Hybrid recombinant human leukocyte interferon inhibits differentiation in murine B-16 melanoma cells

We have investigated the effects of recombinant human leukocyte interferons (IFN-alpha A and IFN-alpha D) and various hybrid recombinant human leukocyte interferons on differentiation in B-16 mouse melanoma cells. Inhibition of both spontaneous and melanocyte hormone stimulated differentiation was observed with one hybrid construct, IFN-alpha A/D (Bgl) consisting of amino acids 1 to 62 from IFN-alpha A and amino acids 64 to 166 from IFN-alpha D. In contrast, the parental human interferons, IFN-alpha A and IFN-alpha D, when used alone or in combination, as well as other hybrid human leukocyte interferons, did not cause significant inhibition of melanogenesis in B-16 mouse cells. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also inhibited B-16 differentiation and the combination of TPA with IFN-alpha A/D (Bgl) or mouse L-cell interferon was synergistic in delaying melanogenesis. These studies indicate that the IFN-alpha A/D (Bgl) hybrid that exhibits antiviral activity on mouse cells can also inhibit differentiation of murine cells.     

Human leukocyte interferon has no structural or biological relationship to corticotropin

In view of recent reports that certain preparations of human leukocyte interferons are structurally and biologically related to the pituitary hormones corticotropin (ACTH) and β-endorphin, we have investigated the properties of two human leukocyte interferons (IFN-α) prepared by recombinant DNA technology. The antiviral activities of purified IFN-αA and IFN-αD were not affected by a large molar excess of ACTH antiserum nor did ACTH interfere in interferon immunoassays. Neither IFN-αA, IFN-αD nor pepsin digests of these proteins were able to stimulate steroidogenesis in adrenocortical cells. There was no cross reaction between ACTH antiserum and the two leukocyte interferons or the pepsin digests of the interferons. These results cast doubt on recent proposals that some of interferon's biological effects are mediated by ACTH or β-endorphin-related fragments of the interferon molecule.     

Ⅲ型干扰素——新型抗病毒细胞因子

干扰素（IFN）是抗病毒感染的第一道防线,Ⅰ型和Ⅱ型干扰素不仅可抑制病毒,而且还能参与天然免疫反应和获得性免疫反应。最近干扰素家族增添一位新成员：Ⅲ型干扰素,即IFN-λ,因其具有类似干扰素的抗病毒活性且能诱导干扰素相关基因的表达而命名。IFN-λ受体与Ⅰ型干扰素的受体不同,但具有与Ⅰ型干扰素类似的诱导表达方式和信号转导通路,并能激活一系列相似的干扰素刺激基因。就IFN-λ家族及其受体、基因表达和信号转导机制、抗病毒作用等进行综述。     

Purification and structural analysis of interferon

Studies with crude or partly purified interferon have provided a significant amount of structural information. However, complete biochemical characterization required purification to homogeneity. Earlier work on fractionation has met with many difficulties because interferon was available only in minute quantities. A scale-up of production, adaptation of multi-step purification schemes, use of high-resolution separation techniques and highly sensitive analytical methods have yielded pure interferons and hence many structural data. Specific activities, amino-acid compositions, partial sequences and structural homologies of many interferons were determined. Finally, cloned copy DNA (cDNA) fragments derived from specific interferon mRNA, as well as isolated interferon genes, have been sequenced and the data were used to elucidate complete sequences of many interferons with a high degree of confidence.     

Some biological properties of the human amniotic membrane interferon.

Human amniotic interferon was investigated to define the species specificity of its antiviral action and to compare its anti-cellular and NK cell stimulating activities with those of other human interferons. The antiviral effect was titrated in bovine (RV-IAL) and monkey (VERO) cells. Amniotic interferon exhibited, in bovine cells, 5% of the activity seen in monkey cells, while alpha interferon displayed 200%. No effect was detected with either beta or gamma interferon in bovine cells. Daudi cells were exposed to different concentrations of various interferons and the cell numbers were determined. The anticellular effect of the amniotic interferon reached its peak on the third day of incubation. Results suggested a higher activity for alpha and gamma interferons and a lower activity for beta when compared to amniotic interferon. Using total mononuclear cells as effector cells and K 562 as target cells in a 51Cr release assay, it was demonstrated that low concentrations of amniotic interferon consistently stimulated NK cell activity in cells derived from several donors, the results indicating a higher level of activity with this interferon than with alpha and beta interferons.     

Monoclonal antibodies can discriminate between some active and inactive forms of leukocyte interferon

Antiviral activity of recombinant human leukocyte A interferon was inactivated by heating at 65 degrees C or by reduction of disulfide bonds. The specific immunoreactivity, as measured by radioimmunoassays measuring binding to monoclonal antibodies, decreased concomitantly with the antiviral activity. Although the monoclonal antibodies did bind to inactivated interferon, their binding affinity to inactivated interferon was in general very much lower than their binding affinity to active interferon. Therefore, this immunoassay could replace the antiviral assay for detection of biologically active interferon. In addition, most of these antibodies should be especially useful for purification of the interferons since they discriminate between the native active and inactive denatured species. Screening for such antibodies is convenient and simple. The general use of antibodies that preferentially interact with native molecules provides a powerful new principle for choosing monoclonal antibodies with extraordinary potential in assay and purification.     

