Human conjunctiva organoids to study ocular surface homeostasis and disease.

The conjunctival epithelium covering the eye contains two main cell types: mucus-producing goblet cells and water-secreting keratinocytes, which present mucins on their apical surface. Here, we describe long-term expanding organoids and air-liquid interface representing mouse and human conjunctiva. A single-cell RNA expression atlas of primary and cultured human conjunctiva reveals that keratinocytes express multiple antimicrobial peptides and identifies conjunctival tuft cells. IL-4/-13 exposure increases goblet and tuft cell differentiation and drastically modifies the conjunctiva secretome. Human NGFR+ basal cells are identified as bipotent conjunctiva stem cells. Conjunctival cultures can be infected by herpes simplex virus 1 (HSV1), human adenovirus 8 (hAdV8), and SARS-CoV-2. HSV1 infection was reversed by acyclovir addition, whereas hAdV8 infection, which lacks an approved drug therapy, was inhibited by cidofovir. We document transcriptional programs induced by HSV1 and hAdV8. Finally, conjunctival organoids can be transplanted. Together, human conjunctiva organoid cultures enable the study of conjunctival (patho)-physiology.

Pediatric Graves' orbitopathy: a multicentre study.

PURPOSE: Graves' orbitopathy (GO) is a rare condition in children often considered to be a less severe condition than at an older age. The aim of our study was to analyse if there are any factors that distinguish paediatric from adult GO in order to provide guidelines for assessing and managing paediatric GO. METHODS: Study design is a multicentre retrospective observational case series; 115 paediatric patients diagnosed with GO who visited our university medical centres in the Netherlands and Iran between 2003 and 2019 were submitted for complete ophthalmological examinations, serological testing and/or orbital imaging. Main outcome measures focussed on the natural course and clinical picture as well as medical and surgical treatment in paediatric GO. RESULTS: Clinical findings included proptosis (n = 97; 84.3%), eyelid retraction (n = 77; 67%) and diplopia (n = 13; 11.3%). Ninety-two patients (80%) presented with mild disease, 21 (18.3%) with moderate-severe disease and two (1.7%) with severe GO. Five patients (4.3%) underwent intravenous glucocorticoids and 25 patients underwent orbital decompression surgery. Strabismus surgery due to primary involvement of extraocular muscles was performed in two patients (1.7%). Overall, rehabilitative surgical treatment was planned in 31 patients (26.9%) with inactive disease. Two patients experienced reactivation of the disease. CONCLUSION: Despite the fact that paediatric and adult GO are considered two separate entities, they might be the same disease with two different clinical phenotypes. Paediatric GO population presents with a comparable clinical picture regarding both soft tissue involvement and proptosis, which may require surgical intervention. Proptosis was present in the majority of paediatric GO patients. Orbital decompression was performed in 21.7% of patients.

Orbital Inflammation and Dacryoadenitis after COVID-19 Vaccination.

The aim of this paper was to report the case of a patient with orbital inflammation and dacryoadenitis after COVID-19 vaccination. During the COVID-19 pandemic, we noticed an increase of post-viral syndromes, both linked to infection and to vaccination. A 53-year-old male presented with proptosis, chemosis, hypotropia, and ophthalmoplegia of the right eye, 1 day after his COVID-19 booster shot. Anecdotally, he suffered similar symptoms after his initial two vaccinations. Idiopathic orbital inflammation and dacryoadenitis were diagnosed, and the patient was successfully treated with oral steroids. Orbital inflammation and dacryoadenitis after infection or vaccination are no new entities, but due to the scale of the current pandemic and the associated vaccination programs, these rare ocular diseases could be encountered more often.

Exploring the human lacrimal gland using organoids and single-cell sequencing.

The lacrimal gland is essential for lubrication and protection of the eye. Disruption of lacrimal fluid production, composition, or release results in dry eye, causing discomfort and damage to the ocular surface. Here, we describe the establishment of long-term 3D organoid culture conditions for mouse and human lacrimal gland. Organoids can be expanded over multiple months and recapitulate morphological and transcriptional features of lacrimal ducts. CRISPR-Cas9-mediated genome editing reveals the master regulator for eye development Pax6 to be required for differentiation of adult lacrimal gland cells. We address cellular heterogeneity of the lacrimal gland by providing a single-cell atlas of human lacrimal gland tissue and organoids. Finally, human lacrimal gland organoids phenocopy the process of tear secretion in response to neurotransmitters and can engraft and produce mature tear products upon orthotopic transplantation in mouse. Together, this study provides an experimental platform to study the (patho-)physiology of the lacrimal gland.

cDC2 and plasmacytoid dendritic cells diminish from tissues of patients with non-Hodgkin orbital lymphoma and idiopathic orbital inflammation.

