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M Fabbi

Publications and source records attributed to M Fabbi.

52 records · Page 3Linked to original sources
PubMed1986

The sheep erythrocyte receptor and both alpha and beta chains of the human T-lymphocyte antigen receptor bind the mitogenic lectin (phytohaemagglutinin) from Phaseolus vulgaris.

We have studied the interaction of mitogenic lectins such as phytohaemagglutinin (PHA) and concanavalin A (Con A) with both surface molecules which, by the use of monoclonal antibodies, are known to trigger T-cell mitogenesis. Monoclonal antibodies recognizing the T-lymphocyte receptor for antigen (Ti) and/or its associated structure, CD3, activate T cells. More recently, a second pathway of activation has been described which involves the sheep erythrocyte binding glycoprotein CD2, a surface molecule distinct from Ti-CD3. Lysates from surface-iodinated T-leukaemia cell lines were treated with lectin and affinity purified anti-lectin antibodies coupled to protein A-Sepharose. We have shown that eluates from Con A/anti-Con A or PHA/anti-PHA immunoprecipitates contained Ti, since a rabbit anti-T alpha serum, which recognizes the native and denatured forms of the constant region of the alpha chain, immunoprecipitated Ti from these eluates. Furthermore, Ti immunoprecipitated by anti-T alpha serum from lysates of surface iodinated E+ lymphocytes was binding to PHA after elution from the immunoprecipitate. When the purified Ti molecule was reduced and alkylated, allowing the permanent dissociation of its alpha and beta subunits, PHA interacted with both chains, whereas anti-T alpha serum immunoprecipitated the alpha chain only. Altogether, these results demonstrate that PHA interacts with both chains of the T cell receptor for antigen on human peripheral T lymphocytes. With the HPB-ALL tumour line, a similar approach showed that both alpha and beta chains of Ti bind to Con A and Ulex europaeus 1 but not Helix pomatia. Affinity chromatography on immobilized lectins and immunoprecipitation with lectin/anti-lectin antibodies were employed to test whether CD2 binds to PHA and Con A. The results show that CD2 from human peripheral T lymphocytes binds both lectins but with a lower affinity for PHA than Con A.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1986

The ontogeny, structure and function of the human T lymphocyte receptor for antigen and major histocompatibility complex.

Recent studies using cloned antigen-specific T lymphocytes and monoclonal antibodies directed at their various surface glycoprotein components have led to identification of the human T cell antigen receptor as a surface complex comprised of a clonotypic 90 kDa Ti heterodimer and the invariant 20 and 25 kDa T3 molecules. Approximately 30,000-40,000 Ti and T3 molecules exist on the surface of human T lymphocytes. These glycoproteins are acquired and expressed during late thymic ontogeny, thus providing the structural basis for immunological competence. The alpha and beta subunits of Ti bear no precursor-product relationship to one another and are encoded by separate germline V, D, J and C segments which rearrange during intrathymic differentiation to form an active gene set. Triggering of the T3-Ti receptor complex induces a rapid increase in free cytoplasmic Ca2+ and gives rise to specific antigen-induced proliferation through an autocrine pathway involving endogenous interleukin-2 production, release and subsequent binding to interleukin-2 receptors. The implications of these findings for understanding of human T cell growth and its regulation in disease states are discussed.

Amino Acid Sequence↗
PubMed1985

Production and characterization of antibody probes directed at constant regions of the alpha and beta subunit of the human T cell receptor.

To generate antibodies directed at constant regions of the human T cell receptor, purified alpha and beta subunits of a human T cell antigen/major histocompatibility complex receptor from the REX tumor (Ti-REX) were isolated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and utilized to immunize rabbits. H36 (anti-alpha subunit) and H38 (anti-beta subunit) antisera were strongly reactive with the denatured subunits and also immunoprecipitated the Ti heterodimer from 125I surface-labeled lysates of REX, inducer, suppressor and cytotoxic T cell clones, peripheral T lymphocytes and thymocytes. Moreover, immunodepletion experiments showed that such antisera recognized antigenic determinant(s) shared by all Ti molecules expressed in the thymus. Several observations were made with these anticonstant region antibodies. First, peptide map analysis showed that the T cell receptor molecules recognized by the anti-clonotype and the anti-constant region heteroantisera on a given T cell clone are identical, thus supporting the view that the T cell receptor undergoes allelic exclusion. Second, since the individual antisera were weakly cross-reactive with the other denatured subunit, such subunits probably share conserved sequences. Third, the absence of antisera reactivity with intact cells implies that most of these constant region epitopes must be obscured by associated molecules, perhaps including one or more of the 20-25-kDa T3 subunits. Fourth, the extensive difference in two-dimensional peptide maps of Ti alpha subunits from clones of differing specificities makes it likely that the subunit contributes in a major way to antigen/major histocompatibility complex binding.

