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G Garotta

Publications and source records attributed to G Garotta.

At least 37 records · Page 2Linked to original sources
PubMed1996

Monocyte chemotactic protein 4 (MCP-4), a novel structural and functional analogue of MCP-3 and eotaxin.

A novel human CC chemokine complementary DNA was identified in a library constructed from human fetal RNA, cloned into a baculovirus vector, and expressed in Sf9 insect cells. The mature recombinant protein that was released had the NH2-terminal sequence pyro-QPDALNVPSTC...and consisted of 75 amino acids. Minor amounts of two variants of 77 and 82 residues (NH2 termini: LAQPDA...and FNPQGLAQPDA...) were released as well. The novel chemokine was designated monocyte chemotactic protein 4 (MCP-4) and the variants were designated (LA)MCP-4 and (FNPQGLA)MCP-4. MCP-4 shares the pyroglutamic acidproline NH2-terminal motif and 56-61% sequence identity with the three known monocyte chemotactic proteins and is 60% identical to eotaxin. It has marked functional similarities to MCP-3 and eotaxin. Like MCP-3, MCP-4 is a chemoattractant of high efficacy for monocytes and T lymphocytes. On these cells, it binds to receptors that recognize MCP-1, MCP-3, and RANTES. On eosinophils, MCP-4 has similar efficacy and potency as MCP-3, RANTES, and cotaxin. It shares receptors with eotaxin and shows full cross-desensitization with this cosinophil-selective chemokine. Of the two variants, only (LA)MCP-4 could be purified in sufficient quantities for testing and was found to be at least 30-fold less potent than MCP-4 itself. This suggests that the 75-residue form with the characteristic NH2 terminus of an MCP is the biologically relevant species.

Acetylglucosaminidase↗
PubMed1996

IFN-gamma restores HIV- and non-HIV-specific cell mediated immune response in vitro and its activity is neutralized by antibodies from patients with AIDS.

The addition of IFN-gamma to cultures of peripheral blood mononuclear cells (PBMCs) obtained from asymptomatic HIV-infected patients increased cell proliferation in response to HIV envelope synthetic peptides (Env), influenza A virus (VIRUS), and allogeneic lymphocytes (ALLO) but not to phytohaemagglutinin (PHA) stimulation. F(Ab)2 fragments of IgG purified from the sera of HIV-seropositive patients specifically interfered with IFN-gamma-induced cell proliferation in response to recall antigens. Neutralization of the lymphokine activity was found to be sustained by specific IFN-gamma antibodies. Data obtained demonstrate that IFN-gamma can restore the cell-mediated immunity of a number of asymptomatic HIV+ individuals in vitro, while IFN-gamma antibodies present in sera of patients with AIDS interfere with the activity of the lymphokine.

Acquired Immunodeficiency Syndrome↗
PubMed1996

The intracellular domain of the second chain of the interferon-gamma receptor is interchangeable between species.

In this report we show that the mouse interferon (IFN)-gamma R1 and IFN-gamma R2 subunits expressed in hamster cells are capable of rendering the cells sensitive to mouse IFN-gamma as measured by induction of class I MHC antigens and the activation of the transcription factor Stat1 alpha. However, these cells showed no antiviral protection in response to IFN-gamma when challenged with vesicular stomatitis virus (VSV) but limited protection when challenged with encephalomyocarditis virus (EMCV). Furthermore, the cytoplasmic domains of the IFN-gamma R2 subunits, like the cytoplasmic domains of the IFN-gamma R1 chains, can be interchanged between species with no loss of biologic activity, demonstrating that the species-specific interaction of the IFN-gamma R1 and IFN-gamma R2 chains involves only the extracellular domains of the two proteins.

Amino Acid Sequence↗
PubMed1996

The effects of a nonimmunogenic form of murine soluble interferon-gamma receptor on the development of autoimmune diabetes in the NOD mouse.

