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Biomedical subjects

G Garotta

Publications and source records attributed to G Garotta.

At least 55 records · Page 3Linked to original sources
PubMed1995

Human PBMC proliferative response to Leishmania infantum promastigotes is inhibited by anti-IFN-gamma mAb gamma 123.

PBMC from individuals both exposed and non-exposed to leishmaniae proliferative and produce interferon-gamma (IFN-gamma) following stimulation with Leishmania antigens. We studied the kinetics of the proliferative response of PBMC from non-exposed individuals and from patients recovering from visceral leishmaniasis due to Leishmania infantum, using heat-killed stationary-phase promastigotes of L. infantum as stimulating agent. The kinetics of both groups followed a similar temporal pattern, with higher values in the patient's group. Moreover, we observed that in both groups the activation was dose-dependently inhibited following the addition of gamma 123 anti-IFN-gamma monoclonal antibody. These results indicate the need for IFN-gamma in the activation process of PBMC induced by Leishmania antigens and stress the role of IFN-gamma in the immune response to leishmaniasis. The relevance of the elucidation of the immune response mechanism in human leishmaniasis for therapy and vaccination is briefly discussed.

Animals↗
PubMed1994

Interleukin 12, interferon gamma, and tumor necrosis factor alpha are the key cytokines of the generalized Shwartzman reaction.

The Shwartzman reaction is elicited by two injections of lipopolysaccharide (LPS) in mice. The priming LPS injection is given in the footpad, whereas the lethal LPS challenge is given intravenously 24 h later. The injection of interferon gamma (IFN-gamma) or interleukin 12 (IL-12) instead of the LPS priming injection induced the lethal reaction in mice further challenged with LPS. Antibodies against IFN-gamma when given together with the priming agent, prevented the lethal reaction in mice primed with either LPS, IL-12, or IFN-gamma. Antibodies against IL-12, when given together with the priming agent, prevented the lethal reaction in mice primed with either LPS or IL-12 but not with IFN-gamma. These results strongly suggest that LPS induces the release of IL-12, that IL-12 induces the production of IFN-gamma, and that IFN-gamma is the cytokine that primes macrophages and other cell types. Upon LPS challenge, the lethal Shwartzman reaction is induced by a massive production of inflammatory cytokines that act on the target sites already sensitized by IFN-gamma. If mixtures of TNF and IL-1 or mixtures of TNF and IFN-gamma are used to challenge mice previously primed with IFN-gamma or IL-12, mortality is induced. In the same conditions, the individual cytokines or a mixture of IL-1 and IFN-gamma do not replace the LPS challenge. When the mice are primed with LPS, the combination of TNF, IL-1, and IFN-gamma induced only a partial mortality incidence suggesting that the involvement of other LPS-induced factors.

Animals↗
PubMed1994

Radiation inactivation of human gamma-interferon: cellular activation requires two dimers.

gamma-Interferon (IFN-gamma) is a 17-kDa broad-spectrum cytokine which exerts its effects on a variety of target cells through its interaction with the IFN-gamma receptor. Although physicochemical studies of Escherichia coli-derived IFN-gamma, as well as its crystal structure, demonstrate that it is a homodimer in solution (M(r) 34,000), previous radiation inactivation studies yielded a functional size for IFN-gamma of 63-73 kDa in an antiviral assay. To understand the relationship between the solution form of IFN-gamma and the moiety that actually binds to the cellular receptor and activates cells, we examined irradiated nonradioactive and 32P-labeled IFN-gamma for its migration in SDS/polyacrylamide gels (to determine its physical integrity), its binding to cells, its reactivity in an ELISA, and its antiviral activity. The functional size of IFN-gamma differed in the assays, being 22 +/- 2 kDa for the physical destruction of IFN-gamma, 56 +/- 2 kDa for the cellular binding assay, 45-50 kDa for reactivity in the ELISA, and 72 +/- 6 kDa for antiviral activity. The results from the binding assays constitute direct evidence that IFN-gamma binds to its cellular receptor as a dimer. However, for antiviral activity, the functional mass is equivalent to a tetramer. This is consistent with models involving ligand-induced receptor dimerization, whereby two dimers acting in concert (equivalent to the target size of a tetramer) are required to activate cells in the antiviral assay.

Adenosine Triphosphate↗
PubMed1994

Interferon-gamma induces tyrosine phosphorylation of interferon-gamma receptor and regulated association of protein tyrosine kinases, Jak1 and Jak2, with its receptor.

