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Biomedical subjects

L Celis

Publications and source records attributed to L Celis.

13 recordsLinked to original sources
PubMed2001

Building novel binding ligands to B7.1 and B7.2 based on human antibody single variable light chain domains.

Ligands specific for B7.1 (CD80) and B7.2 (CD86) have applications in disease indications that require inhibition of T-cell activity. As we observed significant sequence and structural similarity between the B7-binding ligand, cytotoxic T-lymphocyte associated protein-4 (CTLA-4), and antibody variable light chain domains (VLs), we have explored the possibilities of making novel B7 binding molecules based on single VL domains. We first describe the "rational" design and construction of a VL/CTLA-4 hybrid molecule in which we have grafted both the CDR1 and CDR3-like loops of CTLA-4 onto a single VL light chain, at sites determined by sequence and structure-based alignment. This molecule was secreted as a soluble product from Escherichia coli, but did not show any binding to B7.1 and B7.2. In a second approach we constructed a VL library in which human VL genes derived from B-cells were spiked with the CDR3-like loop of CTLA-4 and further diversified by DNA shuffling. This library was displayed on phage, and after selection gave B7.1 binding ligands which competed with CTLA-4. In order to evaluate the possible general utility of VL domains as binding ligands, we have constructed a non-biased VL library. From this DNA-shuffled human VL library we have selected single VL domains specific for B7.1, B7.2 or human IgG. Two B7.1-specific VL ligands and one B7.2-specific VL ligand showed competition with CTLA-4. One candidate VL domain-specific for B7.1 was affinity matured by simultaneous randomisation of all CDR loops using DNA shuffling with degenerate CDR-spiking oligonucleotides. From this library, a single VL domain with affinity of 191 nM for B7.1 was obtained, which also showed binding to B7.1 in situ. This VL had mutations in CDR1 and CDR3, indicating that antigen recognition for this single VL is most likely mediated by the same regions as in the VL domain of whole antibodies. The B7.1 and B7.2-specific VL domains described in this study may form the basis of a new family of immunomodulatory recombinant molecules. Furthermore, our studies suggest that it is feasible to create specific single VL domains to diverse targets as is the case for single VH domains.

Amino Acid Sequence↗
PubMed2001

Expression and purification of monospecific and bispecific recombinant antibody fragments derived from antibodies that block the CD80/CD86-CD28 costimulatory pathway.

The development of recombinant techniques for rapid cloning, expression, and characterization of cDNAs encoding antibody (Ab) subunits has revolutionized the field of antibody engineering. By fusion to heterologous protein domains, chain shuffling, or inclusion of self-assembly motifs, novel molecules such as bispecific Abs can be generated that possess the subset of functional properties designed to fit the intended application. We describe the engineering of Ab fragments produced in bacteria for blocking the CD28-CD80/CD86 costimulatory interaction in order to induce tolerance against transplanted organs. We designed single-chain Fv antibodies, monospecific and bispecific diabodies, and a bispecific tetravalent antibody (BiTAb) molecule directed against the CD80 and/or CD86 costimulatory molecules. These recombinant Ab molecules were expressed in Escherichia coli, followed by purification and evaluation for specific interaction with their respective antigen in an enzyme-linked immunosorbent assay (ELISA). A specific sandwich ELISA confirmed the bispecificity of the bispecific diabodies and the BiTAb.

Animals↗
PubMed1997

Clonal expansion of mycobacterial heat-shock protein-reactive T lymphocytes in the synovial fluid and blood of rheumatoid arthritis patients.

OBJECTIVE: To examine the reactivity pattern and T cell receptor (TCR) characteristics of mycobacterial heat-shock protein 65 (hsp65)-reactive T cells generated from paired synovial fluid (SF) and peripheral blood (PB) samples obtained from rheumatoid arthritis (RA) patients and from healthy subjects. METHODS: The reactivity pattern of hsp65-reactive T cell clones generated under limiting-dilution conditions was analyzed in 3H-thymidine incorporation assays. The TCR variable regions of these hsp65-reactive T cells were characterized by polymerase chain reaction with TCR AV- and BV-specific primers and by DNA sequence analysis of the third complementarity-determining region (CDR3). RESULTS: The hsp65-reactive T cells derived both from RA patients and controls preferentially recognized the 1-170 and 303-540 regions of hsp65 and did not cross-react with human hsp60. The hsp65-reactive T cell clones derived from RA patients displayed a restricted TCR AV and BV gene usage, which can be attributed to the limited clonal origin(s) of the independent T cell clones, as evidenced by CDR3 sequence analysis. These clonally expanded T cells were found in both PB and SF and in different inflamed joints of RA patients. CONCLUSION: Our study suggests that there is in vivo clonal activation and expansion of mycobacterial hsp65-reactive T cells in patients with RA.

Antigens, Bacterial↗
PubMed1995

[Capillary testicular hemangioma: a case report].

Testicular tumors in infants are rare and most of them are malignant. Hemangioma of the testis is an extremely rare tumor in infants. We report on an intratesticular capillary hemangioma that appeared in a 10 month-old child subsequently undergoing orchidectomy and on the patient's evolution at 4 years. The histological characteristics showed proliferation of small blood vessels, lined with endothelial cells. Mitoses and atypias were not found. Permitting differential diagnosis with other vascular tumors: Masson's vegetant intravascular hemangio-endothelioma and angiosarcoma.

Capillaries↗
PubMed1994

Nuclear extracts enhance the interaction of fusion proteins containing the DNA-binding domain of the androgen and glucocorticoid receptor with androgen and glucocorticoid response elements.

