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Importance: Adjuvant therapy is an important and effective treatment for retinoblastoma. However, there is a lack of head-to-head clinical trials comparing 3 vs 6 cycles of CEV chemotherapy (carboplatin, etoposide, and vincristine) for enucleated unilateral retinoblastoma with high-risk pathological features., Objective: To assess whether 3 cycles of CEV chemotherapy is noninferior to 6 cycles for enucleated unilateral retinoblastoma with high-risk pathological features., Design, Setting, and Participants: This double-center, randomized, open-label, noninferiority trial was conducted at 2 premier eye centers in China and included 187 patients who had undergone enucleation for unilateral retinoblastoma with high-risk pathological features (massive choroidal infiltration, retrolaminar optic nerve invasion, or scleral infiltration) between August 2013 and March 2024. The final date of follow-up was March 21, 2024., Interventions: Patients were randomly assigned to receive either 3 (n = 94) or 6 (n = 93) cycles of CEV chemotherapy regimen after enucleation., Main Outcomes and Measures: The primary end point was disease-free survival, with a noninferiority margin of 12%. Secondary end points encompassed overall survival, safety, economic burden, and the quality of life of children., Results: All 187 patients (median [IQR] age, 25.0 [20.0-37.0] months; 83 [44.4%] female) completed the trial. Median (IQR) follow-up was 79.0 (65.5-102.5) months. Five-year disease-free survival was 90.4% for the 3-cycle group vs 89.2% for the 6-cycle group (difference, 1.2% [95% CI, -7.5% to 9.8%]), which met the noninferiority criterion (P = .003 for noninferiority). The 6-cycle group experienced a higher frequency of adverse events, greater reduction in quality of life scores, and increased costs compared with the 3-cycle group., Conclusions and Relevance: Among patients with unilateral pathologic high-risk retinoblastoma, 3 cycles of CEV chemotherapy resulted in 5-year disease-free survival that was noninferior to 6 cycles of CEV chemotherapy., Trial Registration: ClinicalTrials.gov Identifier: NCT01906814.

Introduction: MicroRNAs (miRNAs) have been recognized as promising diagnostic biomarkers for Diabetic Retinopathy (DR) due to their notable upregulation in individuals with the condition. However, the development of highly sensitive miRNAs assays for the rapid diagnosis of DR in clinical settings remains a challenging task., Methods: In this study, we introduce an enhanced CRISPR/Cas12a assay, leveraging suboptimal PAM (sPAM)-mediated Cas12a trans-cleavage in conjunction with rolling circle amplification (RCA). sPAM was found to perform better than canonical PAM (cPAM) in the detection of Cas12a-mediated ssDNA detection at low concentrations and was used instead of canonical PAM (cPAM) to mediate the detection. The parameters of reactions have also been optimized., Results and Discussion: In comparison with cPAM, sPAM has higher sensitivity in the detection of ssDNA at concentrations lower than 10 pM by Cas12a. By replacing cPAM with sPAM in the padlock template of RCA, ultra-high sensitivity for miR-183 detection is achieved, with a detection limit of 0.40 aM. within 25 min and a linear range spanning from 1 aM. to 1 pM. Our assay also exhibits exceptional specificity in detecting miR-183 from other miRNAs. Furthermore, the applicability of our assay for the sensitive detection of miR-183 in clinical serum samples is also validated. This study introduces a groundbreaking assay with excellent performance through a simple modification, which not only addresses existing diagnostic challenges, but also opens exciting new avenues for clinical diagnosis in the realm of DR., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Lu, Wang, Li, Yang, Han, Zhang and Ma.)

Background: Thyroid eye disease (TED) is a vision-threatening autoimmune disorder. Orbital tissue fibrosis leading to intractable complications remains a troublesome issue in TED management. Exploration of novel therapeutic targets and agents to ameliorate tissue fibrosis is crucial for TED. Recent work suggests that Ca 2+ signaling participates in tissue fibrosis. However, whether an alteration of Ca 2+ signaling has a role in fibrogenesis during TED remains unclear. In this study, we aimed to investigate the role of Ca 2+ signaling in the fibrogenesis process during TED and the potential therapeutic effects of a highly selective inhibitor of the L-type calcium channel (LTCC), nimodipine, through a TGF-β1 induced in vitro TED model., Methods: Primary culture of orbital fibroblasts (OFs) were established from orbital adipose connective tissues of patients with TED and healthy control donors. Real-time quantitative polymerase chain reaction (RT-qPCR) and RNA sequencing were used to assess the genes expression associated with LTCC in OFs. Flow cytometry, RT-qPCR, 5-ethynyl-2'-deoxyuridine (EdU) proliferation assay, wound healing assay and Western blot (WB) were used to assess the intracellular Ca 2+ response on TGF-β1 stimulation, and to evaluate the potential therapeutic effects of nimodipine in the TGF-β1 induced in vitro TED model. The roles of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) and signal transducer and activator of transcription 1 (STAT1) in fibrogenesis during TED were determined by immunohistochemistry, WB, flow cytometry and co-immunoprecipitation assay. Selective inhibitors were used to explore the downstream signaling pathways., Results: LTCC inhibitor nimodipine blocked the TGF-β1 induced intracellular Ca 2+ response and further reduced the expression of alpha-smooth muscle actin (α-SMA), collagen type I alpha 1 (Col1A1) and collagen type I alpha 2 (Col1A2) in OFs. Besides, nimodipine inhibited cell proliferation and migration of OFs. Moreover, our results provided evidence that activation of the CaMKII/STAT1 signaling pathway was involved in fibrogenesis during TED, and nimodipine inhibited the pro-fibrotic functions of OFs by down-regulating the CaMKII/STAT1 signaling pathway., Conclusions: TGF-β1 induces an LTCC-mediated Ca 2+ response, followed by activation of CaMKII/STAT1 signaling pathway, which promotes the pro-fibrotic functions of OFs and participates in fibrogenesis during TED. Nimodipine exerts potent anti-fibrotic benefits in vitro by suppressing the CaMKII/STAT1 signaling pathway. Our work deepens our understanding of the fibrogenesis process during TED and provides potential therapeutic targets and alternative candidate for TED., (© 2024. The Author(s).)

Retinoblastoma (RB) is the most common intraocular malignancy in childhood. The causal variants in RB are mostly characterized by previously used short-read sequencing (SRS) analysis, which has technical limitations in identifying structural variants (SVs) and phasing information. Long-read sequencing (LRS) technology has advantages over SRS in detecting SVs, phased genetic variants, and methylation. In this study, we comprehensively characterized the genetic landscape of RB using combinatorial LRS and SRS of 16 RB tumors and 16 matched blood samples. We detected a total of 232 somatic SVs, with an average of 14.5 SVs per sample across the whole genome in our cohort. We identified 20 distinct pathogenic variants disrupting RB1 gene, including three novel small variants and five somatic SVs. We found more somatic SVs were detected from LRS than SRS (140 vs. 122) in RB samples with WGS data, particularly the insertions (18 vs. 1). Furthermore, our analysis shows that, with the exception of one sample who lacked the methylation data, all samples presented biallelic inactivation of RB1 in various forms, including two cases with the biallelic hypermethylated promoter and four cases with compound heterozygous mutations which were missing in SRS analysis. By inferring relative timing of somatic events, we reveal the genetic progression that RB1 disruption early and followed by copy number changes, including amplifications of Chr2p and deletions of Chr16q, during RB tumorigenesis. Altogether, we characterize the comprehensive genetic landscape of RB, providing novel insights into the genetic alterations and mechanisms contributing to RB initiation and development. Our work also establishes a framework to analyze genomic landscape of cancers based on LRS data., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Purpose: The aim of this study was to analyze the characteristics of CT-measured intersection angle (FB-BNLD) between the frontal bone and bony nasolacrimal duct and to provide suggestions for treating primary acquired nasolacrimal duct obstruction (PANDO) patients in West China., Methods: Three hundred and nine participants' CT were, respectively, evaluated with RadiAnt DICOM Viewer. We defined the FB-BNLD angle >0° as the anterior type and the FB-BNLD angle ≤0° as the posterior type., Results: The mean FB-BNLD was -2.52° (95% CI, -3.16° to -1.88°) across all participants, of whom 37.2% were of the anterior type and 62.8% of the posterior type. Approximately 65.0% of the female patients had a posterior FB-BNLD type, and 54.2% of the male patients had an anterior FB-BNLD type ( p  = .002). Posterior FB-BNLD was the dominant type in the PANDO and control groups ( p  = .011), and the angle of FB-BNLD was statistically different in both groups (PANDO group, -2.54° to -0.71°; control group, -4.42° to -2.67°; p  < .001). Among the male participants, the type of FB-BNLD differed between the two groups ( p  = .036), with differences in the angle of FB-BNLD (PANDO group, 0.59° to 5.13°; control group, -4.08° to 1.89°; p  = .034). There was no difference in the type of FB-BNLD in female participants between the two groups ( p  = .051)., Conclusion: The present study revealed individual differences in the type of FB-BNLD, with anterior-type majority in males and posterior-type dominance in females. Evaluating the FB-BNLD type on CT can provide a fast method for knowing the nasolacrimal duct condition during planning for lacrimal manipulation.

