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M Zanetti

Publications and source records attributed to M Zanetti.

At least 163 records · Page 9Linked to original sources
PubMed1993

The cDNA of the neutrophil antibiotic Bac5 predicts a pro-sequence homologous to a cysteine proteinase inhibitor that is common to other neutrophil antibiotics.

Bac5 is a 5-kDa proline- and arginine-rich antibiotic, stored as inactive precursor (proBac5) in the large granules of bovine neutrophils. A full-length cDNA encoding the precursor form of Bac5 has been cloned. The encoded protein (pre-proBac5) has a calculated mass of 20,031 Da and a pI of 9.21. This comprises a putative signal peptide of 29 amino acid residues and a 101-residue pro-sequence that precede the mature antibiotic. The pro-sequence is acidic and may neutralize the highly cationic Bac5, thus accounting for the inactivation of the antibiotic activity observed in in vitro experiments. The structure of mature Bac5 agrees closely with the amino acid sequence previously determined, with an additional tripeptide tail predicting carboxyl-terminal amidation. A valyl residue is deduced at the cleavage site for the proteolytic maturation of proBac5, consistent with a previous observation showing elastase as the enzyme involved in this processing step. The region upstream of Bac5 reveals high identity to corresponding regions of two neutrophil antimicrobial polypeptides, CAP18 from rabbit and bovine indolicidin. The COOH-terminal sequences of these antibiotics are completely unrelated. The proregion also exhibits remarkable similarity to pig cathelin, an inhibitor of cathepsin L, indicating a common evolutionary origin.

Amino Acid Sequence↗
PubMed1993

Expression of conformationally constrained adhesion peptide in an antibody CDR loop and inhibition of natural killer cell cytotoxic activity by an antibody antigenized with the RGD motif.

We report that an antibody engineered to express three Arg-Gly-Asp (RGD) repeats in the third complementarity-determining region of the heavy chain (antigenized antibody) efficiently inhibits the lysis of human erythroleukemia K-562 cells by natural killer (NK) cells. Synthetic peptides containing RGD did not inhibit. Inhibition was specific for the (RGD)3-containing loop and required simultaneous occupancy of the Fc receptor (CD16) on effector cells. The antigenized antibody inhibited other forms of cytotoxicity mediated by NK cells but not cytotoxicity mediated by major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL). A three-dimensional model of the engineered antibody loop shows the structure and physicochemical characteristics probably required for the ligand activity. The results indicate that an RGD motif is involved in the productive interaction between NK and target cells. Moreover, they show that peptide expression in the hypervariable loops of an antibody molecule is an efficient procedure for stabilizing oligopeptides within a limited spectrum of tertiary structures. This is a new approach towards imparting ligand properties to antibody molecules and can be used to study the biological function and specificity of short peptide motifs, including those involved in cell adhesion.

Amino Acid Sequence↗
PubMed1993

Idiotypes of pre-existing human anti-carcinoma anti-T and anti-Tn antibodies.

All humans normally possess antibodies, predominantly IgM, that react specifically with the Thomsen-Friedenreich (T) and the Tn antigens which are present in immunoreactive form on > 85% of all human carcinomas, but not in healthy and otherwise diseased tissues. We report here a serological study of idiotype expression and antigen reactivity of the anti-T and anti-Tn antibodies. Idiotypy was analyzed with rabbit antibodies raised against, and made specific for, affinity-purified polyclonal anti-T and anti-Tn antibodies from blood group A1B healthy adult donors. Anti-T and anti-Tn antibodies cross-reacted idiotypically in spite of their distinct epitope specificities. By adsorbing anti-T antibodies on insolubilized synthetic T carbohydrate we could firmly link idiotype expression with antigen reactivity. The relation of idiotype expression to the antigen-binding site of plant seed lectins was also studied; one originated from Arachis hypogaea [peanut agglutinin (PNA)], the other from Artocarpus integrifolia (Jacalin). PNA inhibited only anti-T antibodies. Jacalin inhibited both anti-T and anti-Tn antibodies in a dose-dependent manner. Neither idiotypic nor anti-idiotypic antibodies diminished the binding of lectins to T and Tn epitopes. The shared idiotypes on natural anti-T and anti-Tn antibodies permit consideration of application of their anti-idiotypes in treatment and/or prevention of human carcinoma.