Non-Hodgkin orbital lymphoma (NHOL) and idiopathic orbital inflammation (IOI) are common orbital conditions with largely unknown pathophysiology. To investigate the immune cell composition of these diseases, we performed standardized 29 parameter flow cytometry phenotyping in peripheral blood mononuclear cells of 18 NHOL patients, 21 IOI patients, and 41 unaffected controls. Automatic gating by FlowSOM revealed decreased abundance of meta-clusters containing dendritic cells in patients, which we confirmed by manual gating. A decreased percentage of (HLA-DR+ CD303+ CD123+ ) plasmacytoid dendritic cells (pDC) in the circulation of IOI patients and decreased (HLA-DR+ CD11c+ CD1c+ ) conventional dendritic cells (cDC) type-2 for IOI patients were replicated in an independent cohort of patients and controls. Meta-analysis of both cohorts demonstrated that pDCs are also decreased in blood of NHOL patients and highlighted that the decrease in blood cDC type-2 was specific for IOI patients compared to NHOL or controls. Deconvolution-based estimation of immune cells in transcriptomic data of 48 orbital biopsies revealed a decrease in the abundance of pDC and cDC populations within the orbital microenvironment of IOI patients. Collectively, these data suggest a previously underappreciated role for dendritic cells in orbital disorders.

A pan-inflammatory microRNA-cluster is associated with orbital non-Hodgkin lymphoma and idiopathic orbital inflammation.

Non-Hodgkin orbital lymphoma (NHOL) and idiopathic orbital inflammation (IOI) are common orbital conditions with largely unknown pathophysiology that can be difficult to diagnose. In this study we aim to identify serum miRNAs associated with NHOL and IOI. We performed OpenArray® miRNA profiling in 33 patients and controls. Differentially expressed miRNAs were technically validated across technology platforms and replicated in an additional cohort of 32 patients and controls. We identified and independently validated a serum miRNA profile of NHOL that was remarkably similar to IOI and characterized by an increased expression of a cluster of eight miRNAs. Pathway enrichment analysis indicated that the miRNA-cluster is associated with immune-mediated pathways, which we supported by demonstrating the elevated expression of this cluster in serum of patients with other inflammatory conditions. The cluster contained miR-148a, a key driver of B-cell tolerance, and miR-365 that correlated with serum IgG and IgM concentrations. In addition, miR-29a and miR-223 were associated with blood lymphocyte and neutrophil populations, respectively. NHOL and IOI are characterized by an abnormal serum miRNA-cluster associated with immune pathway activation and linked to B cell and neutrophil dysfunction.

Zirconium-89-labelled rituximab PET-CT in orbital inflammatory disease.

BACKGROUND: Orbital inflammatory diseases are a heterogenic group of conditions that often entail a difficult diagnostic process and many patients are treatment resistant. Inflammatory diseases can be visualized by Zirconium-89-labelled rituximab PET-CT (89Zr-rituximab PET/CT). In this study, we describe our experience and possible potential of the 89Zr-rituximab PET/CT for diagnostic and therapeutic management of refractory orbital inflammation. RESULTS: Retrospectively, 89Zr-rituximab uptake was assessed and related to clinical data. The main outcome measures were the characteristics of the scan and the clinical relation of uptake with the diagnostic process and treatment effectivity. Twelve patients with thyroid eye disease (TED) and suspected idiopathic orbital inflammation (IOI) were scanned. Six patients had a strong 89Zr-rituximab uptake and showed a focal distribution within the lesion. Four patients (one TED, three IOI) responded well to rituximab treatment after a positive scan. 89Zr-rituximab PET/CT was essential to the diagnosis of optic nerve meningioma in one patient. CONCLUSION: 89Zr-rituximab PET/CT has the potential to be a powerful tool for the detection of B cell-mediated disease within the orbit and ocular adnexa. This technique can be a valuable addition for diagnosing diseases around the eye and can potentially predict rituximab treatment response in patients with refractory inflammation.