Gasdermin D (GSDMD) is a key executor which triggers pyroptosis as well as an attractive checkpoint in various inflammatory and autoimmune diseases but it has yet to prove its function in Graves'orbitopathy (GO). Our aim was to investigate GSDMD levels in orbital connective tissue and serum of GO patients and then assess the association between serum levels and patients' clinical activity score (CAS). Further, GSDMD-mediated pyroptosis and the underlying mechanism in inflammatory pathogenesis in the cultured orbital fibroblasts (OFs) of GO patients were examined. OFs were collected after tumor necrosis factor (TNF)-α or interferon (IFN)-γ treatment or combination treatment at different times, and the expression of GSDMD and related molecular mechanisms were analyzed. Then, we constructed the GSDMD knockout system with siRNA and the system was further exposed to the medium with or without IFN-γ and TNF-α for a specified time. Finally, we evaluated the production of interleukin (IL)-1β and IL-18. We found that serum GSDMD levels were elevated and positively correlated with the CAS in GO patients. Meanwhile, the expression of GSDMD and N-terminal domain (NT-GSDMD) in orbital connective tissue of GO patients was augmented. Also, increased expression of GSDMD and related pyroptosis factors was observed in vitro model of GO. We further demonstrated that GSDMD-mediated pyroptosis induced inflammation via the nuclear factor kB (NF-κB)/absent in melanoma-2 (AIM-2)/caspase-1 pathway. In addition, blocking GSDMD suppressed proinflammatory cytokine production in GO. We concluded that GSDMD may be a biomarker as well as a potential target for the evaluation and treatment of inflammation related with GO., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Purpose: To evaluate the expression of sry-box transcription factor 9 (SOX9) in orbital fibroblasts (OFs) of thyroid eye disease (TED) and to find its potential role and underlying mechanism in orbital fibrosis., Methods: OFs were cultured from orbital connective tissues obtained from patients with TED (n = 10) and healthy controls (n = 6). SOX9 was depleted by small interfering RNA or overexpressed through lentivirus transduction in OFs. Fibroblast contractile activity was measured by collagen gel contraction assay and proliferation was examined by EdU assay. Transcriptomic changes were assessed by RNA sequencing., Results: The mRNA and protein levels of SOX9 were significantly higher in OFs cultured from patients with TED than those from healthy controls. Extracellular matrix-related genes were down-regulated by SOX9 knockdown and up-regulated by SOX9 overexpression in TED-OFs. SOX9 knockdown significantly decrease the contraction and the antiapoptotic ability of OFs, whereas the overexpression of SOX9 increased the ability of transformation, migration, and proliferation of OFs. SOX9 knockdown suppressed the expression of phosphorylated ERK1/2, whereas its overexpression showed the opposite effect. Epidermal growth factor receptor (EGFR) is one of the notably down-regulated genes screened out by RNA sequencing. Chromatin immunoprecipitation-qPCR demonstrated SOX9 binding to the EGFR promoter., Conclusions: A high expression of SOX9 was found in TED-OFs. SOX9 can activate OFs via MAPK/ERK1/2 signaling pathway, which in turn promotes proliferation and differentiation of OFs. EGFR was a downstream target gene of SOX9. SOX9/EGFR can be considered as therapeutic targets for the treatment of orbital fibrosis in TED.

Objective: For unilateral Dodge Class Ⅰ optic pathway glioma (OPG-uDCⅠ) without neurofibromatosis type 1, unilateral isolated optic nerve gliomas before the optic chiasm have been confirmed to possibly cause visual deterioration and poor prognosis. For this type of highly selective localized tumor, we explored surgery as the only treatment method. This article retrospectively analyzed and summarized the clinical data of this case series, with the aim of exploring the main technical details and clinical prognosis., Methods: Included were patients with OPG-uDCⅠ without neurofibromatosis type 1 and experiencing vision loss on the affected side. The fronto-orbital approach was used, which was mainly divided into 3 parts: intraorbital, optic canal, and intracranial. All patients underwent prechiasmatic resection without any adjuvant treatments. The follow-up period was 3 months after surgery, and magnetic resonance imaging and contralateral visual acuity were reviewed annually after surgery., Results: All OPG-uDCⅠ cases were completely removed without any adjuvant treatments, and there was no recurrence during the follow-up period. Pathological results showed that, except for 1 adult patient with pilomyxoid astrocytoma (World Health Organization grade Ⅱ), the others all had pilocytic astrocytoma (World Health Organization grade Ⅰ). Five patients experienced transient ptosis, and all recovered 3 months after surgery., Conclusions: For OPG-uDCⅠ without neurofibromatosis type 1, radical prechiasmatic resection of the tumor is possible, without the need for postoperative radiotherapy and chemotherapy., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: We aimed to explore the frequencies of islet β-cell autoantibodies and insulin resistance (IR) in thyroid-associated ophthalmopathy (TAO) and identify specific diabetes mellitus (DM) indicators as early predictors for dysthyroid optic neuropathy (DON)., Methods: Ninety-eight TAO patients (57 DON and 41 non-DON patients) and 48 healthy control (HC) participants were recruited for this prospective cross-sectional study. Serum thyroxine, serum thyroid autoantibodies, serum humoral immune markers against islet β-cell, fasting plasma glucose (FPG), fasting serum insulin (FINS), fasting c-peptide (FCP), and glycosylated hemoglobin A1 (HbA1c) were measured. Logistic regression analysis was used to evaluate the correlation of patients' age, body mass index (BMI), FPG, HbA1c, and related indexes of islet β-cell function to the occurrence of DON., Results: The DON group had higher FPG (P<0.001, 0.016) and HbA1c (P<0.0001, P<0.001) levels than the HC and non-DON groups. The homeostasis model assessment (HOMA)-IR level was the highest in the DON group (HC 2.15 ± 0.89, non-DON 2.41 ± 1.24, and DON 2.82 ± 2.65), while the HOMA-β level was the lowest (HC 101.8 ± 44.75%, non-DON 102.9 ± 54.61%, and DON 88.29 ± 52.75%), with no significant differences (P=1, P>0.05). On univariate analysis, age (P=0.006), BMI (P=0.022), history of steroid use (P=0.014), FPG (P=0.013), and HbA1c (P=0.001) levels were significantly associated with the presence/absence of DON. In addition, after adjusting for potential confounds, the HbA1c level was an independent factor associated with DON (P=0.009, OR=4.012)., Conclusions: HbA1c is an independent risk factor for DON. Given the interconnected link between thyroid dysfunction and DM, the use of HbA1c as a potential biomarker for DON warrants further investigation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wang, Ye, Chen, Yang, Zhang, Xiao and Yang.)

Lutein (LU) is a carotenoid that has recently been implicated in multiple roles in fibrosis, inflammation, and oxidative stress. Thyroid-associated ophthalmopathy (TAO) is particularly relevant to these pathological changes. We thus aim to probe the potential therapeutic effects of TAO in an in vitro model. We used LU pre-treating OFs derived from patients with TAO or not, then treated with TGF-β1(or IL-1β)to induce fibrosis (or inflammation). We analyzed the different expressions of related genes and proteins, and the molecular mechanism pathway on TAO OFs was screened by RNA sequencing, which is identified in vitro. We found that LU attenuates fibrotic and inflammatory effects in TAO. LU inhibited ACTA2, COL1A1, FN1, and CTGF mRNA expression and suppressed α-SMA, and FN1 protein expression induced by TGF-β1. Besides, LU suppressed OFs migration. Besides, it is shown that LU suppressed inflammation-related genes, such as IL-6, IL-8, CXCL1, and MCP-1. Moreover, LU inhibited oxidative stress induced by IL-1β, which is analyzed by DHE fluorescent probe staining. RNA sequencing suggested ERK/AP-1 pathway may be the molecular mechanism of LU protective effect on TAO, which is identified by RT-qPCR and western-blot. In summary, this study provides the first evidence that LU significantly attenuates the pathogenic manifestations of TAO by inhibiting the expression of fibrotic and inflammation-related genes and ROS produced by OFs. These data suggested that LU may be a potential medicine for TAO., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Purpose: We investigated the therapeutic potential of ABBV744, a bromodomain and extra-terminal (BET) inhibitor with selectivity for the second bromodomain (BD2) in thyroid eye disease (TED). The anti-fibrotic effects of ABBV744 and its underlying mechanism were explored in cultured orbital fibroblasts (OFs) from patients with TED., Methods: Immunohistochemistry (IHC) and real-time quantitative polymerase chain reaction (RT-qPCR) assays were conducted on orbital connective tissues from TED and controls. RT-qPCR, Western blot, Cell-counting Kit-8 (CCK-8), and 5-ethynyl-2'-deoxyuridine (EdU) cell proliferation assays were conducted on OFs isolated from patients with TED., Results: The expression of BRD4 was upregulated in the orbital tissues of patients with TED relative to controls and in TED OFs stimulated with TGF-β1. Further, we showed that BRD4 modulated the profibrotic process through the interaction with Forkhead Box M1 (FoxM1) and its downstream molecule Polo-like kinase 1 (Plk1) in cultured TED OFs. Inhibition of BRD4 both by BD2 selective inhibitor ABBV744 and pan-BET inhibitor JQ1 exerted anti-fibrotic effects, whereas ABBV744 displayed superior anti-fibrotic effects and acceptable safety compared to JQ1., Conclusions: We conclude that BDR4 may modulate the profibrotic process in OFs of patients with TED via the FoxM1/Plk1 axis, and that selectively targeting BD2 domain of BRD4 may therefore be a potential therapeutic option for treating patients with TED.

