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C Wagener

Publications and source records attributed to C Wagener.

At least 73 records · Page 4Linked to original sources
PubMed1994

Distinction of highly homologous pregnancy-specific glycoprotein (PSG) isoforms by differential absorption of antisera with recombinant PSG fusion protein domains.

The N-terminal domains of two different highly homologous isoforms of pregnancy-specific beta 1 glycoproteins (PSGs) were expressed in bacteria. The N-terminal domain of PSG1 (PSG1-N) and PSG3 (PSG3-N) were chosen since PSG3-N, but not PSG1-N, contains an RGD sequence. Immunosorbents were prepared using bacterially expressed fusion proteins with the respective N domains. Antibodies from a polyclonal antiserum against native PSG were eluted from PSG1-N and were subsequently absorbed against PSG3-N. Using this procedure, antibodies were generated that were able to bind to native PSG and PSG1-N, but not to PSG3-N. These results show that the antiserum against native PSG crossreacts with PSG isoforms of two subgroups. From the PSG antiserum, antibodies can be isolated that differentially bind to V-like PSG domains which differ by eight non-conservative amino acid substitutions, three of which are clustered in a position corresponding to the CDR III of immunoglobulin V region domains. Purification of antibody populations by this technique should make it possible to distinguish rapidly between highly homologous PSG isoforms in tissues and body fluids.

Amino Acid Sequence↗
PubMed1994

Specific detection of carcinoembryonic antigen-expressing tumor cells in bone marrow aspirates by polymerase chain reaction.

PURPOSE: To establish a sensitive assay for the specific detection of carcinoembryonic antigen (CEA)-expressing tumor cells in the bone marrow of patients with colorectal cancer and other CEA-positive carcinomas. PATIENTS AND METHODS: A CEA-specific nested reverse transcriptase (RT) polymerase chain reaction (PCR) assay was developed and optimized using limiting dilutions of a CEA-positive cancer cell line mixed with normal bone marrow cell specimens. The optimized test was then used to examine bone marrow samples obtained from 15 patients with abdominal carcinomas (colorectal, n = 10; pancreatic, n = 3; gastric, n = 2) and six patients with breast cancer. Specificity was assessed by examination of 56 negative controls (malignant hematologic disease, n = 28; nonmalignant disease conditions, n = 5; healthy bone marrow donors, n = 8; normal peripheral-blood samples, n = 15). For 11 patients with abdominal carcinomas, immunostaining evaluations were performed using an anti-CEA and an anticytokeratin antibody, and the results compared with the nested PCR assay. RESULTS: In the sensitivity calibration system, single CEA-expressing tumor cells were detected in 2 to 5 x 10(7) normal bone marrow cells. All 56 control samples failed to amplify. This demonstrates that mRNAs coding for highly homologous CEA-related antigens expressed by various lineages of blood cells do not interfere. Bone marrow samples from 10 of 15 patients with abdominal cancers and four of six breast cancer patients scored positive, indicating micrometastatic bone disease. Four of 11 samples from the gastrointestinal cancer patients were found to be positive by the PCR method, but were negative with the immunocytology method. CONCLUSION: Since approximately 30% of the colorectal carcinoma patients that score negative in immunocytology staining of bone marrow samples have been reported to relapse, earlier diagnosis of the presence of malignant cells is needed. Our result that samples scoring positive in the described CEA-specific PCR test remained negative by two immunostaining methods suggests a higher sensitivity. We conclude that PCR amplification of CEA mRNA may lead to an earlier diagnosis of micrometastatic bone disease in patients with CEA-expressing carcinomas.

Base Sequence↗
PubMed1994

Molecular biology techniques in the diagnosis of monogenic diseases.

Monogenic diseases are defined by their patterns of inheritance: autosomal dominant, autosomal recessive, or X-linked. This article examines the molecular biology techniques available for their study and detection. These techniques comprise direct detection of mutations (including trinucleotide repeats) and linkage analysis. The usefulness of these techniques is compared to conventional biochemical tests for the diagnosis of various inherited diseases. The authors stress the need for caution in the interpretation of test results and the importance of genetic counseling.

Forecasting↗
PubMed1993

Biliary glycoprotein, a potential human cell adhesion molecule, is down-regulated in colorectal carcinomas.

