Whole Genome Sequencing Highlights the Pathogenic Profile in Nocardia Keratitis.

Purpose: Nocardia keratitis is a serious and sight-threatening condition. This study aims to reveal the virulence and antimicrobial resistance gene profile of Nocardia strains using whole genome sequencing. Methods: Whole-genome sequencing was performed on 23 cornea-derived Nocardia strains. Together with genomic data from the respiratory tract and the environment, 141 genomes were then utilized for phylogenetic and pan-genome analyses, followed by virulence and antibiotic resistance analysis. The correlations between virulence genes and pathogenicity were experimentally validated, including the characteristics of Nocardia colonies and clinical and histopathological evaluations of Nocardia keratitis mice models. Results: Whole-genome sequencing of 141 Nocardia strains revealed a mean of 220 virulence genes contributed to bacterial pathogenesis. The mce gene family analysis led to the categorization of strains from the cornea into groups A, B, and C. The colonies of group C had the largest diameter, height, and fastest growth rate. The size of corneal ulcers and the clinical scores showed a significant increase in mouse models induced by group C. The relative expression levels of pro-inflammatory cytokines (CD4, IFN-γ, IL-6Rα, and TNF-α) in the lesion area exhibited an increasing trend from group A to group C. Antibiotic resistance genes (ARGs) spanned nine distinct drug classes, four resistance mechanisms, and seven primary antimicrobial resistance gene families. Conclusions: Whole genome sequencing highlights the pathogenic role of mce gene family in Nocardia keratitis. Its distribution pattern may contribute to the distinct characteristics of the growth of Nocardia colonies and the clinical severity of the mice models.

SAW: an efficient and accurate data analysis workflow for Stereo-seq spatial transcriptomics.

The basic analysis steps of spatial transcriptomics require obtaining gene expression information from both space and cells. The existing tools for these analyses incur performance issues when dealing with large datasets. These issues involve computationally intensive spatial localization, RNA genome alignment, and excessive memory usage in large chip scenarios. These problems affect the applicability and efficiency of the analysis. Here, a high-performance and accurate spatial transcriptomics data analysis workflow, called Stereo-seq Analysis Workflow (SAW), was developed for the Stereo-seq technology developed at BGI. SAW includes mRNA spatial position reconstruction, genome alignment, gene expression matrix generation, and clustering. The workflow outputs files in a universal format for subsequent personalized analysis. The execution time for the entire analysis is ∼148 min with 1 GB reads 1 × 1 cm chip test data, 1.8 times faster than with an unoptimized workflow.

Ferroptosis as a Potential Therapeutic Target for Reducing Inflammation and Corneal Scarring in Bacterial Keratitis.

Purpose: Bacterial keratitis (BK) is a serious ocular infection that can cause severe inflammation and corneal scarring, leading to vision loss. In this study, we aimed to investigate the involvement of ferroptosis in the pathogenesis of BK. Methods: Transcriptome analysis was performed to evaluate ferroptosis-related gene expression in human BK corneas. Subsequently, the ferroptosis in mouse models of Pseudomonas aeruginosa keratitis and corneal stromal stem cells (CSSCs) were validated. The mice were treated with levofloxacin (LEV) or levofloxacin combined with ferrostatin-1 (LEV+Fer-1). CSSCs were treated with lipopolysaccharide (LPS) or LPS combined Fer-1. Inflammatory cytokines, α-SMA, and ferroptosis-related regulators were evaluated by RT-qPCR, immunostaining, and Western blot. Iron and reactive oxygen species (ROS) were measured. Results: Transcriptome analysis revealed significant alterations in ferroptosis-related genes in human BK corneas. In the BK mouse models, the group treated with LEV+Fer-1 exhibited reduced inflammatory cytokines (MPO, TNF-α, and IFN-γ), decreased corneal scarring and α-SMA expression, and lower Fe3+ compared to the BK and LEV groups. Notably, the LEV+Fer-1 group showed elevated GPX4 and SLC7A11 in contrast to the BK and LEV group. In vitro, Fer-1 treatment effectively restored the alterations of ROS, Fe2+, GPX4, and SLC7A11 induced by LPS in CSSCs. Conclusions: Ferroptosis plays a crucial role in the pathogenesis of BK. The inhibition of ferroptosis holds promise for mitigating inflammation, reducing corneal scarring, and ultimately enhancing the prognosis of BK. Consequently, this study provides a potential target for innovative therapeutic strategies for BK, which holds immense potential to transform the treatment of BK.