Interferon-gamma (IFN-gamma) induces the expression of a set of early response genes by tyrosine phosphorylation of latent transcription factors such as p91. Although the tyrosine kinases, Jak1 and Jak2, have recently been shown to be critical for signal transduction by IFN-gamma, evidence is lacking for both tyrosine phosphorylation of the IFN-gamma receptor (IFN-gamma R) and the interaction between Jak1, Jak2, and the IFN-gamma R. In this report, we show that binding of IFN-gamma to HeLa cells initiated a series of events that resulted in the extremely rapid (15 s) tyrosine phosphorylation of not only Jak1, Jak2, and p91 but also the IFN-gamma R. Coimmunoprecipitation experiments revealed that Jak1 was associated with the IFN-gamma R prior to ligand binding, whereas Jak2 became part of the IFN-gamma R-Jak1 complex immediately after ligand binding. H2O2/vanadate treatment of cells for 15 min resulted in only the tyrosine phosphorylation of Jak1 and IFN-gamma R. Only after 60 min of this treatment did we observe tyrosine phosphorylation of Jak2 and p91 and assembly of the transcription factor complex FcRF gamma that binds to the promoter of the fcgr1 gene. These data suggest that JAK1 associates with the IFN-gamma R prior to ligand binding. IFN-gamma treatment of cells results in recruitment of JAK2 into the IFN-gamma R-Jak1 complex followed by assembly of the transcription factor FcRF gamma complex.

Electrophoresis, Polyacrylamide Gel↗
PubMed1994

Environmental signals influencing expression of the IFN-gamma receptor on human T cells control whether IFN-gamma promotes proliferation or apoptosis.

IFN-gamma R expression is subject to contrasting modulation on human T cells. IFN-gamma R constitutive expression is low on three human malignant T cells (ST4, PF382, and Jurkat) growing in medium supplemented with serum. The addition of IFN-gamma down-modulates IFN-gamma R expression and increases both proliferation and MHC class I Ag expression. By contrast, when malignant T cells are cultured in medium without serum, IFN-gamma R expression dramatically increases and the cells undergo a slow apoptotic death. The addition of IFN-gamma enhances apoptosis and inhibits cell rescue in serum-supplemented medium. This opposite ability of IFN-gamma to stimulate malignant T cell proliferation or death correlates with the intensity of IFN-gamma R cell expression, high expression being a marker for cell apoptosis. IFN-gamma R up-modulation also occurs on malignant T cells undergoing apoptosis after treatment with dexamethasone, on irradiated normal CD3+ PBL, and on cultured normal CD3+ thymocytes. Moreover, the ability of IFN-gamma to augment apoptosis of highly IFN-gamma R-positive thymocytes suggests that its role in promoting T cell apoptosis is also important in physiologic conditions.

Apoptosis↗
PubMed1994

First trimester human trophoblast expresses both interferon-gamma and interferon-gamma-receptor.

Interferon-gamma (IFN-gamma) is a lymphokine, produced by activated T lymphocytes, which plays a key regulatory role in the host immunological responses. In addition, IFN-gamma is expressed by human and porcine trophoblast. As IFN-gamma exerts its biological functions through specific cell surface receptors and a great number of IFN-gamma receptors (IFN-gamma R) have been purified from human placenta, we have examined the relative distribution of IFN-gamma and IFN-gamma R in human placental tissues at different stages of pregnancy. By using immunohistochemical analysis and monoclonal antibodies, it was found that IFN-gamma expression is intense in the first trimester but almost imperceptible at term, whereas the expression of IFN-gamma R is present at both stages of pregnancy. For both lymphokine and receptor, the most intense expression was observed in villous syncytiotrophoblast and in extravillous interstitial trophoblast. From these results it appears that the expression of IFN-gamma R in trophoblast is related to the presence of the lymphokine in the early phase of gestation but not later. On this basis, it can be argued that IFN-gamma exerts its functional role via an autocrine and/or a paracrine loop mainly during the first trimester.

Embryonic and Fetal Development↗
PubMed1993

IFN-gamma involvement in the severity of gram-negative infections in mice.

The role of IFN-gamma in the regulation of inflammation leading to gram-negative septic shock is still poorly understood. IFN-gamma blockade has been shown to improve the survival of animals challenged with i.v. bolus injections of LPS and gram-negative bacteria. We have investigated a model of focal Escherichia coli infection leading to peritonitis and septic shock. Mice were challenged i.p. with an inoculum near the LD50. The addition of rIFN-gamma together with bacteria increased the mortality and the level of blood TNF-alpha and IL-6. Conversely blockade of IFN-gamma with a neutralizing mAb significantly improved the survival of the mice. This beneficial effect was not associated with a stringent decrease in blood bacterial counts and TNF-alpha and IL-6 levels in survivors. In this model, the protective effect of anti-IFN-gamma mAb contrasted with the ineffectiveness of a neutralizing anti-TNF-alpha mAb. These findings suggest that overproduction of IFN-gamma might have a more detrimental role than overproduction of TNF-alpha during focal gram-negative infections.