Previous studies have shown that in vivo treatment with antiinterferon-gamma (anti-IFNgamma) monoclonal antibodies (mAbs) prevents the development of autoimmune diabetes in NOD mice. Although these findings anticipate that specific anti-IFNgamma therapies may be useful for the prevention/treatment of human insulin-dependent diabetes mellitus, there are several reasons why the use of anti-IFNgamma mAb may be difficult in the clinical setting. With the aim to develop alternative forms of specific anti-IFNgamma therapies, we recently produced a nonimmunogenic form of the soluble IFNgamma receptor (sIFNgammaR) that binds and neutralizes murine IFNgamma with an affinity higher than that of anti-IFNgamma mAb. In this study we compared the efficacy of sIFNgammaR to that of two anti-IFNgamma mAbs (XMG 1.2 and AN-18) in the prevention of spontaneous and accelerated (cyclophosphamide-induced) forms of autoimmune diabetes in NOD mice. The results show that in the spontaneous model, sIFNgammaR could prevent histological and clinical signs of autoimmune diabetes as efficiently as the two mAbs. Under ex vivo conditions, sIFNgammaR exhibited a more powerful modulatory effect than XMG 1.2 mAb on cytokine secretion from splenic lymphoid cells, which resulted in a significant reduction of Concanavalin A-induced IL-2 secretion and an augmented release of both unstimulated and lipopolysaccharide-induced IL-6. Moreover, although both mAbs were immunogenic and elicited formation of high titers of anti-rat IgG, sIFNgammaR did not induce antibody formation. Unexpectedly, in the cyclophosphamide-induced model, sIFNgammaR turned out to be less effective than either of the two anti-IFNgamma mAbs. Taken together, these data support the role of IFNgamma in the pathogenesis of NOD mice, but, more importantly, suggest that a nonimmunogenic approach is possible to the diminution of the effects of IFNgamma in this model.

Animals↗
PubMed1995

CD4+ and CD8+ lymphocytes of patients with AIDS synthesize increased amounts of interferon-gamma.

Individual cells capable of interferon-gamma (IFN-gamma) synthesis are easily detected by immunofluorescence and flow cytometric analysis using an anti-IFN-gamma monoclonal antibody as specific reagent. By IFN-gamma flow cytometry assay, we demonstrated that HIV-seropositive patients, starting at the early stage of viral infection, generally have an increased percentage of lymphocytes potentially able to produce IFN-gamma, compared with healthy blood donors. IFN-gamma expression in patient lymphocytes was observed to increase with the progressive stages of HIV infection, with the highest figures occurring in stage C patients. Such increased IFN-gamma expression involved both CD4+ and CD8+ T cell subsets. Most interestingly, we found patients at the same stage of HIV infection who had similar numbers of total and CD4+ lymphocytes but highly different percentages of lymphocytes potentially capable of producing IFN-gamma.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Interaction between the components of the interferon gamma receptor complex.

Interferon gamma (IFN-gamma) signals through a multimeric receptor complex consisting of two different chains: the IFN-gamma receptor binding subunit (IFN-gamma R, IFN-gamma R1), and a transmembrane accessory factor (AF-1, IFN-gamma R2) necessary for signal transduction. Using cell lines expressing different cloned components of the IFN-gamma receptor complex, we examined the function of the receptor components in signal transduction upon IFN-gamma treatment. A specific IFN-gamma R2:IFN-gamma cross-linked complex was observed in cells expressing both IFN-gamma R1 and IFN-gamma R2 indicating that IFN-gamma R2 (AF-1) interacts with IFN-gamma and is closely associated with IFN-gamma R1. We show that the intracellular domain of IFN-gamma R2 is necessary for signaling. Cells coexpressing IFN-gamma R1 and truncated IFN-gamma R2, lacking the COOH-terminal 51 amino acids (residues 286-337), or cells expressing IFN-gamma R1 alone were unresponsive to IFN-gamma treatment as measured by MHC class I antigen induction. Jak1, Jak2, and Stat1 alpha were activated, and IFN-gamma R1 was phosphorylated only in cells expressing both IFN-gamma R1 and IFN-gamma R2. Jak2 kinase was shown to associate with the intracellular domain of the IFN-gamma R2.

