Impact of ibrutinib on inflammation in a mouse model of Graves' orbitopathy.

Introduction: Bruton's tyrosine kinase (BTK) and interleukin (IL)-2 Inducible T-cell Kinase (ITK) inhibitors have anti-inflammatory properties. We investigated the therapeutic effect of ibrutinib, an orally bioavailable BTK/ITK inhibitor, in a mouse model of Graves' orbitopathy (GO). Methods: Genetic immunization was performed through intramuscular administration of the recombinant plasmid, pCMV6-hTSHR cDNA, to 8-week-old female BALB/c mice. Serum levels of T3, T4, and thyroid-stimulating hormone receptor (TSHR) antibodies (TRAbs) were quantified using enzyme-linked immunosorbent assay. Histopathological changes in orbital tissues were examined using immunohistochemistry (IHC) staining for TSHR and various inflammatory markers. Following successful genetic immunization, ibrutinib was orally administered daily for 2 weeks in the GO model mice. After treatment, the mRNA and protein expression levels of BTK, ITK, IL-1ß, and IL-6 in orbital tissues were evaluated using real-time PCR and Western blotting. Results: In total, 20 mice were sacrificed to confirm successful genetic immunization. The GO mouse group exhibited significantly increased serum T3, T4, and TRAb levels. IHC revealed increased expression of TSHR, IL-1ß, IL-6, transforming growth factor-ß1, interferon-γ, CD40, CD4, BTK, and ITK in the GO mouse model. The orbital inflammation was significantly attenuated in ibrutinib-treated mice. The mRNA and protein expression levels of BTK, ITK, IL-1ß, and IL-6 in orbital tissue were lower in ibrutinib-treated GO mouse group compared to the phosphate-buffered saline-treated GO mouse group. Conclusion: The GO mouse model demonstrated enhanced BTK and ITK expression. Ibrutinib, a BTK/ITK inhibitor, suppressed the inflammatory cytokine production. These findings highlight the potential involvement of BTK/ITK in the inflammatory pathogenesis of GO, suggesting its role as a novel therapeutic target.

A Review of Pathophysiology and Therapeutic Strategies Targeting TGF-ß in Graves' Ophthalmopathy.

TGF-ß plays a pivotal role in the pathogenesis of GO by promoting orbital tissue remodeling and fibrosis. This process involves the stimulation of orbital fibroblasts, leading to myofibroblast differentiation, increased production of inflammatory mediators, and hyaluronan accumulation. Studies have elucidated TGF-ß's role in driving fibrosis and scarring processes through both canonical and non-canonical pathways, particularly resulting in the activation of orbital myofibroblasts and the excessive accumulation of extracellular matrix. Additionally, recent in vitro and in vivo studies have been summarized, highlighting the therapeutic potential of targeting TGF-ß signaling pathways, which may offer promising treatment interventions for GO. This review aims to consolidate the current understanding of the multifaceted role of TGF-ß in the molecular and cellular pathophysiology in Graves' ophthalmopathy (GO) by exploring its contributions to fibrosis, inflammation, and immune dysregulation. Additionally, the review investigates the therapeutic potential of inhibiting TGF-ß signaling pathways as a strategy for treating GO.

Therapeutic role of histone deacetylase inhibition in an in vitro model of Graves' orbitopathy.

Graves' orbitopathy (GO), a manifestation of Graves' disease, is characterized by orbital fibroblast­induced inflammation, leading to fibrosis or adipogenesis. Histone deacetylase (HDAC) serves a central role in autoimmune diseases and fibrosis. The present study investigated HDAC inhibition in orbital fibroblasts from patients with GO to evaluate its potential as a therapeutic agent. Primary cultured orbital fibroblasts were treated with an HDAC inhibitor, panobinostat, under the stimulation of IL­1ß, TGF­ß or adipogenic medium. Inflammatory cytokines, and fibrosis­ and adipogenesis­related proteins were analyzed using western blotting. The effects of panobinostat on HDAC mRNA expression were measured in GO orbital fibroblasts, and specific HDACs were inhibited using small interfering RNA transfection. Panobinostat significantly reduced the IL­1ß­induced production of inflammatory cytokines and TGF­ß­induced production of fibrosis­related proteins. It also suppressed adipocyte differentiation and adipogenic transcription factor production. Furthermore, it significantly attenuated HDAC7 mRNA expression in GO orbital fibroblasts. In addition, the silencing of HDAC7 led to anti­inflammatory and anti­fibrotic effects. In conclusion, by inhibiting HDAC7 gene expression, panobinostat may suppress the production of inflammatory cytokines, profibrotic proteins and adipogenesis in GO orbital fibroblasts. The present in vitro study suggested that HDAC7 could be a potential therapeutic target for inhibiting the inflammatory, adipogenic and fibrotic mechanisms of GO.

