injection of each type of IFN-beta. Both were well tolerated and provoked similar changes in clinical indices. Serum neopterin (Np) values increased significantly from the 24th to 72nd h post-injection of rHuIFN-beta and Frone. beta 2-Microglobulin (beta 2-M) serum levels were statistically above baseline 24-96 h after rHuIFN-beta, and from the 24th to the 120th h with Frone. Both IFNs provoked a rise in intracellular 2',5'-adenylate (2-5A) levels from the 10th to the 48th h, as well as in Hu-Mx synthesis, which was significant from the 10th to the 96th h. Serum levels of 2-5A, interleukin-1 alpha (IL-1 alpha), and interleukin-1 beta (IL-1 beta) remained unchanged. There were no statistical differences in the changes provoked by the two differently derived IFN-beta in any of the biological parameters studied. Overall, the results of this study indicate that rHuIFN-beta and Frone have similar pharmacodynamics.

The alpha/beta (type I) interferon-inducible human MxA protein confers resistance to vesicular stomatitis virus (VSV) and influenza A virus in MxA-transfected mouse 3T3 cells (3T3/MxA). We investigated the inhibitory effects of the MxA protein on measles virus (MV) and VSV in the human monocytic cell line U937. In transfected U937 clones which constitutively express MxA (U937/MxA), the release of infectious MV and VSV was reduced approximately 100-fold in comparison with control titers. Transcription of VSV was inhibited similar to that observed for 3T3/MxA cells, whereas no difference was detected for MV in the rates of transcription or the levels of MV-specific mRNAs. In contrast, analysis of MV protein expression by immunofluorescence and immunoprecipitation revealed a significant reduction in the synthesis of MV glycoproteins F and H in U937/MxA cells. These data demonstrate a virus-specific effect of MxA which may, in the case of MV, contribute to the establishment of a persistent infection in human monocytic cells.

The clinical tolerance of and the effects recombinant human interferon-beta (rHuIFN-beta) obtained from mammalian cells (Chinese hamster ovary cells) exerts on 2',5'-oligoadenyl (2-5A) synthetase activity, human-Mx protein, neopterin, beta 2-microglobulin, interleukin-1 (IL-1) alpha and beta synthesis were compared to those of natural IFN-beta in 12 healthy volunteers. Each subject received a single i.m. injection of 6 x 10(6) IU rHuIFN-beta and natural IFN-beta according to a randomized double-blind cross-over study design. Both were well tolerated and provoked similar changes in clinical indices. Moreover, rHuIFN-beta and natural IFN-beta induced significant and similar increases in 2'-5' adenylates, human Mx protein, and neopterin levels, but neither modulated beta 2-microglobulin, IL-1 alpha or beta synthesis. The sum of these findings indicates that rHuIFN-beta and natural IFN-beta are biologically equivalent. In view of these results, we are of the opinion that these two types of IFN are probably also therapeutically equivalent and, in consequence, that trials to evaluate the response of viral and neoplastic disease patients to rHuIFN-beta are fully justified.

SKCO 1 human colon carcinoma cells have been shown to be synergistically inhibited in their growth by the combinations of alpha-interferon (IFN-alpha) or beta-interferon (IFN-beta ser) and gamma-interferon (IFN-gamma). To determine if a correlation could be established between this synergistic antiproliferative effect and a synergistic induction in IFN-inducible proteins, or a unique perturbation in the cell cycle, we studied the effects of IFN-beta ser and IFN-gamma, alone and in combination, on 2',5'-oligoadenylate (2-5A) synthetase, indoleamine-2,3-dioxygenase (IDO), a human analogue of the murine Mx protein (p78), and the phases of cell cycle. 2-5A synthetase was maximally induced after a 24-h exposure to both IFN-beta and IFN-gamma. A synergistic enhancement of 2-5A synthetase activity was observed only with low concentrations of each IFN (0.05 ng/ml). IDO activity was induced by IFN-gamma and the combination of IFN-beta ser and IFN-gamma, but not IFN-beta ser alone. The differences in IDO activity between IFN-gamma and the combination, however, were not statistically significant. The p78 protein was induced in a dose-dependent manner by IFN-alpha and IFN-beta ser. IFN-gamma enhanced the expression of p78 induction by IFN-alpha or IFN-beta ser, even at concentrations of IFN-gamma that did not induce the protein when administered as a single agent. The combination of IFN-alpha and IFN-beta ser, which results in an antagonistic antiproliferative effect, also resulted in an antagonistic induction of p78. No changes in the cell cycle were observed following exposure to IFN-beta ser, IFN-gamma, or the combination, and treatment with IFN-gamma did not inhibit the accumulation of cells in G2M caused by colchicine. Thus, the synergistic antiproliferative effect produced by IFN-beta ser and IFN-gamma in SKCO 1 cells could not be correlated with a synergistic enhancement in 2-5A synthetase or IDO activity, or with a perturbation in the cell cycle. In contrast, the combination of IFN-gamma and IFN-alpha or IFN-beta ser synergistically enhanced the expression of p78 protein in these cells.