Amino Acid Sequence↗
PubMed1995

The Jak kinases differentially associate with the alpha and beta (accessory factor) chains of the interferon gamma receptor to form a functional receptor unit capable of activating STAT transcription factors.

Interferon gamma (IFN gamma) induces the expression of early response genes by tyrosine phosphorylation of Jak kinases and transcription factors referred to as STAT proteins. The topology of the IFN gamma receptor is partially understood and the relationship between the alpha chain that binds the ligand and the beta chain that is required for signal transduction is undefined. In a cell line which expresses only the human alpha chain, we show that these cells did not activate Jak kinases or STAT proteins with human IFN gamma, even though Jak1 co-immunoprecipitated with the alpha chain. In cells unexposed to IFN gamma, Jak1 preferentially associated with the alpha chain, while Jak2 associated with the beta chain. There was evidence for Jak1 kinase activity in untreated cells. For Jak2, kinase activity was IFN gamma-dependent. Although the alpha chain was tyrosine-phosphorylated in response to ligand, we found no evidence for tyrosine phosphorylation of the beta chain. These data are consistent with a model of the IFN gamma receptor in which Jak1 associates with the alpha chain, whereas Jak2 associates with the beta chain. IFN gamma clusters at least two receptor units which results in the tyrosine phosphorylation of Jak1 and Jak2, the activation of Jak2 kinase activity, and the recruitment of STAT1 alpha resulting in its activation by tyrosine phosphorylation.

Base Sequence↗
PubMed1995

Interferon gamma receptor extracellular domain expressed as IgG fusion protein in Chinese hamster ovary cells. Purification, biochemical characterization, and stoichiometry of binding.

Agents that antagonize the functions of interferon gamma (IFN gamma) are potential pharmaceuticals against several immunological and inflammatory disorders. IFN gamma receptor-immunoglobulin G fusion proteins (IFN gamma R-IgG) function as antagonists of endogenous IFN gamma and have longer half-lives in vivo in comparison with soluble IFN gamma receptors (sIFN gamma R), consisting of the extracellular region of the native sequence. A fusion protein comprising the extracellular domain of the human IFN gamma receptor and the hinge, CH2 and CH3 domains of the human IgG3 constant region, was expressed in Chinese hamster ovary cells. The IFN gamma R-IgG3 fusion protein was secreted into the culture medium as a 175-kDa glycoprotein and was purified over Protein G-Sepharose, DEAE-Sepharose, and size exclusion chromatography. IFN gamma R-IgG3 bound IFN gamma in solid phase assays and ligand blots, competed for the binding of radiolabeled IFN gamma to the cell surface receptor of Raji cells, and inhibited the IFN gamma-mediated antiviral activity with an efficiency at least one order of magnitude higher than that of the soluble receptor produced in the same expression system. Two IFN gamma R-IgG3 fusion proteins bound two IFN gamma dimers forming a complex of approximately 380 kDa. In immunodiffusion assays, the IFN gamma R-IgG3 fusion protein did not precipitate IFN gamma. Dissociation of bound IFN gamma from IFN gamma R-IgG3 was 2-fold slower than from the sIFN gamma R produced in insect cells.

Animals↗
PubMed1995

Dissection of the extracellular human interferon gamma receptor alpha-chain into two immunoglobulin-like domains. Production in an Escherichia coli thioredoxin gene fusion expression system and recognition by neutralizing antibodies.

