Mouse Gamma Interferon Produced by a Cloned T-Cell Lymphoma. I. Purification and Physicochemical Characterization

Gribaudo, Giorgio; F, Cofano; Negro-Ponzi, Alessandro; Landolfo, Santo

doi:10.1089/jir.1984.4.91

Cited by 8 publications

(9 citation statements)

References 16 publications

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“…1, lane 4). Such a heterogeneity of the IFN-y molecule should not be surprising, being probably due to different degrees of glycosylation as reported by other investigators (18,26).…”

Section: Methodssupporting

confidence: 68%

“…For these reasons some investigators, in the attempt to make monoclonal antibodies against human IFN-a or -,B, were forced to immunize animals with materials subjected to extensive low-yielding purification procedures. By contrast, we could use as immunogen crude cell supernatants harvested from PMA-stimulated L12-R4 cells, which are particularly rich in IFN-y and do not contain other contaminating lymphokines as detected by biological assays (17,18). This allowed us to avoid long purification procedures, with the exception of concentration by two sequential ammonium sulfate precipitations.…”

Section: Discussionmentioning

confidence: 99%

“…It has been reported that the IFN-y molecule is a glycoprotein (18,25). Thus, in order to know against which portion of the molecule the AN-18.17.24 monoclonal antibody reacted, we exposed IFN-y preparations to different physicochemical or enzymatic treatments, degrading either the protein or the carbohydrate moiety.…”

Section: Methodsmentioning

confidence: 99%

“…In previous studies we established in vitro a murine T-cell lymphoma line producing, upon stimulation with phorbol 12-myristate 13-acetate (PMA), huge amounts of homogeneous IFN-y apparently not contaminated by other known lymphokines (17,18). Taking advantage of this system, we now have produced a rat monoclonal antibody recognizing murine IFN-y but not murine IFN-a or -,3.…”

mentioning

confidence: 99%

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Monoclonal antibodies against murine gamma interferon.

Prat¹,

Gribaudo²,

Comoglio³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

136

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Monoclonal antibodies against murine immune interferon (IFN-y) were produced by fusing the murine nonsecreting myeloma cell line P3.X63.Ag8.653 with spleen cells from rats immunized with IFN-y-containing supernatants obtained by stimulating a T-cell lymphoma, L12-R4, with phorbol 12-myristate 13-acetate. Supernatants from a twicecloned hybridoma were found to neutralize and to adsorb in depletion experiments up to 27 units of mouse IFN-y but not equivalent amounts of mouse leukocyte or fibroblast IFNs. The AN-18.17.24 monoclonal antibody neutralized to the same extent mouse IFN-y from different sourcesnamely, (i) concanavalin A-stimulated spleen cells, (ii) alloantigen-stimulated spleen cells, and (iii) monkey fibroblasts transfected with the cloned gene of murine IFN-y. Moreover, the monoclonal antibody displayed species specificity, since it did not neutralize IFN-y of human origin. Binding inhibition experiments with murine IFN-y preparations exposed to enzymatic or physicochemical degradation demonstrated that the protein moiety and not the carbohydrate residues were responsible for the binding to the Activation of T lymphocytes by alloantigens (1, 2), mitogens (3), and conventional antigens (4) induces the release of an interferon (IFN) type classified as immune (IFN-y) that acts on a variety of somatic and immunorelated target cells in a genetically unrestricted manner (5). Some in vitro and in vivo studies indicate that IFN-y has a greater antiproliferative effect on neoplastic cells than do leukocyte and fibroblast IFNs (IFN-a and IFN-p, respectively) (6).Recently, genes of both human and murine IFN-y have been successfully cloned by recombinant DNA technology. The results of these experiments show that the molecular weights of human and mouse IFN-'y are 17,100 and 15,900, respectively (7-9). A limit of this kind of technology, however, is that it does not take into account the possible posttranslational modifications of the synthesized molecules. In fact, as far as IFN-y is concerned, discrepancies on molecular weights have been reported in studies using conventional physicochemical techniques for purification (10) as opposed to those using recombinant DNA technology (7-9).Monoclonal antibodies have proved to be valuable reagents for the unambiguous identification, quantitative analysis, and large-scale purification of a variety of proteins (11). Whereas different monoclonal antibodies have already been produced and successfully used for the purification and biological characterization of IFN-a and -,/ (12-15), few and conflicting reports exist in the case of monoclonal antibodies against human 16).In previous studies we established in vitro a murine T-cell lymphoma line producing, upon stimulation with phorbol 12-myristate 13-acetate (PMA), huge amounts of homogeneous IFN-y apparently not contaminated by other known lymphokines (17, 18). Taking advantage of this system, we now have produced a rat monoclonal antibody recognizing murine IFN-y but not murine IFN-a or -,3. MATERIALS AND METHODSIm...