Adult↗
PubMed1993

Analysis of human antitopoisomerase-I idiotypes.

Antibodies to topoisomerase-I are present in approximately 26% of patients with scleroderma and are rarely found in patients with other diseases. In the current study, the expression of the antitopoisomerase-I (antitopo-I) idiotype from two scleroderma patients (E.M. and S.G.) and from a healthy individual (N.M.) were studied. Idiotype EM-SCL was restricted to the three classes of antitopo-I, whereas idiotypes SG-SCL and NM were found in all classes of antitopo-I as well as in their non-antitopo-I Igs. Sera from 9 of 10 antitopo-I-positive unrelated scleroderma patients expressed idiotype SG-SCL and some also expressed idiotype NM. Sera from N.M.'s 3 daughters and from 7 of 18 nonrelated normals expressed idiotype NM in the three immunoglobulin classes of non-antitopo-I. Two of the antitopo-I antibodies expressed a cross-reacting idiotype (CRI) that is present in non-antitopo-I antibodies from the same donor. Contrary to the natural CRI, SG-SCL's CRI is closely associated with the antigen binding site. Antitopo-I idiotypes are on the heavy chains. Like many other autoantibodies, Id-SG-SCL use VH4.2-1, DXP1, and JH4 in germline configuration.

Aged↗
PubMed1993

Antigenicity and immunogenicity of antigenized antibodies. Studies on B and T cells.

This laboratory has been testing the possibility of using the complementarity-determining region (CDR) loops of the antibody molecule to express oligopeptide epitopes in an immunologically-accessible and conformationally-suitable way. The new process consists in grafting peptides epitopes derived from antigens other than immunoglobulins into antibody CDR loops. This process, "antibody antigenization," utilizes the immunoglobulin fold as a scaffold to immobilize and present oligopeptide epitopes to the immune system as the integral part of the immunoglobulin molecule. Here we describe some of the initial results with antigenized antibodies (AgAbs).

Amino Acid Sequence↗
PubMed1992

cDNA sequence analysis of an antibiotic dodecapeptide from neutrophils.

The full-length cDNA of a neutrophil antibiotic dodecapeptide has been cloned by reverse transcription/PCR from bovine bone marrow RNA. This peptide was originally isolated from bovine neutrophils, and shown to exert a potent antimicrobial activity in vitro on both Escherichia coli and Staphylococcus aureus. The cDNA codes for a polypeptide of 155 amino acid residues with a predicted mass of 17,629 Da and a pI of 8.03. The deduced sequence comprises a putative signal peptide of 29 amino acids, a 114 residue pro-region, and a carboxy-terminal dodecapeptide corresponding to the mature antibiotic. The pro-sequence displays extensive identity to corresponding regions of other structurally unrelated antibiotic peptides of bovine neutrophils recently cloned.

Amino Acid Sequence↗
PubMed1992

cDNA cloning of the neutrophil bactericidal peptide indolicidin.

A structurally novel, tryptophan-rich antimicrobial tridecapeptide amide, named indolicidin, has recently been purified from bovine neutrophils (Selsted et al. (1992) J. Biol. Chem. 267, 4292-4295). Here we describe the molecular cloning of this endoantibiotic, which is synthesised in bone marrow cells as a 144 amino acid residue precursor. The encoded protein has a predicted mass of 16479 Da and a pI of 6.51. A putative signal peptide of 29 amino acids precedes a 101 residue pro-region. The mature peptide is at the 3' end of the open reading frame. A glycine, not found in purified indolicidin, is present at the carboxyl terminus of the deduced sequence and is very likely involved in post-translational peptide amidation.

Amino Acid Sequence↗
PubMed1992

Synthetic peptides of human CD4 enhance binding of Ig to monocyte/macrophage cells. I. Characterization and mapping studies.

