Plasma exosomes from patients with active thyroid-associated orbitopathy induce inflammation and fibrosis in orbital fibroblasts.

BACKGROUND: The pathogenesis of thyroid-associated orbitopathy (TAO) remains incompletely understand. The interaction between immunocytes and orbital fibroblasts (OFs) play a critical role in orbital inflammatory and fibrosis. Accumulating reports indicate that a significant portion of plasma exosomes (Pla-Exos) are derived from immune cells; however, their impact upon OFs function is unclear. METHODS: OFs were primary cultured from inactive TAO patients. Exosomes isolated from plasma samples of patients with active TAO and healthy controls (HCs) were utilized for functional and RNA cargo analysis. Functional analysis in thymocyte differentiation antigen-1+ (Thy-1+) OFs measured expression of inflammatory and fibrotic markers (mRNAs and proteins) and cell activity in response to Pla-Exos. RNA cargo analysis was performed by RNA sequencing and RT-qPCR. Thy-1+ OFs were transfected with miR-144-3p mimics/inhibitors to evaluate its regulation of inflammation, fibrosis, and proliferation. RESULTS: Pla-Exos derived from active TAO patients (Pla-ExosTAO-A) induced stronger production of inflammatory cytokines and hyaluronic acid (HA) in Thy-1+ OFs while inhibiting their proliferation. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and single sample gene set enrichment analysis (ssGSEA) suggested that the difference in mRNA expression levels between Pla-ExosTAO-A and Pla-ExosHC was closely related to immune cells. Differential expression analysis revealed that 62 upregulated and 45 downregulated miRNAs in Pla-ExosTAO-A, with the elevation of miR-144-3p in both Pla-Exos and PBMCs in active TAO group. KEGG analysis revealed that the target genes of differentially expressed miRNA and miR-144-3p enriched in immune-related signaling pathways. Overexpression of the miR-144-3p mimic significantly upregulated the secretion of inflammatory cytokines and HA in Thy-1+ OFs while inhibiting their proliferation. CONCLUSION: Pla-Exos derived from patients with active TAO were immune-active, which may be a long-term stimulus casual for inflammatory and fibrotic progression of TAO. Our finding suggests that Pla-Exos could be used as biomarkers or treatment targets in TAO patients.

A case of bilateral amyloidosis localized to extraocular muscles mimicking thyroid eye disease.

BACKGROUND: Amyloidosis is a rare condition characterized by the abnormal deposition of amyloid proteins in various tissues and organs. While systemic amyloidosis has been well-documented, amyloid deposition in extraocular muscles is an exceptionally rare occurrence, with only 35 reported cases. This case report sheds light on the importance of considering amyloidosis in the differential diagnosis of patients presenting with proptosis and diplopia, which are often associated with thyroid eye disease. CASE PRESENTATION: A woman in her twenties sought medical attention due to a complaint of diplopia. Her ocular examination revealed almost normal findings except for exotropia and proptosis. Orbital magnetic resonance imaging displayed fusiform enlargement of nearly all eye muscles, a presentation typically observed in thyroid eye disease. However, despite corticosteroid therapy, her symptoms showed no improvement. Given the unusual lack of response to conventional treatment, and inhomogeneous enhancement of the muscle, an extraocular muscle biopsy was conducted. This biopsy yielded a unique finding-amyloid deposition within the muscle tissue. This discovery was particularly intriguing due to the extreme rarity of amyloidosis affecting extraocular muscles, with fewer than three dozen documented cases worldwide. CONCLUSION: This unique case underscores the critical need for a comprehensive approach to diagnosing patients with proptosis and diplopia. While these symptoms are commonly attributed to thyroid eye disease, it is essential to consider alternative diagnoses such as amyloidosis, especially when standard treatments fail to yield results. The discovery of amyloid deposition in the extraocular muscles, although exceedingly rare, emphasizes the significance of a thorough differential diagnosis. In conclusion, this case report highlights the importance of vigilance in clinical practice, encouraging ophthalmologists to explore less common diagnostic possibilities when faced with challenging cases. Further research and clinical investigation are warranted to better understand the mechanisms and potential treatments for amyloidosis affecting the extraocular muscles.