Summary Inborn resistance to orthomyxoviruses due to the allele Mx was investigated at the molecular level in macrophages from A2G mice. In the absence of interferon, Mx-bearing and control cells were equally permissive for M-TUR, a macrophage-adapted strain of influenza A virus. After treatment with various doses of mouse interferon type I, Mx-bearing cells were fully resistant to this virus whereas cells lacking Mx were only marginally protected. Virus attachment and penetration were equally efficient in Mx-carrying and control cells and were not influenced by interferon. However, virus protein synthesis was blocked in Mx-bearing macrophages expressing resistance. These findings indicate that the resistance mechanism specific for influenza viruses realized by Mx in cooperation with interferon inhibits influenza virus replication in macrophages at an early stage following attachment and penetration of the virus.

Interferon-alpha + beta (IFN-alpha + beta) plays a central role in the specific resistance to influenza virus infection of those mice carrying the gene Mx (for review, see Haller, O. (1981) Curr. Topics Microbiol. Immun. 92, 25). Particularly, mouse IFN-alpha + beta induces a unique protein in cultivated Mx-bearing cells which is associated with a highly efficient and specific antiviral resistance to influenza viruses (Horisberger, M. A., Staeheli, P., and Haller, O. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 1910). In this report, a procedure is described for the induction of this protein in several organs of Mx-bearing mice and a method for its purification from liver tissue. The protein Mx is nucleophilic and has a Mr approaching 78,000. It is not concentrated in nucleoli and it is not tightly bound to chromatin or nuclear matrices. Polyclonal antibodies to the protein Mx were raised in BALB/c mice. They recognized the protein Mx immobilized on nitrocellulose in a dot immunoassay and they immunoprecipitated the IFN-induced protein Mx from cultivated Mx-bearing cells labeled with a radioactive tracer.

In mice, the combined action of alpha and beta interferons (IFNs) against influenza viruses is modulated by the host gene Mx. High concentrations of IFN fail to prevent efficiently the replication of influenza A virus in cultured macrophages lacking the gene Mx, whereas cultured macrophages carrying Mx develop strong antiviral activity even at low concentrations of IFN. Several steps in the replication cycle of influenza virus were compared in Mx/Mx and +/+ mouse macrophages treated with IFN-alpha + beta. Uncoating was not affected. A twofold reduction in the accumulation of primary transcripts was observed in IFN-treated macrophages at the highest concentration of IFN regardless of the genetic constitution of the host cell. No evidence was obtained for inhibition of influenza virus translation in macrophages which lacked Mx when treated with IFN-alpha + beta. In contrast, a marked shut-off of influenza virus polypeptide synthesis occurred in Mx-bearing macrophages treated with these IFNs, although the primary transcripts were active in directing the synthesis of viral polypeptides in a cell-free system. We concluded that a specific inhibitory mechanism for influenza virus translation was induced by IFN-alpha + beta in macrophages bearing the resistance gene Mx.

Interferon-alpha + beta (IFN-alpha + beta) plays a central role in the specific resistance to influenza virus infection of those mice carrying the gene Mx (for review, see Haller, O. (1981) Curr. Topics Microbiol. Immun. 92, 25). Particularly, mouse IFN-alpha + beta induces a unique protein in cultivated Mx-bearing cells which is associated with a highly efficient and specific antiviral resistance to influenza viruses (Horisberger, M. A., Staeheli, P., and Haller, O. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 1910). In this report, a procedure is described for the induction of this protein in several organs of Mx-bearing mice and a method for its purification from liver tissue. The protein Mx is nucleophilic and has a Mr approaching 78,000. It is not concentrated in nucleoli and it is not tightly bound to chromatin or nuclear matrices. Polyclonal antibodies to the protein Mx were raised in BALB/c mice. They recognized the protein Mx immobilized on nitrocellulose in a dot immunoassay and they immunoprecipitated the IFN-induced protein Mx from cultivated Mx-bearing cells labeled with a radioactive tracer.

