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A Roseto

Publications and source records attributed to A Roseto.

At least 19 recordsLinked to original sources
PubMed2000

Production and functional characterization of two mouse/human chimeric antibodies with specificity for the tumor-associated Tn-antigen.

In this work, we have constructed two functional mouse/human chimeric antibodies (IgMkappa and IgG1kappa isotypes) by inserting genomic DNA fragments encoding VH and Vkappa variable regions of the murine monoclonal antibody IgMK-83D4 into mammalian expression vectors containing human mu, gamma1, and kappa constant exons, and by transfecting them into the nonsecreting mouse myeloma X-63 cell line. In previous works, we have demonstrated that 83D4 murine mAb reacts with Tn determinant (GalNAcalpha-O-Ser/Thr) expressed in 90% of breast, ovary, and colon carcinomas. Both expressed chimeric antibodies were purified from the transfected cell line supernatant by affinity chromatography, and their reactivities against Tn antigen were confirmed by ELISA on asialo ovine submaxilar mucin and immunofluorescence studies on MCF-7 breast carcinoma cell line. We have demonstrated by gel filtration chromatography, that the principal secreted forms were monomers for IgG1kappa and pentamers for IgMkappa. The binding affinities of these chimeric antibodies against synthetic Tn glycopeptides, were evaluated by surface plasmon resonance showing an affinity constant similar to that of 83D4 native antibody for IgMkappa and a lower affinity constant for IgG1kappa chimeric antibody. On the other hand, the replacement of mouse C regions with human C regions confers both chimeric antibodies the ability to activate human complement. These mouse/human chimeric antibodies should be much less immunogenic and could play an important role in the lysis of tumor cell expressing Tn-antigen. Therefore, these anti-Tn chimeric antibodies could be considered as potential tools for human in vivo studies.

Animals↗
PubMed1999

Efficiency of Ber-EP4 antibody for isolating circulating epithelial tumor cells before RT-PCR detection.

We compared the efficacy of 4 methods for isolating circulating tumor cells: immunocapture with Ber-EP4-coated magnetic beads, density gradient separation, ammonium chloride, and distilled water-mediated erythrocyte lysis. Human blood from healthy volunteers was mixed with serial dilutions of prostate (LNCaP) and liver (HepG2) derived tumor cells. Isolation of circulating tumor cells was followed by reverse transcriptase-polymerase chain reaction with primers specific for prostate-specific antigen and alpha-fetoprotein. Ber-EP4 antigen expression was evaluated by immunohistochemistry in 27 hepatocellular carcinomas and 34 prostate adenocarcinomas. Peripheral blood samples from 12 patients with hepatocellular carcinoma and 10 with prostate adenocarcinoma also were tested. Density gradient separation and Ber-EP4 immunocapture were the most sensitive techniques for isolating circulating tumor cells in in vitro tests. Isolation by density gradient separation was significantly more sensitive than Ber-EP4 immunocapture when applied to peripheral blood samples of patients with cancer, a result consistent with the variable expression of Ber-EP4 antigen that we found by immunohistochemistry in prostate adenocarcinomas and hepatocellular carcinomas.

Adenocarcinoma↗
PubMed1997

Amino acid sequence and three-dimensional structure of the Tn-specific isolectin B4 from Vicia villosa.

The partial amino acid sequence of the tetrameric isolectin B4 from Vicia villosa seeds has been determined by peptide analysis, and its three-dimensional structure solved by molecular replacement techniques and refined at 2.9 A resolution to a crystallographic R-factor of 21%. Each subunit displays the thirteen-stranded beta-barrel topology characteristic of legume lectins. The amino acid residues involved in metal- and sugar-binding are similar to those of other GalNAc-specific lectins, indicating that residues outside the carbohydrate-binding pocket modulate the affinity for the Tn glycopeptide. Isolectin B4 displays an unusual quaternary structure, probably due to protein glycosylation.