Animals↗
PubMed1993

Mouse soluble IFN gamma receptor as IFN gamma inhibitor. Distribution, antigenicity, and activity after injection in mice.

With the purpose to use a soluble receptor as an IFN gamma antagonist in vivo, we assessed the immunogenicity, the half-life, the bioavailability, and the activity of the soluble receptor after injection into mice. No significant immunogenicity was detected after multiple chronic injections of mouse (Mo)IFN gamma-R or injections of MoIFN gamma-R in emulsion with CFA. Pharmacokinetic experiments using radiolabeled MoIFN gamma-R revealed that the half-life of the soluble MoIFN gamma-R is 3 h in the blood and 6 h in the lymphoid organs. When the MoIFN gamma-R protein was traced by the capacity of the animal sera to displace the binding of radiolabeled MoIFN gamma-R from affinity purified rabbit anti-MoIFN gamma-R antibodies, a blood half-life of 1 h was determined. Finally, the capacity of the injected receptor to bind and neutralize the IFN gamma-mediated antiviral activity was assessed in vivo. Treatment with IFN-gamma or IFN-alpha A/D protected the mice from the lethal infection with encephalomyocarditis virus. Mice treated with MoIFN-gamma-R neutralized the protective effect of IFN-gamma.

Animals↗
PubMed1993

Alignment of disulfide bonds of the extracellular domain of the interferon gamma receptor and investigation of their role in biological activity.

The extracellular ligand binding domain of the human interferon gamma receptor includes eight cysteine residues forming four disulfide bonds. Only the nonreduced protein binds interferon gamma. We investigated the alignment of the disulfide bonds, using an enzymatically deglycosylated form of a soluble interferon gamma receptor, produced in baculovirus-infected insect cells. The soluble receptor was digested with endoproteinase Glu-C and proteinase K, and the proteolytic fragments were characterized by amino acid sequence analysis and mass spectrometry. It was found that four consecutive disulfide bonds are formed between residues Cys60-Cys68, Cys105-Cys150, Cys178-Cys183, and Cys197-Cys218. We also investigated the role of the disulfide bonds in biological activity of the receptor, using site-directed mutagenesis and by exchanging the cysteine residues for serines. The mutated proteins were expressed in Escherichia coli and analyzed for ligand binding capacity on protein blots. The assays showed that all disulfide bonds are essential for full ligand binding capacity. Double or quadruple mutations at cysteine residues 60 and 68, and residues 178, 183, 197, and 218, respectively, resulted in complete loss of the activity, whereas double mutations at residues 105 and 150, 178 and 183, and 197 and 218, respectively, resulted in a residual activity about 1 order of magnitude lower than that of the wild type. The specific antibodies gamma R38 and gamma R99 detected conformational epitopes stabilized by disulfide bonds involving cysteine residues 60 and 68, and 178 and 183, respectively.

Amino Acid Sequence↗
PubMed1993

A monoclonal antibody to the NH2-terminal segment of human IFN-gamma selectively interferes with the antiproliferative activity of the lymphokine.

To gain more information about the relationship between the structure of IFN-gamma and its activity, a peptide corresponding to a hydrophilic peak between amino acids 4 and 16 was used to immunize mice and generate mAb. mAb IGMB-15 reacts to both native and rIFN-gamma and neutralizes the antiproliferative activity of IFN-gamma without affecting its antiviral activity or its ability to up-regulate HLA-DR Ag expression. Moreover, we observed that mAb IGMB-15 was unable to inhibit the binding of radiolabeled IFN-gamma to its cellular receptor. These findings show that the NH2-terminal region may somehow be involved in the biologic activity of IFN-gamma. Besides, the capability of mAb IGMB-15 to inhibit the antiproliferative but not the antiviral activity of IFN-gamma in the same cell (HEp-2) suggests the presence of different elements involved in signal transduction, which may account for the multiple activities of the lymphokine.

Amino Acid Sequence↗
PubMed1993

The effects of a monoclonal antibody to interferon-gamma on experimental autoimmune thyroiditis (EAT): prevention of disease and decrease of EAT-specific T cells.