Fibrosis is the late stage of thyroid-associated ophthalmopathy (TAO), resulting in serious complications. Effective therapeutic drugs are still lacking. We aimed to explore the mechanism of TAO fibrosis and to find a targeted drug. High-throughput RNA sequencing was performed on orbital connective tissues from twelve patients with TAO and six healthy controls. Protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes (STRING) database and we identified the hub gene by Cytoscape software. Additionally, the RNA sequencing results were validated by quantitative real-time polymerase chain reaction (qRT-PCR). Bioinformatic prediction identified the functions of differentially expressed genes (DEGs). Further orbital connective tissue and serum samples of the TAO and control groups were collected for subsequent experiments. Histologic staining, Western blotting (WB), qRT-PCR, enzyme-linked immunosorbent assays (ELISAs), gene overexpression through lentiviral infection or silencing gene by short interfering RNA (siRNA) were performed. We found that the relaxin signaling pathway is an important regulatory pathway in TAO fibrosis pathogenesis. Serelaxin exerts antifibrotic and anti-inflammatory effects in TAO. Furthermore, the downstream Notch pathway was activated by serelaxin and was essential to the antifibrotic effect of serelaxin in TAO. The antifibrotic effect of serelaxin is dependent on RXFP1.

Early detection of visual impairment is crucial but is frequently missed in young children, who are capable of only limited cooperation with standard vision tests. Although certain features of visually impaired children, such as facial appearance and ocular movements, can assist ophthalmic practice, applying these features to real-world screening remains challenging. Here, we present a mobile health (mHealth) system, the smartphone-based Apollo Infant Sight (AIS), which identifies visually impaired children with any of 16 ophthalmic disorders by recording and analyzing their gazing behaviors and facial features under visual stimuli. Videos from 3,652 children (≤48 months in age; 54.5% boys) were prospectively collected to develop and validate this system. For detecting visual impairment, AIS achieved an area under the receiver operating curve (AUC) of 0.940 in an internal validation set and an AUC of 0.843 in an external validation set collected in multiple ophthalmology clinics across China. In a further test of AIS for at-home implementation by untrained parents or caregivers using their smartphones, the system was able to adapt to different testing conditions and achieved an AUC of 0.859. This mHealth system has the potential to be used by healthcare professionals, parents and caregivers for identifying young children with visual impairment across a wide range of ophthalmic disorders., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Techniques used to suture the rectus muscle to the implant can influence the implant-related complications which is still a major problem following retinoblastoma enucleation. The goals of this work were to report the efficacy among patients with retinoblastoma who underwent enucleation followed by porous implant placement with the rectus muscles sutured with 5-0 polyester suture., Methods: This was a retrospective study of consecutive patients with retinoblastoma who underwent primary enucleation and porous implant placement with the rectus muscles tagged and sutured to the implant with polyester 5-0 suture. All the patients were followed up for a minimum of 2 years. The main outcome measure was implant exposure. The secondary efficacy measures were other implant-related complications., Results: Between May 2016 and December 2018, a total of 120 patients (120 eyes) underwent primary enucleation and porous implant placement were included. Postoperatively, 10/120 (8.3%) eyes developed exposure or conjunctival granuloma. Exposure was the most common postoperative complication (7/10, 70.0%). There were no cases of implant extrusion, migration, or infection., Conclusions: Polyester 5-0 sutures are successful in patients with retinoblastoma who underwent enucleation followed by porous implant placement. Complications are minimal. Polyester 5-0 sutures were not associated with unacceptable complications in this pediatric population., (© 2023. The Author(s).)

Background: In a previous study, we found that traditional Chinese medicine (TCM) alleviated the clinical symptoms and improved the quality of life (QoL) in patients with hepatocellular carcinoma (HCC)., Objectives: A cohort was continuously followed up to determine the impact of the TCM adjuvant therapies on the prognosis of HCC after conventional treatments., Methods: We did a retrospective monocentric cohort study including 175 eligible patients. The participants who received TCM adjuvant therapies were termed as TCM group. For the purpose of stratification analysis, the patients who received TCM adjuvant therapies over 3 months per year were further classified into the high frequency group, while the rest of the TCM users were classified into the low frequency group. Non-users were recorded as the control group. The primary outcome was overall survival (OS) and the secondary outcome was the mean progression-free survival (mPFS) primarily introduced in this study, referring to the time from initial diagnosis to the latest progression over the number of disease progressions. Analyses used Cox proportional hazards and Kaplan-Meier (K-M) methods, adjusted for stratification factors., Results: Until June 30, 2021, 56 patients survived, 21 patients were lost to follow-up, and 98 patients died from the disease. Each disease progression of every individual was recorded, and most of the PFS was within 1 year. The baseline data of the allocated groups were balanced, the result revealed that TCM adjuvant therapies might have little influence on OS ( P  = .129). However, the 1, 3, and 5-year progression-free survival rates of the patients in TCM and control group were 68.75%, 37.50%; 25.00%, 8.33% and 8.33%, 2.08%, respectively, indicating TCM use significantly extended the mPFS, and decreased the risk of disease progression by a factor of 0.676 ( P  = .006). In the patients with BCLC stage B HCC, compared with controls, a 37-month median OS advantage in the high frequency group was noted ( P  = .045); and the high frequency of TCM use significantly suppressed disease progression ( P  = .001)., Conclusions: The present study revealed that TCM adjuvant therapies could postpone disease progression in HCC. Furthermore, using TCM over 3 months per year might extend OS in patients with intermediate HCC.

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-β-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.

Purpose: The purpose of this study was to investigate the molecular mechanism underlying thyroid-associated ophthalmopathy (TAO) clinical subtypes, to do so, we performed transcriptomic analysis to reveal the expression profile of circular RNAs (circRNAs) in TAO subtypes., Methods: High-throughput RNA-sequencing was performed in six pairs of type I and type II orbital connective tissue samples from patients with TAO. The expression levels of circRNAs and mRNAs in type I and type II samples were measured by quantitative real-time polymerase chain reaction (qRT-PCR) in another three pairs of type I and type II TAO connective tissue samples. We used bioinformatics predictions to construct a circRNA-microRNA (miRNA)-mRNA network. A protein-protein interaction (PPI) network was constructed based on differential mRNA expression, and the hub genes were determined by the Cytoscape software plugin. Functional and pathway enrichment analyses were performed to elucidate circRNA function. Lentiviral-mediated overexpression of hsa&#95;circ&#95;0007006 and the relationship between hsa&#95;circ&#95;0007006 with COL1A1 and MMP2 were evaluated by Western blotting (WB). Moreover, the differential pathways were assessed by WB., Results: RNA sequencing results predicted a total of 7489 circRNAs and 15,803 mRNAs, with 94 upregulated and 76 downregulated circRNAs and 488 upregulated and 138 downregulated mRNAs. The qRT-PCR analysis of seven dysregulated circRNAs and two major mRNAs validated the RNA-sequencing data. The competing endogenous RNA (ceRNA) network included 7 circRNAs, 23 miRNAs, and 262 mRNAs. Functional analysis revealed several important pathways. Overexpression of hsa&#95;circ&#95;0007006 led to decreased expression levels of COL1A1 and MMP2. Activation of the relaxin signaling pathway differed between the two subtypes., Conclusion: We showed that circRNAs are differentially expressed between type I and type II TAO. We speculate that the hsa&#95;circ&#95;0007006-COL1A1 and MMP2-relaxin signaling pathways are important regulatory axes in the pathogenesis of this disease type and could be considered promising diagnostic and therapeutic targets in the future.