Lacrimal gland enlargement in inactive thyroid eye disease: a case series and literature review.

PURPOSE: This study aimed to evaluate the morphometric and volumetric dimensions of the lacrimal gland in patients with inactive thyroid eye disease and compare them with the values reported in the literature. METHODS: This case series evaluated consecutive patients with inactive thyroid eye disease treated at a tertiary eye hospital from 2015 to 2020. The patients' baseline demographics and clinical characteristics were obtained. The axial and coronal length, width, and volume of the lacrimal gland were measured on computed tomography scan images, and the results were statistically analyzed. RESULTS: A total of 21 patients (42 orbits) with inactive thyroid eye disease were evaluated. Their mean age was 49.0 ± 14.6 years, and 12 (57.1%) of them were men. The main complaint was dryness, and the majority of the patients had good vision and mild proptosis. The mean axial length and width of the lacrimal gland were 19.3 ± 3.9 mm and 7.5 ± 2.1 mm, respectively; coronal length and width, 20.4 ± 4.5 mm and 7.5 ± 2.1 mm, respectively; and lacrimal gland volume, 0.825 ± 0.326 mm3. Age, sex, or laterality were not found to be determinants of lacrimal gland enlargement. CONCLUSION: Patients with thyroid eye disease have enlarged lacrimal gland even in the nonactive phase of the disease multifactorial aspects influence the lacrimal gland in thyroid eye disease, making it difficult to establish a clear correlation with predisposing factors. Further studies are warranted to better understand the association between thyroid eye disease and the lacrimal gland.

Vascular changes of the peripapillary choroidal area in the thyroid orbitopathy.

PURPOSE: The aim of this study was to investigate the peripapillary choroidal vascular changes in thyroid orbitopathy (TO). METHODS: The study included 20 eyes of 10 patients with active TO (aTO), 30 eyes of 15 patients with inactive TO (inaTO) and 30 eyes of 30 healthy subjects. The peripapillary choroidal vascular change was assessed with peripapillary choroidal vascular index (pCVI), peripapillary choroidal luminal area (pLA), peripapillary choroidal stromal area (pSA), peripapillary total choroidal area (pTCA). RESULTS: Compared to the control group, there was a reduction in the nasal and temporal areas of pCVI in both the aTO and inaTO groups (aTO vs control: nasal p = 0.001 and temporal p = 0.004; inaTO vs control: nasal p = 0.007 and temporal p < 0.001), while the inferior area was lower only in the inaTO group (p = 0.001). Compared to the other groups, the inaTO group exhibited a decrease pSA (vs aTO: total p = 0.004, inferior p = 0.02 and vs control: total p = 0.01, inferior p = 0.03), pLA (vs aTO: total p = 0.02, inferior p = 0.02, temporal p < 0.001 and vs control: total p = 0.002, inferior p < 0.001, temporal p < 0.001) and pTCA (vs aTO: total p = 0.009, inferior p = 0.01, temporal p < 0.001 and vs control: total p = 0.003, inferior p = 0.001, temporal p < 0.001). CONCLUSION: The horizontal area (nasal and temporal area) of the peripapillary choroidal vascular structure may be more sensitive than the vertical area in TO patients. The first affected quadrant of RPC-VD in the active TO may be the inferior quadrant. Structural or vascular choroidal changes may occur during the chronic or post-active phase of the disease.

Role of Inositol-Requiring Enzyme 1 and Autophagy in the Pro-Fibrotic Mechanism Underlying Graves' Orbitopathy.

PURPOSE: Orbital fibroblasts play key roles in the pathogenesis of Graves' orbitopathy (GO), and previous findings have shown that endoplasmic reticulum (ER) stress and autophagy also contribute to GO. In this study, we investigated the presently unclear roles of inositol-requiring enzyme 1 (IRE1) and related autophagy processes in the pro-fibrotic mechanism of GO. MATERIALS AND METHODS: Orbital adipose/connective tissues were obtained from eight GO patients and six normal individuals during surgery. GO fibroblasts were transfected with IRE1 small-interfering RNA and treated with bafilomycin A1 (Baf-A1) to evaluate the inhibitory effects of ER stress and autophagy, and protein-expression levels were analyzed through western blotting after stimulation with transforming growth factor (TGF)-ß. RESULTS: TGF-ß stimulation upregulated IRE1 in GO orbital fibroblasts, whereas silencing IRE1 suppressed fibrosis and autophagy responses. Similarly, Baf-A1, an inhibitor of late-phase autophagy, decreased the expression of pro-fibrotic proteins. CONCLUSION: IRE1 mediates autophagy and the pro-fibrotic mechanism of GO, which provides a more comprehensive interpretation of GO pathogenesis and suggests potential therapeutic targets.