CBA/J mice immunized with thyroglobulin (Tg) develop an experimental autoimmune thyroiditis (EAT) with lymphocytic infiltration of the thyroid glands, autoantibodies to Tg and occurrence of EAT-specific T cells. When these mice were treated for 4 weeks after immunization with 1 mg/week of a monoclonal antibody (mAb) that neutralizes the activity of interferon-gamma (IFN) a beneficial effect on the onset of EAT was observed. Characteristic features of EAT were significantly reduced, including the lymphocytic infiltrations of the thyroid glands and the serum levels of autoantibodies to Tg. Moreover, in lymphoid organs, mAb to IFN-gamma significantly reduced the percentages of Tg-specific CD8+ cells, labeled by the anti-clonotypic mAb AG7. These Tg-specific T cells seem responsible for thyroid damages and disease development, since EAT was simultaneously abrogated. These results show that IFN-gamma plays an essential role in the pathophysiology of EAT and suggest the possibility to treat autoimmune thyroid diseases with mAb to IFN-gamma or drugs able to antagonize the production and/or the action of this cytokine.

Animals↗
PubMed1993

Modulation of interferon-gamma receptor during human T lymphocyte alloactivation.

Previous work has shown that neutralization of physiologically secreted interferon(IFN)-gamma or blockade of its receptor during T lymphocyte activation inhibits both proliferation and cytotoxic T lymphocyte generation, suggesting that IFN-gamma plays a crucial role in T lymphocyte induction and differentiation. In this study, the kinetics of the surface expression of the 90-kDa IFN-gamma receptor (IFN-gamma R) was followed during human mixed lymphocyte reaction (MLR) to alloantigens. IFN-gamma R mRNA is constitutively expressed on resting peripheral blood lymphocytes emerging from nylon wood column (NW-PBL) and its expression increases two- to threefold on alloactivated NW-PBL. IFN-gamma R protein is poorly expressed on the membrane of resting CD3+ cells, but up-modulates after 3-day MLR and sharply down-modulates at day 6. Both the p55 and the p75 chains of interleukin-2 receptor (IL-2R) were shown to up-modulate in parallel with IFN-gamma R, whereas they were still highly expressed at day 6. After alloactivation, IFN-gamma and IL-2 secretion starts at 24 h, peaks at day 3 and decreases just when IFN-gamma R and IL-2R begin to up-modulate. Proliferation peaks at day 6. Lastly, stimulation with distinct cell populations showed that the intensity of lymphocyte proliferation, IFN-gamma R membrane up-modulation, and IFN-gamma and IL-2 secretion are regulated in a parallel manner, thus suggesting that they are interrelated. Taken as whole these results demonstrate that increased expression of IFN-gamma R on T lymphocytes can be a critical event during their activation, and strongly support the hypothesis that IFN-gamma/IFN-gamma R interaction provides a signal for its progression.

Cell Line↗
PubMed1993

On the relative roles of interleukin-2 and interleukin-10 in the generation of lymphokine-activated killer cell activity.

Induction of cytokine gene transcription by recombinant human IL-2 (rhIL-2) in peripheral blood mononuclear cells (PBMC) from healthy donors was studied by qualitative polymerase chain reaction. In all donors tested, optimal doses of rhIL-2-induced transcription of genes encoding for IL-5, GM-CSF, IFN-gamma, and TNF-alpha whereas transcription of IL-1-alpha, IL-3, IL-4, and IL-6 genes could only be detected in about half of the donors. Moreover, we observed that different doses of rhIL-2 were needed to induce transcription of different cytokine genes. In contrast, transcription of IL-2 and IL-10 genes was only observed in a minority of donors, irrespective of the concentration of rhIL-2 used. Since IL-10 displays a well-characterized inhibitory activity on the synthesis of cytokines possibly involved in the generation of lymphokine-activated killer (LAK) cells, we asked whether the absence of IL-10 gene transcription plays a role in the induction of LAK cells. Thus, we tested the effects of different doses of rhIL-10 on the rhIL-2-driven generation of LAK activity. Interestingly, rhIL-10 dose-dependently inhibited the production of IFN-gamma and TNF-alpha induced by IL-2, but had no effects on PBMC proliferation and generation of LAK activity. Similarly, purified CD3-/CD16+ lymphocytes, the precursors of LAK effector cells, could be optimally induced by low doses of rhIL-2 to proliferate and generate MHC-unrestricted cytotoxic activity against NK-resistant targets in the presence of rhIL-10. Altogether, our results indicate that rhIL-2 induces transcription of a preferential pattern of cytokine genes, with the IL-10 gene being infrequently transcribed. On the other hand, rhIL-10 shows diverse effects on rhIL-2-triggered PBMC activation, in that it inhibits IFN-gamma and TNF-alpha production but does not affect PBMC proliferation or generation of LAK activity.