Development and application of animal models to study thyroid-associated ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO), also known as Graves' ophthalmopathy, is an autoimmune disease that is usually accompanied by hyperthyroidism. Its pathogenesis involves the activation of autoimmune T lymphocytes by a cross-antigen reaction of thyroid and orbital tissues. The thyroid-stimulating hormone receptor (TSHR) is known to play an important role in the development of TAO. Because of the difficulty of orbital tissue biopsy, the establishment of an ideal animal model is important for developing novel clinical therapies of TAO. To date, TAO animal modeling methods are mainly based on inducing experimental animals to produce anti-thyroid-stimulating hormone receptor antibodies (TRAbs) and then recruit autoimmune T lymphocytes. Currently, the most common methods are hTSHR-A subunit plasmid electroporation and hTSHR-A subunit adenovirus transfection. These animal models provide a powerful tool for exploring the internal relationship between local and systemic immune microenvironment disorders of the TAO orbit, facilitating the development of new drugs. However, existing TAO modeling methods still have some defects, such as low modeling rate, long modeling cycles, low repetition rate, and considerable differences from human histology. Hence, the modeling methods require further innovation, improvement, and in-depth exploration.

Ceruloplasmin regulating fibrosis in orbital fibroblasts provides a novel therapeutic target for Graves' orbitopathy.

PURPOSE: In diagnosing the pathogenesis of Graves' orbitopathy (GO), there is a growing interest in fibrosis generated by orbital fibroblasts (OFs); nevertheless, the involvement of ceruloplasmin (CP) in OFs remains unknown. METHODS: Differentially expressed genes (DEGs) were identified through bioinformatic analysis. OFs were isolated from orbital tissue and identified with immunofluorescent staining. The levels of DEGs were validated in GO tissue samples and TGF-ß-challenged OFs, and CP was selected for the following laboratory investigations. CP overexpression or knockdown was achieved, and cell viability and fibrosis-associated proteins were investigated to assess the cell phenotype and function. Signaling pathways were subsequently investigated to explore the mechanism of CP function in OFs. RESULTS: CP and cathepsin C (CTSC) are two overlapped DEGs in GSE58331 and GSE105149. OFs were isolated and identified through fibrotic biomarkers. CP and CTSC were downregulated in GO tissue samples and TGF-ß-challenged OFs. CP overexpression or knockdown was achieved in OFs by transducing a CP overexpression vector or small interfering RNA against CP (si1-CP or si2-CP) and verified using a qRT-PCR. CP overexpression inhibited cell viability and reduced the levels of α-SMA, vimentin, fibronectin, and collagen I, whereas CP knockdown exerted opposite effects on OFs. CP overexpression inhibited the phosphorylation of Smad3, Erk1/2, p38, JNK, and AKT; conversely, CP knockdown exerted opposite effects on the phosphorylation of factors mentioned above. CONCLUSION: CP was downregulated in GO and suppressed the expression of fibrosis-associated proteins in both GO and normal OFs. CP might serve as a promising therapeutic agent in the treatment regimens for GO.

The role and molecular mechanism of gut microbiota in Graves' orbitopathy.

PURPOSE: Graves' orbitopathy (GO) is an autoimmune orbital disorder. Gut microbiota dysfunction plays a vital role in autoimmune diseases, including Graves' disease (GD) and GO. In the present study, we aimed to investigate the change of gut microbiota in GD/GO using mouse model. METHODS: The murine model of GD/GO was established by the challenge of adenovirus expressing thyroid-stimulating hormone (TSH) receptor (TSHR) (Ad-TSHR). The histological changes of orbital and thyroid tissues were analyzed by hematoxylin and eosin (H&E), Masson staining, and immunohistochemistry (IHC) staining. The fecal samples were collected for 16S rRNA gene sequencing and bioinformatics analysis. RESULTS: The GD/GO model was established successfully, as manifested as the broadened eyelid, exophthalmia and conjunctive redness, severe inflammatory infiltration among thyroid glands and between extraocular muscle space, hypertrophic extraocular muscles, elevated thyroxine (T4) and decreased TSH, and positive CD34, CD40, collagen I, and α-SMA staining. A total of 222 operational taxonomic units (OUTs) were overlapped between mice in the Ad-NC and Ad-TSHR groups. The microbial composition of the samples in the two groups was mainly Bacteroidia and Clostridia, and the Ad-NC group had a significantly lower content of Bacteroidia and higher content of Clostridia. KEGG orthology analysis results revealed differences in dehydrogenase, aspartic acid, bile acid, chalcone synthase, acetyltransferase, glutamylcyclotransferase, glycogenin, and 1-phosphatidylinositol-4-phosphate 5-kinase between two groups; enzyme commission (EC) analysis results revealed differences in several dehydrogenase, oxidase, thioxy/reductase between two groups; MetaCyc pathways analysis results revealed differences in isoleucine degradation, oxidation of C1 compounds, tricarboxylic acid (TCA) cycle IV, taurine degradation, and biosynthesis of paromamine, heme, colonic acid building blocks, butanediol, lysine/threonine/methionine, and histidine/purine/pyrimidine between two groups. CONCLUSION: This study induced a mouse model of GD/GO by Ad-TSHR challenge, and gut microbiota characteristics were identified in the GD/GO mice. The Bacteroidia and Clostridia abundance was changed in the GD/GO mice. These findings may lay a solid experimental foundation for developing personalized treatment regimens for GD patients according to the individual gut microbiota. Given the potential impact of regional differences on intestinal microbiota, this study in China may provide a reference for the global overview of the gut-thyroid axis hypothesis.