Amino Acid Sequence↗
PubMed1997

Molecular cloning of a monoclonal anti-tumor antibody specific for the Tn antigen and expression of an active single-chain Fv fragment.

We report here the first amino acid sequence of an anti-Tn monoclonal antibody raised against human breast cancer cells and show that a single chain Fv fragment of this IgM retains the Tn-binding specificity as defined by functional assays with asialo-OSM and membrane extracts from MCF-7 cells. Sequence comparisons and molecular modeling of 83D4 indicate that the antibody combining site displays a cavity-like feature primarily defined by the CDR H1 and H2 loops. This pocket could accommodate a single Tn molecule, thus, suggesting a structural explanation for the predominant expression of a particular VH gene segment in a group of antibodies that recognize tumor-associated antigens arising from an aberrant O-glycosylation.

Amino Acid Sequence↗
PubMed1996

Multicenter evaluating of a commercially available PCR assay for diagnosing enterovirus infection in a panel of cerebrospinal fluid specimens.

Thirteen laboratories participated in blind tests of a panel of 20 coded cerebrospinal fluid specimens (7 uninfected samples, 3 samples infected with 1 50% tissue culture infective dose [TCID50]/0.1 ml [nonenterovirus strains], and 10 samples infected with 10, 1, or 0.1 TCID50/0.1 ml [three different enterovirus serotypes]) on the Amplicor enterovirus PCR assay (Roche Diagnostic Systems). The panel was also evaluated by in-house PCR (two nested-PCR and three one-step PCR assay) or tissue culture (eight laboratories). The viral load was shown to influence greatly the sensitivity of the assay. The average sensitivity of the Amplicor test ranged from 67 to 98% for viral titers of 1 to 10 TCID50/0.1 ml, respectively; titers of 0.1 TCID50/0.1 ml resulted in a sensitivity of only 16%. The overall specificity of the Amplicor test was 98%. The Amplicor assay compared favorably to the five in-house PCR tests (no significant difference in either sensitivity or specificity) and was much more sensitive than tissue culture (P < 0.001), even for high viral loads. It was easy to perform, rapid (about 6 h), well-standardized, and appeared to be suitable for the diagnosis of enterovirus meningitis on a routine basis in laboratories trained in molecular biology techniques.

Base Sequence↗
PubMed1995

Immunocytological analysis of the Tn associated antigen 83D4 in serous effusions from patients with cancer: comparison with Tn soluble glycoprotein.

AIMS: To determine whether the monoclonal antibody (MoAb) 83D4, previously shown to be highly specific for carcinoma cells, can be used as an immunocytological marker to discriminate between benign and malignant cells in serous effusions; and to test for a correlation between expression of the antigen reacting with MoAb 83D4 on effusion cells and the amount of soluble 83D4 antigen in effusion fluids. METHODS: Thirty three pleural and 23 peritoneal effusions from 56 cancer patients with metastatic disease were tested for the presence of Tn associated 83D4 antigen by immunocytochemical staining, and for the presence of soluble antigen in supernatants. The patients had undergone various chemotherapy and radiation therapy protocols. RESULTS: As a result of the various types of treatment, the cytological characteristics of the cells were often modified and the antigenic epitopes may have been altered. Positive staining for 83D4 MoAb was obtained in 36 (97%) of the 37 malignant effusions, eight (73%) of 11 suspect effusions, and three (38%) of the eight apparently benign effusions (free of malignant cells). In these latter cases, cytological reassessment showed a few suspect cells in two cases. 83D4 soluble antigen was detected in 30 of 37 malignant effusions (81%), five of 11 suspected infusions (46%), and five of eight apparently benign effusions (63%). CONCLUSIONS: Immunocytochemical staining with anti-83D4 antibody is useful for differentiating reactive or atypical mesothelial cells from epithelial cells, especially in breast cancer effusions.

Adenocarcinoma↗
PubMed1994

Analysis of a heterogeneous group of human breast carcinoma associated glycoproteins bearing the Tn determinant.

