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Thyroid stimulation does not require antibodies with identical epitopes but does involve recognition of a critical conformation at the N terminus of the thyrotropin receptor A-subunit.
No full textWhether monoclonal antibodies with thyroid-stimulating activity [thyroid-stimulating antibody/antibodies (TSAb)] from immunized animals are identical to human autoantibodies in Graves' disease is unknown. Here, we compared properties of a monoclonal hamster TSAb (MS-1) with human autoantibodies. The epitopes of neither MS-1 nor human autoantibodies can be determined by peptide scanning, indicating their conformational nature. A property of human TSAb is that their epitope is partially obscured on the TSH holoreceptor on the cell surface relative to the TSH receptor (TSHR) ectodomain tethered to the membrane by a glycosylphosphatidyl inositol anchor. On flow cytometry, as for human autoantibodies, MS-1 preferentially recognized the glycosylphosphatidyl inositol-anchored ectodomain vs. the TSH holoreceptor on Chinese hamster ovary cells. Also, as with human autoantibodies, only A-subunits with the active (but not the inactive) conformation adsorbed MS-1 binding activity. This difference localizes antibody binding to a cysteine-rich region at the TSHR N terminus. Remarkably, active TSHR A-subunit more effectively ( approximately 40-fold) neutralized human autoantibodies than it did MS-1. Therefore, MS-1 interacts less well than autoantibodies with the free A-subunit. In summary, we provide evidence that TSAb need not have identical epitopes. However, the TSAb epitope does appear to require involvement of the highly conformational N terminus of the A-subunit
Evidence that the thyrotropin receptor protease is membrane-associated and is not within lipid rafts
No full textThe thyrotropin receptor (TSHR) cleaves to a variable extent within the ectodomain into a ligand-binding A subunit linked by disulfide bonds to the largely transmembrane B subunit. To obtain insight into this variability, we examined the extent of cleavage of TSHR ectodomains tethered to the plasma membrane by different means: (1) the wild-type, serpentine region, (2) a glycosylphosphatidylinositol (GPI) anchor, and (3) a single CD8alpha transmembrane region. For this purpose, we covalently cross-linked(125)I-TSH to the TSHR ectodomain expressed on the surface of intact cell monolayers. The extent of cleavage of the CD8alpha-tethered ectodomain was similar to the wild-type TSHR (approximately 50%) whereas the same ectodomain with a GPI anchor remained almost entirely (approximately 90%) uncleaved. These findings have three possible implications. First, differential cleavage of the TSHR ectodomain depending on its attachment to the plasma membrane suggests that the TSHR protease is membrane-associated and is not a soluble (secreted or shed) protease. Second, because GPI-anchored proteins (unlike CD8alpha) segregate in membrane lipid rafts, the TSHR protease appears not to be associated with lipid rafts. Finally, the similar extent of cleavage of the wild-type TSHR and the CD8alpha (not the GPI) tethered ectodomain supports the concept that the wild-type TSHR resides largely outside lipid rafts
Progression of autoimmune damage in primary biliary cirrhosis: an immunohistochemical study.
No full textAberrant MHC Class II antigen expression and the nature of the infiltrating lymphoid cells were studied by immunohistochemical techniques in liver biopsies from 37 patients with Primary biliary cirrhosis (PBC) (11 histological stage I, 13 stage II-III, 13 stage IV) and 15 patients with chronic non autoimmune liver disease. Bile duct epithelial cells expressed HLA-DR, DP and DQ antigens in biopsies from patients with early (Stage I) PBC and less frequently in the late cirrhotic phases of the disease (Stage IV); these observations support the hypothesis that induction of Class II antigens on epithelial cells may be involved in initiating autoimmune responses towards bile duct components. The presence of cytotoxic/suppressor T cells around the bile ducts in Stage I suggests a role for cell mediated destruction of the ducts at this early stage. The nature of the chronic inflammatory cell infiltrate in the portal tracts, periportal areas and lobular parenchyma does not establish the mechanism(s) involved in disease progression. However, the lack of Class II antigen expression on hepatocytes is compatible with the hypothesis that hepatocellular damage is non-specific and may be secondary to the initial bile duct injury
Thyroid-stimulating autoantibodies in Graves disease preferentially recognize the free A subunit, not the thyrotropin holoreceptor.
No full textGraves disease is directly caused by thyroid-stimulating autoantibodies (TSAb's) that activate the thyrotropin receptor (TSHR). We observed upon flow cytometry using intact cells that a mouse mAb (3BD10) recognized the TSHR ectodomain with a glycosidylphosphatidylinositol (ECD-GPI) anchor approximately tenfold better than the same ectodomain on the wild-type TSHR, despite the far higher level of expression of the latter. The 3BD10 epitope contains the N-terminal cysteine cluster critical for TSAb action. Consequently, we hypothesized and confirmed that TSAb (but not thyrotropin-blocking autoantibodies [TBAb's]) also poorly recognize the wild-type TSHR relative to the ECD-GPI. Despite poor recognition by TSAb of the holoreceptor, soluble TSHR A subunits (known to be shed from surface TSHR) fully neutralized autoantibody-binding activity. These data indicate that the epitope(s) for TSAb's, but not for TBAb's, are partially sterically hindered on the holoreceptor by the plasma membrane, the serpentine region of the TSHR, or by TSHR dimerization. However, the TSAb epitope on the soluble A subunit is freely accessible. This observation, as well as other evidence, supports the concept that A subunit shedding either initiates or amplifies the autoimmune response to the TSHR, thereby causing Graves disease in genetically susceptible individuals
Why thyroid is major site of thyroid autoantibody synthesis in autoimmune thyroid disease.
No full textEvidence that shed thyrotropin receptor A subunits drive affinity maturation of autoantibodies causing Graves' disease.
No full textCONTEXT:
In Graves' disease, thyroid-stimulating antibodies (TSAb) activate the TSH receptor (TSHR) causing hyperthyroidism. Serum polyclonal TSAb are difficult to study because of their extremely low serum levels.
OBJECTIVE:
Our objective was to determine whether monoclonal TSAb possess characteristics previously reported for polyclonal autoantibodies in Graves' sera.
DESIGN:
We studied monoclonal TSAb from three laboratories: six generated from mice with induced hyperthyroidism; and one, M22, a human autoantibody obtained from Graves' B cells.
RESULTS:
All TSAb with one exception were potent activators of TSHR-mediated cAMP generation, with relatively similar half-maximal stimulatory concentrations. Like polyclonal autoantibodies, monoclonal TSAb were largely neutralized by conformationally "active" (but not "inactive") recombinant TSHR A subunits (the N-terminal cleavage product of the TSHR). Chimeric substitutions of TSHR amino acids 25-30 (the extreme N terminus after removal of the 21 residue signal peptide) abrogated the binding and function of all monoclonal TSAb but with one antibody (TSAb4) revealing a nonidentical epitope. Remarkably, these residues are uninvolved in the M22 epitope determined by x-ray analysis. Finally, flow-cytometric dose-response analyses, not previously possible with polyclonal TSAb, revealed that all monoclonal TSAb, human and murine, bound with lower affinity to their in vivo target, the TSH-holoreceptor, than to the isolated TSHR ectodomain.
CONCLUSIONS:
TSAb function does not require antibodies with identical epitopes, and human autoantibody M22 may, therefore, not represent the full epitopic repertoire of polyclonal TSAb in Graves' disease. Most important, we provide strong evidence that the shed ectodomain (primarily the A subunit) is the primary antigen driving affinity maturation of TSAb producing B cells
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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