Traditional Chinese medicine in thyroid-associated orbitopathy.

PURPOSE: Orbital fibroblasts (OF) are considered the central target cells in the pathogenesis of thyroid-associated orbitopathy (TAO), which comprises orbital inflammation, orbital tissue edema, adipogenesis, fibrosis, oxidative stress and autophagy. Certain active ingredients of traditional Chinese medicine (TCM) demonstrated inhibition of TAO-OF in pre-clinical studies and they could be translated into novel therapeutic strategies. METHODS: The pertinent and current literature of pre-clinical studies on TAO investigating the effects of active ingredients of TCM was reviewed using the NCBI PubMed database. RESULTS: Eleven TCM compounds demonstrated inhibition of TAO-OF in-vitro and three of them (polydatin, curcumin, and gypenosides) resulted in improvement in TAO mouse models. Tanshinone IIA reduced inflammation, oxidative stress and adipogenesis. Both resveratrol and its precursor polydatin displayed anti-oxidative and anti-adipogenic properties. Celastrol inhibited inflammation and triptolide prevented TAO-OF activation, while icariin inhibited autophagy and adipogenesis. Astragaloside IV reduced inflammation via suppressing autophagy and inhibited fat accumulation as well as collagen deposition. Curcumin displayed multiple actions, including anti-inflammatory, anti-oxidative, anti-adipogenic, anti-fibrotic and anti-angiogenic effects via multiple signaling pathways. Gypenosides reduced inflammation, oxidative stress, tissue fibrosis, as well as oxidative stress mediated autophagy and apoptosis. Dihydroartemisinin inhibited OF proliferation, inflammation, hyaluronan (HA) production, and fibrosis. Berberine attenuated inflammation, HA production, adipogenesis, and fibrosis. CONCLUSIONS: Clinical trials of different phases with adequate power and sound methodology will be warranted to evaluate the appropriate dosage, safety and efficacy of these compounds in the management of TAO.

Magnetic resonance imaging parameters on lacrimal gland in thyroid eye disease: a systematic review and meta-analysis.

BACKGROUND: Thyroid eye disease is an extrathyroidal manifestation of Graves' disease and is associated with dry eye disease. This is the first systematic review and meta-analysis to evaluate the role of magnetic resonance imaging lacrimal gland parameters in thyroid eye disease diagnosis, activity grading, and therapeutic responses prediction. METHODS: Up to 23 August, 2022, 504 studies from PubMed and Cochrane Library were analyzed. After removing duplicates and imposing selection criteria, nine eligible studies were included. Risk of bias assessment was done. Meta-analyses were performed using random-effect model if heterogeneity was significant. Otherwise, fixed-effect model was used. Main outcome measures include seven structural magnetic resonance imaging parameters (lacrimal gland herniation, maximum axial area, maximum coronal area, maximum axial length, maximum coronal length, maximum axial width, maximum coronal width), and three functional magnetic resonance imaging parameters (diffusion tensor imaging-fractional anisotropy, diffusion tensor imaging-apparent diffusion coefficient or mean diffusivity, diffusion-weighted imaging-apparent diffusion coefficient). RESULTS: Thyroid eye disease showed larger maximum axial area, maximum coronal area, maximum axial length, maximum axial width, maximum coronal width, diffusion tensor imaging-apparent diffusion coefficient/ mean diffusivity, and lower diffusion tensor imaging-fractional anisotropy than controls. Active thyroid eye disease showed larger lacrimal gland herniation, maximum coronal area, diffusion-weighted imaging-apparent diffusion coefficient than inactive. Lacrimal gland dimensional (maximum axial area, maximum coronal area, maximum axial length, maximum axial width, maximum coronal width) and functional parameters (diffusion tensor imaging-apparent diffusion coefficient, diffusion tensor imaging-apparent diffusion coefficient) could be used for diagnosing thyroid eye disease; lacrimal gland herniation, maximum coronal area, and diffusion-weighted imaging-apparent diffusion coefficient for differentiating active from inactive thyroid eye disease; diffusion tensor imaging parameters (diffusion tensor imaging-fractional anisotropy, diffusion tensor imaging-mean diffusivity) and lacrimal gland herniation for helping grading and therapeutic responses prediction respectively. CONCLUSIONS: Magnetic resonance imaging lacrimal gland parameters can detect active thyroid eye disease and differentiate thyroid eye disease from controls. Maximum coronal area is the most effective indicator for thyroid eye disease diagnosis and activity grading. There are inconclusive results showing whether structural or functional lacrimal gland parameters have diagnostic superiority. Future studies are warranted to determine the use of magnetic resonance imaging lacrimal gland parameters in thyroid eye disease.

