Changes in the ocular surface microbiome of patients with coronavirus disease 2019 (COVID-19).

Purpose: To elucidate the reasons behind the increased incidence of ocular disease in patients with coronavirus disease 2019 (COVID-19), this study delved deeper into the specific effects of COVID-19 on patients' ocular surface microbiome (OSM) and investigated its relationship with the increased incidence of ocular disease. Methods: In this study, conjunctival sac swabs were collected from 43 participants for 16S rRNA amplicon sequencing. The participants were categorized into three groups based on their COVID-19 status: the control group (C group) consisted of 15 participants who showed no evidence of COVID-19, the experimental group (E group) included 15 participants who tested positive for COVID-19, and the COVID-19 recovery period group (R group) comprised 13 participants. Results: In the comparison of alpha diversity, group E had a higher Shannon, Chao1 and Goods coverage index. When comparing beta diversity, groups E and R were more similar to each other. At the phylum level, although the OSM of the three groups was dominated by Proteobacteria, Actinobacteriota, Bacteroidota and Firmicutes, the compositional proportions were significantly different. At the genus level, the dominant species in the three OSM groups were significantly different, with Pseudomonas becoming the dominant genus in groups E and R compared to group C, and the abundance of Ralstonia decreasing significantly. Conclusion: This study provides additional evidence supporting the association between the OSM and COVID-19, which contributes to our understanding of the potential mechanisms underlying ocular symptoms and complications associated with COVID-19 in the future.

Loose suture-related ocular surface inflammation and activation of conjunctiva-associated lymphoid tissue in patients after keratoplasty.

The purpose of this study is to evaluate loose suture-related inflammation and activation of conjunctiva-associated lymphoid tissue (CALT) in patients after keratoplasty. The patients who were treated with keratoplasty at the First Affiliated Hospital of Harbin Medical University between 2015 and 2022 were recruited into the study. We evaluated the time and location of loose suture development in patients after keratoplasty. In addition, in vivo confocal microscopy was used to evaluate the activation of CALT and the accumulation of inflammatory cells around loose sutures. Meso Scale Discovery assay detection kits were used to evaluate the inflammatory cytokines in the tears of patients before and after the loose suture was removed. In this study, we collected the information from 212 cases (212 eyes) who had PK (126 eyes) and DALK-treated (86 eyes) for corneal transplantation, including 124 males and 88 females, aged 14-84 years old. The average age was 50.65 ± 16.81 years old. Corneal sutures were more prone to loose at 3 months and 6 months after keratoplasty, and the frequent sites were at 5 and 6 o'clock. An increased number of inflammatory cells could be observed around the loose sutures than normal sutures (P < 0.001). In CALT, the density of diffuse lymphocytes (P < 0.001), follicles (P < 0.001), and parafollicular lymphocytes (P < 0.001) were higher and the central reflection of the follicles (P < 0.001) was stronger when suture loosening happened. The levels of inflammatory cytokines such as IL-1ß (P = 0.003), IL-8 (P = 0.012), and TNF-α (P < 0.001) were higher in the tears of the patients with loose sutures. The activation of CALT was partly settled after removing the loose sutures. In conclusion, loose sutures after corneal transplantation can lead to increased infiltration of inflammatory cells, activation of CALT, and increased secretion of inflammatory cytokines in the tears of patients. Regular follow-up to identify and solve the problem in time can avoid suture-related complications.

Identification and Verification of Ferroptosis-Related Genes in Keratoconus Using Bioinformatics Analysis.

