Biomedical subjects

M Zanetti

Publications and source records attributed to M Zanetti.

At least 145 records · Page 8Linked to original sources

PubMed1995

Major histocompatibility complex class I-restricted presentation of influenza virus nucleoprotein peptide by B lymphoma cells harboring an antibody gene antigenized with the virus peptide.

We analyzed the capacity of B cells to process and present a peptide from the variable region of an endogenous immunoglobulin heavy (H) chain to a major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocyte (CTL) clone. The H-chain gene was engineered to express 14-amino acid peptide from the sequence of the influenza virus nucleoprotein (NP) antigen in the third complementarity-determining region (CDR3). This NP peptide is presented in association with the Db allele in H-2b mice. We demonstrate that B lymphoma cells (H-2b) harboring the antigenized H-chain gene process and present the NP peptide in association with the Db molecule and are lysed by a CTL clone specific for that peptide in an MHC-restricted way. In contrast, the soluble antigenized antibody failed to mediate lysis of H-2b target cells. The endogenously processed immunoglobulin CDR3 peptide could be eluted from surface Db molecules in transfected cells. This study formally demonstrates that peptides from the hypervariable loops of endogenous immunoglobulin are processed through the endogenous degradative pathway and are presented to CD8+ T cells in the context of MHC class I molecules. The implication of these findings for processing and presentation of endogenous immunoglobulin peptides in B cells and network regulation by idiopeptides is discussed.

Amino Acid Sequence↗

PubMed1995

Engineered idiotypes. Immunochemical analysis of antigenized antibodies expressing a conformationally constrained Arg-Gly-Asp motif.

We report on the immunochemical characterization of two antibodies engineered to express RGD, a peptide from adhesive proteins of the extracellular matrix. One or three RGD motifs were introduced in the third complementarity-determining region (CDR) of a murine heavy (H) chain variable (V) region gene yielding two antibodies, gamma 1RGD and gamma 1(RGD)3. A murine monoclonal antibody (mAb) raised against an RGD-containing synthetic peptide bound in Western blot the H chain of both gamma 1RGD and gamma 1(RGD)3. Pronectin F, a genetically-engineered polymer containing RGD, abrogated this binding. Anti-idiotypic antibodies against the (RGD)3 loop were generated in a rabbit by immunization with gamma 1(RGD)3. Anti-idiotype antibodies purified by affinity-chromatography on the synthetic peptide GRGDSPC reacted in ELISA with gamma 1(RGD)3 and human fibronectin. Adhesive proteins, unlike RGD-containing synthetic peptides, were able to interfere with the interaction between gamma 1(RGD)3 and the anti-idiotypic antibodies. These results suggest that it is possible to genetically engineer the hypervariable loops of immunoglobulins and confer them new idiotypic characteristics. These results support the concept of antibody mimicry.

Amino Acid Sequence↗

PubMed1995

Human recombinant CD4 and CD4-derived synthetic peptides agglutinate immunoglobulin-coated latex particles. Evidence that residues 25-28 and 35-38 of human CD4 form two separate immunoglobulin binding sites.

It has been shown previously that amino acid residues 21-49 of the first extracellular domain of human CD4 form the core of an immunoglobulin (Ig) binding site. Synthetic peptides of human CD4 that encompass this region also bind Ig and, with higher affinity, antigen/antibody complexes. Synthetic peptides also enhance binding of both monomeric and aggregated Ig to monocytic U937 cells and Staphylococcus aureus Protein A. To better characterize the nature of the Ig binding site on CD4, we tested the ability of human recombinant CD4 (rCD4) to agglutinate polystyrene particles coated with Ig. Evidence is presented that soluble rCD4 and CD4 peptide p21-49 were capable of specific agglutination of polystyrene particles coated with polyclonal Ig of either human or sheep origin. Agglutination could be blocked by soluble human polyclonal IgG or F(ab')2 fragments. Both heparin and sulfated dextrans also inhibit agglutination, suggesting that charged residues on rCD4 played an important role in agglutination mediated by rCD4 or CD4 peptide. Similarly, aurintricarboxylic acid (ATA) also blocked agglutination of Ig-coated particles by rCD4. Agglutination mapping studies performed using truncated peptides revealed the existence of two discrete, closely related Ig binding sites (residues 25-28 and 35-38).