The storage of facial images in medical records poses privacy risks due to the sensitive nature of the personal biometric information that can be extracted from such images. To minimize these risks, we developed a new technology, called the digital mask (DM), which is based on three-dimensional reconstruction and deep-learning algorithms to irreversibly erase identifiable features, while retaining disease-relevant features needed for diagnosis. In a prospective clinical study to evaluate the technology for diagnosis of ocular conditions, we found very high diagnostic consistency between the use of original and reconstructed facial videos (κ ≥ 0.845 for strabismus, ptosis and nystagmus, and κ = 0.801 for thyroid-associated orbitopathy) and comparable diagnostic accuracy (P ≥ 0.131 for all ocular conditions tested) was observed. Identity removal validation using multiple-choice questions showed that compared to image cropping, the DM could much more effectively remove identity attributes from facial images. We further confirmed the ability of the DM to evade recognition systems using artificial intelligence-powered re-identification algorithms. Moreover, use of the DM increased the willingness of patients with ocular conditions to provide their facial images as health information during medical treatment. These results indicate the potential of the DM algorithm to protect the privacy of patients' facial images in an era of rapid adoption of digital health technologies., (© 2022. The Author(s).)

Purpose: To perform orbital magnetic resonance imaging (MRI) three-dimensional (3D) reconstruction based on volume rendering and analyse changes in soft tissues in dysthyroid optic neuropathy (DON) patients., Methods: Using MRI 3D reconstruction based on volume rendering, orbital model parameters were compared with measurements made by observers. The fat volume (FV) and extraocular rectus muscle volume (EOMV) of subjects were calculated via MRI 3D reconstruction. Visual functions were assessed for all thyroid-associated ophthalmology (TAO) patients. Receiver operating characteristic (ROC) curves were analysed to evaluate DON in soft tissues. Correlations between visual function parameters and 3D reconstruction measurement were analysed., Results: All interclass correlation coefficients between the 3D reconstruction and observer measurements were above 0.950. A total of 21 healthy orbits, 38 TAO orbits without DON and 23 TAO orbits with DON were studied. The FV and EOMV were greater in the DON group than in the healthy and non-DON groups (all p  < 0.05). EOM enlargement contributed the most to the DON (odds ratio = 2.79, 95% confidence interval = 1.53, 5.07). The areas under the ROC curves of the reconstruction measurements were as follows: EOMV, 0.850; FV, 0.674; whole volume, 0.726; and EOMV/FV, 0.712. Visual function impairment was positively associated with EOM enlargement. When the EOMV was above 4.035 ml, the occurrence of DON was probable., Conclusions: MRI 3D reconstruction based on volume rendering is a reliable method for analysing orbital soft tissues. A larger the EOMV was the most relevant factor in DON.

Objectives: To comprehensively analyse the tear cytokine levels of patients with extranodal marginal zone B-cell lymphoma (EMZL) of the ocular adnexa (OA), and the association with clinical characteristics., Methods: Tear cytokine concentrations of 21 OA-EMZL patients and 14 age- and sex-matched healthy controls were measured using a 27-multiplex bead analysis on a Luminex system. Tear break-up time, corneal fluorescent staining and other clinical and demographic data were collected as well. The diagnosis of OA-EMZL was established based on the incisional biopsy and histopathology., Results: The concentrations of interleukin-1 receptor antagonist (IL-1RA) and IL-8, and the ratio of IL-1RA/IL-1β were significantly increased in OA-EMZL tear samples (all P < 0.05), while the levels of three cytokines (FGF-2, IL-2 and IL-4), as well as IL-10/IL-6 ratio were significantly decreased (all P < 0.05). The American Joint Committee on Cancer Tumour stage was significantly associated with tear concentrations of FGF-2 (r = -0.44, P = 0.043), GM-CSF (r = -0.49, P = 0.025) and IL-2 (r = -0.45, P = 0.042), while lacrimal gland lymphoma invasion was related to levels of IL-8 (r = 0.53, P = 0.012), FGF-2 (r = -0.43, P = 0.049) and IL-10/IL-6 ratio (r = -0.48, P = 0.026). Receiver operating characteristic (ROC) curve analysis revealed moderate diagnostic accuracy of these indices in differentiating OA-EMZL from normal eyes (area under ROC: 0.69-0.74)., Conclusions: Multiple tear cytokines were significantly dysregulated in OA-EMZL patients. These cytokines could potentially serve as diagnostic biomarkers and therapeutic targets in future., (© 2021. The Author(s), under exclusive licence to The Royal College of Ophthalmologists.)

Graves' ophthalmopathy (GO) is a common orbital disease that threatens visual function and appearance. Orbital fibroblasts (OFs) are considered key target and effector cells in GO. In addition, hyaluronan (HA) production, inflammation, and orbital fibrosis are intimately linked to the pathogenesis of GO. In this study, we explored the therapeutic effects of dihydroartemisinin (DHA), an antimalarial drug, on GO-derived, primary OFs. CCK8 and EdU assays were applied to evaluate the antiproliferative effect of DHA on OFs. Wound healing assays were conducted to assess OF migration capacity, while qRT-PCR, western blotting, ELISA, and immunofluorescence were used to determine the expression of fibrosis-related and pro-inflammatory markers in these cells. Moreover, RNA sequencing was conducted to identify differentially expressed genes (DEGs) in DHA-treated OFs, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs was performed to explore potential mechanisms mediating the antifibrotic effect of DHA on GO-derived OFs. Results showed that DHA dose-dependently inhibited OF proliferation and downregulated, at the mRNA and protein levels, TGF-β1-induced expression of fibrosis markers, including alpha smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF). Furthermore, DHA inhibited TGF-β1 induced phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and signal transducer and activator of transcription 3 (STAT3), which suggested that DHA exerted antifibrotic effects via suppression of the ERK and STAT3 signaling pathways. In addition, DHA suppressed the expression of pro-inflammatory cytokines and chemokines, including IL-6, IL-8, CXCL-1, MCP-1, and ICAM-1, and attenuated HA production induced by IL-1β in GO-derived OFs. In conclusion, our study provides first-time evidence that DHA may significantly alleviate pathogenic manifestations of GO by inhibiting proliferation, fibrosis- and inflammation-related gene expression, and HA production in OFs. These data suggest that DHA may be a promising candidate drug for treatment of GO., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yang, Wang, Xiao, Xu, Ye, Sha and Yang.)

Fibrosis of extraocular muscles (EOMs) is a marker of end-stage in Graves' orbitopathy (GO). To determine the antifibrotic and anti-inflammatory therapeutic effects and the underlying molecular mechanisms of disulfiram (DSF) on perimysial orbital fibroblasts (pOFs) in a GO model in vitro, primary cultures of pOFs from eight patients with GO and six subjects without GO (NG) were established. CCK-8 and EdU assays, IF, qPCR, WB, three-dimensional collagen gel contraction assays, cell scratch experiments, and ELISAs were performed. After TGF-β1 stimulation of pOFs, the proliferation rate of the GO group but not the NG group increased significantly. DSF dose-dependently inhibited the proliferation, contraction, and migration of pOFs in the GO group. Additionally, DSF dose-dependently inhibited fibrosis and extracellular matrix production markers (FN1, COL1A1, α-SMA, CTGF) at the mRNA and protein levels. Furthermore, DSF mediates antifibrotic effects on GO pOFs partially through the ERK-Snail signaling pathway. In addition, DSF attenuated HA production and suppressed inflammatory chemokine molecule expression induced by TGF-β1 in GO pOFs. In this in vitro study, we demonstrate the inhibitory effect of DSF on pOFs fibrosis in GO, HA production, and inflammation. DSF may be a potential drug candidate for preventing and treating tissue fibrosis in GO.