Human T cell glycoprotein CD4 binds to class II MHC molecules and to HIV envelope protein gp120. We have shown that CD4 and synthetic peptides corresponding to amino acid residues 21-49 of the first extracellular domain of CD4, also bind Ig and, with greater avidity, antibody:Ag complex. We investigated the effect of CD4 synthetic peptides on the binding and uptake of human Ig by monocyte/macrophage U937 cells. We found that a synthetic peptide corresponding to amino acid residues 21-49 enhanced binding to U937 cells of both aggregated and nonaggregated Ig. The enhancement was concentration dependent, occurred both in normal and low ionic strength conditions, and varied with the time and the temperature of the preincubation step. The enhancement was maximal after preincubation for 3 h at 37 degrees C. A peptide concentration of 20 micrograms/ml was sufficient for optimal binding of both nonaggregated and aggregated Ig. CD4 peptide 21-49 also enhanced binding of Ig to Staphylococcus aureus protein A. These studies open a new perspective in the way monocyte/macrophage cells handle Ig, antibody:Ag or Id:anti-Id complex, in particular when present at threshold amounts in a nonprecipitating form.

Amino Acid Sequence↗
PubMed1992

Antigenized antibodies.

A new process, antigenization of antibodies, consisting of the expression of oligopeptides in the hypervariable loops of an antibody molecule is described. The potential applications of antigenized antibodies are discussed.

Animals↗
PubMed1992

A method to analyze the interaction between gp120 of human immunodeficiency virus and CD4.

The study presents a new in vitro method to investigate the interaction between the glycoprotein (gp)120 of human immunodeficiency virus (HIV) and its receptor, CD4. The method is based on the binding of soluble recombinant CD4 to a human T cell line, 8E5, which constitutively expresses gp120 at its surface as a result of infection with HIV (LAV) and lacks reverse transcriptase activity. The binding of CD4 to gp120 on the cell surface is revealed by immunofluorescence using a murine monoclonal antibody to CD4. Binding can be inhibited by different substances like dextran sulfate, heparin, pentosan polysulfate, but not Leu3a. The reasons for this discrepancy are discussed. We propose this assay as a simple, reproducible, and rapid new method to screen new, pharmacological inhibitors of the gp120/CD4 interaction.

CD4 Antigens↗
PubMed1992

Ontogeny of the immune system and the invisible frontier to immune regulation.

The data presented focus on three topics: 1) Self-reactivity of early B cells as a constitutive feature of the immune system; 2) Self-reactive V regions and their possible involvement in immune regulation; and 3) Autoantibodies directed at T cell surface molecules as a new form of direct regulation of the B cell repertoire on the T cell compartment. Evidence is provided for lack of substantial difference in the reactivity of neonatal hybridomas from normal and autoimmune mice, and the proposal is made that the immune systems of normal and autoimmune neonatal mice start with similar characteristics implying that avoidance of autoimmune disease is matter of active regulation through a process learned in ontogeny. Two general possibilities for immune regulation are discussed. One is based on the V regions of self-reactive antibodies and their antigenic determinants. The other is through natural autoantibodies able to interfere with the state of activation of T cells. It is concluded that the role of highly conserved structures like self antigens is to maintain immunoglobulin genes and favor their expression in the incipient immune system so that simple patterns of regulation can be set in motion and made available.

Animals↗
PubMed1991

Stimulus-induced maturation of probactenecins, precursors of neutrophil antimicrobial polypeptides.

The antimicrobial polypeptides Bac7 and Bac5 (bactenecins) are stored in the large granules of bovine neutrophils as precursor forms, or probactenecins. Maturation of probactenecins has been investigated by studying the effects of stimulus-induced degranulation on this process. Stimulation of neutrophils with PMA, which is a secretagogue for specific and large granules but not for azurophils, induces a substantial discharge of uncleaved probactenecins in the extracellular medium, as revealed by Western blot analysis. When neutrophils are exposed to opsonized bacteria in the presence of cytochalasin B, resulting in exocytosis of the content of azurophils in addition to that of specific and large granules, probactenecins are secreted and rapidly converted into the corresponding mature antimicrobial peptides. Such a conversion is prevented if serine proteases, stored in the azurophils, are inhibited by pretreatment of neutrophils with PMSF. Phagocytosis, while causing a rapid discharge of the contents of azurophil and of the large granules into phagocytic vacuoles, as indicated by immunogold electron microscopy, also induces cleavage of probactenecins into mature peptides, as revealed by Western blot analysis. We conclude that the final processing of the storage forms of bactenecins arises from their interaction with the serine protease(s) of azurophils during bacteria-induced degranulation of neutrophils.