Inflammation and Fibrosis in Orbital Inflammatory Disease: A Histopathologic Analysis.

PURPOSE: The purpose of this study was to compare the histopathologic inflammation and fibrosis of orbital adipose tissue in orbital inflammatory disease (OID) specimens. METHODS: In this retrospective cohort study, inflammation, and fibrosis in orbital adipose tissue from patients with thyroid-associated orbitopathy (TAO), granulomatosis with polyangiitis (GPA), sarcoidosis, nonspecific orbital inflammation (NSOI), and healthy controls were scored by 2 masked ocular pathologists. Both categories were scored on a scale of 0 to 3 with scoring criteria based on the percentage of specimens containing inflammation or fibrosis, respectively. Tissue specimens were collected from oculoplastic surgeons at 8 international centers representing 4 countries. Seventy-four specimens were included: 25 with TAO, 6 with orbital GPA, 7 with orbital sarcoidosis, 24 with NSOI, and 12 healthy controls. RESULTS: The mean inflammation and fibrosis scores for healthy controls were 0.0 and 1.1, respectively. Orbital inflammatory disease groups' inflammation (I) and fibrosis (F) scores, formatted [I, F] with respective p -values when compared to controls, were: TAO [0.2, 1.4] ( p = 1, 1), GPA [1.9, 2.6] ( p = 0.003, 0.009), sarcoidosis [2.4, 1.9] ( p = 0.001, 0.023), and NSOI [1.3, 1.8] ( p ≤ 0.001, 0.018). Sarcoidosis had the highest mean inflammation score. The pairwise analysis demonstrated that sarcoidosis had a significantly higher mean inflammation score than NSOI ( p = 0.036) and TAO ( p < 0.0001), but no difference when compared to GPA. GPA had the highest mean fibrosis score, with pairwise analysis demonstrating a significantly higher mean fibrosis score than TAO ( p = 0.048). CONCLUSIONS: Mean inflammation and fibrosis scores in TAO orbital adipose tissue samples did not differ from healthy controls. In contrast, the more "intense" inflammatory diseases such as GPA, sarcoidosis, and NSOI did demonstrate higher histopathologic inflammation and fibrosis. This has implications in prognosis, therapeutic selection, and response monitoring in orbital inflammatory disease.

Prostaglandin F2α Regulates Adipogenesis by Modulating Extracellular Signal-Regulated Kinase Signaling in Graves' Ophthalmopathy.