Antiviral and protein-inducing activities of recombinant human leukocyte interferons and their hybrids.

The antiviral activities of recombinant human leukocyte interferons IFN-alpha A and IFN-alpha D as well as five hybrids of these interferons against retroviruses, vesicular stomatitis virus, and encephalomyocarditis virus were studied in feline, human, and murine cells. Although these interferon species had widely different potencies, their activities against these viruses were, in general, proportional. The IFN-alpha A/D (Bgl) hybrid was the most potent species, and the IFN-alpha D/A (Bgl) hybrid was the least potent. However, the latter species did not interfere with the action of the former species. Like natural human leukocyte interferon, each of the seven species of recombinant interferons induced the synthesis of at least five proteins in human fibroblasts, whereas induction of only one such protein was readily detected in a feline fibroblast line in which these interferon species inhibited the replication of all three viruses.     

Immunological functions of macrophages and their regulation by interferons

Important progress has been recently made on the identification, physico-chemical properties, purification and production by recombinant technology of a series of immunoregulatory mediators, including interferons (IFNs). It is now possible to appreciate which part play IFNs in the regulation and activation of monocyte-macrophage (MO) functions. The present review discusses old concepts, such as Macrophage-Activating Factor (MAF) activity, at the light of recent data on the effects of IFN alpha, beta and gamma on four main immunological properties of MO, namely intracellular killing of microorganisms, non specific lysis of tumor cells, antigen presentation and secretion of soluble immunoregulatories molecules such as interleukin 1. All three IFN species enhance such functions, whereas enhancement of membrane class II major histocompatibility complex antigens is a unique property of IFN gamma. The role of IFNs, especially IFN gamma, as major MAF in vitro and in vivo is discussed.     

Interferon levels in human pulmonary tumors are lower than plasma levels

It is uncertain whether interferon levels in the interstitial fluid of tumors are equivalent to interferon plasma levels and we have investigated this problem in human pulmonary tumors by infusing human recombinant interferon alpha A and natural interferon Beta for about three hours before surgery. By determining the hematocrit and hemoglobin content it was possible to calculate interferon values (International Units/g wet tissue) present in the interstitial fluid of tumor and lung samples, simultaneously. In 14 patients (epidermoids, n = 9 and adenocarcinomas, n = 5) interferon levels in tumor and "normal" lung expressed as percentages of interferon plasma levels were: 9.5 +/- 3.9 and 29.8 +/- 6.9 for recombinant interferon alpha A and 3.1 +/- 0.4 and 10.1 +/- 2.4 for natural interferon Beta, respectively. Differences for both interferons are statistically significant (p less than 0.05). To our knowledge these are the first data indicating that interferon levels in pulmonary tumor interstitial fluid are markedly lower than those in normal lung although they do not clarify the main factor responsible for the decrease, they explain at least in part the negligible therapeutic activity of interferons in these tumors and emphasize the need for new approaches for improving the therapeutic index of interferons.     

Synthesis of recombinant gamma interferons resistant to proteolysis in the yeast Pichia pastoris

Genes encoding truncated versions of bovine and chicken gamma interferon genes lacking predicted protease cleavage sites at the C-terminus were constructed and expressed in the yeast Pichia pastoris. The recombinant proteins possessed increased stability in comparison with the corresponding wild-type gamma interferons while retaining biological activity. The recombinant strains provide a useful tool for the purification of bovine and chicken gamma interferons for their use in veterinary applications.     

Absence of functional and structural homology of natural and recombinant human leukocyte interferon (IFN-α) with human α-ACTH and β-endorphin

A comparison of the amino acid sequence of one human recombinant IFN-α (IFLrA) with either human β-endorphin or ACTH reveals only a minimal and insignificant degree of homology. Also, synthetic ACTH, β-endorphin and β-endorphin-(1–15) have no antiviral protective effects on human fibroblasts and cannot inhibit the neutralization of the antiviral effects of natural IFN-α by an antiserum directed against the interferon. Anti ACTH and Anti β-endorphin do not neutralize the antiviral effects of IFLrA, and radioimmunoassays of partially purified natural IFN-α and pure IFLrA do not reveal any evidence of α-MSH or β-endorphin-like material in the interferons. These results demonstrate an absence of functional and structural homology of natural and recombinant IFN-α with ACTH and β-endorphin.     

The neuraminidase yield-reduction bioassay of human other interferons.

The production of neuraminidase by the recombinant influenza virus X7(F1) in human monkey, rabbit, hamster, mouse, and chicken cell cultures is inhibited by interferon. Described is a new enzyme assay for neuraminidase that can be applied to the bioassay of interferons. The advantages of this interferon bioassay are its sensitivity, reproducibility, rapidity, and convenience.     