Antibody Formation↗
PubMed1985

The human T-cell receptor.

Recent studies using cloned antigen-specific T lymphocytes and monoclonal antibodies directed at their various surface glycoprotein components have led to the identification of the human T-cell antigen receptor as a surface complex comprised of a clonotypic 90-kD Ti heterodimer and the invariant 20- and 25-kD T3 molecules. Approximately 30,000-40,000 Ti and T3 molecules exist on the surface of human T lymphocytes. These glycoproteins are acquired and expressed during late thymic ontogeny, thus providing the structural basis for immunologic competence. The alpha and beta subunits of Ti bear no precursor-product relationship to one another and are encoded by separate genes. Moreover, the presence of unique peptides following proteolysis of different Ti molecules isolated by non-cross-reactive anticlonotypic monoclonal antibodies supports the notion that variable regions exist within both the alpha and the beta subunits. N-Terminal amino acid sequencing and molecular cloning of the Ti beta subunit further show that it bears an homology to the first V-region framework of immunoglobulin light chains and represents the product of a gene that rearranges specifically in T lymphocytes. Triggering of the T3-Ti receptor complex gives rise to specific antigen-induced proliferation through an autocrine pathway involving endogenous IL-2 production, release, and subsequent binding to IL-2 receptors. The implications of these findings for understanding human T-cell growth and its regulation in disease states are discussed.

Amino Acid Sequence↗
PubMed1984

The same human alloreactive T cell clone can help both B lymphocytes and specific cytotoxic precursors.

Human alloreactive proliferating T cell clones have been compared for their capacity to provide help for B cell activation and the generation of a specific cytotoxic response. The results demonstrate that, when triggered by the relevant alloantigen, the same T cell clone can induce a strong polyclonal B cell activation and serve as the only source of helper cells for the generation of a specific cytotoxic response by any source of CTL precursors against any stimulator cell present in culture.

B-Lymphocytes↗
PubMed1984

Genes encoding the Ti beta subunit of the antigen/MHC receptor undergo rearrangement during intrathymic ontogeny prior to surface T3-Ti expression.

To obtain further information about the ontogeny of the T-cell antigen/MHC receptor, a Ti beta subunit cDNA probe and heteroantisera specific for the Ti alpha and Ti beta subunits were utilized to characterize human T-lineage cells. Analysis of thymic tumors and normal thymocytes at both the DNA and protein levels demonstrates that Ti beta gene rearrangement is evident in stage II (T11+T6+T3-) and stage III (T11+T6-T3+), but not stage I (T11+T6-T3-) thymocytes. In contrast, surface expression of Ti alpha and Ti beta molecules is exclusively restricted to stage III thymocytes. Thus human T-lineage ontogeny is characterized by an orderly series of differentiation steps wherein Ti beta gene rearrangement precedes surface expression of the T3-Ti molecular complex.

Cloning, Molecular↗
PubMed1984

An alternative pathway of T-cell activation: a functional role for the 50 kd T11 sheep erythrocyte receptor protein.

A series of seven monoclonal antibodies was produced against the T-lineage-specific 50 kd T11 sheep erythrocyte rosette (SRBC) receptor protein in order to define the function of the molecule. Three distinct epitopes were detected: T11(1), the SRBC binding site expressed on all T lymphocytes and thymocytes; T11(2), an epitope unrelated to the SRBC binding site but with a similar distribution; and T11(3), a neo-epitope expressed only upon T-cell activation. Simultaneous triggering of T11(2) and T11(3) epitopes by monoclonal antibodies induces T lymphocytes to proliferate and mediate their functional programs in the absence of antigen and/or antigen-presenting cells. This antigen-independent mode of triggering is distinct from that involving the T3-Ti antigen receptor complex and represents an alternate pathway of T-cell activation. Given that T11 is the earliest T-lineage surface glycoprotein to appear in thymic ontogeny and is thus expressed before T3-Ti, the former may be involved in clonal expansion and/or differentiation during early development.