Evaluation of classification systems for nonspecific idiopathic orbital inflammation.

PURPOSE: To systematically analyze existing classification systems for idiopathic orbital inflammation (IOI) and propose and test a new best practice classification system. METHODS: A systematic literature search was conducted to find all studies that described and applied a classification system for IOI. Classification categories used in more than two studies were extracted, and criteria for these categories were defined using common descriptors. Using patient data, these newly defined classification systems were evaluated. Reliability was tested by inter- and intrarater agreement of two raters and distinction tested by evaluating clinical differences among classification categories. Feasibility, face validity, and content validity were qualitatively tested. RESULTS: The most frequently encountered IOI classification systems were based on onset (acute, chronic), histopathology (classic, granulomatous, sclerosing), or localization (diffuse, extraocular muscle, lacrimal gland, sclera, optic nerve). Systems based on histopathology and localization showed good reliability (κ values range 0.74-0.89), were easy to apply (feasibility), and described the biologic process (face validity). Because of static sampling, histopathology-based systems had moderate content validity and moderate distinction between classification categories. Being a static measure, localization had moderate content validity, but good distinction. It was found that content validity was improved by combining histopathology and localization into a two-dimensional classification system. CONCLUSIONS: This combined histopathology and localization-based classification system provides a repeatable, easy to use, plausible, and complete classification system that can be used to further advance the research of IOI.

The role of biopsy in diagnosing patients suspected of idiopathic orbital inflammation.

PURPOSE: To provide a framework when to biopsy patients suspected of idiopathic orbital inflammation (IOI). MATERIALS AND METHODS: One-hundred seventeen patients were selected with signs of orbital inflammation in whom after history and physical examination, no definite diagnosis was made. In this cohort, the role of biopsy in making a diagnosis was evaluated and compared to a therapeutic trial of corticosteroids. RESULTS: In 67 of 117 patients, a diagnosis was evident after imaging and laboratory testing. In 50 remaining patients, a decision had to be made to biopsy or administer a therapeutic trial of corticosteroids. Thirty-nine patients underwent biopsy as the next step. Nine patients underwent a therapeutic trial of corticosteroids of which one was subsequently subjected to biopsy. Two patients spontaneously resolved. No patients with a malignancy were inadvertently treated with corticosteroids. One patient experienced a biopsy related complication but did not lose vision. CONCLUSIONS: This study suggests using a therapeutic trial of corticosteroids only in patients with low suspicion of malignancy with muscular and apical mass localizations, or with optic-nerve compression. This framework was demonstrated to be safe in not delaying diagnosis of malignancies and efficient in providing a rapid diagnosis.

Orbital mass as manifestation of Wegener's granulomatosis: an ophthalmologic diagnostic approach.

OBJECTIVES: Orbital manifestation of Wegener's granulomatosis is diverse and diagnosis is often difficult. This study aims to improve the diagnostic strategy in orbital Wegener. METHODS: A review of the diagnostic process in patients in whom a diagnosis of orbital WG was considered. RESULTS: Thirty-three patients were analysed, consisting of 15 patients with orbital WG, 11 with idiopathic orbital inflammation, 6 with orbital sarcoidosis and one with aspergillosis. Diagnostic findings indicating orbital WG were ear/nose/throat involvement, multiple organ system involvement, a positive ANCA, and on histology vasculitis, whereas granulomatous inflammation without signs of vasculitis was more indicative of another orbital disease. CONCLUSIONS: The diagnostic process of orbital WG should include CT scanning of the orbit and sinuses, ANCA blood testing, consultation of a rheumatologist, an ophthalmologist, and an ear-nose-throat specialist, and biopsy of an easily accessible, active inflammatory lesion.

Treatment of severe idiopathic orbital inflammation with intravenous methylprednisolone.

