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Binding of thyroglobulin (Tg) to the low-density lipoprotein receptor-associated protein (RAP) during the biosynthetic pathway prevents premature Tg interactions with sortilin
No full textSortilin, a Vps10p family member, is expressed by thyroid epithelial cells (TEC), where it binds to internalized thyroglobulin (Tg) molecules. Premature binding of Tg to sortilin during biosynthesis may cause intracellular retention of Tg. Such a premature interaction may be prevented by one or more inhibitor/s. Because both sortilin and Tg bind to the low-density lipoprotein receptor-associated protein (RAP), we investigated whether RAP serves such a function
Intracellular retention of thyroglobulin in the absence of the low-density lipoprotein receptor-associated protein (RAP) is likely due to premature binding to megalin in the biosynthetic pathway
No full textObjective: The low-density lipoprotein receptor associated protein (RAP) is expressed by thyroid epithelial cells (TEC) in a TSH-dependent manner. In the thyroid RAP functions as a molecular chaperone for the thyroglobulin (Tg) endocytic receptor megalin/LRP2, which is retained intracellularly in RAP KO mice rather than being expressed on the apical membrane of TEC, its usual location. RAP binds also to Tg, which is also retained intracellularly in RAP KO mice, thereby suggesting a role of RAP in Tg secretion. Here we investigated whether Tg intracellular retention in the absence of RAP is due to premature Tg-megalin interactions during the biosynthetic pathway or to a direct action of RAP on Tg secretion. Methods: We performed immunoprecipitation experiments in thyroid extracts from RAP KO and WT mice. In addition, we investigated Tg secretion in COS-7 cells co-transfected with human RAP (hRAP) and mouse Tg (mTg). Results: An anti-megalin megalin precipitated greater amounts of Tg in thyroid extracts from RAP KO than from WT mice, suggesting increased intracellular interactions between megalin and Tg in the absence of RAP. COS-7 cells transiently transfected with hRAP, mTg or both, expressed the two proteins accordingly. RAP was found almost exclusively in cell extracts, whereas Tg was found both in extracts and media, as expected from the knowledge that RAP is ER-resident and that Tg is secreted. Regardless of whether cells were transfected with mTg alone or were co-transfected with hRAP, similar proportions of the total Tg synthesized were detected in cell extracts and media. Conclusions: The intracellular retention of Tg in the absence of RAP is likely due to its premature interaction with megalin, whereas RAP does not seem to affect Tg secretion directly
Antioxidant effects of β-carotene, but not of retinol and vitamin E, in orbital fibroblasts from patients with Graves' orbitopathy (GO)
No full textBackground: Oxidative stress is involved in the pathogenesis of Graves' orbitopathy (GO) and several antioxidant agents, namely, selenium, quercetin, enalapril, vitamin C, N-acetyl-l-cysteine, and melatonin, have been shown to reduce oxidative stress and its consequences in primary culture of orbital fibroblasts. In addition, selenium is effective for the treatment of mild GO. Here, we investigated the action of three additional antioxidants in orbital fibroblasts, namely, retinol, Î2-carotene, and vitamin E. Methods: Primary cultures of orbital fibroblasts were established from GO patients and control subjects. To induce oxidative stress, cells were treated with H2O2, after which glutathione disulfide (GSSG) (a parameter of oxidative stress), cell proliferation, hyaluronic acid, TNFα, IFNÎ3, and IL1Î2 were measured. Results: H2O2-dependent oxidative stress (augmented GSSG) was associated with increased cell proliferation and cytokine release. All the three antioxidant substances reduced GSSG in both GO and control fibroblasts.β-carotene reduced proliferation in GO, but not in control fibroblasts. IL1Î2 was reduced by all three substances. Retinol reduced IFNÎ3 in GO and control fibroblasts. Conclusions: Our study supports an antioxidant role of retinol, β-carotene, and vitamin E in orbital fibroblasts from patients with GO and provides a basis for a possible clinical use these substances
Action of three bioavailable antioxidants in orbital fibroblasts from patients with Graves’ orbitopathy (GO): a new frontier for GO treatment?
