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Biomedical subjects

F Sinigaglia

Publications and source records attributed to F Sinigaglia.

At least 109 records · Page 6Linked to original sources
PubMed1992

Identification of a CD4 binding site on the beta 2 domain of HLA-DR molecules.

The CD4 and CD8 molecules are transmembrane glycoproteins expressed by functionally distinct subsets of mature T cells. CD4+ and CD8+ T cells recognize antigens on major histocompatibility complex (MHC) class II-bearing and class I-bearing target cells respectively. The ability of monoclonal antibodies against CD4 and CD8 to block antigen recognition by T cells, as well as cell-cell adhesion assays, indicate that CD4 and CD8 bind to nonpolymorphic determinants of class II or class I MHC. Here we demonstrate that soluble recombinant HLA-DR4 molecules from insect cells and HLA-DR-derived peptides bind to immobilized recombinant soluble CD4. CD4 binds recombinant soluble DR4 heterodimers, as well as the soluble DR4-beta chain alone. Furthermore, two out of twelve DR4-beta peptides could interact specifically with CD4. These findings show that CD4 interacts with a region of MHC class II molecules analogous to a previously identified loop in class I MHC proteins that binds CD8 (refs 8, 9).

Amino Acid Sequence↗
PubMed1992

Stimulation of human platelets with concanavalin A involves phospholipase C activation.

In response to concanavalin A, cytoplasmic calcium movement was observed in human platelets, both in the presence of 1 mM Ca2+ or 1 mM EGTA in the medium. Concanavalin A also caused the activation of inositide turnover and the production of inositol phosphates, suggesting that activation of phospholipase C occurs. The mechanism by which concanavalin A stimulates phospholipase C does not depend on GTP-binding transducers, because it was not inhibited by GDP beta S, while experiments performed in the presence of cytochalasin B suggested a role for membrane glycoprotein IIb-IIIa-cytoskeleton interaction in this process. Ca(2+)-proteases and Na+/H+ antiport also seemed to be related to concanavalin A-induced phospholipase C activation, as suggested by experiments performed in the presence of leupeptin and amiloride.

Biological Transport↗
PubMed1992

Protection against malaria in Aotus monkeys immunized with a recombinant blood-stage antigen fused to a universal T-cell epitope: correlation of serum gamma interferon levels with protection.

The major surface antigen p190 of the human malaria parasite Plasmodium falciparum contains nonpolymorphic, immunogenic stretches of amino acids which are attractive components for a subunit vaccine against malaria. One such polypeptide, termed 190L, is contained in the 80-kDa processing product of p190, which constitutes the major coat component of mature merozoites. We report here that immunization of Aotus monkeys with 190L gives only poor protection against P. falciparum challenge. However, addition by genetic engineering of a universal T-cell epitope (CS.T3) to 190L improved immunity, and as a result three of four monkeys were protected following challenge infection with blood-stage parasites. Neither antibody against the immunizing antigens or against blood-stage parasites nor the capacity of the monkeys' sera to inhibit in vitro parasite invasion correlated with protection. However, in contrast to sera from nonprotected monkeys, sera from protected animals contained elevated levels of gamma interferon. These results suggest that gamma interferon is directly or indirectly involved in the process of asexual parasite control in vivo.

Animals↗
PubMed1992

Gold-specific T cells in rheumatoid arthritis patients treated with gold.

Gold-specific T lymphocyte clones were isolated from a patient with rheumatoid arthritis who developed delayed type hypersensitivity reactions to gold. All of the isolated T cell clones required histocompatible antigen presenting cells as well as gold for induction of proliferation. Using a panel of HLA-homozygous Epstein Barr virus-transformed B (EBV-B) cells and anti-HLA antibodies, the clones were shown to recognize gold in the context of DR1 molecules. Gold recognition did not require active antigen processing since specific proliferation was not affected by glutaraldehyde fixation of the DR1 homozygous antigen presenting cells. Furthermore, we could show that gold salts inhibited peptide-induced responses of a peptide-specific T cell clone. In addition to providing evidence for gold-specific T cells in gold-treated RA patients exhibiting delayed type hypersensitivity responses, these data suggest that gold can alter MHC-peptide complexes. The latter observation may in part explain the mechanism/s responsible for both the therapeutic and the toxic effects of gold.

Antigen-Presenting Cells↗
PubMed1991

Platelet glycoprotein IIb-IIIa is associated with 21-kDa GTP-binding protein.

Platelet membrane glycoprotein IIb-IIIa has been widely studied in the last years because of its role as an activation-dependent, adhesive protein receptor. Recently we demonstrated that occupancy of glycoprotein IIb-IIIa-receptor sites by specific ligands exerts an inhibitory effect on platelet responses induced by mild stimulation, leading us to suppose that this event may interact with activation pathways. Although the mechanisms of signal transduction in human platelets are not completely elucidated, the hypothesis that GTP-binding proteins are involved is generally accepted. Our results demonstrate that platelet ConA receptors, known to be located mainly on GP IIb-IIIa, are able to bind [35S]GTP gamma S; the GTP-binding activity is specific and is due to the association with the receptors of two G-proteins, with apparent molecular masses of 25 and 21 kDa, respectively. After the purification of GP IIb-IIIa, a glycoprotein complex electrophoretically pure was obtained that was still associated with a GTP-binding activity, migrating in SDS-polyacrylamide gel electrophoresis as a narrow band of about 21 kDa.