Biliary glycoprotein (BGP) is the human homologue of a cell adhesion molecule (CAM) of the rat designated Cell-CAM. The BGP gene is a member of the carcinoembryonic antigen gene family, which belongs to the immunoglobulin superfamily. BGP is expressed in cells of epithelial and myeloid origin. In granulocytes, BGP is a main antigen of the CD66 cluster of differentiation antigens that mediate the binding to endothelial E-selectin. Since BGP is a major human CAM, the expression of BGP was studied in 21 colorectal carcinoma tissue specimens and in the respective adjacent normal mucosae. As an internal control for epithelial mRNA, the expression of cytokeratin 18 was evaluated in parallel. In addition, the expression of carcinoembryonic antigen and nonspecific crossreacting antigen, which are highly homologous to BGP, was investigated. Two BGP mRNAs of 3.9 and 1.5 kilobases were detected in the normal colonic mucosa samples. The median of the tumor-to-normal ratios of mRNA expression was 0.2 for both BGP mRNAs. In contrast, the median was 1.2 for cytokeratin, 1.0 for carcinoembryonic antigen, and 1.4 for nonspecific crossreacting antigen. Relative to cytokeratin 18 expression, the expression of BGP was reduced to < or = 0.1 in half of the tumors and to < or = 0.4 in > 80% of the tumors. These findings indicate that the loss or reduced expression of the adhesion molecule BGP is a major event in colorectal carcinogenesis.

Antigens, CD↗
PubMed1993

Identification of the specific oligosaccharide sites recognized by type 1 fimbriae from Escherichia coli on nonspecific cross-reacting antigen, a CD66 cluster granulocyte glycoprotein.

Nonspecific cross-reacting antigen (NCA), a CD66 cluster antigen, is a well characterized glycoprotein on granulocytes, macrophages, and lung epithelium. Structural studies at the protein and genomic levels have revealed that NCA is a member of the immunoglobulin (Ig) supergene family and contains a domain structure similar to Ig with an amino-terminal variable-like domain followed by disulfide loop-containing constant-like domains. Previous work by this laboratory and others has demonstrated that NCA is a receptor for binding of bacteria expressing type 1 fimbriae (pili). This binding is mediated by interaction between lectins on the bacteria fimbriae and carbohydrate chains on NCA. In the present work we further characterize the specificity for bacterial binding by NCA using endoglycosidases and site-directed mutagenesis. Results of these studies demonstrate that Escherichia coli expressing type 1 fimbriae binds to high mannose oligosaccharide structures on NCA and that the functionally relevant sites are located in the variable-like domain of NCA.

Amino Acid Sequence↗
PubMed1993

Molecular characterization of a cloned idiotypic cascade containing a network antigenic determinant specific for the human carcinoembryonic antigen.

The monoclonal anti-idiotypic antibody (maId) 6G6.C4, directed against the carcinoembryonic antigen (CEA)-specific T84.66 immunoglobulin, was recently shown to act as a surrogate antigen for CEA in experimental animals. In this report, we have extended our studies. 1) The kinetics of complex formation in this CEA-specific idiotypic cascade were investigated using Biosensor technology. 2) Bacterial expression studies show that the mimicked epitope can be delimited to the A3 domain of CEA. 3) We cloned and characterized the genes coding for maId 6G6.C4. 4) Comparison of this epitope-bearing domain with the hypervariable region sequences of 6G6.C4 yields substantial amino acid similarity. Sequence homology between maId 6G6.C4 and anti-CEA antibodies binding to the T84.66 epitope led us to investigate the interaction of maId 6G6.C4 and CEA. Surprisingly, 6G6.C4 specifically binds to CEA but not CEA-related antigens in Western blots. 6G6.C4 and T84.66 recognize different epitopes on CEA. Our results suggest that the T84.66 epitope functionally mimicked by maId 6G6.C4 may be involved in the homophilic binding between CEA molecules, and that heterophilic interactions in the CEA-family are mediated by different binding sites. A model for the intermolecular adhesion of CEA is presented.

Amino Acid Sequence↗
PubMed1993

Monoclonal, anti-domain and anti-peptide antibodies assign the molecular weight 160,000 granulocyte membrane antigen of the CD66 cluster to a mRNA species encoded by the biliary glycoprotein gene, a member of the carcinoembryonic antigen gene family.