The extracellular interferon gamma receptor alpha-chain (IFN gamma R) is believed to comprise two discrete approximately 110 amino acid immunoglobulin-like domains, perhaps similar to those seen in the crystal structure of the extracellular human growth hormone receptor [De Vos, A. M., Ultsch, M., & Kossiakoff, A. (1992) Science 255, 306-312], a distant relative in the cytokine receptor superfamily. In accord with this idea, we show that these IFN gamma R immunoglobulin-like domains can be produced separately in a soluble form with a native-like fold. The N-terminal domain (residues 1-108), with a Cys105 to Ser105 mutation, was produced at a high level, in a soluble form, as a thioredoxin-interferon gamma receptor fragment fusion protein in the cytoplasm of Escherichia coli. Upon extraction, the receptor Cys60-Cys68 disulfide bond formed spontaneously, to generate a native-like structure directly without the need for refolding. Cleavage of the fusion protein by enterokinase released the receptor fragment (approximately 12 kDa), which was recognized by several neutralizing antibodies with affinities, measured using surface plasmon resonance technology, that were essentially indistinguishable from those seen with the full length extracellular IFN gamma R produced in eukaryotic cells. Circular dichroism and 1D 1H nuclear magnetic resonance spectra indicated that the receptor fragment adopts a folded state, with mainly beta-sheet and reverse turn secondary structure. The second membrane-proximal Ig-like domain of the IFN gamma R (residues 90-229) was produced, albeit less efficiently, and characterized in a similar way. The production of these two independently folded proteins provides experimental support for the two domain organization of the IFN gamma R and opens new avenues for structural studies on these Ig-like molecules by NMR and crystallographic methods.

Amino Acid Sequence↗
PubMed1995

Experimental therapy of systemic lupus erythematosus: the treatment of NZB/W mice with mouse soluble interferon-gamma receptor inhibits the onset of glomerulonephritis.

Female NZB/W F1 mice develop an autoimmune disease similar to human systemic lupus erythematosus (SLE), and ultimately die of glomerulonephritis. Starting at the age of 16 weeks NZB/W F1 mice were treated for a period of 19 weeks with soluble interferon-gamma receptor (sIFN-gamma R), anti-IFN-gamma monoclonal antibody (mAb) or IFN-gamma. All mice treated with sIFN-gamma R or anti-IFN-gamma mAb were alive 4 weeks after the treatment was discontinued, whereas 50% of mice died in the placebo groups and 78% of the mice died in the IFN-gamma-treated group. Histologically, there was severe membrano-proliferative glomerulonephritis in IFN-gamma- and placebo-treated mice, and minimal or no mesangioproliferative disease in mice receiving sIFN-gamma R or anti-IFN-gamma mAb. The renal mononuclear infiltrate (T lymphocytes and monocytes), expression of major histocompatibility complex class II antigen and glomerular immunoglobulin and complement deposition were reduced in those mice. These data suggest that an IFN-gamma inhibitor, such as the soluble IFN-gamma R, can be used for SLE therapy in the early stages of the disease.

Animals↗
PubMed1995

Interleukin-12 is required for interferon-gamma production and lethality in lipopolysaccharide-induced shock in mice.

Several cytokines, in particular tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), have been shown to be responsible for pathological reactions which may lead to shock and death observed in infection with Gram-negative bacteria and in response to endotoxins (lipopolysaccharides, LPS). Priming of mice with the avirulent Bacille Calmette Guérin (BCG) vaccine strain of Mycobacterium bovis increases the sensitivity of mice to the lethal effect of LPS and results in an efficient priming for cytokine production. In response to low doses (1 microgram/mouse) of LPS, BCG-primed mice produce interleukin-12 (IL-12) which controls IFN-gamma production, as demonstrated by the ability of neutralizing anti-IL-12 antibodies to suppress IFN-gamma production. However, the concentration of the biologically active IL-12 p70 heterodimer is similar in the serum of both BCG-primed or unprimed mice, reaching levels of 1-3 ng/ml at 3-6 h after LPS injection, whereas IFN-gamma production was observed only in BCG-primed mice. The priming effect of BCG on IFN-gamma production appears to be mostly due to its ability to increase TNF-alpha production, which acts as cofactor with LPS-induced IL-12 in inducing IFN-gamma production, as shown by the ability of injection of TNF-alpha and LPS (1 microgram/mouse), but not LPS alone, to induce IFN-gamma production. However, in addition to TNF-alpha, other LPS-induced cofactor(s) are required in cooperation with IL-12 to induce optimal IFN-gamma production, because co-injection of TNF-alpha and IL-12, sufficient to induce serum concentrations of both cytokines higher and more persistent than those obtained by injection of LPS, was not sufficient to induce IFN-gamma production in vivo. Neutralizing anti-IL-12 antibodies, in addition to inhibiting the in vivo LPS-induced IFN-gamma production, also completely protect BCG-primed mice injected with up to 10 micrograms of LPS from shock-induced death. Thus, IL-12 is required for IFN-gamma production and lethality in an endotoxic shock model in mice.