Animals↗
PubMed1991

Immunogenicity of an engineered internal image antibody.

We engineered an antibody expressing in the third complementarity-determining region of its heavy chain variable region a "foreign" epitope, the repetitive tetrapeptide Asn-Ala-Asn-Pro (NANP) of the circumsporozoite protein of Plasmodium falciparum parasite, one of the etiologic agents of malaria in humans. A monoclonal antibody to P. falciparum specific for the (NANP)n amino acid sequence bound to the engineered antibody, and a synthetic (NANP)3 peptide blocked this interaction. Immunization of rabbits and mice with the engineered antibody resulted in the elicitation of a humoral response to (NANP)3 synthetic peptide and P. falciparum parasite. In mice, in which immunity to the (NANP)n epitope is highly restricted by immune response genes, antibodies were induced in responder and nonresponder haplotypes of the major histocompatibility complex. Rabbit antibodies efficiently inhibited the in vitro invasion of cultured liver cells by P. falciparum parasite. Collectively, this study indicates that immunity to malaria in the absence of the parasite can be induced using antibody variable regions engineered to mimic the parasite's molecular structure. In general terms, the results suggest that antibody (idiotype) mimicry of an exogenous antigen is possible and may only require a discrete stretch of identity between the two molecules. The implication for the preparation of antibody-based vaccines and idiotype regulation of immunity are discussed.

Amino Acid Sequence↗
PubMed1991

CD4/immunoglobulin interaction: implications for immune physiology and autoimmunity.

CD4 has an important role in T cell activation events that depend on its binding to non-polymorphic MHC class II determinants on antigen-presenting cells. Here, we provide evidence that CD4 also interacts with immunoglobulins (Ig). The Ig-binding region lies within residues 21-49 of V1 domain of CD4. Immunochemical studies suggest that this property of CD4 does not depend on the three-dimensional folding of the CD4 molecule. Synthetic peptides (p) encompassing amino acid residues 16-49 and 21-49 of CD4 bind immunoglobulins in comparable way to the intact molecule. In vitro p 16-49 enhances significantly idiotype/anti-idiotype and some weak antigen-antibody interactions. Antigen antibody complexes formed in antigen excess bind CD4 peptides with much higher avidity then non-complexed antibodies. The possible role of the CD4/Ig interaction in T-B cell cooperation is discussed.

Animals↗
PubMed1991

Idiotypic analysis of human anti-topoisomerase I autoantibodies.

Anti-topoisomerase I autoantibodies (anti-topo I) are associated with proximal scleroderma and are of prognostic significance in patients with Raynaud's phenomenon. Polyclonal anti-idiotypic sera were raised against affinity-purified anti-topo I from 2 patients with scleroderma (EM, SG) and 1 healthy individual (NM). All 3 anti-topo I preparations expressed immunodominant private Ids in or near the antigen binding site of the autoantibody. Further analysis of Id-EM showed isotypic restriction to IgG and a stable Id-expression over the course of 9 years. Id-SG and Id-NM were expressed on IgG and on IgA. The idiotypic character of anti-topo I closely resembles that of anti-centromere autoantibodies which are associated with the CREST syndrome of scleroderma. The data suggest an antigen-driven process in the origin of autoantibodies in scleroderma.

Antigens↗
PubMed1991

Idiotypic analysis of human anticentromere autoantibodies.

The idiotypes (Ids) of anticentromere antibodies (ACA) have been studied using a fusion protein obtained from cloned cDNA of the major centromere antigen, CENP-B, for isolation of the autoantibodies. IgG-ACA were affinity purified from 4 patient sera and anti-Ids prepared in rabbits. Analysis revealed the existence of two distinct types of immunodominant Ids. One Id is near the antibody combining site and one is framework associated. A longterm longitudinal study of Id expression in a patient who seroconverted from ACA (-) to ACA (+) when she developed Raynaud's phenomenon showed a close correlation between Id expression and ACA titers (r = 0.94). These results may be interpreted as evidence for an autoantigen driven process in the anticentromere immune response.

Adult↗