Prostaglandin F2α (PGF2α), the first-line anti-glaucoma medication, can cause the deepening of the upper eyelid sulcus due to orbital lipoatrophy. However, the pathogenesis of Graves' ophthalmopathy (GO) involves the excessive adipogenesis of the orbital tissues. The present study aimed to determine the therapeutic effects and underlying mechanisms of PGF2α on adipocyte differentiation. In this study primary cultures of orbital fibroblasts (OFs) from six patients with GO were established. Immunohistochemistry, immunofluorescence, and Western blotting (WB) were used to evaluated the expression of the F-prostanoid receptor (FPR) in the orbital adipose tissues and the OFs of GO patients. The OFs were induced to differentiate into adipocytes and treated with different incubation times and concentrations of PGF2α. The results of Oil red O staining showed that the number and size of the lipid droplets decreased with increasing concentrations of PGF2α and the reverse transcription-polymerase chain reaction (RT-PCR) and WB of the peroxisome proliferator-activated receptor γ (PPARγ) and fatty-acid-binding protein 4 (FABP4), both adipogenic markers, were significantly downregulated via PGF2α treatment. Additionally, we found the adipogenesis induction of OFs promoted ERK phosphorylation, whereas PGF2α further induced ERK phosphorylation. We used Ebopiprant (FPR antagonist) to interfere with PGF2α binding to the FPR and U0126, an Extracellular Signal-Regulated Kinase (ERK) inhibitor, to inhibit ERK phosphorylation. The results of Oil red O staining and expression of adipogenic markers showed that blocking the receptor binding or decreasing the phosphorylation state of the ERK both alleviate the inhibitory effect of PGF2a on the OFs adipogenesis. Overall, PGF2α mediated the inhibitory effect of the OFs adipogenesis through the hyperactivation of ERK phosphorylation via coupling with the FPR. Our study provides a further theoretical reference for the potential application of PGF2α in patients with GO.

Long-term outcomes in corticosteroid-refractory Graves' orbitopathy treated with tocilizumab.

BACKGROUND: Up to 20% of patients with moderate to severe Graves' orbitopathy (GO) do not respond to high-dose glucocorticoids (GC). A few studies, including a randomized trial, have demonstrated the efficacy of interleukin-6 (IL-6) blockade with tocilizumab (TCZ) in GC-refractory GO. However, data on predictors of response to TCZ and long-term outcomes are lacking. METHODS: Observational single-center study on ten consecutive patients treated with TCZ for GC-refractory GO, between 2016 and 2020. Median (interquartile range) follow-up was 24 (12-36) months. RESULTS: Inflammation and exophthalmos improved dramatically in all patients within months after starting TCZ. Mean Clinical Activity Score decreased from 4.80 ± 1.13 to 0.70 ± 0.82 points at 6 months (mean change: -4.10 ± 1.52; p < .0001). Proptosis improved from 23.2 ± 2.1 to 20.6 ± 2.0 mm at 6 months (mean change: -2.9 ± 1.4 mm; p < .0001). Diplopia resolved in 7 patients. Thyroid receptor antibodies decreased markedly during TCZ treatment. Baseline serum IL-6 levels did not predict clinical response. TCZ was well-tolerated. During follow-up, 3 patients were diagnosed with cancer (breast cancer in 2 and urothelial cancer in 1). CONCLUSIONS: TCZ was rapidly effective and well-tolerated in our patients with GC-refractory GO. Four patients experienced mild/moderate adverse events as neutropenia, hyperlipidemia, and infections; nearly a third developed cancer during the follow-up. The increased incidence observed could be explained by the high prevalence of smokers, that are at higher risk for Graves' orbitopathy and solid malignancies as breast cancer. Thus, regular cancer screening could be proposed to this vulnerable population receiving high doses of immunosuppressants.

MRI in the assessment of thyroid-associated orbitopathy activity.

Management of patients with thyroid-associated orbitopathy (also called Graves' disease) is dependent on the assessment of the disease activity. Evaluation of disease activity is based on ophthalmological examination. Magnetic resonance imaging (MRI) is an auxiliary method that may help quantify the activity and is also helpful in obtaining anatomical information concerning muscle thickness, exophthalmos, or optic neuropathy. We present a review of MRI techniques of the orbits with emphasis on the evaluation of disease activity. The most convincing seems to be the group of T2-weighted techniques such as conventional T2 weighting, T2 relaxometry, and T2 mapping. Dynamic contrast-enhanced MRI is another promising method.

2021 update on thyroid-associated ophthalmopathy.

