Biomedical subjects

B Ober

Publications and source records attributed to B Ober.

9 recordsLinked to original sources

PubMed2001

Generation and characterization of recombinant vascular targeting agents from hybridoma cell lines.

Vascular targeting agents (VTAs) can be produced by linking antibodies or antibody fragments directed against endothelial cell markers to effector moieties. So far, it has been necessary to produce the components of VTAs (antibody, antibody fragment, linker, and effector) separately and, subsequently, to conjugate them by biochemical reactions. We devised a cloning and expression system to allow rapid generation of recombinant VTAs from hybridoma cell lines. The VTAs consist of a single chain Fv antibody fragment as a targeting moiety and either truncated Pseudomonas exotoxin (resulting in immunotoxins) or truncated human tissue factor (resulting in coaguligands) as effectors. The system was applied to generate recombinant immunotoxins and coaguligands directed against endoglin, vascular endothelial growth factor (VEGF):VEGF receptor (VEGFR) complex and vascular cell adhesion molecule 1 (VCAM-1). The fusion proteins exhibited similar functional activity to analogous biochemical constructs. This is the first report to describe the generation and characterization of recombinant coaguligands.

Animals↗

PubMed1999

Characterization of the interaction of a TCR alpha chain variable domain with MHC II I-A molecules.

The alphabeta TCR recognizes peptides bound to MHC molecules. In the present study, we analyzed the interaction of a soluble TCR alpha chain variable domain (Valpha4.2-Jalpha40; abbreviated to Valpha4.2) with the MHC class II molecule I-Au. Valpha4.2 bound specifically to I-Au expressed on the surface of a transfected thymoma cell line. Modifications in the amino acid residues located within the three complementarity-determining regions (CDRs) of the Valpha domain did not markedly affect this interaction. However, mutation of glutamic acid to alanine at position 69 of the fourth hypervariable region (HV4alpha) significantly increased the binding. Antibody inhibition studies suggested that the binding site was partly contributed by a region of the beta chain of I-Au. Furthermore, the binding of Valpha4.2 to the MHC molecule was dependent on the nature of the peptide bound in the groove. Soluble Valpha4.2 specifically inhibited the activation of TCR transfectants by I-Au-expressing cells pulsed with an N-terminal peptide of myelin basic protein. Valpha4.2 also bound to MHC class II-expressing spleen cell populations from mice of the H-2(u) and H-2(d) haplotypes. The binding of Valpha4.2 to I-A molecules might explain the immunoregulatory effects reported previously for TCR alpha chains. This Valpha4.2 interaction may also be relevant to models of antigen presentation involving the binding of intact proteins to MHC class II molecules followed by their processing to generate epitopes suitable for T cell recognition.

Amino Acid Substitution↗

PubMed1997

Recombinant T cell receptor molecules can prevent and reverse experimental autoimmune encephalomyelitis: dose effects and involvement of both CD4 and CD8 T cells.

Autoimmune diseases are often characterized by spontaneous remission followed by relapses. Although the mechanism(s) controlling pathogenic self-reactive T cells are not fully understood, recent data in experimental autoimmune encephalomyelitis (EAE), a prototype for CD4 T cell-mediated autoimmune diseases, indicate that spontaneous recovery is mediated by regulatory T cells (Treg) specific for peptides derived from the beta-chain of the TCR. Here we have tested whether recombinant single-chain TCRs (scTCRs) containing Vbeta domains can be used as vaccines for efficient priming of Treg. A single injection of mice with these recombinant proteins leads to efficient in vivo priming of Treg and almost complete protection from Ag-induced EAE. Significantly, administration of scTCRs during ongoing disease at a 10-fold lower dose than that required for prophylactic treatment also reverses established EAE. However, if a higher dose of scTCR is administered during ongoing disease, paralytic symptoms become exacerbated and the majority of treated animals die from severe and chronic EAE. Furthermore, we demonstrate that regulatory determinants are processed and presented from scTCRs resulting in the recruitment of both CD4 and CD8 regulatory T cells which are required for efficient regulation induced by scTCR. Reversal of established disease following an optimum dose of recombinant TCRs suggests that proteins expressing appropriate Vbeta domains could be used for the treatment of a variety of T cell-mediated pathologic conditions.

Animals↗

PubMed1996

The stoichiometry and affinity of the interaction of murine Fc fragments with the MHC class I-related receptor, FcRn.

The binding of recombinant wild type and mutant Fc-hinge fragments to soluble, FcRn expressed in insect cells has been analysed. The mutant Fc-hinge fragments are derived from murine IgG1 with mutation of residues located at the CH2-CH3 domain interface (Ile253, His31O, Gln311, His433 and Asn434; EU numbering). These mutant Fc-hinge fragments have previously been shown to be deficient in neonatal transcytosis in suckling mice and also have abnormally short serum half lives. The mutated residues are highly conserved in human and rodent gammaglobulins (IgGs) and are also involved in binding to staphylococcal protein A. This study demonstrates that the Fc mutants have lower binding affinities for recombinant FcRn and mutations in the CH2 domain have a greater effect than those in the CH3 domain. There is an excellent correlation between affinity and transcytosis or the control of catabolism, and this provides further evidence in support of the close overlap of the sites of IgG/Fc involved in these processes. The stoichiometry of the FcRn:Fc interaction has also been investigated and has been found to be 1:1, indicating that binding of FcRn to one CH2-CH3 domain interface site precludes an FcRn:Fc interaction at the second site.