BACKGROUND: Prednisone pulse therapy is used to treat active non-infectious orbital inflammatory disease to attain faster clinical improvement and to shorten the duration of prednisone treatment. This study addresses the use of intravenous methylprednisolone (IVMP) pulse therapy, in addition to oral prednisone (OP), in the treatment of severe idiopathic orbital inflammation (IOI). METHODS: This was a multicentre retrospective cohort study. Patients with severe IOI treated with IVMP pulse and OP therapy (IVMP+OP) were compared with patients with IOI who were treated only with OP. Main outcome measures were duration of prednisone treatment, symptom-free outcome and complications. RESULTS: Between 2000 and 2007, 12 patients with severe IOI were treated with IVMP+OP and 15 patients were treated with OP only. The median treatment duration was 160 (range 34-680) days in the IVMP+OP group and 110 (range 27-730) days in the OP-only group. In patients who had severe IOI, 73% in the IVMP+OP group and 87% in the OP-only group were symptom-free after treatment. No patients developed complications related to prednisone therapy. CONCLUSION: In our study there was no advantage of treating patients with severe IOI with IVMP+OP in terms of shortened treatment duration, lower cumulative dose or decrease in persistent symptoms. We suggest that the indication of IVMP in the treatment of severe IOI is limited to speeding symptom relief and recovery from optic nerve dysfunction.

Risk factors for idiopathic orbital inflammation: a case-control study.

OBJECTIVE: To identify risk factors involved in the development of idiopathic orbital inflammation (IOI). METHODS: Case-control study of 69 adults who had had a first episode of IOI and 296 adult controls with rhegmatogenous retinal detachment (RD) selected from three orbital centres in The Netherlands between 2000 and 2006. Participants filled out a questionnaire on demographic factors, medical history, health status and exposures for the 2 years prior to disease presentation. In addition, women were questioned about previous or current pregnancies and their hormonal status. ORs and accompanying 95% CIs for IOI in relation to potential risk factors such as body mass index (BMI), bisphosphonates and autoimmune disease were estimated. ORs were adjusted for age, sex, socio-economic status, smoking and blunt orbital trauma using logistic regression. Analyses were carried out both with and without multiple imputation of missing values. RESULTS: The risk of IOI was increased in participants who had a higher BMI (third vs first tertile: OR, 2.88; 95% CI 1.32 to 6.32) and in participants who used bisphosphonates (OR 8.68; 95% CI 1.16 to 65.0). The risk was decreased in participants with a higher socio-economic status (third vs first tertile: OR 0.38; 95% CI 0.17 to 0.84) and in women who were older at first childbirth (third vs first tertile: OR 0.14; 95% CI 0.03 to 0.64). An almost significant association was found for IOI and autoimmune disease (OR 2.56; 95% CI 0.93 to 7.05). CONCLUSIONS: IOI is associated with lower socio-economic status, higher BMI and use of oral bisphosphonates. In women, IOI is also associated with younger age at first childbirth.

Methylprednisolone pulse therapy for patients with moderately severe Graves' orbitopathy: a prospective, randomized, placebo-controlled study.

OBJECTIVE: To assess whether methylprednisolone (MP) pulse therapy is efficacious in the treatment of moderately severe Graves' orbitopathy (GO). DESIGN: Prospective, placebo (PL)-controlled, double-blind, randomized study. METHODS: Fifteen previously untreated patients with active, moderately severe GO participated in the study; 6 patients received MP and 9 patients a PL. Moderately severe disease was defined using the NOSPECS classification of clinical signs of GO . Activity was measured with the clinical activity score (CAS). A dose of 500 mg MP or only solvent was administered intravenously, over three consecutive days, in four cycles at 4 weekly intervals (6 g of MP in total). Qualitatively, a successful treatment outcome was defined as an improvement in one major and/or two minor criteria in the worst eye at week 48. The major criteria were: improvement in diplopia grade; improvement in eye movement; a decrease in CAS of three points. The minor criteria were: decrease of eyelid retraction; decrease of proptosis; improvement in grade of soft tissue swelling; a decrease in CAS of two points. RESULTS: The qualitative treatment outcome was successful at the end of the trial in five out of six (83%) patients receiving MP and in one out of nine (11%) patients given the PL (relative risk=7.5; (95% confidence interval 1.1-49.3), P=0.005). The treatment was well tolerated. CONCLUSIONS: In spite of the small number of patients, a significant difference in outcome was observed between MP- and PL-treated patients. We conclude that MP pulse therapy appears to be an effective treatment for active, moderately severe GO.