Objective: Keratoconus is a commonly progressive and blinding corneal disorder. Iron metabolism and oxidative stress play crucial roles in both keratoconus and ferroptosis. However, the association between keratoconus and ferroptosis is currently unclear. This study aimed to analyze and verify the role of ferroptosis-related genes (FRGs) in the pathogenesis of keratoconus through bioinformatics. Methods: We first obtained keratoconus-related datasets and FRGs. Then, the differentially expressed FRGs (DE-FRGs) associated with keratoconus were screened through analysis, followed by analysis of their biological functions. Subsequently, the LASSO and SVM-RFE algorithms were used to screen for diagnostic biomarkers. GSEA was performed to explore the potential functions of the marker genes. Finally, the associations between these biomarkers and immune cells were analyzed. qRT‒PCR was used to detect the expression of these biomarkers in corneal tissues. Results: A total of 39 DE-FRGs were screened, and functional enrichment analysis revealed that the DE-FRGs were closely related to apoptosis, oxidative stress, and the immune response. Then, using multiple algorithms, 6 diagnostic biomarkers were selected, and the ROC curve was used to verify their risk prediction ability. In addition, based on CIBERSORT analysis, alterations in the immune microenvironment of keratoconus patients might be associated with H19, GCH1, CHAC1, and CDKN1A. Finally, qRT‒PCR confirmed that the expression of H19 and CHAC1 was elevated in the keratoconus group. Conclusion: This study identified 6 DE-FRGs, 4 of which were associated with immune infiltrating cells, and established a diagnostic model with predictive value for keratoconus.

Tetrahedral DNA-Based Functional MicroRNA-21 Delivery System: Application to Corneal Epithelial Wound Healing.

Corneal injury occurs frequently which may lead to serious visual impairment. Rapid and efficient re-epithelialization after corneal epithelial injury is the key issue for maintaining corneal homeostasis. Among various treatment strategies, microRNA (miR)-based therapy shows great potential. However, structural limitations of miRNAs hinder its biomedical functionality. Nucleic acid nanotechnology is an appealing candidate for gene delivery because of its flexible modification and excellent biocompatibility. Herein, modified 3D tetrahedral framework nucleic acids (tFNAs) utilized as gene carriers for miR-21 delivery are constructed. TFNAs-miR-21 (T-21) shows great enzymatic resistance in extracellular environment and payload delivery into human corneal epithelial cells (HCECs) via clathrin-mediated endocytosis. T-21 facilitates proliferation and migration in HCECs via activating PI3K/AKT and ERK1/2 signaling pathways in vitro. In vivo studies, T-21 can be internalized by corneal epithelium in mice. In the mice corneal scratch model, T-21 ophthalmic solutions used as eye drops show no apparent side effects on the ocular surface histologically and exert great potential in accelerating corneal wound healing. These findings demonstrate that modified tFNAs are promising candidates for miRNA delivery for corneal wound healing. The convenient administration and great biocompatibility of tetrahedral DNA nanoparticles highlight its potential as gene transporter in solving ocular problems.

Misdiagnosis and Treatment of Corneal Complications Caused by Suture Exposure After Buried-Suture Double-Eyelid Blepharoplasty.

PURPOSE: The purpose of this study was to summarize the misdiagnosis and treatment of corneal complications associated with suture exposure in cases of buried-suture double-eyelid blepharoplasty. METHODS: This study retrospectively analyzed 14 patients with palpebral conjunctival and corneal complications due to suture exposure after buried-suture double-eyelid blepharoplasty at the First Affiliated Hospital of Harbin Medical University from January 2020 to July 2022. The patients' clinical symptoms included photophobia, lacrimation, pain, foreign body sensation, swelling of the eyelids, conjunctival hyperemia, secretion, etc. We recorded the patient's sex, age, surgical method, length of exposed suture, suture type, number of double-eyelid surgeries, surgical site, timepoint when eye discomfort occurred, misdiagnosed disease and treatment. RESULTS: Three patients were misdiagnosed with dry eye, nine patients were misdiagnosed with viral keratitis, and two patients were misdiagnosed with allergic conjunctivitis. All 14 patients had manifestations of photophobia, lacrimation, pain, foreign body sensation and conjunctival hyperemia. Eight patients had manifestations of swelling of the eyelids. Five patients had manifestations of eye secretions. There were 8 patients with corneal epithelial injuries and 6 patients with corneal ulcers. All patients underwent suture removal without further progression. Ten patients were treated with artificial tears, and 4 patients were treated with calf serum deproteinized gel after suture removal. CONCLUSION: If there is postoperative eye discomfort caused by eyelid and corneal complications in patients after buried-suture double-eyelid blepharoplasty, clinicians should carefully check whether there is suture exposure and determine the cause in a timely manner. Suture removal is the best way to treat this complication. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Activation of Conjunctiva-Associated Lymphoid Tissue in Diabetic Patients.