PURPOSE: Our understanding of thyroid-associated ophthalmopathy (TAO, A.K.A Graves' orbitopathy, thyroid eye disease) has advanced substantially, since one of us (TJS) wrote the 2010 update on TAO, appearing in this journal. METHODS: PubMed was searched for relevant articles. RESULTS: Recent insights have resulted from important studies conducted by many different laboratory groups around the World. A clearer understanding of autoimmune diseases in general and TAO specifically emerged from the use of improved research methodologies. Several key concepts have matured over the past decade. Among them, those arising from the refinement of mouse models of TAO, early stage investigation into restoring immune tolerance in Graves' disease, and a hard-won acknowledgement that the insulin-like growth factor-I receptor (IGF-IR) might play a critical role in the development of TAO, stand out as important. The therapeutic inhibition of IGF-IR has blossomed into an effective and safe medical treatment. Teprotumumab, a ß-arrestin biased agonist monoclonal antibody inhibitor of IGF-IR has been studied in two multicenter, double-masked, placebo-controlled clinical trials demonstrated both effectiveness and a promising safety profile in moderate-to-severe, active TAO. Those studies led to the approval by the US FDA of teprotumumab, currently marketed as Tepezza for TAO. We have also learned far more about the putative role that CD34+ fibrocytes and their derivatives, CD34+ orbital fibroblasts, play in TAO. CONCLUSION: The past decade has been filled with substantial scientific advances that should provide the necessary springboard for continually accelerating discovery over the next 10 years and beyond.

Role of the mononuclear cell infiltrate in Graves' orbitopathy (GO): results of a large cohort study.

OBJECTIVE: The extent to which mononuclear cells and TSH-receptor autoantibodies (TRAb) contribute to Graves' orbitopathy (GO) is not completely defined. Here we investigated the relationship between the immunohistochemical phenotype of orbital infiltrating cells and GO features in a large number of patients. METHODS: We conducted an observational cohort study in 76 consecutive patients with GO (16 men and 60 women) who underwent orbital decompression over a period of 18 consecutive months. An ophthalmological evaluation was performed in all patients, as well as immunohistochemistry for CD3, CD4, CD8, CD56 (T-cell markers), CD25 (T and B-cell marker), CD20, CD19 (B-cell markers), and CD138 (plasmacell marker) in specimens collected at decompressive surgery. RESULTS: Having established cutoff values for each marker, cell infiltrates were found in 60 patients (78.9%; CD3: 39.4%, CD4 55.2%, CD8 50%, CD56: 0%, CD25: 28.9%, CD20: 51.3%, CD19: 25%, CD138: 26.3%). Eleven (14.4%) stained exclusively for CD138 (plasmacells). Patients with CD4-positive mononuclear cells had a significantly greater GO clinical activity score (CAS) (mean difference 1.07, 95% CI - 0.33 to - 1.82, P = 0.004 by univariate, P = 0.05 by multivariate analysis). CAS as well as the remaining GO features were not affected significantly by the mononuclear cell subpopulations in multivariate analyses. CONCLUSIONS: Mononuclear cell infiltrates are present in the majority of GO patients, with a small percentage represented exclusively by plasmacells. CD4 cells exert a major role on GO activity. These findings may represent a further advancement in the comprehension of GO pathogenesis.

A case of Graves' disease presenting with internal ophthalmoplegia during methylmercaptoimidazole treatment.

A 28-year-old Japanese woman positive for TSH receptor antibody and anti-nuclear antibody complained of difficulty seeing nearby objects, severe throbbing retro-orbital pain, diplopia, blepharoptosis and upward gaze palsy when she became hypothyroid during treatment with 30 mg methylmercaptoimidazole for Graves' hyperthyroidism. Brain magnetic resonance imaging revealed slightly swollen bilateral inferior rectus muscles, suggesting the external ophthalmoplegia due to the muscle pathology commonly encountered in Graves' disease. The retro-orbital pain was associated with marked accommodation failure and the pupillary abnormalities. The left and/or right eye showed intermittent, asymmetric and fluctuating mydriasis, being unresponsive to ordinary light but slowly responsive to strong sunlight and slowly responsive in a dark room. During the 5-year period, mydriasis was observed 9 times on both sides, 11 times only on the right side and 4 times only on the left side. Internal ophthalmoplegia with tonic pupils and accommodation failure affecting both the pupillary sphincter muscle and ciliary muscle due to damage to the parasympathetic outflow to these muscles was suggested. Autoimmune mechanism and/or the mechanism underlying channelopathy affecting the ciliary ganglion or short ciliary nerves might be responsible for this fluctuating complication. This very rare panophthalmopathy affecting both external and internal muscles occurred when the patient was suffering from iatrogenic hypothyroidism during the 30 mg methylmercaptimidazole treatment for Graves' disease.

Metformin Attenuates Inflammation and Fibrosis in Thyroid-Associated Ophthalmopathy.