Therapeutic effect of selective interleukin-2-inducible tyrosine kinase inhibitor in orbital fibroblasts from patients with Graves' orbitopathy.

Interleukin-2-inducible tyrosine kinase (ITK) is a crucial cytoplasmic protein in the T-cell signaling pathway. Here, we aimed to demonstrate the anti-inflammatory effect of the selective IL-2-induced tyrosine kinase inhibitor BMS-509744 (BMS) on Graves' orbitopathy (GO) in an in vitro model. ITK mRNA expression in orbital tissues from GO and normal controls was compared using real-time polymerase chain reaction (RT-PCR) and immunohistochemistry. Primary cultured orbital fibroblasts from each group were pretreated with BMS and stimulated with interleukin (IL)-1ß to induce inflammatory reaction. ITK mRNA expression was evaluated using western blotting, and inflammatory cytokine production and downstream transcription factor expression were analyzed after pretreatment with BMS. ITK mRNA expression in GO tissues was significantly higher than that in normal control tissues. After stimulation with IL-1ß, ITK phosphorylation significantly increased in both GO orbital and normal control tissues. BMS inhibited IL-1ß-induced IL-8 expression in the GO orbital fibroblasts. BMS pretreatment significantly suppressed NF-κB phosphorylation in both GO and normal controls. The selective ITK inhibitor attenuates proinflammatory cytokine production and proinflammatory transcription factor phosphorylation in in vitro model of GO.

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.

Performance of T2 mapping in the staging of Graves' ophthalmopathy based on different region of interest selection methods.

BACKGROUND: Discriminating the stage of Graves' ophthalmopathy (GO) is crucial for clinical decision. Application of conventional T2-weighted imaging in the staging is still limited. PURPOSE: To evaluate the performance of T2 mapping based on two different regions of interest (ROIs) for staging GO. MATERIAL AND METHODS: In total, 56 GO patients were retrospectively enrolled and divided into two groups according to the clinical activity score (CAS). T2 relaxation time (T2RT) of extraocular muscle (EOM) on T2 mapping based on two different ROIs (T2RTROI-1: ROIs were drawn separately in the four EOMs; T2RTROI-2: ROI was drawn in the most inflamed EOM) was measured and compared between active and inactive groups. RESULTS: Both T2RTROI-1 and T2RTROI-2 values in the active GO were significantly higher than those of inactive GO (P <0.001). T2RTROI-1 and T2RTROI-2 values were positively correlated with CAS (rs=0.73, 0.69; P <0.001). When the T2RTROI-1 value of 83.3 ms and T2RTROI-2 value of 106.3 ms were used as cutoff values for staging GO, respectively, the best results were obtained with areas under the curve (AUCs) of 0.822 and 0.827. There was no significant difference for AUCs between T2RTROI-1 and T2RTROI-2 (P = 0.751). Excellent and good inter-observer agreements were achieved in quantitative measurements for T2RTROI-1 and T2RTROI-2 values, respectively, with intraclass correlation coefficients of 0.954 and 0.882. CONCLUSION: The T2RT values derived from two different ROIs were useful for assessment of disease activity. Taking reproducibility and diagnostic performance into consideration, T2RTROI-1 would be an ideal image biomarker for staging GO compared to T2RTROI-2.

Immune checkpoints: new insights into the pathogenesis of thyroid eye disease.