Animals↗

PubMed1995

Crystal structure of the V alpha domain of a T cell antigen receptor.

The crystal structure of the V alpha domain of a T cell antigen receptor (TCR) was determined at a resolution of 2.2 angstroms. This structure represents an immunoglobulin topology set different from those previously described. A switch in a polypeptide strand from one beta sheet to the other enables a pair of V alpha homodimers to pack together to form a tetramer, such that the homodimers are parallel to each other and all hypervariable loops face in one direction. On the basis of the observed mode of V alpha association, a model of an (alpha beta)2 TCR tetramer can be positioned relative to the major histocompatibility complex class II (alpha beta)2 tetramer with the third hypervariable loop of V alpha over the amino-terminal portion of the antigenic peptide and the corresponding loop of V beta over its carboxyl-terminal residues. TCR dimerization that is mediated by the alpha chain may contribute to the coupling of antigen recognition to signal transduction during T cell activation.

Animals↗

PubMed1995

Self tolerance to T cell receptor V beta sequences.

T cell tolerance to self is achieved by deletion or inactivation of clones recognizing peptides of self proteins presented by major histocompatibility complex molecules. A considerable fraction of self proteins accessible to the immune system is contributed by the system itself, for example, the receptors used for antigen recognition (antibodies and T cell receptors [TCRs]). Thus far, it has remained unclear, whether antigen receptors are subject to self tolerance, or on contrary, engage into network interactions implying immunity rather than tolerance. In this study, we demonstrate self tolerance to synthetic peptides corresponding to the first hypervariable region of the V beta 8.1 and V beta 8.2 TCR proteins. We also show that the tolerogenic synthetic peptide corresponds to a fragment produced by processing of the V beta protein, and conversely, that a V beta peptide not produced by processing is also not subject to self tolerance. Thus, the rules of tolerance seem to apply to antigen receptors, at least to their germline-encoded portions, in a similar fashion as to other self proteins. This finding has important implications for studies of natural and artificially induced immune networks.

Amino Acid Sequence↗

PubMed1992

Endocytosis of different members of the small chondroitin/dermatan sulfate proteoglycan family.

The family of small interstitial chondroitin/dermatan sulfate proteoglycans consists of at least three different molecular species: biglycan (proteoglycan I), decorin (proteoglycan II), and proteoglycan-100, which has a glycosylated core protein of about 100 kDa. The core protein of decorin has been shown to be responsible for receptor-mediated endocytosis of this proteoglycan species by a variety of mesenchymal cells. It is now demonstrated that skin fibroblasts and articular chondrocytes endocytose biglycan with an efficiency similar to that of decorin. Uptake of biglycan is also mediated by its core protein and can be inhibited by decorin in a partially competitive manner. In human fibroblasts, endosomal proteins of 51 and 26 kDa, which are known to bind decorin core protein, also interact with biglycan. This interaction can be inhibited by decorin. Bovine articular chondrocytes contained binding proteins of 48 and 25 kDa. Proteoglycan-100 can be distinguished from biglycan and decorin by its low clearance rate, which however, exceeds the rate of fluid phase endocytosis.

Blotting, Western↗

PubMed1992

Influence of continuous infusion of interleukin-1 alpha on the core protein and the core protein fragments of the small proteoglycan decorin in cartilage.

Decorin, a collagen-binding small proteoglycan, is considered to have a specific function in the organization or stability of the collagen network. Therefore, alteration of its molecular properties may be of pathophysiological relevance during the development of cartilage damage. It is shown here that normal cartilage from rabbit knee-joint contains glycosaminoglycan chain-bearing core protein fragments of 39, 23, and 18 kDa, each one amounting to approximately 5-6% of the intact decorin core protein. Continuous infusion of human recombinant interleukin-1 alpha for 14 days (200 ng/day) into a knee-joint led in condylar cartilage to a reduction in the amount of intact core protein from 2 micrograms/mg wet tissue to about 1.1 micrograms/mg. The increase in its quantity found after infusion of heat-inactivated interleukin-1 was not statistically significant. The concentration of all three core protein fragments became reduced to a similar extent as the intact core protein under the influence of the cytokine, and additional fragments were not found. Surprisingly, there was a much smaller response to interleukin-1-treatment in patellar cartilage.

Animals↗

PubMed1988

Longitudinal studies of regional cerebral metabolism in Alzheimer's disease.

Measurement of cerebral glucose metabolism in six patients with Alzheimer's disease using positron emission tomography demonstrated that hypometabolism remained relatively more severe in parietal cortex than in frontal cortex over time. Lateral metabolic asymmetries were preserved in less severely involved brain regions, but were less stable in parietal cortex.

Aged↗