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“…1, lane 4). Such a heterogeneity of the IFN-y molecule should not be surprising, being probably due to different degrees of glycosylation as reported by other investigators (18,26).…”

Section: Methodssupporting

confidence: 68%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Monoclonal antibodies against murine gamma interferon.

Prat¹,

Gribaudo²,

Comoglio³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

136

View full text Add to dashboard Cite

show abstract

“…Mouse IFN-y (3.6 x 106 IU/mg) purified according to Gribaudo et al (1984) was kindly donated by Dr S. Landolfo (Institute of Microbiology, University of Turin, Italy). Macrophage IFN (IFN-Mq~; 1 x 10 s IU/mg) was induced in bone marrow macrophages by 10-carboxymethyl-9-acridanone as described by Storch & Kirchner (1982).…”

mentioning

confidence: 99%

Inhibition of Replication of Herpes Simplex Virus in Mouse Macrophages by Interferons

Domke

Straub

Jacobsen

et al. 1985

Journal of General Virology

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SUMMARYThe replication of herpes simplex virus (HSV) type 1 in macrophages grown from spleen cells of mouse strains susceptible to HSV infection in vivo was very sensitive to interferon (IFN). Different types of mouse IFN (c~, fl, ~,) exhibited similar antiviral activities. However, treatment of cells with IFN-~ in combination with IEN-~ or IFN-/3 resulted in a synergistic inhibition of virus growth. As shown by assaying HSV DNA polymerase, IFN inhibited expression of the/3-genes. Inhibition of enzyme induction correlated well with the reduction of viral yield. Induction of HSV DNA polymerase was delayed by IFN in a dose-dependent manner. These results show that IFN inhibits HSV replication at an early step prior to or during the synthesis of/3-proteins.

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Interferon‐activated tumor inhibition in vivo. Small amounts of interferon‐gamma inhibit tumor growth by eliciting host systemic immunoreactivity

Giovarelli

Vecchi

et al. 1986

Intl Journal of Cancer

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Ten international units (IU) of recombinant (r) or natural (n) murine interferon (MuIFN)-gamma were used for the in vivo immunotherapy of a chemically induced fibrosarcoma (CE-2) of BALB/c mice. In vitro, doses of r-IFN-gamma below 100 units have a marginal antiproliferative effect on CE-2 cells and do not induce expression of H-2d class-II antigens, whereas they do increase that of class-I antigens. In vivo, 10 daily injections of 10 IU of r- or n-MuIFN-gamma at the challenge site provide significant protection against increasing doses of CE-2 tumor cells. This protection was enhanced when non-reactive T-lymphocytes from CE-2 tumor-bearing mice were admixed at a 10:1 ratio with the CE-2 tumor cells. Combined lymphocyte and r-MuIFN-gamma treatment also inhibited the growth of already established tumors when it was started before these reached a mean diameter of 5 mm. Tumor inhibition depends upon activation of the host immune system. The antitumor activity of r-MuIFN-gamma and T-lymphocytes was null when mice were first irradiated with 450 rads. Moreover, host leukocytes massively infiltrated the area of tumor growth and the small r-MuIFN-gamma doses injected daily activated various host immunoreactivity mechanisms.

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Mouse Gamma Interferon Produced by a Cloned T-Cell Lymphoma. I. Purification and Physicochemical Characterization

Cited by 8 publications

References 16 publications

Monoclonal antibodies against murine gamma interferon.

Monoclonal antibodies against murine gamma interferon.

Inhibition of Replication of Herpes Simplex Virus in Mouse Macrophages by Interferons

Interferon‐activated tumor inhibition in vivo. Small amounts of interferon‐gamma inhibit tumor growth by eliciting host systemic immunoreactivity

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