The Tn determinant (GalNAc alpha-O-Ser/Thr) is expressed by about 90% of human carcinomas, but is cryptic in most normal human tissues. A murine monoclonal antibody (MAb) 83D4, developed following immunization with human breast carcinoma cells, reacts with a Tn-related epitope. In the present study we characterized the glycoprotein antigen identified by 83D4 in the human breast carcinoma cell line MCF-7. We further showed that the 83D4 antigenic determinant is masked in human milk fat globule membranes (HMFGM), and can be exposed upon mild m-periodate treatment after desialylation. Western-blot analysis resolved the 83D4 antigen from MCF-7 into two main components of 120-190 kD and > 500 kD respectively. Non equilibrium pH gradient electrophoresis/SDS PAGE revealed the acidic nature of the reactive glycoproteins (pI 4.43-4.70). 83D4 antigenic activity resolved by CsCl gradient ultracentrifugation layered on a wide range of densities (1.30-1.46 g/ml) including typical densities of mucin-like glycoproteins but also lower densities. The amino acid composition of the antigen, relatively rich in serine but poor in threonine and proline, confirmed the divergence from other mucin-like carcinoma-associated glycoproteins. Dicarboxylic amino acids were abundant, accounting in part for the acidic nature of the molecules. ELISA and Western-blot analysis of the subcellular fractions from MCF-7 cells revealed that the 83D4 antigen is mainly contained in plasma membranes (85%) from which it may be resolved into two broad bands (slow and fast migrating components). These results provide information on a group of breast carcinoma associated glycoproteins related to but different from typical mucins, and provide data on alteration of O-glycosylation in tumor cells.

Adenocarcinoma↗
PubMed1994

Neutralization epitopes on HIV pseudotyped with HTLV-I: conservation of carbohydrate epitopes.

One mechanism for expanding the cellular tropism of human immunodeficiency virus (HIV) in vitro is through formation of phenotypically mixed particles (pseudotypes) with human T lymphotropic virus type I (HTLV-I). In this study we found that pseudotypes allow penetration of HIV particles into CD4-negative cells, previously nonsusceptible to HIV infection. The infection of CD4-negative cells with pseudotypes could be blocked with anti-HTLV-I serum but failed to be significantly inhibited with anti-HIV serum or a V3-neutralizing anti-gp120 monoclonal antibody. This may represent a possibility for pseudotypes to escape neutralization by the immune system in vivo. Previous reports have suggested that carbohydrate structures may be conserved neutralization epitopes on retroviruses. In this study, the neutralizing capacity of lectins and anti-carbohydrate monoclonal antibodies was found to block infection by cell-free pseudotypes in CD4-negative cells. We suggest that although viral cofactors might expand the tropism of HIV in vivo, HIV and HTLV-I seem to induce common carbohydrate neutralization epitopes.

Antibodies, Monoclonal↗
PubMed1993

Monoclonal anti-idiotypic antibodies as functional internal images of enzyme active sites: production of a catalytic antibody with a cholinesterase activity.

Monoclonal antibody 9A8 was selected by immunizing mice with AE-2, a monoclonal antibody directed against the active site of acetylcholinesterase. In accordance with the idiotypic network theory, monoclonal anti-idiotypic antibody 9A8 displayed internal-image properties of the original immunogen, the acetylcholinesterase active site. Hydrolysis of acetylthiocholine and related esters of thiocholine by 9A8 follows saturation kinetics and kinetic parameters were determined. The hydrolytic activity is characterized by a lowered kcat value (81 s-1) and an increased Km value (0.6 mM) when compared with the original enzyme. However, the rate acceleration (kcat/kuncat = 4.15 x 10(8) remains higher than for the esterase activities usually described for catalytic antibodies directed against transition-state analogs. The 9A8 activity exhibits a relaxation of specificity toward both substrates and inhibitors. This specificity does not correspond to a known enzymatic activity. The anti-idiotypic approach should be valuable for producing different structural and functional copies of the same enzyme active site. This should allow further insights into structure-activity relationships. Furthermore, use of chemically modified enzymes as immunogens may result in anti-idiotypic antibodies with catalytic activities not found in the native enzymes.