Animals↗
PubMed1984

Purification and NH2-terminal amino acid sequencing of the beta subunit of a human T-cell antigen receptor.

To obtain information about the structural basis for T-cell antigen recognition, a T3-associated Ti receptor molecule was isolated from crude membranes of the REX human thymic tumor line and purified by affinity chromatography with an anti- clonotypic monoclonal antibody in conjunction with preparative gel electrophoresis. NH2-terminal amino acid sequencing of the beta subunit unambiguously identified the amino acids in positions 2-12. Comparative protein sequence analysis by computer search demonstrated that this Ti beta sequence bore weak, but definite, homology to the first framework of the variable region of human lambda light chain. Anti-sera to a synthetic peptide corresponding to positions 2-11 precipitated the denatured Ti beta subunit from REX, thus confirming the above sequence. This information suggests that the Ti beta subunit is distantly related to human immunoglobulin lambda light chain and, moreover, should be of use in the molecular cloning of the Ti beta gene.

Amino Acid Sequence↗
PubMed1984

Clonotypic surface structure on human T lymphocytes: functional and biochemical analysis of the antigen receptor complex.

Recent studies using cloned antigen-specific T lymphocytes and monoclonal antibodies directed at their various surface glycoprotein components have led to identification of the human T cell antigen receptor as a surface complex comprised of a clonotypic 90KD Ti heterodimer and the monomorphic 20/25KD T3 molecules. Approximately 30,000-40,000 Ti and T3 molecules exist on the surface of human T lymphocytes. These glycoproteins are acquired and fully expressed during late thymic ontogeny, thus providing the structural basis for immunologic competence. The alpha and beta subunits of Ti bear no precursor-product relationship to one another and are encoded by separate genes. The presence of unique peptides following proteolysis of different Ti molecules isolated by noncrossreactive anticlonotypic monoclonal antibodies supports the notion that variable regions exist within both the alpha and beta subunits. Moreover, N-terminal amino acid sequencing of the Ti beta subunit shows that it bears homology to the first V-region framework of immunoglobulin light chains and represents the product of a gene that rearranges specifically in T lymphocytes. Soluble or Sepharose-bound anti-Ti monoclonal antibodies, like physiologic ligand (antigen/MHC), enhanced proliferative responses to purified IL-2 by inducing a 6-fold increase in surface IL-2 receptor expression. In contrast, only Sepharose-bound anti-Ti or physiologic ligand triggered endogenous clonal IL-2 production and resulted in subsequent proliferation. The latter was blocked by antibodies directed at either the IL-2 receptor or IL-2 itself. These results suggest that induction of IL-2 receptor expression but not IL-2 release occurs in the absence of T3-Ti receptor crosslinking. Perhaps more importantly, the findings demonstrate that antigen-induced proliferation is mediated through an autocrine pathway involving endogenous IL-2 production, release, and subsequent binding to IL-2 receptors.

Antibodies, Monoclonal↗
PubMed1984

Generation and characterization of murine monoclonal antibodies against HLA Class II molecules.

HLA Class II antigens (human la) are coded by Major Histocompatibility Complex and play important biological roles in health and disease. In this report we describe the generation and characterization of nine murine monoclonal antibodies (MoAbs) specific for determinants localized on the human la molecules. The reactivity of these MoAbs inferred from serological analysis along with the data obtained from biochemical characterization of the target structures allowed a classification of these reagents as monomorphic and polymorphic. Two monomorphic MoAbs, identifying different subsets of human la molecules, were studied in detail.

Animals↗
PubMed1983

A monoclonal antibody to platelet glycoproteins IIb and IIIa complex: its use in purifying human megakaryocytes from sternal bone marrow aspirates for immunofluorescence studies of Ia-like antigens.