PURPOSE: To evaluate changes in conjunctiva-associated lymphoid tissues (CALTs) in patients with type 2 diabetic mellitus (T2DM). METHODS: Thirty-two patients with T2DM and 32 healthy volunteers underwent comprehensive examinations. In vivo confocal microscopy and Image J were used to observe and evaluate the patients' CALT-related parameters. Conjunctival impression cytology (CIC) samples of the tarsal conjunctiva were collected from the patients, and CD4+ and CD8+ cells were evaluated by immunofluorescence staining. RESULTS: The diabetes group showed higher diffuse lymphocyte density(p < .001), follicular density(p < .001) and parafollicular lymphocyte density(p < .001). The percentages of CD4+ cells (p < .001) and CD8+ cells (p < .001) in the diabetes group were higher than those in the control group. CALT-related parameters of the diabetic patients with diabetic retinopathy showed higher degrees of activation than those of the diabetic patients without diabetic retinopathy. CONCLUSIONS: CALT activation is observed in patients with T2DM, and the activation is more obvious in patients with diabetic retinopathy. TRIAL REGISTRATION NUMBER: Retrospectively registered, ChiCTR2100046030.

A 5-Year Retrospective Analysis of the Risk Factors, Treatment, and Prognosis of Patients With Fungal Keratitis in Heilongjiang, China.

PURPOSE: To report the epidemiologic features, laboratory findings, and treatment outcomes of patients with fungal keratitis (FK) during a busy farming period (May, June, and September to November) in Heilongjiang Province, China. DESIGN: Retrospective, observational case series. METHODS: In total, 251 patients diagnosed with FK at the Eye Hospital, First Affiliated Hospital of Harbin Medical University, from 2017 to 2021 were identified. Medical records were retrospectively analyzed, and demographic features, risk factors, monthly distributions, clinical characteristics, laboratory findings, treatment strategies, and prognostic data were collected. RESULTS: The number of FK cases in the busy farming period accounted for 74.1% (186/251) of the total. The mean patient age was 58.2±9.5 years. Males (66.7%) were more likely to develop FK than females (33.3%), and plant-related trauma was the main cause in 80.1% (149/186) of the cases. The most common causative fungal species was Fusarium (34.9%). In vivo confocal microscopy had the highest positivity rate for FK diagnosis (94.6%). The depth of hypopyon, depth of hyphae or spores, and infiltrate width were significantly positively correlated with delayed presentation. CONCLUSIONS: Patients with a longer time of presentation to the hospital were more likely to undergo surgery because of topical medical therapy failure. The most important measures for FK prevention are the use of eye protection during outdoor work and visiting the hospital for treatment as soon as possible after eye injury.

Dexamethasone attenuates dry eye-induced pyroptosis by regulating the KCNQ1OT1/miR-214 cascade.

Dry eye disease (DED) is an inflammatory disorder of the ocular surface seriously affecting the quality of life of patients. Topical dexamethasone (Dex) administration protects the cornea from the hyperosmotic stress (HS) induced by tears. Pyroptosis participates in the activation of epithelial inflammation during DED. However, it remains unclear whether Dex attenuates the progression of DED through pyroptosis. In this study, we aimed to investigate the effect of Dex on DED using both cell and animal models and its underlying mechanism. The inflammatory factors contained in tears were detected using a cytokine assay. The pyroptosis in DED mice and human corneal epithelial cells (HCECs) treated with hyperosmotic medium under various treatments was evaluated by immunohistochemical assays (IHC) or western blotting (WB). RNA expression was manipulated with siRNA or agomir microRNAs and measured using a polymerase chain reaction. The scratch assay was used to assess the migration rate of HCECs. Remaining corneal defects were evaluated using fluorescein staining and photographed using a digital camera. Dex could suppress the release of inflammatory factors and notably attenuate pyroptosis, KCNQ1OT1 expression, and NF-κB activation induced by HS injury in vivo and in vitro. KCNQ1OT1 upregulation could activate pyroptosis by sponging miR-214. Furthermore, KCNQ1OT1 knockdown and miR-214 overexpression reversed the effect of HS, promoted the migration of HCECs, and accelerated corneal wound healing. Dex effectively suppressed HS-induced pyroptosis through the KCNQ1OT1/miR-214/caspase-1 signaling axis by inhibiting the NF-κB activation. Our results provide a novel understanding of the mechanism of Dex as an anti-inflammatory drug in DED.