Thyroid eye disease (TED) is a disfiguring autoimmune disease characterized by changes in the orbital tissues and is caused by abnormal thyroid function or thyroid-related antibodies. It is the ocular manifestation of Graves' disease. The expression of thyroid-stimulating hormone receptor (TSHR) and the insulin-like growth factor-1 receptor (IGF-1 R) on the cell membrane of orbital fibroblasts (OFs) is responsible for TED pathology. Excessive inflammation is caused when these receptors in the orbit are stimulated by autoantibodies. CD34+ fibrocytes, found in the peripheral blood and orbital tissues of patients with TED, express immune checkpoints (ICs) like MHC II, B7, and PD-L1, indicating their potential role in presenting antigens and regulating the immune response in TED pathogenesis. Immune checkpoint inhibitors (ICIs) have significantly transformed cancer treatment. However, it can also lead to the occurrence of TED in some instances, suggesting the abnormality of ICs in TED. This review will examine the overall pathogenic mechanism linked to the immune cells of TED and then discuss the latest research findings on the immunomodulatory role of ICs in the development and pathogenesis of TED. This will offer fresh perspectives on the study of pathogenesis and the identification of potential therapeutic targets.

Cyclosporin A inhibits PDGF-BB induced hyaluronan synthesis in orbital fibroblasts.

Orbital connective tissue changes are contributors to the pathogenesis in thyroid eye disease (TED). Activated fibroblasts respond to immune stimuli with proliferation and increased hyaluronan (HA) production. Cyclosporin A (CsA) was reported to be beneficial in the treatment of TED. PDGF isoforms are increased in orbital tissue of TED patients and enhance HA production. We aimed to study the effect of CsA on HA production and hyaluronan synthase (HAS1, 2 and 3) and hyaluronidase (HYAL1 and 2) mRNA expressions in orbital fibroblasts (OFs). Measurements were performed in the presence or absence of CsA (10 µM) in unstimulated or PDGF-BB (10 ng/ml) stimulated OFs. The HA production of TED OFs (n = 7) and NON-TED OFs (n = 6) were measured by ELISA. The levels of mRNA expressions were examined using RT-PCR. The proliferation rate and metabolic activity were measured by BrdU incorporation and MTT assays, respectively. Treatment with CsA resulted in an average 42% decrease in HA production of OFs (p < 0.0001). CsA decreased the expression levels of HAS2, HAS3 and HYAL2 (p = 0.005, p = 0.005 and p = 0.002, respectively.) PDGF-BB increased HA production (p < 0.001) and HAS2 expression (p = 0.004). CsA could reduce the PDGF-BB-stimulated HA production (p < 0.001) and HAS2 expression (p = 0.005) below the untreated level. In addition, CsA treatment caused a decrease in proliferation potential (p = 0.002) and metabolic activity (p < 0.0001). These findings point to the fact that CsA affects HA metabolism via HAS2, HAS3 and HYAL2 inhibition in OFs. In addition to its well characterized immunosuppressant properties, CsA's beneficial effect in TED may be related to its direct inhibitory effect on basal and growth factor stimulated HA production.

Mouse model of Graves' orbitopathy induced by the immunization with TSHR A and IGF-1R α subunit gene.

PURPOSE: The study aimed to establish a mouse model of Graves' disease (GD) with Graves' orbitopathy (GO; GD + GO) that can represent the clinical disease characteristics. METHODS: A eukaryotic expression plasmid of insulin-like growth factor 1 receptor (IGF-1R) α subunit (pcDNA3.1/IGF-1Rα) and a thyrotropin receptor (TSHR) A subunit plasmid (pcDNA3.1/TSHR-289) were injected in female BALB/c mice followed by immediate electroporation to induce a GD + GO model. Grouping was performed according to the frequency of injection (2- to 4-week intervals) and type of injected plasmids: T: pcDNA3.1/TSHR-289( +), I: pcDNA3.1/IGF-1Rα( +), or co-injection T + I: pcDNA3.1/TSHR-289( +) and pcDNA3.1/IGF-1Rα( +). Serum TSH, T4, TSAb, TSBAb, body weight, and blood glucose levels were evaluated. Thyroid 99mTcO4- imaging and retrobulbar magnetic resonance imaging (MRI) were performed, and bilateral eye muscle volumes were measured. Immunohistochemistry and hematoxylin-eosin staining were performed on the relevant tissues, and semi-quantitative analysis was performed. RESULTS: A total of 60% of mice (3/5, one mouse died) in the T group developed GD + GO. In the T + I group, 83.3% of mice (5/6) developed GD + GO. Mice in the I group did not develop GD. Compared with the control group, serum T4, TSAb, and TSBAb of the mice in the GD + GO model groups were increased to varying degrees (P < 0.05), and serum TSH and body weight were significantly lower compared to the control group (P < 0.05). The thyroid uptake capacity of 99mTcO4- and the volume of eye muscle of mice in the GD + GO group were significantly higher compared to the control group (P < 0.05). The thyroid and retrobulbar muscles of these mice showed varying inflammatory infiltration and interstitial muscle edema. The severity of GD + GO in the co-injection group was not related to injection frequency; however, GD and ocular signs in co-injection mice were more severe compared to the T group. CONCLUSIONS: We successfully induced a GD + GO mouse model by a repeated co-injection of pcDNA3.1/IGF-1Rα and pcDNA3.1/TSHR-289 plasmids. Injection of pcDNA3.1/IGF-1Rα alone failed to induce GD. Co-injection of two plasmids induced more severe GD + GO than pcDNA3.1/TSHR-289( +) alone.