Autoradiography↗
PubMed1991

Regulation of human lymphocyte proliferation by a heterodimeric cytokine, IL-12 (cytotoxic lymphocyte maturation factor).

IL-12 is a heterodimeric cytokine that was identified on the basis of its ability to synergize with IL-2 in the induction of cytotoxic effector cells and was originally called cytotoxic lymphocyte maturation factor (CLMF). IL-12 was also found to stimulate the proliferation of PHA-activated lymphoblasts which were greater than 90% CD3+ T cells. In this report we further characterize the effects of IL-12 on lymphocyte proliferation. Studies with purified subpopulations of PHA-activated lymphoblasts and with cloned lines of human T cells indicated that IL-12 caused the proliferation of activated T cells of both the CD4+ and CD8+ subsets. This effect of IL-12 was independent of IL-2 because it was not blocked by antibodies to either IL-2 or IL-2R. The maximum proliferation induced by IL-12 was 31 to 72% of the maximum caused by IL-2; however, IL-12 was active at a lower effective concentration (EC50 = 8.5 +/- 1.3 pM) than IL-2 (EC50 = 52 +/- 8 pM). Combination of suboptimal amounts of IL-12 and IL-2 resulted in additive proliferation, up to the maximum induced by IL-2 alone. IL-12 also caused the proliferation of lymphocytes activated by culture with IL-2 for 6 to 12 days. CD56+ NK cells were among the IL-12-responsive cells in the IL-2-activated lymphocyte population. Unlike IL-2 or IL-7, IL-12 caused little or no proliferation of resting peripheral blood mononuclear cells (PBMC). In this regard, IL-12 was similar to IL-4. However, IL-12 could enhance the proliferation of resting PBMC caused by suboptimal amounts of IL-2, whereas IL-4 inhibited IL-2-induced PBMC proliferation. Thus, IL-12 is a growth factor for activated human T cells and NK cells; however, its spectrum of lymphocyte growth-promoting properties is distinct from that of IL-2, IL-4, or IL-7.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1991

Analysis of the permissive association of a malaria T cell epitope with DR molecules.

In this study we examined the association of a promiscuous malaria T cell epitope, CS.T3, to different HLA-DR alleles. A large series of singly substituted or truncated variants of CS.T3 was prepared and tested for the ability to be recognised in association with, or to bind to, three distinct HLA-DR alleles (DR1, DRw11, and DRw14(w6)) and three natural variants of HLA-DRw11. We found that although association with the different DR molecules mapped to identical or closely overlapping regions of the peptide, distinct substitutions could drastically influence the capacity of the peptide to interact with one but not another of the three DR molecules tested. Based on analysis of the distribution of residues recognized by T cell clones restricted to the different DR alleles, we suggest that the peptide CS.T3 is not bound, at least for the three DR examined, as an alpha-helix. In addition we tested three subtypes of DRw11 as APC for the CS.T3 analogues and observed that the peptide is most likely bound in the same conformation to the three natural variants of the DRw11 molecule.

Amino Acid Sequence↗
PubMed1991

Selective interaction of Ni with an MHC-bound peptide.

T cells generally recognize foreign antigens as peptides associated with self-molecules encoded by genes of the major histocompatibility complex (MHC). However, T cells which are specific for non-peptidic haptens have been described, in particular in patients with contact sensitivity reactions to metals such as nickel (Ni). Previously, we isolated MHC class II-restricted Ni-specific T cell clones from patients with Ni allergy. The experiments reported here examine the molecular basis for the interaction between Ni and peptide-MHC complexes. We find that Ni alters a T cell response to a peptide and show that Ni interacts with this peptide to alter its antigenicity rather than its ability to bind to MHC molecules. These findings hold implications for a model of hapten recognition by T cells.

Amino Acid Sequence↗
PubMed1991

HLA polymorphism and T cell recognition of a conserved region of p190, a malaria vaccine candidate.

We have examined T cell recognition of a recombinant polypeptide (190L), corresponding to a 175-amino-acid-long conserved region of the major surface antigen (p190) of Plasmodium falciparum merozoites. We show that 190L contains a variety of T cell epitopes, and can be recognized in association with many different MHC class II molecules, including HLA-DR, DP, and DQ antigens. Most of the epitope-containing peptides are able to bind to more than one DR, and a single DR molecule can bind to different peptides. These findings, together with the fact that humans are generally heterozygous at the DR, DQ, and DP beta chain loci, suggest that MHC restriction should not be a major constraint in the development of malaria subunit vaccines.