Animals↗
PubMed1995

Biphasic effect of interferon-gamma in murine collagen-induced arthritis.

Interferon-gamma (IFN-gamma) exerts both enhancing and suppressing influences on collagen-induced arthritis (CIA), depending on the route and protocol of administration. To study the role of IFN-gamma on the autoimmune process of CIA, we treated DBA/1 mice with two different rat monoclonal antibodies (mAb) to murine IFN-gamma. Treatments, given twice weekly for 4 weeks, consisted of intraperitoneal injections of either mAb. In early treatments, starting from the day of immunization with type II collagen (CII), the severity of arthritis was reduced in both groups of anti-IFN-gamma-treated mice compared with control groups. Moreover, anti-CII antibody levels decreased in the sera of these mice. CIA was also down-regulated in mice treated from days 14 or 28 post immunization. In contrast, late treatments with anti-IFN-gamma mAb either induced aggravating effects, or did not affect the course of the disease. On the other hand, administration of high doses (8 x 10(4) U three times/week) of rat recombinant IFN-gamma exerted a transient increase of CIA severity. These findings suggest that IFN-gamma may play a critical role during both the induction and the course of CIA, first enhancing the immune response, and then regulating the arthritis process.

Animals↗
PubMed1995

Variable region cDNA sequences and characterization of murine anti-human interferon gamma receptor monoclonal antibodies that inhibit receptor binding by interferon gamma.

Murine monoclonal antibodies (mAbs) are described that recognize the extracellular human interferon gamma receptor alpha-chain (IFN gamma R) and inhibit the binding to it of interferon gamma. The inhibitory activities (IC50s) of these mAbs, quantified by radioimmunoassay using native receptor on human Raji cells, lie in the range 0.5-24 nM, whereas their relative affinities for the immobilised recombinant extracellular receptor, determined using surface plasmon resonance technology, are in the range 0.6-40.9 nM. Nine mAbs derived from one immunization, were shown by variable region cDNA sequencing to be clonally related, with mAb A6 from this group showing the highest affinity for the receptor. Another two mAbs, gamma R38 and gamma R99, derived from a separate immunization, are clonally unrelated to each other and to those in the A6 family. From the V-region sequences, the L-chains of mAbs A6, gamma R38 and gamma R99 were shown to belong to the V kappa 34C, V kappa 34C and V kappa 1 families, whereas the H-chains belong to the 3069, J606 and J558 families, respectively. The mAbs A6 and gamma R38 recognize overlapping epitopes on the N-terminal Ig-like domain of the IFN gamma R, whereas the gamma R99 epitope is located largely in the membrane proximal Ig-like domain. Sequence comparisons with Ig structures solved by X-ray diffraction allowed deductions concerning likely CDR canonical conformations. These studies provide essential information for crystallographic and mutagenesis experiments aimed at understanding the molecular basis of the interactions of these mAbs with the extracellular IFN gamma R.

Amino Acid Sequence↗
PubMed1995

Aspects of molecular interaction between HIV p17 and human gamma interferon.