Amino Acid Sequence↗

PubMed1995

Fast spin-echo inversion-recovery imaging versus fast T2-weighted spin-echo imaging in bone marrow abnormalities.

RATIONALE AND OBJECTIVES: The purpose of this investigation is to compare a fat-suppressed T2-weighted fast spin-echo (FSE) sequence in bone marrow abnormalities with an FSE STIR sequence that recently has become commercially available. METHODS: Fast spin-echo images (repetition time [TR], 3500-5000 mseconds; echo time [TE], 96-114 mseconds) and FSE STIR images (TR, 3000-5000 mseconds; TE, 32-40 mseconds; inversion time [TI], 140-150 mseconds) were compared quantitatively and qualitatively calculating signal-to-noise ratios (SNRs), contrast-to-noise ratios (CNRs), and lesion conspicuity and using a qualitative scoring system. RESULTS: Signal-to-noise ratio (mean +/- standard deviation) was 36.4 +/- 19.3 for the FSE and 29.0 +/- 15.9 for the FSE STIR images (P = .002). Contrast-to-noise ratio (mean +/- standard deviation) was 18.7 +/- 14.3 for the FSE and 20.3 +/- 16.0 for the FSE STIR images (P = .45). Lesion conspicuity (mean +/- standard deviation) was 1.7 +/- 1.5 for the FSE and 3.5 +/- 4.0 for the FSE STIR images (P = .025). The most important difference in the qualitative evaluation related to the better signal homogeneity on the FSE STIR images. CONCLUSIONS: Fast spin-echo STIR images may be preferable to FSE images with fat suppression due to better image homogeneity and lesion conspicuity.

Adolescent↗

PubMed1995

A model of skeletal muscle leucine kinetics measured across the human forearm.

We propose a new six-compartment model of intracellular muscle kinetics of leucine and of its transamination product alpha-ketoisocaproic acid (KIC) by combining systemic tracer infusions of [14C]- and [15N]leucine with the arterial-deep venous catheterization of the human forearm. Venous [14C]KIC specific activity (SA) is taken as representative of intracellular [14C]leucine SA, whereas net [15N]leucine disposal is used to calculate leucine inflow and outflow across forearm cell membrane(s). In post-absorptive normal subjects, model-derived rates of intracellular leucine release from and incorporation into protein were approximately 32% (P = 0.03) and approximately 37% greater (P = 0.025), respectively, than those calculated using a conventional arteriovenous approach. Forearm fasting proteolysis exceeded protein synthesis (P < 0.025), whereas leucine oxidation was greater than zero (P < 0.01), suggesting a net negative leucine (i.e., protein) balance. Leucine inflow from blood to cell represented approximately 30% of arterial leucine delivery; therefore approximately 70% of arterial leucine bypassed intracellular metabolism. This model provides a comprehensive description of regional leucine and KIC kinetics and new estimates of protein degradation and synthesis across the human forearm.

Adult↗

PubMed1995

[Ultrasonography and magnetic resonance tomography (MRI) of tendon injuries].

Both ultrasound and MR imaging have become increasingly important for the assessment of tendon abnormalities. Ultrasound is widely available and is mainly suitable for emergency situations in traumatology and as a screening method. For soft tissue imaging only up-to-date sonographic equipment should be used. Moreover, the examiner's experience is crucial for adequate results. MR imaging is not as easily available. However, it is more reproducible and may be indicated in a preoperative situation. Most publications about sonography and MR imaging deal with the rotator cuff and the Achilles tendon. Fewer investigations relate to the posterior tibial tendon, the extensors of the knee and the proximal and distal biceps tendon. The role of ultrasound and MR imaging in abnormalities of these structures is discussed based on the literature and our own experience.