The pathogenesis of thyroid-associated ophthalmopathy (TAO) is still unclear, and therapeutic drugs have great limitations. As metformin has multiple therapeutic effects in many autoimmune diseases, we explored the effects of metformin on TAO in an in vitro fibroblast model. We used orbital connective tissues and fibroblasts that were obtained from TAO patients and normal controls. The activity of adenosine monophosphate-activated protein kinase (AMPK) and the levels of inflammatory or fibrotic factors were examined by immunofluorescence (IF) and immunohistochemistry (IHC). Quantitative real-time polymerase chain reaction (qPCR), cytokine quantification by enzyme-linked immunosorbent sssay (ELISA), IF, and western blotting (WB) were used to measure the expression of factors related to inflammation, fibrosis, and autophagy. To determine the anti-inflammatory and antifibrotic mechanisms of metformin, we pretreated cells with metformin, 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR, an AMPK activator) or compound C (CC, an AMPK inhibitor) for 24 h and used WB to verify the changes in protein levels in the AMPK/mammalian target of rapamycin (mTOR) pathway. We determined that the low activity of AMPK in the periorbital tissue of TAO patients may be closely related to the occurrence and development of inflammation and fibrosis, and metformin exerts multiple effects by activating AMPK in TAO. Furthermore, we suggest that AMPK may be a potential target of TAO therapy.

TGFß-Treated Placenta-Derived Mesenchymal Stem Cells Selectively Promote Anti-Adipogenesis in Thyroid-Associated Ophthalmopathy.

Orbital fibroblasts (OFs) in thyroid-associated ophthalmopathy (TAO) are differentiated from pre-adipocytes and mature adipocytes; increased lipid and fat expansion are the major characteristics of ophthalmic manifestations. Human placental mesenchymal stem cells (hPMSCs) were reported to immunomodulate pathogenesis and suppress adipogenesis in TAO OFs. Here, we prepared transforming growth factor ß (TGFß, 20 ng/mL)-treated hPMSCs (TGFß-hPMSCs) in order to enhance anti-adipogenic effects in vitro and in TAO mice. TAO OFs were grown in a differentiation medium and then co-cultured with hPMSCs or TGFß-hPMSCs. TAO OFs were analyzed via quantitative real-time polymerase chain reaction, Oil red O staining, and western blotting. The results showed that TGFß-hPMSCs reduced the expression of adipogenic, lipogenic, and fibrotic genes better than hPMSCs in TAO OFs. Moreover, the adipose area decreased more in TAO mice injected with TGFß-hPMSCs compared to those injected with hPMSCs or a steroid. Further, TGFß-hPMSCs inhibited inflammation as effectively as a steroid. In conclusion, TGFß-hPMSCs suppressed adipogenesis and lipogenesis in vitro and in TAO mice, and the effects were mediated by the SMAD 2/3 pathways. Furthermore, TGFß-hPMSCs exhibited anti-inflammatory and anti-fibrotic functions, which suggests that they could be a new and safe method to promote the anti-adipogenic function of hPMSCs to treat TAO patients.

Different Effects of Cigarette Smoke, Heated Tobacco Product and E-Cigarette Vapour on Orbital Fibroblasts in Graves' Orbitopathy; a Study by Real Time Cell Electronic Sensing.

Thyroid autoimmunity in Graves' disease (GD) is accompanied by Graves' orbitopathy (GO) in 40% of the cases. Orbital fibroblasts (OF) play a key role in the pathogenesis and cigarette smoking is a known deteriorating factor. Alongside conventional cigarettes (CC) new alternatives became available for smokers, including heated tobacco products (HTP) and E-cigarettes (ECIG). We aimed to study the cellular effects of smoke extracts (SE) in orbital fibroblasts. Primary OF cultures from GO and NON-GO orbits were exposed to different concentrations of SE (1%, 50%) and the changes were followed using Real Time Cell Electronic Sensing (RT-CES). Untreated GO and NON-GO cells had different maximum cell index (CI) values of 3.3 and 2.79 respectively (p < 0.0001). CC, HTP and ECIG treated NON-GO fibroblasts exhibited peak CIs of 2.62, 3.32 and 3.41 while treated GO cells' CIs were higher, 5.38, 6.25 and 6.33, respectively (p < 0.0001). The metabolic activity (MTT) decreased (p < 0.001) and hyaluronan production doubled (p < 0.02) after 50% of CC SE treatment in all cell cultures. GO fibroblasts were more sensitive to low concentration SE then NON-GO fibroblasts (p < 0.0001). The studied SEs exerted different effects. RT-CES is a sensitive technique to detect the effects of very low concentration of SE on fibroblasts.