Retinoblastoma (RB) is the most common primary intraocular malignancy of childhood. It is known that the tumor microenvironment (TME) regulates tumorigenesis and metastasis. However, how the malignant progression in RB is determined by the heterogeneity of tumor cells and TME remains uncharacterized. Here, we conducted integrative single-cell transcriptome and whole-exome sequencing analysis of RB patients with detailed pathological and clinical measurements. By single-cell transcriptomic sequencing, we profiled around 70,000 cells from tumor samples of seven RB patients. We identified that the major cell types in RB were cone precursor-like (CP-like) and MKI67+ cone precursor (MKI67+ CP) cells. By integrating copy number variation (CNV) analysis, we found that RB samples had large clonal heterogeneity, where the malignant MKI67+ CP cells had significantly larger copy number changes. Enrichment analysis revealed that the conversion of CP-like to MKI67+ CP resulted in the loss of photoreceptor function and increased cell proliferation ability. The TME in RB was composed of tumor-associated macrophages (TAMs), astrocyte-like, and cancer-associated fibroblasts (CAFs). Particularly, during the invasion process, TAMs created an immunosuppressive environment, in which the proportion of TAMs decreased, M1-type macrophage was lost, and the TAMs-related immune functions were depressed. Finally, we identified that TAMs regulated tumor cells through GRN and MIF signaling pathways, while TAMs self-regulated through inhibition of CCL and GALECTIN signaling pathways during the invasion process. Altogether, our study creates a detailed transcriptomic map of RB with single-cell characterization of malignant phenotypes and provides novel molecular insights into the occurrence and progression of RB., (© 2022. The Author(s).)

Purpose: To report three-decade changes of clinical characteristics, progress of treatments, and risk factors associated with mortality and enucleation in patients with retinoblastoma in China., Design: Retrospective cohort study., Methods: This multicenter study included 2552 patients diagnosed with retinoblastoma in 38 medical centers in 31 provinces in China from 1989 to 2017, with follow-up data. Kendall's tau-b value was used to describe correlation coefficients between the three eras (between 1989 and 2008, between 2009 and 2013, and between 2014 and 2017) and clinical or demographic features. Hazard ratios and odds ratios were applied to measure risk factors., Results: A total of 324 (13%) patients died and 1414 (42%) eyes were removed. The 1-year, 3-year, and 5-year overall survival rates were 95%, 86%, and 83%, respectively. Patients were diagnosed at a better stage by International Classification for Retinoblastoma over time (Kendall's tau-b value = -0.084, P < .001). Pathological risk factors were also observed less in recent eras. New conservative therapies were adopted and used in more patients. The eye removal rate gradually decreased (Kendall's tau-b value = -0.167, P < .001). The overall survival rates were 81%, 83%, and 91% in the three eras. By multivariate Cox regression, bilateral tumors and extraocular extension were identified as risk factors for death. Among intraocular disease, Group E indicated higher risk of mortality. By multivariate logistics regression, unilateral tumors, earlier era of diagnosis, and extraocular extension were risk factors for eye salvage failure. Among intraocular retinoblastoma, Groups D and E had higher risk of eye salvage failure., Conclusions: Patients were diagnosed at an earlier stage in recent eras. Conservative therapies, including intra-arterial chemotherapy, were increasingly being used. The above changes may contribute to the decreasing enucleation rate. Although no significant impact was identified on the mortality by the three eras, a decreasing trend was shown., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Adipogenesis, glycosaminoglycan hyaluronan (HA) production, inflammation, and fibrosis are the main pathogenic mechanisms responsible for Graves' orbitopathy (GO). We hypothesized that disulfiram (DSF), an aldehyde dehydrogenase (ALDH) inhibitor used to treat alcoholism, would have therapeutic effects on orbital fibroblasts (OFs) in GO. This study aimed at determining the therapeutic effects and underlying mechanisms of DSF on these parameters. Methods: Primary cultures of OFs from six GO patients and six control subjects were established. The OFs were allowed to differentiate into adipocytes and treated with various concentrations of DSF. Lipid accumulation within the cells was evaluated by Oil Red O staining. Real-time polymerase chain reaction (RT-PCR) and Western blotting were used to measure the expression of key adipogenic transcription factors, ALDH1A1, ALDH2, and mitogen-activated protein kinase (MAPK) signaling proteins. Apoptosis assays and reactive oxygen species levels were evaluated by flow cytometry. HA production was measured by using an enzyme-linked immunosorbent assay (ELISA) kit. The mRNA levels of proinflammatory molecules were measured by using RT-PCR after interleukin (IL)-1β stimulation with or without DSF. The mRNA expression of markers associated with fibrosis was examined by using RT-PCR after transforming growth factor (TGF)-β1 stimulation with or without DSF. The wound-healing assay was assessed by phase-contrast microscopy. Results: Under identical adipogenesis conditions, GO OFs effectively differentiated, while normal control (NC) OFs did not. DSF dose dependently suppressed lipid accumulation during adipogenesis in GO OFs. The expression of key adipogenic transcription factors, such as perilipin-1 (PLIN1), PPARγ (PPARG), FABP4, and c/EBPα (CEBPA), was downregulated. Further, DSF inhibited the phosphorylation of ERK by inhibiting ALDH1A1. In addition, DSF attenuated HA production and suppressed inflammatory molecule expression induced by IL-1β in GO OFs and NC OFs. The antifibrotic effects of DSF on TGF-β1 were also observed in GO OFs. Conclusions: In the current study, we provide evidence of the inhibitory effect of DSF on GO OFs adipogenesis, HA production, inflammation, and fibrosis in vitro . The results of this study are noteworthy and indicate the potential use of DSF as a therapeutic agent for the treatment of GO.

Orbital fibrosis is a hallmark of tissue remodeling in thyroid-associated ophthalmopathy (TAO). Previous studies have shown that interleukin (IL)-11 plays a pivotal profibrotic role in various inflammatory and autoimmune diseases. However, the expression pattern of IL-11 in patients with TAO and whether IL-11 is mechanistically linked with pathological fibrosis remains unknown. In this study, we investigated IL-11 levels in the serum and orbital connective tissue of patients with TAO, and evaluated the correlation of these levels with the patient's clinical activity score. We also evaluated the expression pattern of IL-11Rα in orbital connective tissue. Furthermore, we elucidated the regulatory factors, profibrotic function, and downstream signaling pathways for IL-11 in TAO using in vitro studies. IL-11 levels in serum and orbital connective tissues were increased in patients with TAO, as compared with healthy controls. In addition, both levels were positively correlated with disease activity. Single-cell RNA sequencing of orbital connective tissue indicated that IL-11Rα was dominantly expressed in orbital fibroblasts (OFs). RNA sequencing of paired unstimulated and transforming growth factor (TGF)-β1-stimulated samples demonstrated that upregulation of IL-11 expression defined the dominant transcriptional response. IL-11 signaling was also confirmed to be downstream of TGF-β1 and IL-1β. Therefore, we deduced that IL-11 protein is secreted in an autocrine loop in TAO. We also indicated that IL-11 mediated the profibrotic phenotype switch by inducing the expression of myofibroblast differentiation markers, including α-smooth muscle actin and collagen type I α1, which could be abrogated by an anti-IL-11 neutralizing antibody. Furthermore, we revealed that extracellular regulated protein kinase may be a crucial factor in the pro-fibrotic, translationally specific signaling activity of IL-11. These data demonstrate that IL-11 plays a crucial role in orbital fibroblast phenotype switching and may be a potential therapeutic target candidate for the treatment of TAO., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wu, Lin, Huang, Meng, Zhang, Zhou, Hei, Ke, Yang and Huang.)

Purpose: Teprotumumab, a blocking antibody to the insulin like growth factor 1 receptor (IGF-1R) has been shown to significantly reduce proptosis in recent phase 2 and 3 trials in patients with inflammatory thyroid eye disease (TED). Herein, we investigate the impact of teprotumumab on patients with non-inflammatory TED. We also investigate the expression of the IGF-1R on orbital tissues from patients with inflammatory and non-inflammatory TED compared to controls., Methods: Consecutive patients with non-inflammatory TED (clinical activity score, CAS ≤ 1, for at least 4 months, were treated with teprotumumab. They received a complete course (total eight infusions) of teprotumumab (10 mg/kg for the first infusion and 20 mg/kg for subsequent infusions every 3 weeks). The primary outcome was a proptosis response at week 24. Further, IGF-1R α and β expression was evaluated on orbital tissue from patients with inflammatory and non-inflammatory TED, as well as healthy controls. Non-biased histological analysis of IGF-1R expression was performed using ImageJ., Results: Four patients met eligibility criteria for the clinical study, with a mean (SD) CAS of 0 (0). Following teprotumumab treatment, there was a mean (SD) reduction in proptosis of 2.6 mm (1.2). Five patients were included for each group of the histological study; inflammatory TED, non-inflammatory TED and controls. IGF-1Rα and IGF-1Rβ expression was significantly greater in the orbital tissues of patients with inflammatory TED and non-inflammatory TED, when compared to controls., Conclusion: Our findings demonstrate for the first time, that teprotumumab, a blocking antibody to the IGF-1R reduces proptosis in a series of patients with non-inflammatory TED. Overexpression of the IGF-1R in orbital tissue from patients with non-inflammatory disease compared to controls may be an important consideration for effect., (© 2020. The Author(s), under exclusive licence to The Royal College of Ophthalmologists.)

