Biomedical subjects

Nadia Terrazzini

Publications and source records attributed to Nadia Terrazzini.

4 recordsLinked to original sources

PubMed2006

The TCR Vbeta signature of bacterial superantigens spreads with stimulus strength.

Superantigens (Sags) induce large-scale stimulation of T lymphocytes by a mechanism distinct from conventional antigen presentation, involving direct MHC binding and stimulation of TCR families based on Vbeta gene usage. The specific Vbeta targets of a given Sag have, since the earliest studies in murine models, been considered a hallmark of that toxin. Bacterial Sags are implicated in the aetiology of a wide range of human diseases, although their role has been most clearly defined in toxic shock syndrome. While Sags have been defined by the Vbeta-specific changes in T cell repertoire they induce, human studies of in vitro stimulation or analysis of cells from infected patients have produced inconsistent findings. Here we have evaluated the contribution of HLA allelic polymorphisms and strength of stimulus to this response. We show that there are differences in binding and presentation of the staphylococcal Sag, staphylococcal enterotoxin A (SEA), by different HLA-DR alleles. We also show that the TCR Vbeta response, previously thought to be a fixed property defining a given Sag, varies with stimulus strength such that a broader repertoire of response is seen at higher concentrations or following presentation by high-binding class II types. Responses of human Vbeta8 and Vbeta1 to SEA, Vbeta5 to SEB and of Vbeta12 and Vbeta13 to streptococcal pyrogenic exotoxin A are absolutely dependent on stimulus strength. These findings have important implications for heterogeneity in the response to Sags and the consequent differences in susceptibility to severe toxic shock.

Antigen Presentation↗

PubMed2005

The human chorionic gonadotropin-beta arginine68 to glutamic acid substitution fixes the conformation of the C-terminal peptide.

Wild-type human chorionic gonadotropin (hCG) has been used as a contraceptive vaccine. However, extensive sequence homology with LH elicits production of cross-reactive antibodies. Substitution of arginine(68) of the beta-subunit (hCG(beta)) with glutamic acid (R68E) profoundly reduces the cross-reactivity while refocusing the immune response to the hCG(beta)-specific C-terminal peptide (CTP). To investigate the molecular basis for this change in epitope usage, we immunized mice with a plasmid encoding a truncated hCG(beta)-R68E chain lacking the CTP. The animals produced LH-cross-reactive antibodies, suggesting that the refocused immunogenicity of R68E is a consequence of epitope masking by a novel disposition of the CTP in the mutant rather than a structural change in the cross-reactive epitope region. This explanation was strongly supported by surface plasmon resonance analysis using a panel of anti-hCG(beta)-specific and anti-hCG(beta)/LH cross-reactive monoclonal antibodies (mAbs). Whereas the binding of the LH cross-reactive mAbs to hCG(beta)-R68E was eliminated, mAbs reacting with hCG(beta)-specific epitopes bound to hCG(beta) and hCG(beta)-R68E with identical affinities. In a separate series of experiments, we observed that LH cross-reactive epitopes were silent after immunization with a plasmid encoding a membrane form of hCG(beta)-R68E, as previously observed with the soluble mutant protein itself. In contrast, the plasmid encoding the soluble secreted form of hCG(beta)-R68E evoked LH cross-reactive antibodies, albeit of relatively low titer, suggesting that the handling and processing of the proteins produced by the two constructs differed.

Amino Acid Substitution↗

PubMed2005

The expression and function of cathepsin E in dendritic cells.

Cathepsin E is an aspartic proteinase that has been implicated in Ag processing within the class II MHC pathway. In this study, we document the presence of cathepsin E message and protein in human myeloid dendritic cells, the preeminent APCs of the immune system. Cathepsin E is found in a perinuclear compartment, which is likely to form part of the endoplasmic reticulum, and also a peripheral compartment just beneath the cell membrane, with a similar distribution to that of Texas Red-dextran within 2 min of endocytosis. To investigate the function of cathepsin E in processing, a new soluble targeted inhibitor was synthesized by linking the microbial aspartic proteinase inhibitor pepstatin to mannosylated BSA via a cleavable disulfide linker. This inhibitor was shown to block cathepsin D/E activity in cell-free assays and within dendritic cells. The inhibitor blocked the ability of dendritic cells from wild-type as well as cathepsin D-deficient mice to present intact OVA, but not an OVA-derived peptide, to cognate T cells. The data therefore support the hypothesis that cathepsin E has an important nonredundant role in the class II MHC Ag processing pathway within dendritic cells.

Animals↗

PubMed2004

DNA immunization with plasmids expressing hCGbeta-chimeras.

Human chorionic gonadotropin has been used as an anti-fertility vaccine and as a target for cancer immunotherapy. We have explored the use of DNA immunization with the aim of improving the immunogenicity of this hormone. Stimulating the muscle with electric pulses following intramuscular injection of plasmids expressing hCGbeta resulted in higher levels of human chorionic gonadotropin (hCG)-specific antibodies, which could be further enhanced following a protein boost with hCG mixed with adjuvant. DNA vaccines encoding a membrane attached or a secreted form of hCGbeta produced similar-albeit relatively modest-antibody responses. Providing hCGbeta with additional T cell help by vaccinating with a plasmid encoding a hCGbeta-hFc fusion protein did not further increase the antibody levels in the immunized animals. However, immunization of mice with a construct encoding hCGbeta fused to C3d(3) produced significantly lower antibody levels relative to mice immunized with the hCGbeta-alone expression plasmid, even though the hCGbeta-C3d(3) chimera was expected to facilitate cross-linking of the antigen-specific B-cell receptor and CR2 thereby lowering the threshold of activation. Thus the limiting factor determining the antibody levels following hCGbeta immunization, at least for DNA immunization, is related to the amount of protein available rather than the form of protein produced or lack of T cell epitopes.

Adjuvants, Immunologic↗