Acetylcholinesterase↗
PubMed1993

Crystallization and preliminary crystallographic analysis of a tetrameric isolectin from Vicia villosa, specific for the Tn antigen.

Isolectin B4 isolated from Vicia villosa seeds is specific for the Tn antigen, a carcinoma-associated molecular marker. Crystals of the isolectin grown in the presence of carbohydrate are tetragonal, space group P4(1) (or P4(3), with a = 91.3 A, c = 151.7 A and one tetramer in the asymmetric unit. The crystals diffract X-rays to 2.8 A resolution and are suitable for high-resolution structural analysis.

Acetylgalactosamine↗
PubMed1992

Detection of a soluble antigen defined by monoclonal antibody 83D4 in serous effusions associated with breast carcinoma.

Monoclonal antibody (MoAb) 83D4 was generated by immunization with cell suspensions obtained from sections of formol-fixed paraffin embedded human breast cancer. It recognized an antigen expressed in breast carcinomas but not in normal breast tissue. Pleural and ascitic fluids from 66 patients were studied by an 83D4 heterologous sandwich radioimmunoassay (SRIA) using solid-phase immobilized wheat germ agglutinin to detect the 83D4 soluble antigen. Using a cutoff level of 5 units/ml of 83D4 antigen, higher values were found in 22 of 27 breast cancer-associated effusions (mean = 10.72 +/- 6.80 units/ml). The 20 nonmalignant effusion fluids tested showed lower values (mean = 1.16 +/- 1.49 units/ml, P less than 0.001). The antigen was undetectable or present in low levels in effusions from patients with hematologic malignancies. When SRIA results were compared with conventional cytologic diagnosis in breast-cancer effusions, elevated levels of 83D4 soluble antigen were found in all patients (8 of 8) in whom malignant cells had been detected, in 4 of 8 patients with the diagnosis of "suspected malignancy," and in 10 of 11 patients with negative cytologic findings. Using an immunoglucosidase method on cell smears of various origins, MoAb 83D4 stained metastatic cells of breast and ovary carcinomas but did not reactive with mesothelial cells and other normal or malignant cell types. These results suggest that quantitation of the 83D4 soluble antigen may be used to improve the diagnosis of cancer in serous effusions.

Antibodies, Monoclonal↗
PubMed1991

Purification and characterisation of a breast-cancer-associated glycoprotein not expressed in normal breast and identified by monoclonal antibody 83D4.

Monoclonal antibody (mAb) 83D4 was generated using formol-fixed paraffin-embedded human breast carcinoma tissue as the immunogen. Previous studies demonstrated that it was reactive with breast carcinoma tissues, but not with normal breast. The antigen identified by mAb 83D4 was detected, using ELISA, in MCF7 breast carcinoma cell line membrane extracts, in primary breast and colon carcinoma tissue extracts and in pleural effusion fluid from patients with metastatic breast cancer. No reactivity with 83D4 was found in either human milk fat globule membranes or skimmed milk. 83D4 reactive antigen was found to be a heterogeneous high molecular weight (MW) protein (apparent Mr:300-400 to over 1000 kDa) by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The antigen was purified from MCF7 cells, breast and colon carcinomas and effusion fluid, by perchloric acid solubilisation followed by immunoaffinity chromatography with 83D4. The immunopurified antigen from MCF7 cells and pleural effusion fluid was further analysed by gel filtration and ion-exchange chromatography, which confirmed the high MW and indicated the charge heterogeneity of the reactive molecules. The 83D4 reactive antigen strongly bound to wheat-germ agglutinin and weakly to peanut lectin. No binding was found with lentil lectin or concanavalin A. Antigenic activity was strongly reduced by trypsin and subtilysin digestion and by treatment with sodium periodate, but it was not affected by neuraminidase. These results imply the glycoprotein nature of the 83D4-defined antigen and the involvement of carbohydrate, but probably not sialic acid, in the epitope. Purified 83D4 antigen did not display reactivity for mAb HMFG-1, directed against a polymorphic epithelial mucin, PEM, using ELISA, but bound mAb CC49 and weakly mAb B72.3, antibodies which define a tumour associated glycoprotein, TAG-72. Moreover CC49 and 83D4 showed similar reactivity pattern in immunoblotting assays. A double determinant radioimmunoassay confirmed that 83D4 antigen carries epitopes for mAb B72.3 and CC49. Competition radioimmunoassays clearly distinguished the 83D4 defined epitope from those recognised by B72.3 and CC49, demonstrating that antibody 83D4 identifies a unique epitope. It is suggested that the antigens identified by mAb 83D4 and by mAb B72.3 and CC49 may form part of the same family of carcinoma associated glycoproteins.