Context: Thyroid-associated ophthalmopathy (TAO) is an organ-specific autoimmune disease closely associated with Graves' disease. IL-38, a novel cytokine in the IL-1 superfamily, has been reported to be involved in the pathogenesis of various autoimmune diseases., Objective: We aimed to evaluate the relationship between IL-38 and TAO disease activity and its role in inflammation and fibrosis in TAO., Methods: Blood samples and orbital connective tissues were collected from TAO patients and controls. Orbital fibroblasts were isolated from patients with TAO. Enzyme-linked immunosorbent assay, immunohistochemistry, flow cytometry, immunofluorescence, quantitative real-time PCR and Western blot analysis were performed., Results: Here, we demonstrated that IL-38 levels decreased in the circulation and orbital connective tissues of patients with TAO compared with the controls, and levels were negatively correlated with the clinical activity score. In vitro, potent anti-inflammatory and antifibrotic effects of IL-38 were observed. Furthermore, we revealed that IL-38 can counteract the phosphorylation of star molecules in multiple classical pathways., Conclusion: IL-38 plays a protective role in TAO and is associated with its pathogenesis. Our data suggest that IL-38 may be a promising marker of TAO disease activity and a potential target for TAO therapy., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Purpose : To evaluate the morphologic and hemodynamic changes of bulbar conjunctival vessels in thyroid-associated ophthalmopathy (TAO) patients and the correlations with the activity. Methods : Patients diagnosed as TAO with different clinical activity scores (CAS) and healthy participants were recruited. All subjects underwent a complete ophthalmic examination and functional slit-lamp biomicroscope. Vascular variables including the vessel density, vessel complexity, average diameter, blood flow velocity and blood flow rate in microvascular networks were measured. The correlations among microvascular parameters, CAS and exophthalmos were analyzed. Areas under the receiver operating characteristic curves (AUROCs) were applied to evaluate the diagnostic accuracy of microvascular alterations for active TAO. Results : A total of 46 eyes were enrolled in our study. The vessel complexity and blood flow velocity increased in the active TAO group significantly compared with the inactive group and healthy controls ( P < .05). Meanwhile, the vessel complexity and blood flow rate were positively correlated with CAS (r = 0.641 and r = 0.526). Bulbar conjunctival microvascular parameters performed a good ability in distinguishing the active stage of TAO (AUROC = 0.793). Conclusions : Increasing bulbar conjunctival vessel complexity and blood flow were evident in TAO with severe inflammation. The measurements of bulbar conjunctival microvasculature could be a reference to evaluate activity in TAO.

Background: Idiopathic orbital inflammation (IOI) is a disfiguring and vision-threatening fibroinflammatory disorder. The pathogenesis of IOI has not been elucidated. We sought to clarify the regulatory T cell (Treg) distribution and function in patients with IOI., Methods: The frequency, phenotype and function of Tregs were identified by multicolor flow cytometry and in vitro cell functional assays. Plasma and tissue samples were obtained to investigate cytokines, chemokines and their receptors of interest by relative real-time polymerase chain reaction (PCR) and Luminex assays., Results: Compared with healthy subjects, patients with IOI exhibited obvious increases of Tregs in peripheral blood and affected orbital tissues. Circulating Tregs from patients with IOI were significantly more polarized to a Th17-like phenotype with defective regulatory function, whereas orbit-derived Tregs were polarized to a Th2-like phenotype. Furthermore, ST2 expression levels in circulating Tregs and interleukin (IL)-33 mRNA levels in orbital tissues were decreased in IOI. IL-33 restored the suppressive function of Tregs, reduced interferon (IFN)-γ production by Tregs and decreased the activation of orbital fibroblasts (OFs) cocultured with Tregs in IOI., Conclusion: Increased Tregs with proinflammatory and profibrotic polarization were first identified in IOI, suggesting that Treg plasticity and heterogeneity plays an essential role in IOI pathogenesis. Additionally, our study identified a regulatory effect of IL-33 on inflammation and fibrosis in IOI. Reversing the plastic Tregs via IL-33 might be a potential option for IOI patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chen, Ye, Xiao, Mao, Ai, Chen, Lian, Shi, Wang, Bi, Yang, Ji, Zhang and Yang.)

Purpose: To investigate the clinical features and treatment outcomes of IgG4-related ophthalmic disease (IgG4-ROD) among idiopathic orbital inflammatory disease (IOID) patients., Methods: The medical records of 165 biopsy-proven IOID patients were retrospectively reviewed. Biopsy specimens were immunostained to detect IgG4 and IgG, and data regarding the clinicopathologic features, treatment outcomes, and recurrence were analyzed., Results: Among the 165 IOID patients enrolled, 100 (60.6%) were histopathologically IgG4-positive. The IgG4-positive patients had a lower rate of painful swelling or mass (17.0% versus 33.8%, p = 0.013), a longer symptom duration (p = 0.070), and a lower proportion of eyelid hyperemia (39.0% versus 58.5%, p = 0.014) than the IgG4-negative patients. Increased Ki-67 expression (15.02 ± 6.86%, p < 0.001) was observed in the IgG4-positive patients with characteristic pathological manifestations (more lymphocyte infiltration, nodular plasma cell infiltration, and follicular hyperplasia). IgG4-positive group had a higher recurrence rate in the subgroup of patients treated with surgery plus oral glucocorticoids (p = 0.046), and combined radiotherapy group has a higher cumulative proportion with recurrence (p = 0.011)., Conclusion: Over 60% of biopsy-proven IOID were classified as IgG4-ROD with a stronger proliferation potential. Additional radiotherapy after surgical debulking with oral corticosteroids still has a higher relapse rate, and more effective treatments are needed to prevent recurrence.

Background: The aim of this study is to evaluate the optic nerve sheath diameter (ONSD) in eyes with dysthyroid optic neuropathy (DON) and its relationship with clinical characteristics and disease severity., Methods: Patients diagnosed as thyroid-associated ophthalmopathy (TAO), with or without DON, and healthy participants were recruited. Vertical and horizontal sectional images of the optic nerve were collected by B-scan ultrasonography. ONSDs at 3 mm and 6 mm behind the eyeball were measured independently by two researchers. Multivariate regression analysis was performed to evaluate the association of ONSD with demographic and ocular parameters in TAO patients. Areas under the receiver operating characteristic curves (AUROCs) were applied to evaluate the diagnostic accuracy of ONSD for DON., Results: A total of 47 healthy eyes, 36 TAO eyes without DON, and 33 eyes with DON were studied. ONSDs at 3 mm and 6 mm of DON eyes were significantly higher than those in non-DON and healthy eyes (all P < 0.05). There was no significant difference in ONSDs between clinically active and inactive eyes (both P > 0.05). DON occurrence showed a positive association with both ONSDs at 3 mm (β = 0.49, 95% CI: 0.14-0.83, P = 0.007) and 6 mm (β = 0.58, 95% CI: 0.20-0.96, P = 0.003). ONSDs at 3 mm and 6 mm showed a desirable diagnostic capacity to distinguish DON from non-DON eyes (AUROC was 0.77 and 0.75, respectively)., Conclusions: An increase in ONSD is evident in DON eyes independent of clinical activity. Ultrasound-based ONSD has sufficient ability to distinguish DON from non-DON eyes.

Objectives  Cranioorbital lesions present a great challenge for neurosurgeons and ophthalmologists. There is no consensus on the choice of surgical approach. The aims of this study were to investigate 49 cases of cranioorbital lesions and evaluate surgical approaches and outcomes. Patients and Methods  A retrospective study was done on 49 patients (51 operations) from 2009 to 2018. Information about the lesion was used to decide whether the supraorbital eyebrow approach (SEA) or pterional approach (PA) was performed. Results  Twenty-eight patients had surgical resection using SEA, 21 patients received PA, each group included one case of recurrence, who underwent reoperation via the same approach. SEA provided better cosmetic satisfaction, and a shorter incision than PA ( p  < 0.05). There was no significant difference in total resection rates, visual outcomes, recovery of ptosis, and other new surgical-related complications between SEA group and PA group ( p  > 0.05). Forty-nine cases of proptosis (94.1%, 49/51) were improved. Thirty-three patients (33/37, 89.2%) who underwent follow-up for longer than 12 weeks had a modified Rankin Scale (mRS) score ≤ 3. Conclusion  Surgery is the preferred treatment for cranioorbital lesions, but total resection is difficult. SEA may be a more minimally invasive option for some more limited lesions superior to optic nerve. PA may be more reasonable for the lesion with obvious hyperostosis and more extensive lesions., Competing Interests: Conflict of Interest None., (Thieme. All rights reserved.)