Achilles Tendon↗

PubMed1994

[Magnetic resonance tomography of the hand: current status].

Magnetic resonance imaging (MRI) is an established imaging method in abnormalities of the wrist. Inherent advantages of this imaging method such as the lack of ionizing irradiation, the free choice of imaging planes and its excellent contrast resolution have been enhanced by the introduction of surface coils and new sequences. These developments result in an improved spatial resolution, allowing imaging of small structures such as the triangular fibrocartilage. There is a number of established indications for MRI of the wrist, e.g. diagnosis of avascular necrosis, follow-up after carpal tunnel surgery and imaging of soft tissue masses. Increasingly, MRI can be used for the diagnosis of intercarpal ligament tears as well. The diagnostic capabilities of MRI of the wrist are discussed, based on personal experience and a review of the literature.

Carpal Tunnel Syndrome↗

PubMed1994

Molecular cloning of Bac7, a proline- and arginine-rich antimicrobial peptide from bovine neutrophils.

Bac7 is a 7 kDa proline- and arginine-rich antimicrobial peptide which was purified from bovine neutrophils. We have used PCR to clone the cDNA of Bac7 precursor, a polypeptide of 21,569 Da. This cDNA is highly conserved in the 5' region, with respect to the corresponding region in the precursors of several other structurally unrelated myeloid antimicrobial peptides. Furthermore, a 148 nt non-coding region at the 3' end is 75% homologous to a corresponding region of the cDNA of the precursor of PR-39, a porcine antibacterial peptide which is also proline- and arginine-rich.

Amino Acid Sequence↗

PubMed1994

Molecular cloning and chemical synthesis of a novel antibacterial peptide derived from pig myeloid cells.

A group of myeloid precursors of defense peptides has recently been shown to have highly homologous N-terminal regions. Using a strategy based on this homology, a novel cDNA was cloned from pig bone marrow RNA and found to encode a 153-residue polypeptide. This comprises a highly conserved region encompassing a 29-residue signal peptide and a 101-residue prosequence, followed by a unique, 23-residue, cationic, C-terminal sequence. A peptide corresponding to this C-terminal sequence was chemically synthesized and shown to exert antimicrobial activity against both Gram positive and negative bacteria at concentrations of 2-16 microM. The activity of this potent and structurally novel antibacterial peptide appears to be mediated by its ability to damage bacterial membranes, as shown by the rapid permeabilization of the inner membrane of Escherichia coli.

Amino Acid Sequence↗

PubMed1994

Chemical synthesis and biological activity of a novel antibacterial peptide deduced from a pig myeloid cDNA.

Several myeloid precursors of antibacterial peptides have recently been shown to share homologous pre- and pro-regions. Taking advantage of this homology, a novel cDNA was cloned from pig bone marrow RNA. This encodes a 166-residue polypeptide with highly conserved pre- (29 residues) and pro- (101 residues) sequences, followed by a unique, 36-residue C-terminal sequence. Structure analyses of this C-terminal region have identified a highly cationic sequence predicted to adopt an amphipathic alpha-helical conformation. A peptide corresponding to this sequence was chemically synthesized and shown to arrest the growth of both Gram-positive and Gram-negative bacteria. At least for Escherichia coli, the activity of this peptide appears to be mediated by its ability to permeabilize the bacterial membranes.

Amino Acid Sequence↗

PubMed1994

Synthetic peptides of human CD4 enhance binding of immunoglobulins to monocyte/macrophage cells. II. Mechanisms of enhancement.