Acanthamoeba Keratitis in China: Genotypic and Clinical Correlations.

Purpose: To investigate the relationship between Acanthamoeba genotypes, clinical manifestations, and outcomes in Acanthamoeba keratitis (AK) patients. Methods: This retrospective study included 159 culture-confirmed AK patients. Patients' data were collected, including demographics, initial diagnosis, treatments, and clinical features. The genotype of Acanthamoeba was identified through sequencing the Diagnostic Fragment 3 (DF3) region in the small ribosomal subunit RNA genes. The phylogenetic tree was constructed using the ClustalW model and maximum likelihood method. Cases with "poor outcome" were defined based on specific clinical criteria, including corneal perforation, keratoplasty, other eye surgery, duration of anti-amoebic therapy ≥8.0 months, and final visual acuity ≤20/80. "Better outcome" cases were the remainder. The correlation between T4 subtypes, clinical phenotypes, and clinical prognosis were further analyzed. Results: In this study, AK was primarily attributed to the T4A genotype, with a positive correlation between geographical and genetic distances. The primary clinical associated with T4 subtypes was deep stromal infiltration. Results was also showed a significant association between T4 subtypes and clinical outcomes (P = 0.021). Further analysis revealed that T4C was closely associated with a better prognosis (P = 0.040) and T4D with worse outcomes (P = 0.013). Conclusions: In China, AK was predominantly caused by the T4A subtype. Geographical distance positively correlated with genetic distance. Clinical prognosis varied among different subtypes, notably in T4C and T4D. Translational Relevance: This study demonstrated the association between T4 subtypes and clinical phenotypes, as well as the effects of T4 subtypes on clinical prognosis.

In Vivo Corneal Confocal Microscopy for Detecting Corneal Nerve Fiber Changes in Patients with Different Types of Optic Neuritis: A Cross-Sectional Study.

PURPOSE: Optic neuritis (ON), a demyelinating disease of the central nervous system, is often a precursor manifestation of neuromyelitis optica spectrum disorders (NMOSD) or multiple sclerosis (MS). Reduced corneal nerve fiber counts have been found in patients with NMOSD or MS. This study aimed to observe and compare the corneal subbasal nerve plexus in patients with three types of ON and controls without ON using in vivo corneal confocal microscopy (IVCM). METHODS: Data were analyzed for 77 eyes of 48 patients with ON, grouped according to seropositivity for anti-aquaporin-4 IgG, myelin oligodendrocyte glycoprotein antibody, or no seropositivity, and 35 healthy eyes in the control group. Corneal parameters were quantified based on IVCM images. Visual function indicators were recorded, following which their correlations with IVCM parameters were analyzed. RESULTS: Significant differences in IVCM parameters were detected among the different groups. Reductions in corneal nerve fiber counts were negatively correlated with visual acuity. Corneal nerve fibers were significantly more damaged in the affected eye than in the unaffected eye in patients with ON. CONCLUSION: IVCM revealed corneal nerve fiber loss of varying degrees, depending on the type of ON. This indicates that, although ON primarily affects the central nervous system, peripheral nerves, such as the trigeminal nerve, which innervates the corneal subbasal nerve plexus may also be damaged in affected patients.

Corneal Epithelial Dendritic Cells: An Objective Indicator for Ocular Surface Inflammation in Patients with Obstructive Meibomian Gland Dysfunction?

PURPOSE: To examine whether corneal epithelial dendritic cells (CEDC) could serve as an indicator to distinguish obstructive meibomian gland dysfunction (MGD) with or without ocular surface inflammation (OSI). METHODS: We performed a case-control study on patients with diagnosed obstructive MGD between August 2017 and November 2019. RESULTS: 30 MGD cases and 25 healthy controls were recruited. The classification of MGD patients with and without OSI was based on the tear pro-inflammatory cytokine levels. Compared with the MGD without OSI and the control group, a higher CEDC density was detected in the MGD with OSI subgroup. The presence of >15.6 cells/mm2 CEDC had a sensitivity of 73% and specificity of 75% for the diagnosis of MGD with OSI. CONCLUSIONS: OSI is not present in all patients with obstructive MGD. Evaluation of CEDC density in the central cornea may help identify whether MGD is concomitant with OSI.