Comparable fragments of the androgen receptor (AR) (amino acids 540-607) and of the glucocorticoid receptor (GR) (amino acids 412-515) were expressed in E. coli as fusion proteins with protein A. Both fusion proteins, denoted ARF1 and GRF1, contain the DNA-binding domain and some flanking amino acids. In vitro binding assays have shown that both fusion proteins interact with androgen/glucocorticoid response elements (ARE/GREs) in an intron fragment of the C3(1) gene of the androgen-regulated rat prostatic binding protein and in the typically glucocorticoid-responsive long terminal repeat (LTR) promoter of mouse mammary tumour virus. Present results indicate that the interaction of both ARF1 and GRF1 with the C3(1) as well as the LTR fragments is enhanced in the presence of nuclear extract. The factor that gives rise to this enhancement appears to be ubiquitous and sensitive to trypsin and temperature treatment. In the C3(1) fragment, the enhancing effect requires the presence of an intact functional ARE/GRE (Core II) as well as a region spanning the ARE/GRE half-site Core I.

Androgens↗
PubMed1993

Intronic androgen response elements of prostatic binding protein genes.

Transcription of the genes that code for the different components of Prostatic Binding Protein is dependent on androgens. DNA-cellulose competition assays have demonstrated the presence of regions with affinity for the androgen receptor located immediately upstream and in the first intron of these genes. DNase I footprinting in vitro now reveals the exact binding site of a receptor dimer in the first intron of the C3(1), C3(2) and C1 genes. Gene transfer experiments resulted in the description of functional androgen response elements in the C3 genes. The C1 intronic binding site, however, has a much weaker affinity for the DNA-binding domain of the androgen receptor, a characteristic that might explain its inability to function as an androgen response element in chimeric gene constructs.

Androgen-Binding Protein↗
PubMed1993

Proteins interacting with an androgen-responsive unit in the C3(1) gene intron.

The expression of the three genes encoding the components C1, C2 and C3 of prostatic binding protein (PBP) is under androgen control and restricted to the rat ventral prostate. The SstI-PvuII fragment of the first intron of the C3(1) gene displays two binding sites for ubiquitous transcription factors and one for a tissue-specific factor in a 80-bp region upstream of its androgen response element (ARE). The octamer transcription factor 1 (OTF-1) binds to the most distal element (site 1) while a member of the nuclear factor I (NF-I) family recognizes site 2. A third unidentified prostate-specific factor, which also occurs in castrated rats, interacts with the proximal element (site 3). In T-47D cells, both the OTF-1 and the NF-I-like factor can modulate the androgen response of the promoter in a reporter gene construct containing the C3(1) intronic fragment.

Androgen-Binding Protein↗
PubMed1991

Interaction of androgen response elements with the DNA-binding domain of the rat androgen receptor expressed in Escherichia coli.

A fragment of the rat androgen receptor (amino acids 533-637) containing the DNA-binding domain was produced in Escherichia coli as a fusion product with protein A of Staphylococcus aureus. The fusion protein was purified on IgG-Sepharose, a method that does not involve the use of denaturing agents. Approximately 4 mg of fusion protein was obtained from 500 ml of bacterial culture. In gel shift assays, the recombinant DNA-binding domain displays an affinity for a fragment of the long terminal repeat of mouse mammary tumor virus and for an intronic fragment of the gene coding for the C3 component of the androgen-regulated rat prostatic binding protein. In a DNase I footprinting assay, the fusion protein protects a sequence in the C3 fragment that has previously been shown to act as a functional androgen response element. Interestingly, a single base pair mutation in the response element, which abolishes androgen inducibility, also destroys the ability to interact with the recombinant androgen receptor DNA-binding domain.

Androgens↗
PubMed1990

Sequence-specific binding of androgen-receptor complexes to prostatic binding protein genes.

Prostatic binding protein is a complex glycoprotein comprising three components, C1, C2 and C3, organized into two different heterodimers (C1-C3 and C2-C3). The rat ventral prostate genes encoding all three constituent polypeptides are expressed under androgenic control. Analysis of genomic fragments containing the genes and flanking sequences revealed in each case one androgen receptor-binding region upstream of or within the promoter and another in the first intron. The effect of androgens on the expression of these genes may, therefore, be mediated by these direct receptor-DNA interactions. The genomic fragments which contain androgen receptor-binding regions all contain nucleotide sequences reminiscent of glucocorticoid response elements (GRE). Mutations in these sequences in restriction fragments and in synthetic oligonucleotides significantly decreased their affinity for androgen-receptor complexes and their introduction into nonspecific sequences conferred affinity for androgen-receptor complexes. Based on these data, a consensus sequence for putative androgen response elements (ARE) is proposed. However, despite the specific recognition of these sequences by the androgen receptor in vitro, only the C3(1) intronic fragment could confer significant androgen responsiveness on a heterologous promoter. While this could be due to the fact that the GRE-like sequences present in the other fragments are not strong AREs, alternative hypotheses are being investigated currently. Not least of these is that the similar localization of the binding sites in each gene might underlie a more complex androgen regulation mechanism.

Androgen-Binding Protein↗
PubMed1989

Functional characterization of an androgen response element in the first intron of the C3(1) gene of prostatic binding protein.

We demonstrate that the 204 bp intronic gene fragment of C3(1), which has a specific in vitro affinity for the androgen receptor, is able to confer androgen responsiveness to a heterologous promoter. This characteristic is completely destroyed by a single G----T substitution, affecting a 5'-TGTTCT-3' element that closely resembles the consensus sequence of the glucocorticoid and progesterone response elements (GRE/PRE). In fact we could show that this androgen response element (ARE) also acts as a similarly weak GRE or PRE in T-47D cells.

Androgen-Binding Protein↗