Antibodies, Monoclonal↗
PubMed1991

Two monoclonal antibodies identify antigens preferentially expressed on normal human breast cells versus breast cancer cells.

In order to obtain antibodies with specificity toward normal mammary epithelial antigenic determinants, we immunized BALB/c mice with normal milk cells and screened the hybridomas against an undifferentiated breast cancer cell line H466B, peripheral blood lymphocytes and normal fibroblasts. Two hybridomas were generated, which produced BA6 (IgG1) and CA4 (IgM) monoclonal antibodies (MAbs). These MAbs did not react with 5 breast cancer cell lines. In cryostat sections of normal human breast tissue, BA6 was reactive with 6/6 and CA4 was reactive with 12/13 specimens both showing an apical staining of epithelial cells. Conversely staining of malignant cells in breast cancer biopsies was observed in 4/33 specimens with BA6 and in 4/19 specimens with CA4. Computerized image analysis (SAMBA) of immunostained sections showed homogeneous distribution of staining, with a high percentage of stained cell surfaces in normal breast (mean percentages of positive surfaces : BA6 : 75% and CA4 : 82%) while, in malignant samples, staining was heterogeneous, with a mean percentage of positive surface of 25% for BA6 and 12% for CA4. Both MAbs reacted strongly with human milk fat globule membranes (HMFGM) and skimmed milk. FPLC size exclusion chromatography of skimmed milk showed that CA4 and BA6 reactive materials eluted in distinct peaks in high molecular weight ranges. Electrophoretic separation of HMFGM followed by CA4 staining detected a high molecular weight reactive band (Mr 380-600 kDa). CA4 and BA6 reactivity was reduced by protease treatment of the antigen but was not affected by neuraminidase digestion, by methanol extraction or by Na-metaperiodate oxidation. After perchloric acid treatment of HMFGM, BA6 activity was lost while the CA4 activity was found in the soluble fraction. The results reported suggest that the two MAbs identify two distinct novel epitopes of normal breast cells.

Animals↗
PubMed1991

[Production of a monoclonal antibody (B1N) recognizing human nuclear antigen associated with cell proliferation].

This report describes the preliminary characterization of a novel antigen reactive with a murine monoclonal antibody designated B1N produced in our laboratory. This antibody (IgM) reacts in IFI with mammals and also insect cells, by staining in a speckled fashion the nucleus of these cells. Immunoblotting analysis of Hela and murine D55 nuclear extracts revealed a polypeptide with an apparent molecular weight of 120kD (p120). In this work we demonstrated that: 1. this polypeptide appeared in human peripheral blood lymphocytes only when they were induced to proliferate in vitro after phytohemagglutinin stimulation; 2. this polypeptide was no longer detected in D55 resting cells, following serum deprivation; 3. the MAb B1N specifically revealed the nucleus of proliferating cells on frozen sections of uterine tissue. These data strongly suggest that the p120 nuclear antigen expression is associated with the proliferation state of cells.

Animals↗