Novel insights into the pathogenesis of thyroid eye disease through ferroptosis-related gene signature and immune infiltration analysis.

Thyroid eye disease (TED) has brought great physical and mental trauma to patients worldwide. Although a few potential signaling pathways have been reported, knowledge of TED remains limited. Our objective is to explore the fundamental mechanism of TED and identify potential therapeutic targets using diverse approaches. To perform a range of bioinformatic analyses, such as identifying differentially expressed genes (DEGs), conducting enrichment analysis, establishing nomograms, analyzing weighted gene correlation network analysis (WGCNA), and studying immune infiltration, the datasets GSE58331, GSE105149, and GSE9340 were integrated. Further validation was conducted using qPCR, western blot, and immunohistochemistry techniques. Eleven ferroptosis-related DEGs derived from the lacrimal gland were originally screened. Their high diagnostic value was proven, and diagnostic prediction nomogram models with high accuracy and robustness were established by using machine learning. A total of 15 hub gene-related DEGs were identified by WGCNA. Through CIBERSORTx, we uncovered five immune cells highly correlated with TED and found several special associations between these immune cells and the above DEGs. Furthermore, EGR2 from the thyroid sample was revealed to be closely negatively correlated with most DEGs from the lacrimal gland. High expression of APOD, COPB2, MYH11, and MYCN, as well as CD4/CD8 T cells and B cells, was verified in the periorbital adipose tissues of TED patients. To summarize, we discovered a new gene signature associated with ferroptosis that has a critical impact on the development of TED and provides valuable insights into immune infiltration. These findings might highlight the new direction and therapeutic strategies of TED.

Early diagnosis of thyroid-associated ophthalmopathy using label-free Raman spectroscopy and multivariate analysis.

Thyroid-associated ophthalmopathy (TAO) is the most common orbital disease in adults, with complex clinical manifestations and significant impacts on the life quality of patients. The current diagnosis of TAO lacks reliable biomarkers for early and non-invasive screening and detection, easily leading to poor prognosis. Therefore, it is essential to explore new methods for accurately predicting TAO development in its early stage. In this study, Raman spectroscopy, with non-destructive, label-free, and high-sensitivity characteristics, was used to analyze the differences in biochemical components of orbital adipocyte and tear samples between TAO and control groups. Furthermore, a multivariate analysis method (i.e., Principal Component Analysis-Linear Discriminant Analysis (PCA-LDA)) was applied for data processing and analysis. Compared with controls, PCA-LDA yielded TAO diagnostic accuracies of 72.7% and 75.0% using orbital adipocytes and tears, respectively. Our proof-of-concept results suggest that Raman spectroscopy holds potential for exploring the underlying pathogenesis of TAO, and its potential application in early screening of other thyroid-associated diseases can be further expanded.

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.

Structural and Functional Brain Changes After Glucocorticoid Therapy in Thyroid-Associated Ophthalmopathy.

OBJECTIVE: To investigate the brain structural and functional alterations in patients with thyroid-associated ophthalmopathy (TAO) before and after glucocorticoid therapy, using voxel-based morphometry (VBM) as well as resting-state functional magnetic resonance imaging (MRI) with amplitude of low-frequency fluctuation (ALFF) and regional homogeneity (ReHo). METHODS: Between 2019 and 2022, 32 patients with TAO and 23 healthy controls underwent pre-therapy MRI in Nanjing, China. Intravenous glucocorticoid therapy was administered to all patients. At 3 months after end of therapy, 26 patients were available for rescanned MRI. VBM, ALFF, and ReHo were used to evaluate the brain structural and functional differences. RESULTS: Before therapy, TAO patients showed significantly decreased gray matter volume (GMV) in the left orbital part of superior frontal gyrus (ORBsup) and medial superior frontal gyrus (SFGmed) than healthy controls. Patients had higher ALFF values in bilateral gyrus rectus and olfactory cortex and lower values in bilateral cuneus. Patients also showed decreased ReHo values in bilateral lingual gyrus. After therapy, increased GMV in the left anterior cingulate gyrus and SFGmed, increased ALFF values in bilateral cuneus and superior occipital gyrus, and increased ReHo values in bilateral SFGmed were found in TAO patients compared to the pre-therapy cohort. Compared to controls, decreased GMV in left ORBsup was observed in post-therapy TAO patients. CONCLUSION: Our results indicated that TAO might cause functional and structural deficits in the visual and emotional regions of the brain, with recovery in the former and partial restoration in the latter after effective glucocorticoid therapy. These findings may lead to deeper understanding of the pathophysiological mechanism behind TAO.