Base Sequence↗
PubMed1993

Differential effects of interleukin-2 and CD3 triggering on cytokine gene transcription and secretion in cultured tumor infiltrating lymphocytes.

The therapeutic potential of tumor-infiltrating lymphocytes (TIL) is currently under investigation. TIL have been reported to display characteristic functional defects, including impairments of activation mechanisms. In animal models, therapeutic efficacy of adoptive TIL transfer has been correlated with their capacity to produce cytokines rather than with their in vitro cytotoxic potential. In this work we assayed cytokine gene transcription and protein production in eight cultured TIL populations stimulated with recombinant human IL-2 (rhIL-2), solid-phase-bound anti-CD3 monoclonal antibodies (mAb), or a combination of the two. By using a sensitive reverse polymerase chain reaction technique, we observed that transcription of IL-5, GM-CSF, and TNF-alpha could be detected in unstimulated, IL-2-starved TIL from all cultures, while IL-4 and IFN-gamma genes were found to be transcribed in two cultures out of eight. A 50 U/ml dose of rhIL-2 was sufficient to induce IL-4 and IFN-gamma gene transcription in the remaining six cultures and in four more TIL populations, respectively. In contrast, rhIL-2 could not induce IL-2 gene transcription in five of eight TIL cultures. Furthermore, it could not induce IL-10 gene transcription in any TIL population. Anti-CD3 mAb triggering, however, induced transcription of all these cytokine genes. On the other hand, IL-6, IL-7, or TGF-beta 2 gene transcription could not be induced by any of the stimuli used, including the combination of anti-CD3 and rhIL-2. Despite detection of their gene transcripts, GM-CSF, IFN-gamma, or TNF-alpha was never detectable, at the protein level, in unstimulated TIL. Stimulation by rhIL-2 induced GM-CSF secretion, albeit to different extents, in all TIL populations. In contrast, rhIL-2 induced IFN-gamma and TNF-alpha production in three and one TIL population, respectively. CD3 triggering however, induced cytokine production in all TIL populations. Addition of rhIL-2 significantly increased production of GM-CSF, but not of IFN-gamma and TNF-alpha. These data underline that stimulation with a moderate dose of rhIL-2 reveals considerable heterogeneity of TIL populations regarding cytokine gene transcription and secretion. On the other hand, triggering of the CD3-T-cell receptor complex induces transcription of an extended panel of cytokine genes in all TIL populations and secretion of GM-CSF, IFN-gamma and TNF-alpha in virtually all cultures. Thus, if stimulated by properly presented antigenic peptides, TIL are capable of mounting an efficient response in terms of cytokine gene expression and protein production.

Aged↗
PubMed1993

Immunomodulation with soluble IFN-gamma receptor: preliminary study.

Several in vivo experiments support the hypothesis that an IFN-gamma antagonist may have therapeutic applications in autoimmune diseases, hypersensitivities, and alloreactions. IFN-gamma exerts its biological activity through the binding to a single-chain cell surface receptor. The protein that corresponds to the external domain of mouse IFN-gamma receptor was expressed in insect cells infected with recombinant baculovirus; this protein was characterized and used in vivo as a prototype of the IFN-gamma antagonist. This protein does not show any strong antigenicity after in vivo injection in mice. Despite a blood half-life of only 1-3 hr as demonstrated in pharmacokinetic experiments, the mouse soluble IFN-gamma R was able to modify the onset of acute GVHD (alloreaction) and chronic GVHD (lupuslike disease).

Acute Disease↗
PubMed1993

The human gamma interferon receptor accessory factor encoded by chromosome 21 transduces the signal for the induction of 2',5'-oligoadenylate-synthetase, resistance to virus cytopathic effect, and major histocompatibility complex class I antigens.

Mouse fibroblasts, and human-mouse hybrid fibroblasts carrying only human chromosome 21, were transfected with cDNA encoding full-length human gamma interferon (IFN-gamma) receptor or chimeric IFN-gamma receptor (extracellular domain of the human receptor; transmembrane and intracellular domains of mouse origin). These transfected mouse cells were sensitive to human IFN-gamma only when human chromosome 21 was present. These results show that the species-specific accessory protein encoded by human chromosome 21 interacts with the extracellular domain of human IFN-gamma receptor and transduces the IFN-gamma signal not only for up-regulation of mouse major histocompatibility complex class I antigen expression but also for the induction of 2',5'-oligoadenylate-synthetase and resistance to virus cytopathic effect.

2',5'-Oligoadenylate Synthetase↗