Extracellular Vesicle MicroRNAs From Corneal Stromal Stem Cell Enhance Stemness of Limbal Epithelial Stem Cells by Targeting the Notch Pathway.

Purpose: The limbal niche supports the self-renewal of limbal epithelial stem cells (LESCs). The corneal stromal stem cell (CSSC) is an important component in the niche that regulates the LESC phenotype. However, the intercellular communication between LESCs and CSSCs has yet to be elucidated. Methods: A traditional two-dimensional (2D) system, a direct three-dimensional (3D) system, and an indirect 3D coculture system of LESCs and CSSCs were used to elucidate the paracrine pathway effect of CSSCs on LESCs. To reveal the impact of CSSC derived extracellular vesicles (CSSC-EVs) on LESCs, GW4869 and CSSC-EVs were added separately to the LESC culture medium. The outgrowth rate, cell density, differentiation, and stemness maintenance were compared among these methods. The miRNAs in the CSSC-EVs were sequenced, and the targeted Notch pathway was further confirmed by RT‒qPCR and Western blotting. Results: Compared with 2D culture, both the direct and indirect 3D coculture systems yielded a higher outgrowth rate and expression of stem cell markers of LESCs. The phenotypes of LESCs cultivated using the two coculture approaches were also comparable. Nevertheless, GW4869 inhibited the effect of CSSCs on LESCs, and the addition of CSSC-EVs to the 2D culture system could increase cell density, and the proportion of p63αbright cells, which indicated that CSSC-EVs were crucial in regulating LESCs. Furthermore, the EV-AlixKD with reduced miRNA partly lost its regulating function. The abundant miRNAs in CSSC-EVs, such as hsa-miR-663b, hsa-miR-16-5p, and hsa-miR-1290, target the Notch pathway. The LESCs transfected with miR-663b had higher p63 expression via downregulating of the Notch pathway. Conclusions: CSSC-EV played an important role in promoting LESC proliferation and stemness maintenance by targeting Notch signaling via miRNAs, which will increase our understanding of the limbal niche and provide a potential new approach for LESC culture and the treatment of corneal epithelial disorders.

Risk Factors Associated with Multi-Drug Resistance in Neonatal Sepsis Caused by Escherichia coli.

Background and Objective: An increasing number of cases of neonatal sepsis due to extended-spectrum beta-lactamase (ESBL)-producing multi-drug resistant (MDR) Escherichia coli (E. coli) have been reported worldwide. The aim of this study was to explore the risk factors associated with ESBL-producing MDR E. coli among neonates with culture-confirmed E. coli sepsis and thereby to help selection of appropriate empirical antibiotics. Patients and Methods: All newborn infants with a confirmed pathogen isolated from blood or cerebrospinal fluid (CSF) from 2016 to 2021 were identified and those with E. coli infection were included in this analysis. We compared a group of neonatal patients with ESBL-producing MDR E. coli sepsis (n=69) to a group with ESBL-negative E. coli (n=70) based on antimicrobial susceptibility reports. We used multivariable regression analysis to determine the risk factors associated with ESBL-producing MDR E. coli strains among the neonates with culture-confirmed E. coli sepsis. Results: ESBL-producing MDR E. coli sepsis was more common in premature infants and newborns with hospital-acquired late-onset sepsis (HALOS). The mortality rate of neonatal sepsis caused by ESBL-producing E. coli was about twice as that of sepsis caused by ESBL-negative E. coli. Antepartum exposure to cephalosporins (OR=25.191, 95% CI: 3.184-199.326, P<0.01) and parenteral nutrition for more than 1 week (OR=4.495, 95% CI: 2.009-10.055, P<0.01) were independent risk factors for neonatal infection with ESBL-producing stains among infants with E. coli sepsis. Conclusion: E. coli remains the most common Gram-negative bacterial pathogen causing neonatal sepsis. A higher proportion of ESBL-producing MDR E. coli is seen in premature infants and those newborns with HALOS and is associated with higher mortality. Antepartum use of cephalosporins and prolonged use of parenteral nutrition may be important factors to consider in the selection of empirical antibiotics for use in neonatal sepsis caused by gram-negative rods prior to the availability of the results of antimicrobial susceptibility.