A monoclonal antibody (PBM 6.4) to platelet and megakaryocyte glycoproteins IIb and IIIa has been obtained and used to purify human megakaryocytes from sternal bone marrow aspirates by a simple method, consisting of a Percoll gradient centrifugation followed by affinity adherence on PBM 6.4-coated plastic surface, "panning." Megakaryocytes, 80-90% pure and morphologically well preserved, were attached to poly-L-ornithine-coated multi-well microscope slides and immunofluorescence was done using monoclonal antibodies to human Ia-like antigens (DR, DC1). A higher proportion of DR-positive megakaryocytes was found, in comparison to the values reported by others, while DC1 antigen was detected on about 20% of megakaryocytes. The method described has the unique feature of enriching cells of the human megakaryocytic lineage from simple diagnostic sternal aspirates in an amount adequate for immunofluorescent and morphological analysis.

Animals↗
PubMed1981

Analysis of HLA-DR polymorphism by two-dimensional peptide mapping.

Two-dimensional peptide mapping was used to study the polymorphism of DR antigens, membrane glycoproteins composed of two chains, alpha and beta, and encoded by the human major histocompatibility complex (MHC). Four DR antigens were purified by immunoabsorption from four human lymphoblastoid cell lines homozygous at the DR locus. After labeling with 125I, alpha and beta chains were separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and digested with pepsin. Comparison of the peptide maps showed a marked degree of polymorphism among beta chains: only 43% of peptides were common to all four chains and 15-21% of the spots were unique to a given chain. By contrast, only a limited variability was observed among alpha chains. Homology was 75% for the four chains and the percentage of unique peptides was very low. DR7 did not possess even a single unique peptide. The limited variability among alpha chains and the lack of "private" peptides in one of them point to the conclusion that the beta chain is the unique carrier of the alloantigenic specificities. Higher homology within the known crossreactive groups was not observed, suggesting that the determinants responsible for crossreactivity are on different molecules. From a genetic point of view, because beta chains show allele-associated polymorphism, they are likely to be MHC encoded, whereas the minor differences among alpha chains do not allow a similar conclusion. The available data point to an analogy between these DR antigens and the mouse I-E/C antigens.

Alleles↗
PubMedDate not supplied

Homology of Ti alpha-subunit of a T-cell antigen-MHC receptor with immunoglobulin.

Human T-cell receptors for antigen and major histocompatibility complex (MHC) determinants have now been defined on inducer, suppressor, and class 1 and class 2 MHC-specific cytotoxic T lymphocytes as T3-associated clonotypic molecules (Ti) of relative molecular mass 90,000 (90K) composed of one 49-54K alpha- and one 43K beta-subunit which are disulphide-linked. In the case of the Ti beta-subunit, N-terminal amino acid sequencing and molecular cloning techniques led recently to identification of the Ti beta-gene and showed that T-specific V, D, J and C segments fuse to form an active beta-gene. So far, however, there have been little structural data available on the Ti alpha-subunit. Here we have derived the amino acid sequence of a portion of the Ti alpha-subunit by CNBr fragmentation. Sequence analysis reveals approximately 40% homology between the Ti alpha-subunit fragment and the third framework of the variable region of immunoglobulin light and heavy chains, supporting the notion that the Ti alpha-subunit is a member of the immunoglobulin-Ti beta-gene family.

Amino Acid Sequence↗
PubMedDate not supplied

Pvu II polymorphism of low density lipoprotein receptor gene and familial hypercholesterolemia. Study of Italians.

Familial hypercholesterolemia is a metabolic disorder inherited as an autosomal dominant trait characterized by an increased plasma low density lipoprotein (LDL) level. It has been demonstrated that the disease is caused by several different mutations in the LDL receptor gene. Although early identification of individuals carrying the defective gene could be useful in reducing the risk of atherosclerosis and myocardial infarction, the available techniques for determining the number of the functional LDL receptor molecules are not sufficiently accurate. The recent isolation of the LDL receptor gene now makes it possible to use restriction fragment length polymorphisms to study the inheritance of the defective allele in families with familial hypercholesterolemia. In the present study, we report the use of a Pvu II restriction fragment length polymorphism to follow the inheritance of familial hypercholesterolemia in a total of 79 patients from 37 different families. This restriction fragment length polymorphism allowed unequivocal diagnosis in 32.5% of the cases. Furthermore, in the Italians studied, the absence of a polymorphic Pvu II cutting site (P1 allele) was found to be strongly associated with familial hypercholesterolemia.

Adolescent↗