Amino Acid Sequence↗
PubMed1991

Cloned antigens from Plasmodium falciparum as malaria vaccine candidates.

The problems of genetic polymorphism and poor immunogenicity of malaria vaccine candidates are discussed, with emphasis on the Circumsporozoite (CS) and p190 proteins of Plasmodium falciparum. It may be possible to use conserved regions of these proteins to raise protective immune responses against non-polymorphic determinants. Better adjuvants or delivery systems will be a critical factor in development of an effective vaccine.

Amino Acid Sequence↗
PubMed1990

Malaria antigens and MHC restriction.

In the case of the malaria CS protein we have shown that there is at least one T cell determinant which is able to bind to and be recognized by most human MHC class II molecules, while for the 190L polypeptide, derived from a conserved region of the p190 merozoite surface protein, we have identified several epitopes recognized by T cell clones in association with different HLA-class II isotypes and alleles. In addition, binding analysis of these epitopes indicated that most of the peptides are able to bind to multiple allelic forms of class II molecules. Although there are important obstacles to malaria vaccine development we believe that, in the light of these results, unresponsiveness in humans, caused by MHC restriction, might not be a major constraint in development of a subunit vaccine.

Amino Acid Sequence↗
PubMed1990

A way round the 'real difficulties' of malaria sporozoite vaccine development?

Vaccines against the sporozoite stages of malaria, based on circumsporozoite (CS) protein epitopes, have had poor results in clinical trials. It has recently been suggested that attention should be switched to looking for new sporozoite, or hepatioc-stage proteins from which to develop vaccines. Here Francesco Sinigaglia and Richard Pink argue the case for reconsidering CS proteins as vaccines candidates.

Journal Article↗
PubMed1990

An invariant, "universal" T-cell epitope in the P. falciparum circumsporozoite protein.

Although there are important obstacles to malaria vaccine development, we believe they might be overcome by a strategy of searching for conserved regions of a vaccine candidate that are recognized in association with many different HLA molecules and, if necessary, deliberately modifying the conserved sequences to improve their immunogenicity for T cells. This approach is illustrated by work on the circumsporozoite (CS) protein of Plasmodium falciparum, which covers the surface of the malaria sporozoite and is the best characterized of current vaccine candidates.

Animals↗
PubMed1989

Fibronectin modulates the activation of human platelets.

In this paper we studied the effect of plasma fibronectin on platelet responsiveness to low doses of thrombin or ADP. Fibronectin causes: (i) a significant lowered cytoplasmic calcium movement in platelets activated both with low doses of thrombin and with ADP, (ii) a lowered decrease of the cAMP level induced by low thrombin, but not by ADP, (iii) a dramatic decrease of protein phosphorylation in low thrombin-treated platelets. The results reported here demonstrate that plasma fibronectin behaves like an inhibitor of mild activations of human platelets; its effect is probably mediated by the glycoprotein llb-llla complex in thrombin activated platelets, whereas a different and unknown mechanism must be supposed to explain the results obtained with ADP-activated platelets.

Adenosine Diphosphate↗
PubMed1989

The occupancy of glycoprotein IIb-IIIa complex modulates thrombin activation of human platelets.

Platelet membrane glycoprotein (GP IIb-IIIa), besides its activity as adhesive protein receptor, displays a number of properties supporting its involvement in the mechanisms of transduction of the activation signal. Recently we have observed that GP IIb-IIIa ligands, mostly fibrinogen, inhibit Ca2+ movement and cytoskeleton reorganization caused by mild platelet activation. These findings led us to investigate the effect of GP IIb-IIIa ligands on agonist-induced platelet responses, with particular attention to the two major messenger generating systems, involving the activation of phospholipase C and the inhibition of cAMP production. In this paper we demonstrate that the occupancy of the major adhesive protein receptor on the platelet surface modulates the phosphatidylinositol cycle decreasing the amount of IP3, IP2 and IP produced after mild platelet activation as well as the pattern of protein phosphorylation. The platelet cAMP content of activated platelets was also affected and kept higher when evaluated under the same experimental conditions. Our data provide evidence for a role of fibrinogen binding in regulating the degree of activation of circulating platelets.

Blood Platelets↗
PubMed1989

Requirements for screening recombinant DNA libraries for T cell epitope expression.

The possibility of screening cDNA expression libraries with T cell clones was investigated. The model system was based on human T cell clones specific for the recombinant malaria protein 190L, which was expressed fused to beta-galactosidase in lambda gt11. Several membranes were tested for their capacity to bind antigen and stimulate T cell proliferation. Pretreatment of membranes with DMSO and/or sonication to release the antigen improved the sensitivity of the assay. Under optimal conditions, T cell proliferation in response to antigen bound to a low protein binder membrane was comparable to that observed with the antigen in solution. A dot-blot type apparatus was designed for screening large numbers of plaques with T cells. The technical problems of this approach, its requirements and possible applications are discussed.

Clone Cells↗