MicroRNAs of extracellular vesicles derived from mesenchymal stromal cells alleviate inflammation in dry eye disease by targeting the IRAK1/TAB2/NF-κB pathway.

PURPOSE: To investigate the efficacy and mechanisms of human umbilical cord-derived MSC-derived extracellular vesicles (hucMSC-EVs) in a mouse model of desiccation-induced dry eye disease (DED). METHODS: hucMSC-EVs were enriched by ultracentrifugation. The DED model was induced by desiccating environment combined with scopolamine administration. The DED mice were divided into the hucMSC-EVs group, fluorometholone (FML) group, PBS group, and blank control group. Tear secretion, corneal fluorescein staining, the cytokine profiles in tears and goblet cells, TUNEL-positive cell, and CD4+ cells were examined to assess therapeutic efficiency. The miRNAs in the hucMSC-EVs were sequenced, and the top 10 were used for miRNA enrichment analysis and annotation. The targeted DED-related signaling pathway was further verified by using RT‒qPCR and western blotting. RESULTS: Treatment with hucMSC-EVs increased the tear volume and maintained corneal integrity in DED mice. The cytokine profile in the tears of the hucMSC-EVs group presented with a lower level of proinflammatory cytokines than PBS group. Moreover, hucMSC-EVs treatment increased goblet cell density and inhibited cell apoptosis and CD4+ cell infiltration. Functional analysis of the top 10 miRNAs in hucMSC-EVs showed a high correlation with immunity. Among them, miR-125 b, let-7b, and miR-6873 were conserved between humans and mice and were associated with the IRAK1/TAB2/NF-κB pathway that was activated in DED. Furthermore, IRAK1/TAB2/NF-κB pathway activation and the abnormal expression of IL-4, IL-8, IL-10, IL-13, IL-17, and TNF-α were reversed by hucMSC-EVs. CONCLUSIONS: hucMSCs-EVs alleviate DED signs, suppress inflammation and restore homeostasis of the corneal surface by multitargeting the IRAK1/TAB2/NF-κB pathway via certain miRNAs.

Time-Course Transcriptomic Analysis Reveals the Crucial Roles of PANoptosis in Fungal Keratitis.

Purpose: Fungal keratitis (FK) is a serious corneal infection with high morbidity. Host immune responses function as a double-edged sword by eradicating fungal pathogens while also causing corneal damage, dictating the severity, progression, and outcome of FK. However, the underlying immunopathogenesis remains elusive. Methods: Time-course transcriptome was performed to illustrate the dynamic immune landscape in a mouse model of FK. Integrated bioinformatic analyses included identification of differentially expressed genes, time series clustering, Gene Ontology enrichment, and inference of infiltrating immune cells. Gene expression was verified by quantitative polymerase chain reaction (qPCR), Western blot, or immunohistochemistry. Results: FK mice exhibited dynamic immune responses with concerted trends with clinical score, transcriptional alteration, and immune cell infiltration score peaking at 3 days post infection (dpi). Disrupted substrate metabolism, broad immune activation, and corneal wound healing occurred sequentially in early, middle, and late stages of FK. Meanwhile, dynamics of infiltrating innate and adaptive immune cells displayed distinct characteristics. Proportions of dendritic cells showed overall decreasing trend with fungal infection, whereas that of macrophages, monocytes, and neutrophils rose sharply in early stage and then gradually decreased as inflammation resolved. Activation of adaptive immune cells was also observed in late stage of infection. Furthermore, shared immune responses and activation of AIM2-, pyrin-, and ZBP1-mediated PANoptosis were revealed across different time points. Conclusions: Our study profiles the dynamic immune landscape and highlights the crucial roles of PANoptosis in FK pathogenesis. These findings provide novel insights into host responses to fungi and contribute to the development of PANoptosis-targeted therapeutics for patients with FK.

Development and multi-center validation of machine learning model for early detection of fungal keratitis.