We have previously shown that a synthetic peptide corresponding to amino acid residues 21-49 of the first extracellular domain of human CD4 binds immunoglobulins (Ig) and antibody: antigen (Ab:Ag) complexes, and greatly enhances the uptake of aggregated Ig by monocyte/macrophage U937 cells. In this report, we investigated the mechanisms of enhanced uptake, and the contribution of different receptors present on the surface of monocyte/macrophage cells to this phenomenon. Our results indicate that both Fc receptor (FcR) and cell surface CD4 participate in the enhanced uptake of Ig promoted by the synthetic peptide of CD4. The involvement of these two receptors was demonstrated in experiments using monoclonal antibodies to FcR and CD4, as well as monosialoganglioside GM1, a substance known to modulate surface CD4. The participation of CD4 was further confirmed using the CD4 monocyte/macrophage cell line MM-6. Together, the results of these experiments indicate that surface CD4 may cooperate with FcR in handling aggregated Ig and Ab:Ag complexes. The implications of these findings for immunoregulation by Ab:Ag and idiotype:anti-idiotype (Id:anti-Id) complexes, and infection of macrophages by HIV, are discussed.

Antigen-Antibody Complex↗

PubMed1994

Production and characterization of cross-reactive monoclonal antibodies to sweet proteins.

We report on the characterization of a panel of eight murine monoclonal antibodies induced by immunization with the sweet protein monellin and which display cross-reactivity with thaumatin, another sweet natural molecule. Cross-reactivity was determined based on direct binding assays and antigen competition experiments. Interestingly, half of antibodies bound thaumatin more than monellin, the immunizing antigen. The relatedness between the binding sites of the eight monoclonal antibodies was assessed by cross-competition of the binding of monellin to individual monoclonal antibodies. Antibodies that bound in a similar way monellin and thaumatin were on average the best inhibitors. All antibodies also bound Sp1, a genetically-engineered single chain monellin. These studies demonstrate that the immune repertoire encodes for specificities that are common to monellin and thaumatin despite the fact that the two molecules share little, if any, structural homology. These results are discussed in relation to the use of cross-reactive antibodies to generate anti-receptor antibodies via idiotypic immunization.

Animals↗

PubMed1994

Antibodies specific for the neuronal form of the Src protein elicited by an antigenized antibody.

To elicit antibodies directed specifically against the neuron-specific form of the c-src gene product, pp60c-src(+), we used an antigenized antibody comprising a decamer containing the amino acid sequence specific to pp60c-src(+) inserted into the third hypervariable loop of the heavy (H)-chain variable (V)-region. This was used to raise anti-idiotype antibodies reacting with the peptide epitope in rabbits. The antisera reacted with pp60c-src(+), as judged by immune blotting, immunoprecipitation, immune complex kinase assay, and indirect immunofluorescence staining, but did not react with the fibroblast form of the c-src gene product, pp60c-src. Antigenized antibody is a useful approach for producing antibodies able to distinguish between isoforms of the same gene product and specific for the neuronal form of the Src protein.

3T3 Cells↗

PubMed1994

Fasting and postprandial phenylalanine and leucine kinetics in liver cirrhosis.

To investigate body protein turnover and the pathogenesis of increased concentration of plasma phenylalanine in liver cirrhosis, we have studied phenylalanine and leucine kinetics in cirrhotic (diabetic and nondiabetic) patients, and in normal subjects, both in the postabsorptive state and during a mixed meal, using combined intravenous and oral isotope infusions. Postabsorptive phenylalanine concentration and whole body rate of appearance (Ra) were approximately 40% greater (P < 0.05) in patients than in controls. Leucine concentrations were comparable, but intracellular leucine Ra was also increased (P < 0.05), suggesting increased whole body protein breakdown. Postprandial phenylalanine Ra was also greater (P < 0.05) in the patients. This difference was due to a diminished fractional splanchnic uptake of the dietary phenylalanine (approximately 40% lower in the cirrhotics vs. controls, P < or = 0.05). Postprandial leucine Ra was also increased in the patients, but splanchnic uptake of dietary leucine was normal. Thus both increased body protein breakdown and decreased splanchnic extraction of dietary phenylalanine can account for the increased phenylalanine concentrations in liver cirrhosis.