Phospholipase C-γ as a Potential Therapeutic Target for Graves' Orbitopathy.

BACKGRUOUND: Phospholipase C-γ (PLC-γ) plays a crucial role in immune responses and is related to the pathogenesis of various inflammatory disorders. In this study, we investigated the role of PLC-γ and the therapeutic effect of the PLC-specific inhibitor U73122 using orbital fibroblasts from patients with Graves' orbitopathy (GO). METHODS: The expression of phospholipase C gamma 1 (PLCG1) and phospholipase C gamma 2 (PLCG2) was evaluated using polymerase chain reaction in GO and normal orbital tissues/fibroblasts. The primary cultures of orbital fibroblasts were treated with non-toxic concentrations of U73122 with or without interleukin (IL)-1ß to determine its therapeutic efficacy. The proinflammatory cytokine levels and activation of downstream signaling molecules were determined using Western blotting. RESULTS: PLCG1 and PLCG2 mRNA expression was significantly higher in GO orbital tissues than in controls (P<0.05). PLCG1 and PLCG2 mRNA expression was significantly increased (P<0.05) in IL-1ß, tumor necrosis factor-α, and a cluster of differentiation 40 ligand-stimulated GO fibroblasts. U73122 significantly inhibited the IL-1ß-induced expression of proinflammatory molecules, including IL-6, IL-8, monocyte chemoattractant protein-1, cyclooxygenase-2, and intercellular adhesion molecule-1 (ICAM-1), and phosphorylated protein kinase B (p-Akt) and p38 (p-p38) kinase in GO fibroblasts, whereas it inhibited IL-6, IL-8, and ICAM-1, and p-Akt and c-Jun N-terminal kinase (p-JNK) in normal fibroblasts (P<0.05). CONCLUSION: PLC-γ-inhibiting U73122 suppressed the production of proinflammatory cytokines and the phosphorylation of Akt and p38 kinase in GO fibroblasts. This study indicates the implications of PLC-γ in GO pathogenesis and its potential as a therapeutic target for GO.

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.

Tocilizumab improves clinical outcome in patients with active corticosteroid-resistant moderate-to-severe Graves' orbitopathy: an observational study.

Background: Graves' orbitopathy (GO) is an autoimmune disorder affecting the orbital fat and muscles. A significant role of IL-6 in the pathogenesis of GO has been described and tocilizumab (TCZ), an IL-6 inhibitor targeting IL-6R has been given in some patients. The aim of our case study was to evaluate the therapeutic outcome of TCZ in non-responders to first line treatments with corticosteroids. Methods: We conducted an observational study of patients with moderate to severe GO. Twelve patients received TCZ in intravenous infusions at a dose of 8mg/kg every 28 days for 4 months and followed up for additionally 6 weeks. The primary outcome was improvement in CAS by at least 2 points, 6 weeks after the last dose of TCZ. Secondary outcomes included CAS <3 (inactive disease) 6 weeks after TCZ last dose, reduced TSI levels, proptosis reduction by > 2mm and diplopia response. Results: The primary outcome, was achieved in all patients 6 weeks after treatment course. Furthermore all patients had inactive disease 6 weeks after treatment cessation. Treatment with TCZ reduced significantly median CAS by 3 units (p=0.002), TSI levels by 11.02 IU/L (p=0.006), Hertel score on the right eye by 2.3 mm (p=0.003), Hertel score on the left eye by 1.6 mm (p=0.002), while diplopia persisted in fewer patients (25%) after treatment with TCZ (not statistically significant, p=0.250). After treatment with TCZ, there was a radiological improvement in 75% of patients, while 16.7% showed no response, and in 8.3% of patients deterioration was established. Conclusion: Tocilizumab appears to be a safe and cost effective therapeutic option for patients with active, corticosteroid-resistant, moderate to severe Graves' orbitopathy.

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.