mAb directed against the CD66 cluster of granulocyte differentiation Ag recognize Ag of the carcinoembryonic Ag family. A major Ag in extracts from granulocyte membranes bound by CD66 antibodies exhibits a relative molecular mass of 160,000. According to recent data, this Ag may be a product of the biliary glycoprotein (BGP) gene that belongs to the CEA gene family. As a result of alternative splicing, the BGP gene is transcribed into at least seven distinct mRNA species. To identify splice variants of BGP, antisera were raised against the A2 domain expressed in bacteria and to a peptide comprising the C-terminal 23 amino acids encoded by the 3' exon of the BGP gene. The antisera and an mAb specific for members of the BGP family were used to identify potential BGP splice variants in granulocyte membranes. For comparison, the binding of antibodies to Ag purified from human bile was investigated. In the membrane preparation from granulocytes, the only Ag identified by the mAb, the domain antiserum and the peptide antiserum, was the Ag of M(r) 160,000 recognized by a CD66 antibody. These results indicate that the M(r) 160,000 granulocyte membrane Ag of the CD66 cluster is the product of the BGP-specific mRNA containing all coding sequences of the BGP gene. Among two major biliary glycoproteins present in human bile, the M(r) 115,000 Ag contains the A2 domain, whereas the domain is lacking in the "classical" biliary glycoprotein of M(r) 85,000. None of the bile Ag bound the peptide antiserum.

Antibodies, Monoclonal↗
PubMed1992

A monoclonal anti-idiotypic antibody bearing the image of an epitope specific to the human carcinoembryonic antigen.

One concept for immune therapy of patients bearing carcinomas involves monoclonal anti-idiotypic antibodies (Malds) to trigger the immune system of the host into a response against the tumor cells. Current theory states that so-called internal image Malds bearing epitopes specific to a given tumor-associated antigen would be most suited for that purpose. We report here the generation of syngeneic Malds generated against the murine monoclonal immunoglobulin T84.66 (Ab1), which defines a single epitope on the protein moiety of the carcinoembryonic antigen (CEA). This antigenic determinant is unique to CEA, as it is absent in other members of the CEA gene family that are expressed in a variety of normal human tissues, including granulocytes. The Mald 6G6.C4 (Ab2) exhibits the immunochemical features of an internal image antibody mimicking the epitope recognized by the idiotype T84.66. In enzyme immunoassays the binding of Ab1 to Ab2 is completely inhibited by CEA. In addition, Mald 6G6.C4 only binds to native but not to the denatured or reduced idiotype. Immunization of rabbits with F(ab')2-fragments of 6G6.C4 results in antisera (Ab3) that show specificity to CEA in both binding and inhibition enzyme immunoassays as well as in Western blots. Finally, Ab3 did not detect NCA, a major CEA-related glycoprotein in Western blots, either in a purified form or in a crude tumor extract, indicating a high specificity of the anti-anti-idiotypic response. In summary, these immunochemical data show that the monoclonal anti-idiotype 6G6.C4 can functionally mimic a CEA-specific epitope in rabbits and may do so in humans. Therefore, this antibody may have a clinical potential as a network antigen for active immune therapy in patients suffering from CEA-positive carcinomas.

Animals↗
PubMed1992

Specificity and affinity of monoclonal antibodies against carcinoembryonic antigen.

The binding specificities of 52 well-characterized monoclonal antibodies (Mabs) against carcinoembryonic antigen (CEA) from 12 different research groups were studied by immunohistochemistry and immuno flow cytometry. In addition, the binding constant for the interaction between Mab and CEA was determined by a solution-phase assay. Cryostat sections of colon carcinoma and normal colon, stomach, liver, pancreas, and spleen were studied by immunohistochemistry. Peripheral blood granulocytes, monocytes, and lymphocytes were assayed by immuno flow cytometry. The Mabs used here have previously been classified into five essentially nonoverlapping epitope groups (GOLD 1-5) (Cancer Res., 49: 4852-4858, 1989). Most Mabs cross-reacted with different normal tissues, ranging from highly cross-reactive Mabs (positive reaction with 8 of 9 discriminating tissues) to relatively specific Mabs (positive reaction with 1 of 9 discriminating tissues). Five Mabs (10%) were specific, reacting only with colon carcinoma, normal colon mucosa, and normal gastric foveola. There was a correlation between epitope group and binding specificity. Mabs with a high degree of CEA specificity almost exclusively belonged to epitope groups 1, 2, and 3, while highly cross-reactive Mabs belonged to epitope groups 4 and 5. There was no correlation between antibody specificity and affinity for CEA. Specific Mabs with high as well as low affinity were found.

Adult↗
PubMed1992

Guided tissue regeneration in interproximal defects in the monkey.

The potential for guided tissue regeneration was evaluated in one-walled interproximal sites in Macaca fascicularis. Histologic differences were evaluated at 1 and 3 months. Within the experimental (barrier) group, 100% of the root surfaces with potential for regeneration were covered with new cementum, whereas the control specimens had 20% or less new cementum. The amount of regeneration was determined by the position of the barrier membrane; the more coronal the barrier, the greater the regeneration. Observations indicated that the optimal time for barrier removal is between 1 and 3 months.

Animals↗