Mouse cells carrying the dominant resistance gene Mx develop a more efficient antiviral state toward influenza viruses in response to interferon than do Mx-negative cells. We have identified an Mx gene-associated product by labeling cultured peritoneal macrophages and embryonic cells with [35S]methionine in the presence or absence of interferon. The radioactive proteins from unfractionated cytoplasmic extracts were separated electrophoretically in two dimensions and were revealed by fluorography. A protein with a Mr of 72,500 and an isoelectric point of 6.3 was induced by mouse interferon type I (a mixture of alpha and beta interferons) in cells carrying the gene Mx but not in cells lacking Mx. The induction of this protein could be blocked by actinomycin D. The maximal rate of synthesis was reached in embryonic cells 4-5 hr after treatment with 10(3) reference units of interferon per ml. When the allele Mx (present in the inbred mouse strain A2G) was repeatedly backcrossed on different genetic backgrounds (BALB/c, C57BL/6, A/J), a clear correlation between the inducibility by interferon of this protein and the presence of the allele Mx was observed. The results suggest that this protein induced by the interaction of interferon with Mx plays a role in the selective antiviral state against influenza viruses that is observed in interferon-treated Mx-bearing cells.

Two RNA polymerase activities were characterized in the cytoplasm of influenza Ao/NWS infected cells. Their relationship to the virion-associated RNA polymerase was studied.

The purpose of these studies was to examine the antiproliferative properties of 16 recombinant human IFN-alpha B/D hybrids against various human tumor lines of different histological origin and to determine whether any of the hybrid molecules possessed immunomodulating activity that could active antitumor properties in peripheral blood monocytes of normal donors. Hybrids with the B domain at the NH2 terminal end exhibited higher activity for antiviral activity and a higher level of direct antitumor antiproliferative activities as compared with hybrids with the D domain at the NH2 terminal end. The positive hybrids were directly cytostatic to melanoma, glioblastoma, renal carcinoma, colon carcinoma, and prostatic carcinoma cells. Tumor cell sensitivity to IFN-alpha hybrids was independent of sensitivity to IFN-gamma or to Adriamycin. The growth of a normal cell line (human embryo fibroblast) was unaffected by IFN-alpha hybrids but was completely arrested by Adriamycin. Some of the IFN-alpha hybrids were also cytostatic to mouse melanoma, lung carcinoma, and fibrosarcoma cell lines, albeit at lower levels than they were to human cells. The incubation of monocytes with IFN-alpha hybrids with the B domain at the NH2 terminal end was also associated with marked antitumor cytotoxicity. Kinetic studies, however, indicated that this activity was attributable to IFN-alpha carried on monocytes and acting directly on tumor cells. We conclude that recombinant human IFN-alpha B/D hybrids possess potent direct antiproliferative activity against a large variety of human tumor lines.

In a coupled cell-free system for the transcription and translation of the influenza mRNA's, containing detergent-disrupted purified NWS influenza virion and a micrococcal nuclease-preincubated rabbit reticulocyte lysate, five unglycosylated viral proteins (NS1, M, NP, P1, and P3) were easily produced and isolated. Their identification was based on the electrophoretic separation of peptide fragments resulting from their partial digestion with proteases of restricted specificity (D.W. Cleveland, S. G. Fisher, N. W. Kirschner, and U. K. Laemmli, J. Biol. Chem. 252:1102-1106, 1977).

In cells derived from congenic BALB.A2G-Mx mice carrying the resistance gene Mx, but not in cells from BALB/c mice lacking Mx, mouse interferons alpha and beta induced the synthesis of a unique cellular protein that was associated with an efficient antiviral state with selectivity for influenza viruses. In contrast, native or recombinant mouse interferon gamma failed to efficiently protect Mx-bearing cells against influenza viruses and did not noticeably induce the synthesis of the Mx-associated protein, although interferon gamma was as effective as interferons alpha and beta in protecting BALB.A2G-Mx and BALB/c cells against the rhabdovirus VSV. These results demonstrate that different types of interferons differentially regulate the expression of the Mx gene and thereby induce distinct antiviral states.