Potentiation of growth suppression and modulation of the antigenic phenotype in human melanoma cells by the combination of recombinant human fibroblast and immune interferons

Summary Administration of interferon as a single therapeutic regimen in cancer patients with various neoplasias has had only limited efficacy in ameliorating the negative clinical course of their disease. In the present study, we have evaluated the effect of recombinant human fibroblast (IFN) and immune (IFN) interferon, alone and in combination, on growth, differentiation and the expression of class I and II histocompatibility locus antigens (HLA) and melanoma-associated antigens on the human melanoma cell line H0-1. The effect of combinations of interferons on the antigenic profile of human melanoma cells displaying different organ colonization and spontaneous metastatic potential in athymic nude mice was also determined. H0-1 cells were more sensitive to the antiproliferative activity of IFN than to IFN and the combination of interferons resulted in a potentiation of growth suppression. The antiproliferative effect of both interferons was greater in later-passage than in earlier-passage H0-1 cells, possibly reflecting alterations in the evolving tumor cell population as a result of long-term in vitro propagation and/or the selective outgrowth of cells with an increased growth rate. The enhanced growth suppression observed in H0-1 cells treated with the combination of IFN plus IFN was not associated with a significant increase in the level of melanin, a marker of melanoma differentiation, above that observed with either interferon used alone. IFN and IFN differentially modulated the expression of class I and II HLA and melanoma-associated antigens in H0-1 cells and a series of melanoma cells with different organ colonization and metastatic potential, including MeWo, MeM 50-10, MeM 50-17, 3S5 and 70W. No consistent potentiation or antagonism in the expression of any specific antigen was observed in any of the melanoma cell lines exposed to the combination of interferons. The present study demonstrates that the combination of IFN plus IFN can potentiate growth suppression in H0-1 human melanoma cells and that this effect is not associated with an increase in differentiation or a potentiation in antigenic modulation. In addition, no direct correlation between the expression of any specific antigen or its modulation by IFN or IFN, alone or in combination, and organ colonization and metastatic potential in nude mice was observed in the different melanoma cell lines.     

Interferon-γ: A historical perspective

This article reviews the main lines of thinking and exploration that have led to our current conception of the role of IFN-γ in immune defense and autoimmunity. In 1965 the first report appeared describing production of an interferon-like virus inhibitor in cultured human leukocytes following exposure to the mitogen phytohemagglutinin. In the early 1970s the active principle became recognized as being distinct from classical virus-induced interferons, leading to its designation as immune interferon or Type II interferon, and eventually IFN-γ. Up to that point interest in the factor had come almost exclusively from virologists, in particular those among them who were believers in interferon. Evidence first coming forward in the 1980s that IFN-γ is indistinguishable from macrophage-activating factor (MAF), then a prototype lymphokine, was the signal for immunologists at large to become interested. Today IFN-γ ranks among the most important endogenous regulators of immune responses.     

Nanocomplexes of recombinant proteins and polysialic acid: Preparation,characteristics, and biological activity

A preparation of nanocomplexes containing recombinant proteins (interferons α2b and β1b, insulin, and human granulocyte colony stimulating factor) and natural polysialic acid (PSA) has been described. The incorporation of protein into the complex changes its electrophoretic mobility. Atomic force microscopy reveals the average size of 23-kD insulin complexes with PSA of 10–20 nm and demonstrates that more than 60% of glycopolymer molecules carry a single protein molecule. Experiments with cultured cells show that cytokines bound to polysialic acid retain their ability to regulate cell proliferation. Insulin bound to PSA has a prolonged hypoglycemic effect in vivo.     

Purification and characterization of interferons from a continuous myeloblastic cell line

Several leukocyte interferon species have been purified from a continuous human myeloblast cell line. The purification procedure involving selective precipitations, gel chromatography, and several steps of high performance liquid chromatography results in interferons with specific activities of 1 to 4 X 10(8) units/mg on bovine MDBK cells. The total yield of interferon is 23%, with the yield of the individual fractions ranging from 0.2 to 11.4%. Five fractions are homogeneous as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Molecular weights of the interferons were estimated by mobility on the sodium dodecyl sulfate gels and range from 17,600 to 26,200. The species differ in their relative antiviral activities on two cell lines, bovine MDBK and human AG-1732. In addition, the pure species have similar, but distinct, amino acid compositions and tryptic peptide profiles. These result support the conclusion that leukocyte interferon consists of several homologous proteins.     

Ⅰ型干扰素免疫应答作用机制研究进展

I型干扰素(IFN-Ⅰ)是机体固有免疫应答的一类重要的细胞因子,具有广谱的抗病毒及抗肿瘤等作用。近年来IFN-Ⅰ成为病毒学、疫苗学及肿瘤学等研究的热点,对干扰素诱导基因(ISGs)的功能研究进一步揭示了其抗病毒以及抗肿瘤的作用机制。麻疹病毒、流感病毒和肠道病毒71型等病毒均可通过与IFN-Ⅰ或其上、下游调节因子结合阻断IFN-Ⅰ信号通路,从而逃逸IFN-Ⅰ的抗病毒作用,这对病毒性疾病的防治是新的挑战。近期研究发现IFN-Ⅰ是疫苗诱导抗体产生的必要信号,同时参与调节T、B细胞的活化过程,在免疫应答过程中发挥关键作用。对IFN-Ⅰ免疫应答作用机制研究进行了综述。