Adult↗

PubMed1993

Active immunity against the CD4 receptor by using an antibody antigenized with residues 41-55 of the first extracellular domain.

Using the process of "antibody antigenization," we engineered two antibody molecules carrying in the third complementarity-determining region of the heavy chain variable domain a 7-mer or a 15-mer peptide epitope of the first extracellular domain (D1) of human CD4 receptor--namely, Ser-Phe-Leu-Thr-Lys-Gly-Pro-Ser (SFLTKGPS; positions 42 through 49) and Gly-Ser-Phe-Leu-Thr-Lys-Gly-Pro-Ser-Lys-Leu-Asn-Asp-Arg-Ala (GSFLTKGPSKLNDRA; positions 41 through 55). These amino acid sequences are contained in the consensus binding site for the human immunodeficiency virus (HIV) on CD4 receptor. Both antigenized antibodies (AgAbs) bound recombinant gp120 and were recognized by a prototype monoclonal antibody to CD4 whose binding site is within amino acid residues 41-55. AgAbs were then used as immunogens in rabbits and mice to elicit a humoral response against CD4. Only the AgAb carrying the sequence 41GSFLTKGPSKLN-DRA55 induced a response against CD4. The induced antibodies showed specificity for the amino acid sequence of CD4 engineered in the AgAb molecule, were able to inhibit the formation of syncytia between human CD4+ T cells MOLT-3 and 8E5 (T cells that are constitutively infected with HIV), and stained human CD4+ CEM T cells. Four murine monoclonal antibodies were used to analyze the relationship between syncytia inhibition and CD4 binding at the single antibody level, and indicated that recognition of native CD4 is not an absolute requirement for inhibition of syncytia. This study demonstrates that antigenized antibodies can be used as immunogens to elicit site-specific and biologically active immunity to CD4. The importance of this approach as a general way to induce anti-receptor immunity and as a possible new measure to immunointervention in HIV infection is discussed.

Amino Acid Sequence↗

PubMed1993

A cDNA derived from pig bone marrow cells predicts a sequence identical to the intestinal antibacterial peptide PR-39.

We have recently shown that a group of antimicrobial peptides of bovine neutrophils share highly identical pro-sequences. In this paper we report the cDNA sequence of a 172 amino acid residue pig myeloid protein showing a similar pro-sequence of 101 residues. The carboxyl-moiety of the predicted protein is identical to the mature form of the proline- and arginine-rich antibacterial peptide named PR-39, isolated from pig intestine.

Amino Acid Sequence↗

PubMed1993

A novel cDNA sequence encoding a pig leukocyte antimicrobial peptide with a cathelin-like pro-sequence.

It has recently been shown that the precursors of various structurally unrelated leukocyte antimicrobial peptides share similar pro-regions. These, in turn, are highly identical to a cysteine proteinase inhibitor named cathelin, or PLCPI. In this paper we report a novel cDNA sequence of porcine bone marrow origin, encoding a protein characterized by a cathelin-like domain. The putative protein is 147 amino acid residue long, with a calculated mass of 16479 Da and appears to be the precursor of a recently isolated antimicrobial peptide named protegrin PG-2. The unique sequence of the mature PG-2 is located at the C-terminus of the precursor. Similar to the previously reported precursors, both the signal peptide and the pro-sequence of pre-proPG-2 appear highly conserved.

Amino Acid Sequence↗

PubMed1993

Chemotactic and protease-inhibiting activities of antibiotic peptide precursors.

We have recently shown that two antimicrobial peptides (Bac5 and Bac7) and/or their immature forms (proBac5 and proBac7) can be released extracellularly from activated neutrophils. In the present study we have investigated the biological activities of the immature forms, which do not exhibit antimicrobial effects. We show that proBac7 is a monocyte-selective chemoattractant, potentially contributing to the recruitment of these cells to infection sites, whereas proBac5 efficiently inhibits the in vitro activity of cathepsin L, a cysteine proteinase thought to contribute to tissue injury in inflammation.

Animals↗