Low-grade myofibroblastic sarcoma (LGMS) is an exceedingly rare, malignant tumor with myofibroblastic differentiation. It frequently occurs in the oral cavity and extremities, despite being classified as a distinct entity by the World Health Organization (WHO). Here, we report a rare case of orbital LGMS occurring in an 11-month-old baby with a 3.2 × 2.4 × 2.1 cm mass. LGMS was diagnosed based on the histological and immunohistochemical findings. Previous literature suggests that surgical resection with clear margins is an appropriate method for the treatment of LGMS, and combined adjuvant therapy (local radiotherapy with or without chemotherapy) can improve the prognosis, but further studies are needed.

Objective: To establish a predictive model and investigate its value in evaluating short-term prognosis of patients with hepatitis B virus related acute-on-chronic liver failure (HBV-ACLF)., Methods: Patients with HBV-ACLF admitted to Tianjin Second People's Hospital and Beijing Youan Hospital, Capital Medical University from May 2015 to October 2018 were enrolled. The data of gender, age, laboratory markers at admission, model for end-stage liver disease (MELD) score and clinical complications were collected for analysis. According to the prognosis on 12-week, patients were divided into survival group and death group. Univariate analysis and binary Logistic regression analysis were used to test the risk factors for short-term prognosis of the patients with HBV-ACLF, and a prediction model was established. The accuracy of each index and the established model were verified by the receiver operating characteristic (ROC) curve., Results: A total of 148 patients with HBV-ACLF were enrolled in the study, 91 cases survived while 57 cases died during the 12-week period. The age, total bilirubin (TBIL), neutrophil percentage (NEUT%), hepatitis B surface antigen (HBsAg), MELD score of death group were higher than those of survival group [age (years old): 50.00 (44.50, 55.00) vs. 43.00 (34.00, 53.00), TBIL (μmol/L): 310.30 (240.70, 405.70) vs. 266.40 (184.20, 360.20), NEUT%: (74.52±13.05)% vs. (66.64±12.35)%, lgHBsAg (kU/L): 3.72 (3.29, 3.92) vs. 2.97 (2.49, 3.78), MELD score: 24.27 (19.71, 27.40) vs. 21.88 (18.83, 24.38), all P < 0.05], while albumin (ALB), total cholesterol (CHO), prothrombin activity (PTA) and alpha-fetoprotein (AFP) were lower than those of survival group [ALB (g/L): 29.80 (27.05, 31.05) vs. 30.80 (28.00, 33.90), CHO (mmol/L): 1.98 (1.50, 2.38) vs. 2.49 (2.05, 3.01), PTA: (30.37±7.09)% vs. (32.94±6.03)%, AFP (μg/L): 21.54 (9.28, 51.54) vs. 66.16 (24.50, 152.80), all P < 0.05]. Logistic regression analysis showed that NEUT%, HBsAg and AFP were independent risk factors for short-term prognosis of patients with HBV-ACLF [odds ratio (OR) was 77.843, 1.439, 0.995, respectively, all P < 0.05]. According to the results of regression analysis, the NHA-ACLF model (NEUT%+HBsAg+AFP) was established. The formula was logit (NHA-ACLF) = -5.441+5.688×NEUT%+0.430×lgHBsAg-0.005×AFP. The area under the ROC curve (AUC) of the NHA-ACLF model for pred HBV-ACLF patients was 0.790, which was better than NEUT% (AUC = 0.696), lgHBsAg (AUC = 0.670), AFP (AUC = 0.703) and MELD score (AUC = 0.640). When the cut-off value of NHA-ACLF model score was 0.459, the sensitivity was 73.7%, and the specificity was 79.1%., Conclusions: NEUT%, HBsAg and AFP are independent predictive indicator for short-term prognosis in patients with HBV-ACLF. Compared with MELD score, the risk assessment model NHA-ACLF has a greater value in predicting the short-term prognosis of patients with HBV-ACLF.

Context: Graves' orbitopathy (GO) causes infiltrative exophthalmos by inducing excessive proliferation, adipogenesis, and glycosaminoglycan production in orbital fibroblasts (OFs). Interference with OF autophagy is a potential therapy for proptosis., Objectives: Here, we aimed to evaluate the effects of chloroquine (CQ) and hydroxychloroquine (HCQ), the autophagy inhibitors commonly used in clinical practice, on OFs., Design/setting/participants: OFs isolated from patients with GO (GO-OFs) or control individuals (non-GO-OFs) were cultured in proliferation medium (PM) or subjected to differentiation medium. OFs were treated with CQ or HCQ (0, 0.5, 2, and 10 μM), and subsequently examined in vitro., Main Outcome Measures: CCK-8, EdU incorporation, and flow cytometry assays were used to assess cellular viability. Adipogenesis was assessed with Western blot analysis, real-time polymerase chain reaction (PCR) , and Oil Red O staining. Hyaluronan production was determined by real-time PCR and enzyme-linked immunosorbent assay. Autophagy flux was detected through red fluorescent protein (RFP)-green fluorescent protein (GFP)-LC3 fluorescence staining and Western blot analyses., Results: CQ/HCQ halted proliferation and adipogenesis in GO-OFs in a concentration-dependent manner through blockage of autophagy, phenotypes that were not detected in non-GO-OFs. The inhibitory effect of CQ/HCQ on hyaluronan secretion of GO-OFs was also concentration dependent, mediated by downregulation of hyaluronan synthase 2 rather than hyaluronidases. Moreover, CQ (10 μM) induced GO-OF apoptosis without aggravating oxidative stress., Conclusions: The antimalarials CQ/HCQ affect proliferation, adipogenesis, and hyaluronan generation in GO-OFs by inhibiting autophagy, providing evidence that they can be used to treat GO as autophagy inhibitors., (© Endocrine Society 2020.)

Background: Cavernous hemangioma and schwannoma are tumors that both occur in the orbit. Because the treatment strategies of these two tumors are different, it is necessary to distinguish them at treatment initiation. Magnetic resonance imaging (MRI) is typically used to differentiate these two tumor types; however, they present similar features in MRI images which increases the difficulty of differential diagnosis. This study aims to devise and develop an artificial intelligence framework to improve the accuracy of clinicians' diagnoses and enable more effective treatment decisions by automatically distinguishing cavernous hemangioma from schwannoma., Methods: Material: As the study materials, we chose MRI images as the study materials that represented patients from diverse areas in China who had been referred to our center from more than 45 different hospitals. All images were initially acquired on films, which we scanned into digital versions and recut. Finally, 11,489 images of cavernous hemangioma (from 33 different hospitals) and 3,478 images of schwannoma (from 16 different hospitals) were collected. Labeling: All images were labeled using standard anatomical knowledge and pathological diagnosis. Training: Three types of models were trained in sequence (a total of 96 models), with each model including a specific improvement. The first two model groups were eye- and tumor-positioning models designed to reduce the identification scope, while the third model group consisted of classification models trained to make the final diagnosis., Results: First, internal four-fold cross-validation processes were conducted for all the models. During the validation of the first group, the 32 eye-positioning models were able to localize the position of the eyes with an average precision of 100%. In the second group, the 28 tumor-positioning models were able to reach an average precision above 90%. Subsequently, using the third group, the accuracy of all 32 tumor classification models reached nearly 90%. Next, external validation processes of 32 tumor classification models were conducted. The results showed that the accuracy of the transverse T1-weighted contrast-enhanced sequence reached 91.13%; the accuracy of the remaining models was significantly lower compared with the ground truth., Conclusions: The findings of this retrospective study show that an artificial intelligence framework can achieve high accuracy, sensitivity, and specificity in automated differential diagnosis between cavernous hemangioma and schwannoma in a real-world setting, which can help doctors determine appropriate treatments., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/atm.2020.03.150). The series “Medical Artificial Intelligent Research” was commissioned by the editorial office without any funding or sponsorship. HL served as the unpaid Guest Editor of the series. The other authors have no other conflicts of interest to declare., (2020 Annals of Translational Medicine. All rights reserved.)

Purpose: The purpose of this study was to assess the relationship between thyroid eye disease (TED) in patients undergoing orbital decompression and immunoglobulin G4 (IgG4) levels., Methods: A prospective observational cohort study was conducted among 185 consecutive patients who were diagnosed with TED and underwent orbital decompression. Serum levels of IgG4 were measured, and immunohistochemical staining for IgG and IgG4 was performed in orbital adipose tissue. Data related to clinicopathologic features were analyzed., Results: Among the 185 enrolled patients with TED, 64 (34.6%) were IgG4-positive. The IgG4-positive patients were older, had higher clinical activity scores (CAS), and had worse best-corrected visual acuity (BCVA) than the IgG4-negative patients. Higher thyrotropin receptor antibody (TRAb) levels, histopathological IgG4 counts, IgG4/IgG ratios, and dense lymphocyte infiltration were more frequently observed in IgG4-positive than in IgG4-negative patients. Definitive and probable IgG4 subtypes were independently associated with the active stage in patients with TED., Conclusions: Our data suggest that the IgG4 subtype in TED is common. IgG4-positive patients with TED may be older, have more severe disease, and have higher clinical activity scores. IgG4 may play an important role in the pathogenesis of TED.