[Chinese guideline on the diagnosis and treatment of thyroid-associated ophthalmopathy (2022)].

Thyroid-associated ophthalmopathy (TAO) is an orbital disease closely related to thyroid diseases. TAO has a prolonged course and possibility of blindness and disability. Its diagnosis and treatment can be complex with multiple disciplines involved. To improve the standardized diagnosis and treatment of TAO in China, the Oculoplastic and Orbital Disease Group of Chinese Ophthalmological Society of Chinese Medical Association and the Thyroid Group of Chinese Society of Endocrinology of Chinese Medical Association have jointly drawn up this first clinical guideline in China for TAO by complying with the principles, methods and steps of guideline formation. In this guideline, there are 24 recommendations for the major clinical problems in the diagnosis and treatment of TAO, including the clinical diagnostic criteria, disease staging and grading methods, novel treatment based on the pathogenesis, multi-disciplinary treatment, risk factor control, individualized selection of treatment such as drugs, radiation and surgery, therapeutic efficacy evaluation and critical illness management. This guideline provides instructions for clinicians to carry out relevant clinical and research work.

Leptin receptor is a key gene involved in the immunopathogenesis of thyroid-associated ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO), the most common and severe manifestation of Graves' disease (GD), is a disfiguring and potentially blinding autoimmune disease. The high relapse rate (up to 20%) and substantial side effects of glucocorticoid treatment further decrease the life quality of TAO patients. To develop novel therapies, we amid to explore the immunopathogenesis of TAO. To identify the key immune-related genes (IRGs) in TAO, we integrated the IRG expression profiles in thyrocytes from a GD patient set (GD vs healthy control) and a TAO patient set (TAO vs GD). Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein-protein interaction (PPI) and receiver operating characteristic (ROC) curve analyses identified the leptin receptor (LEPR) gene as the key IRG in TAO immunopathogenesis. Gene set enrichment analysis (GSEA) suggested enrichment of the antigen presentation pathway in TAO patients with higher LEPR. Increased LEPR expression was validated in TAO orbital tissues, and weighted gene co-expression network analysis (WGCNA) showed that cell adhesion processes were positively correlated with LEPR. Our study revealed that LEPR is a key gene in TAO immunopathogenesis and plays different roles in thyrocytes and orbital tissues. Our findings provide new insights into diagnostic and therapeutic biomarkers for TAO.

A cyclic peptide significantly improves thyroid function, thyrotropin-receptor antibodies and orbital mucine /collagen content in a long-term Graves' disease mouse model.

BACKGROUND: BALB/c mice which received long-term immunizations of adenovirus (Ad) expressing thyrotropin receptor A-subunits (TSHR) developed stable Graves' disease (GD). TSHR-derived cyclic peptide 19 (P19) was identified as effective therapy in this model. METHODS: In Ad-TSHR mice, we investigated shorter disease intervals up to 4 months for histological alterations of the orbits, fine tuning of anti-TSHR antibodies (Ab) and free thyroxine (fT4) hormone levels by using novel detection methods in an independent laboratory. Therapy (0.3 mg/kg P19 or vehicle) was given intravenously after the fourth Ad-TSHR immunization (week 11) and continued until week 19. RESULTS: Thyrotropin binding inhibitory immunoglobulins (TBII, bridge immunoassay), blocking (TBAb) and stimulating (TSAb) TSHR-Ab (both cell-based bioassays) and serum levels of fT4 were significantly elevated at week 11 in Ad-TSHR-immunized mice versus none in control mice. For the first time, TSAb, TBAb, and thyroperoxidase-Ab were detected in 17 of 19, 12/19 and 6/19 Ad-TSHR immunized mice, respectively at week 21. Also, for the first time, this study showed that P19 treatment markedly reduced serum TBII (p < 0.0001), serum fT4 (p = 0.02), and acidic mucins and collagen content in the orbital tissue of Ad-TSHR-immunized mice. CONCLUSION: P19 significantly improved thyroid function, confirming previous results in an independent second laboratory. A relevant shift of anti-TSHR antibody subpopulations in response to P19 therapy may help explain its immunological effects. Moreover, P19 exerted a beneficial effect on mucine and collagen content of orbital tissue. Hence, P19 offers a potential novel therapeutic approach for GD and associated orbitopathy.