No full textObjective: Oxidative stress is involved in the pathogenesis of Gravesâ orbitopathy (GO) and an antioxidant approach has been advocated for GO treatment. Here, we investigated the action of three antioxidants in orbital fibroblasts, namely, vitamin C, N-acetyl-l-cysteine, and melatonin. Methods: Primary cultures of orbital fibroblasts from six GO patients and six control subjects were established. Cells were treated with H2O2 to induce oxidative stress. Cell vitality assays were performed to determine the non-cytotoxic dose of each antioxidant. The following assays were performed: glutathione disulfide (GSSG), as a measure of oxidative stress, cell proliferation, hyaluronic acid (HA), TNFα, IFNÎ3, and IL1Î2. Results: H2O2 induced oxidative stress (augmented GSSG), increased cell proliferation as well as cytokine release, but did not affect HA release. All of the three antioxidant substances reduced H2O2-dependent oxidative stress. Vitamin C reduced proliferation in GO, but not in control fibroblasts. N-acetyl-l-cysteine reduced proliferation and IFNÎ3 in GO, and HA and IL1Î2 in both GO and control fibroblasts. Melatonin reduced IL1Î2 and HA in GO and control fibroblasts, and IFNÎ3 only in GO fibroblasts. Conclusions: Our study provides evidence in support of an antioxidant role of vitamin C, N-acetyl-l-cysteine and melatonin in orbital fibroblasts. Some of the effects of these compounds are exclusive to GO fibroblasts, whereas some other are observed also in control fibroblasts. Our observations provide a basis for a possible clinical use of these substances in patients with GO
Genetic Profiling of Orbital Fibroblasts from Patients with Graves' Orbitopathy
No full textContext: Graves' orbitopathy (GO) is an autoimmune disease that persists when immunosuppression is achieved. Orbital fibroblasts from GO patients display peculiar phenotypes even if not exposed to autoimmunity, possibly reflecting genetic or epigenetic mechanisms, which we investigated here. Objective: We aimed to explore potential genetic or epigenetic differences using primary cultures of orbital fibroblasts from GO and control patients. Methods: Cell proliferation, hyaluronic acid (HA) secretion, and HA synthases (HAS) were measured. Next-generation sequencing and gene expression analysis of the whole genome were performed, as well as real-time-PCR of selected genes and global DNA methylation assay on orbital fibroblasts from 6 patients with GO and 6 control patients from a referral center. Results: Cell proliferation was higher in GO than in control fibroblasts. Likewise, HA in the cell medium was higher in GO fibroblasts. HAS-1 and HAS-2 did not differ between GO and control fibroblasts, whereas HAS-3 was more expressed in GO fibroblasts. No relevant gene variants were detected by whole-genome sequencing. However, 58 genes were found to be differentially expressed in GO compared with control fibroblasts, and RT-PCR confirmed the findings in 10 selected genes. We postulated that the differential gene expression was related to an epigenetic mechanism, reflecting diverse DNA methylation, which we therefore measured. In support of our hypothesis, global DNA methylation was significantly higher in GO fibroblasts. Conclusions: We propose that, following an autoimmune insult, DNA methylation elicits differential gene expression and sustains the maintenance of GO
Serum antibodies against the insulin-like growth factor-1 receptor (IGF-1R) in Graves’ disease and Graves’ orbitopathy
No full textBackground: A role of the insulin-like growth factor-1 receptor (IGF-1R) in the pathogenesis of Graves’ orbitopathy (GO) has been proposed, but the existence and function of anti-IGF-1R-antibodies (IGF-1R-Abs) are debated. Methods: We designed a cross-sectional investigation to measure serum IGF-1R-Abs by a commercial assay in consecutive patients with Graves’ disease (GD) compared with healthy subjects and patients with autoimmune thyroiditis (AT). A total of 134 subjects were screened including 27 healthy subjects, 80 GD patients (54 of whom with GO), and 27 AT patients. The main outcome measure was the prevalence of positive serum IGF-1R-Abs in GO, compared with GD without GO and with the other study groups. Results: Having established a cut-off value at 55.2 ng/ml for positive tests, positive IGF-1R-Abs were more frequent in GD (25%), than in AT (3.7%, P = 0.003) and healthy subjects (0%, P = 0.006). Within GD, there was no difference between patients with or without GO. Serum levels of IGF-1R-Abs differed across the study population (P < 0.0001), reflecting their higher concentrations in GD (P < 0.0001 vs both AT and healthy subjects), but with no difference between patients with or without GO. In patients with GO, there was an inverse correlation between serum IGF-1R-Abs and CAS (R = − 0.376, 95% CI: from − 0.373 to − 0.631; P = 0.005), the significance of which remains to be investigated. Conclusions: Serum autoantibodies against the IFG-1R are present in one-fourth of GD patients, regardless of the presence of GO. Further functional studies are needed to investigate the significance of their inverse correlation with GO activity
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe 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
Get PDF“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
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