Importance: Early diagnosis of retinoblastoma, the most common intraocular cancer, can save both a child's life and vision. However, anecdotal evidence suggests that many children across the world are diagnosed late. To our knowledge, the clinical presentation of retinoblastoma has never been assessed on a global scale., Objectives: To report the retinoblastoma stage at diagnosis in patients across the world during a single year, to investigate associations between clinical variables and national income level, and to investigate risk factors for advanced disease at diagnosis., Design, Setting, and Participants: A total of 278 retinoblastoma treatment centers were recruited from June 2017 through December 2018 to participate in a cross-sectional analysis of treatment-naive patients with retinoblastoma who were diagnosed in 2017., Main Outcomes and Measures: Age at presentation, proportion of familial history of retinoblastoma, and tumor stage and metastasis., Results: The cohort included 4351 new patients from 153 countries; the median age at diagnosis was 30.5 (interquartile range, 18.3-45.9) months, and 1976 patients (45.4%) were female. Most patients (n = 3685 [84.7%]) were from low- and middle-income countries (LMICs). Globally, the most common indication for referral was leukocoria (n = 2638 [62.8%]), followed by strabismus (n = 429 [10.2%]) and proptosis (n = 309 [7.4%]). Patients from high-income countries (HICs) were diagnosed at a median age of 14.1 months, with 656 of 666 (98.5%) patients having intraocular retinoblastoma and 2 (0.3%) having metastasis. Patients from low-income countries were diagnosed at a median age of 30.5 months, with 256 of 521 (49.1%) having extraocular retinoblastoma and 94 of 498 (18.9%) having metastasis. Lower national income level was associated with older presentation age, higher proportion of locally advanced disease and distant metastasis, and smaller proportion of familial history of retinoblastoma. Advanced disease at diagnosis was more common in LMICs even after adjusting for age (odds ratio for low-income countries vs upper-middle-income countries and HICs, 17.92 [95% CI, 12.94-24.80], and for lower-middle-income countries vs upper-middle-income countries and HICs, 5.74 [95% CI, 4.30-7.68])., Conclusions and Relevance: This study is estimated to have included more than half of all new retinoblastoma cases worldwide in 2017. Children from LMICs, where the main global retinoblastoma burden lies, presented at an older age with more advanced disease and demonstrated a smaller proportion of familial history of retinoblastoma, likely because many do not reach a childbearing age. Given that retinoblastoma is curable, these data are concerning and mandate intervention at national and international levels. Further studies are needed to investigate factors, other than age at presentation, that may be associated with advanced disease in LMICs.

Objective: This study aimed to investigate the expression of collagen (types I, III, and V), heat shock protein 47 (HSP47), matrix metalloproteinase-2 (MMP-2), and tissue inhibitors of metalloproteinase-1 (TIMP-1) in the retrobulbar adipose tissues of patients with thyroid-associated orbitopathy (TAO)., Materials and Methods: The retrobulbar adipose tissues were collected from 4 TAO patients undergoing orbital decompression and 4 ocular enucleation patients with atrophic eyeball caused by ocular trauma between May 2019 and September 2019. Masson staining was performed to analyze the differences in collagen expression and degree of histologic fibrosis in each sample. The protein expressions of collagen (types I, III, and V), HSP47, MMP-2, and TIMP-1 were determined by western blotting. The data of western blotting were analyzed using SPSS version 17.0, with independent t -tests., Results: The results of Masson staining showed that the expression of collagen fibers in the TAO group was significantly higher than that in the control group, and the fibers were diffuse and irregular in distribution. The expression level of collagen (types I, III, and V), HSP47, MMP-2, and TIMP-1 in the TAO group were significantly higher than that in the control group ( P < 0.05)., Conclusion: The proliferation and fibrosis of retrobulbar adipose tissue in TAO patients might be related to the increased expression of collagen (types I, III, and V) and HSP47 and decreased degradation of extracellular matrix., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Hongyang Luo et al.)

Purpose: To evaluate parent knowledge of screening and genetic testing for retinoblastoma and its potential correlation with demographics, clinical features, and socioeconomical factors., Methods: It was a cross-sectional study conducted at the ocular oncology unit of a tertiary ophthalmic center in Southern China. A face-to-face interview was administered to parents of retinoblastoma children during hospitalization. Parent knowledge was assessed using the sum score of a 7-item questionnaire. Demographics and socioeconomic status were collected from the interview, and clinical data were retrieved from the medical records., Results: A total of 126 parents of retinoblastoma children were included. Parents accurately answered 66.7% to 84.9% of each item in the questionnaire. Only 37 (29.4%) parents correctly answered all 7 questions. Parent knowledge was positively correlated with education, but it was not associated with patients' laterality, sex, or household income. Physicians and the Internet were the major sources of parental health-related information. During the median follow-up of 492 days, 13 (61.9%) of 21 patients in the full-score group without genetic testing at baseline actually conducted testing during follow-up compared to 29 of 67 (43.3%) in the less-than-full-score group ( P =0.136)., Conclusion: Overall parent knowledge about retinoblastoma screening and genetic testing was moderate. Higher education was associated with greater parent knowledge. Future studies should validate our findings in other populations, especially in those with different cultural background and healthcare systems., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper., (Copyright © 2020 Wei Xiao et al.)

Hordei Fructus Germinatus (HFG) has been used as a traditional medicine to treat functional dyspepsia (FD) in China. Stir fried HFG (F-HFG) containing Maillard reaction products (MRPs) is used more widely than the raw HFG (R-HFG). However, the exact mechanisms in its functionality remain unclear. This article investigated the effect of R-HFG, F-HFG, and MRPs on brain-gut peptides, gut microbiota, and digestive enzymes using an FD animal mode. After administration of R-HFG, F-HFG, and MRPs, higher mRNA expression level of gastrin (GAS) and lower mRNA expression level of vasoactive intestinal peptide (VIP) were exhibited in F-HFG and MRPs rats than R-HFG rats ( P  < .05). Furthermore, compared with the R-HFG group, the contents of motilin (MTL) and GAS showed an upward tendency, whereas the contents of VIP and chokcystokinin (CCK) showed a downward tendency in the F-HFG group. In addition, bacterial communities in the control, F-HFG, and MRPs groups clustered closely to one another, and bacterial communities in the model and recovery groups clustered together, whereas the bacterial communities in the R-HFG group were clustered into a category. Moreover, there were no apparent differences in brain-gut peptides and gut microbiota between the F-HFG and MRPs groups. However, after the oral administration of R-HFG, F-HFG, and MRPs, the level of digestive enzyme did not show a significant change as compared with the recovery group. These results indicated that the stronger effect of F-HFG could be attributed to the MRPs produced during stir frying, and MRPs possessed the effect of regulating brain-gut peptides and gut microbiota.

Background: Hepatic metastasis develops in ~ 50% of uveal melanoma (UM) patients with no effective treatments. Although GNAQ/GNA11 mutations are believed to confer pathogenesis of UM, the underlying mechanism of liver metastasis remains poorly understood. Given that profound epigenetic evolution may occur in the long journey of circulating tumor cells (CTCs) to distant organs, we hypothesized that EZH2 endowed tumor cells with enhanced malignant features (e.g., stemness and motility) during hepatic metastasis in UM. We aimed to test this hypothesis and explore whether EZH2 was a therapeutic target for hepatic metastatic UM patients., Methods: Expression of EZH2 in UM was detected by qRT-PCR, Western blotting and immunohistochemistry staining. Proliferation, apoptosis, cancer stem-like cells (CSCs) properties, migration and invasion were evaluated under circumstances of treatment with either EZH2 shRNA or EZH2 inhibitor GSK126. Antitumor activity and frequency of CSCs were determined by xenografted and PDX models with NOD/SCID mice. Hepatic metastasis was evaluated with NOG mice., Results: We found that EZH2 overexpressed in UM promoted the growth of UM; EZH2 increased the percentage and self-renewal of CSCs by miR-29c-DVL2-β-catenin signaling; EZH2 facilitates migration and invasion of UM cells via RhoGDIγ-Rac1 axis. Targeting EZH2 either by genetics or small molecule inhibitor GSK126 decreased CSCs and motility and abrogated the liver metastasis of UM., Conclusions: These findings validate EZH2 as a druggable target in metastatic UM patients, and may shed light on the understanding and interfering the complicated metastatic process.