We describe the specific interaction between high-purity recombinant human immunodeficiency virus (HIV) type 1 p17 and human gamma interferon (hIFN-gamma) proteins. This interaction was found to be dose dependent and to involve conformational epitopes on both sides. Specificity was confirmed by competition ELISA, using monoclonal antibodies (MAbs) to hIFN-gamma as specific reagents. By competition experiments we also identified the epitope(s) on the hIFN-gamma molecule involved in p17 binding, very close to the receptor binding site. The kinetic constants were determined by surface plasmon resonance (SPR) analysis. The affinity constant (KA) of the complex was 2.78 x 10(8) M-1, that is, the ratio between a low dissociation rate constant (Koff)(1 x 10(-5)sec-1) and a high association rate constant (Kon) (3 x 10(3) M-1sec-1). However, p17 did not displace the binding of hIFN-gamma to its cellular receptor, nor did it interfere with the capability of the lymphokine to induce de novo expression of HLA-DR antigens on human monocytic cells or to inhibit the proliferation of tumor cells.

Animals↗
PubMed1995

Interferon-gamma receptor extracellular domain-IgG fusion protein produced in Chinese hamster ovary cells as mixture of glycoforms.

Glycosylation of proteins fulfills important functions and because of its diversity contributes to apparent protein heterogeneity. We investigated the heterogeneity of a fusion protein comprising the extracellular domain of the human interferon-gamma (IFN-gamma) receptor and parts of the human IgG3 constant region, a potential IFN-gamma antagonist. The protein was produced in Chinese hamster ovary (CHO) cells and was secreted into the culture medium as an 175 kD glycoprotein. Glycosylation represented approximately one-third of the apparent molecular mass of the fusion protein, consisted of N- and O-linked carbohydrate moieties, and included sialic acid residues as part of both N- and O-linked oligosaccharides. Fusion protein forms with different apparent molecular masses and charges were separated by ion-exchange chromatography. Preparative electrofocusing revealed a wide spectrum of glycoforms. Glycosylation of the fusion protein and of soluble IFN-gamma receptors, comprising the extracellular domain of the native sequence, expressed in insect and CHO cells did not interfere with affinity of ligand binding.

Amino Acid Sequence↗
PubMed1995

Role of interferon-gamma in mediating the antitumor efficacy of interleukin-12.

Although interleukin-12 (IL-12) has marked antitumor activity against the murine Renca renal cell carcinoma in vivo, no antiproliferative activity with IL-12 was observed against these tumor cells in vitro; in contrast, interferon-gamma (IFN-gamma) had growth inhibitory activity. Since one of the properties of IL-12 is its ability to stimulate production of IFN-gamma, the role of IFN-gamma in mediating the antitumor activity of IL-12 was evaluated. Substantially diminished antitumor activity was observed in mice injected with IL-12 and neutralizing antibody to murine IFN-gamma compared with mice receiving IL-12 alone, indicating that IFN-gamma was required for the optimal antitumor efficacy of IL-12. However, several lines of investigation suggest that the antitumor effect of IL-12 is not mediated solely through the induction of IFN-gamma. Exogenous administration of IFN-gamma to Renca tumor-bearing euthymic mice resulted in less antitumor efficacy than that which could be obtained with IL-12. In addition, the antitumor effect of IL-12 was reduced in nude mice compared with euthymic mice, but an approximately 10-fold higher level of serum IFN-gamma was induced in nude than in euthymic mice. Thus, these results indicate that induction of high serum levels of IFN-gamma is not sufficient to mediate the antitumor efficacy of IL-12.

Animals↗
PubMed1995

The in vivo antiviral activity of interleukin-12 is mediated by gamma interferon.

The injection of 20 ng of mouse interleukin-12 (IL-12) protects mice from a lethal infection with encephalomyocarditis virus. In vitro, an anti-gamma interferon (anti-IFN-gamma) monoclonal antibody but not an anti-IL-12 monoclonal antibody neutralizes the antiviral activity present in the supernatants of splenocytes stimulated with IL-12. Finally, IL-12 fails to protect 129 Sv/Ev IFN-gamma R0/0 mice against encephalomyocarditis virus infection. These results demonstrate that IL-12 exerts its antiviral activity through the induction of endogenous IFN-gamma.

Animals↗