Observation of Conjunctiva-Associated Lymphoid Tissue With In Vivo Confocal Microscopy in Healthy Patients and Patients With Meibomian Gland Dysfunction.

PURPOSE: The purpose of this study was to assess the distribution and morphological variation of conjunctiva-associated lymphoid tissue (CALT) in healthy human subjects and patients with meibomian gland dysfunction (MGD) using laserscanningin vivo confocal microscopy. METHODS: A total of 34 healthy subjects and 32 patients with MGD were enrolled. All subjects underwent a conventional examination consisting of slitlamp biomicroscopy, tear film break-up time, and the Schirmer test. In vivo microscopy was applied to analyze the morphological changes in the diffuse lymphoid layer and lymphoid follicles in CALT. Conjunctival impression cytology (CIC) of samples of patients' palpebral conjunctiva and immunofluorescence staining of CD4 and CD8 antibodies were also performed to indicate the immune response status of CALT. RESULTS: In the MGD group, the density of diffuse lymphocytes ( P < 0.001), follicles ( P < 0.001), and perifollicular lymphocytes was higher ( P < 0.001) and the central reflection of the follicles was stronger ( P < 0.001) than in the control group, while there was no difference in the follicle area ( P = 0.758). Besides, diffuse lymphocyte density was correlated with telangiectasia, and follicular center reflection intensity was correlated with plugging. CIC immunofluorescence staining showed a higher percentage of CD4 + ( P < 0.001) and CD8 + ( P < 0.001) cells in the MGD group than in the control group. CONCLUSIONS: Using laser scanning in vivo confocal microscopy and CIC immunofluorescence staining, we observed the activation of CALT in patients with MGD, and some CALT-related parameters correlated with the lid margin findings of patients with MGD.

Activation of Conjunctiva-Associated Lymphoid Tissue in Patients With Infectious Keratitis Using In Vivo Confocal Microscopy.

Purpose: We aimed to evaluate activation of conjunctiva-associated lymphoid tissue (CALT) in patients with keratitis using in vivo confocal microscopy (IVCM) and conjunctival impression cytology (CIC). Methods: In addition to anterior segment photography and corneal fluorescein staining, IVCM revealed the palpebral conjunctiva in all subjects, and CIC and immunofluorescence staining were performed. Results: Diffuse lymphoid tissue cell density in the eyes of patients with keratitis was significantly greater compared with healthy volunteers (P < 0.001). Similar trends were found in perifollicular lymphocyte density (P < 0.001), follicular density (P = 0.029), follicular center reflection intensity (P = 0.011), and follicular area (P < 0.001). Immunofluorescence staining showed that the proportions of CD4+ (61.7% ± 8.0% vs. 17.3% ± 10.2%, respectively, P < 0.001) and CD8+ (46.9% ± 10.0% vs. 19.6% ± 11.5%, respectively, P < 0.001) cells in patients with keratitis was greater compared with healthy volunteers. Interestingly, we also observed changes in the contralateral eye in subjects with keratitis. Conclusions: Our research suggests that CALT, as an ocular immune structure, is activated and plays an important role in the pathogenesis of keratitis. This has been overlooked previously. CALT is also active in the contralateral eye of subjects with keratitis.