BACKGROUND: Fungal keratitis (FK) is a leading cause of corneal blindness in developing countries due to poor clinical recognition and laboratory identification. Here, we aimed to identify the distinct clinical signature of FK and develop a diagnostic model to differentiate FK from other types of infectious keratitis. METHODS: We reviewed the electronic health records (EHRs) of all patients with suspected infectious keratitis in Beijing Tongren Hospital from January 2011 to December 2021. Twelve clinical signs of slit-lamp images were assessed by Lasso regression analysis and collinear variables were excluded. Three models based on binary logistic regression, random forest classification, and decision tree classification were trained for FK diagnosis and employed for internal validation. Independent external validation of the models was performed in a cohort of 420 patients from seven different ophthalmic centers to evaluate the accuracy, specificity, and sensitivity in real world. FINDINGS: Three diagnostic models of FK based on binary logistic regression, random forest classification, and decision tree classification were established and internal validation were achieved with the mean AUC of 0.916, 0.920, and 0.859, respectively. The models were well-calibrated by external validation using a prospective cohort including 210 FK and 210 non-FK patients from seven eye centers across China. The diagnostic model with the binary logistic regression algorithm classified the external validation dataset with a sensitivity of 0.907 (0.774, 1.000), specificity 0.899 (0.750, 1.000), accuracy 0.905 (0.805, 1.000), and AUC 0.903 (0.808, 0.998). INTERPRETATION: Our model enables rapid identification of FK, which will help ophthalmologists to establish a preliminary diagnosis and to improve the diagnostic accuracy in clinic. FUNDING: The Open Research Fund from the National Key Research and Development Program of China (2021YFC2301000) and the Open Research Fund from Beijing Advanced Innovation Center for Big Data-Based Precision Medicine, Beijing Tongren Hospital, Beihang University &Capital Medical University (BHTR-KFJJ-202001) supported this study.

Medical expenditure and its influencing factors of patients with hypertension in Shanxi Province, China: a study based on 'System of Health Accounts 2011' and multiple-layer perceptron neural network.

OBJECTIVES: To study the medical expenditure and influencing factors of patients with hypertension in Shanxi Province, China. DESIGN: A cross-sectional study. SETTING: 1088 medical institutions, including general hospitals, traditional Chinese medicine hospitals, special hospitals, basic medical institutions and public health institutions. PARTICIPANTS: 180 441 hypertensive outpatients and 14 763 inpatients from 1 January to 31 December 2017. PRIMARY AND SECONDARY OUTCOME MEASURES: Curative care expenditure for hypertension (CCEht) was measured based on System of Health Accounts 2011. Influenced factors were analysed by univariate analysis and multiple layer perceptron neural network. RESULTS: In 2017, CCEht was US$307.71 million, accounting for 3.63% of total CCE and 0.14% of gross domestic product (GDP) in Shanxi Province of China. CCE of hypertensive outpatients (CCEht-out) and inpatients (CCEht-in) accounted for 44.49% and 55.51% of CCEht. Drug fee accounted for 81.55% of CCEht-out and 22.50% of CCEht-in, respectively. The top three influencing factors were drug fee, surgical fee and hospitalisation days for inpatients, and drug fee, examination fee and test fee for outpatients. CONCLUSIONS: The medical expenditure of hypertension is still heavy for individuals and society. The diagnosis and treatment capacities of primary healthcare system has been enhanced. New rural cooperation medical insurance and urban employee basic medical insurance have a trend of overusing, and the burden of family healthcare expenditure is still heavy. To ease the economic burden of patients with hypertension and improve the efficiency of social resources utilisation, policymakers should pay more attention to key groups, further increase support for primary healthcare system, standardise the treatment and reimbursement of hypertension and incline the reimbursement policy to outpatient service.

Dry Eye Disease in Patients With Schizophrenia: A Case-Control Study.

OBJECTIVE: To evaluate the clinical features and inflammatory cytokines of dry eye disease (DED) in patients with schizophrenia. METHODS: This is a case-control study. The modified self-rating depression scale (M-SDS) and the ocular surface disease index (OSDI) were used to evaluate the symptoms of depression and DED, respectively. Lipid layer thickness (LLT), partial blink rate (PBR), meibomian gland loss (MGL), tear break-up time (TBUT), corneal fluorescein staining, Schirmer I-test, and eyelid margin abnormalities were also measured. A multiplex ELISA Quantibody array was used to detect the inflammatory cytokines in the tears of all participants. RESULTS: Forty schizophrenic patients and 20 control subjects were included. The mean age was 45.0 ± 9.5 years (range, 22-63 years) in schizophrenic patients and 45.4 ± 16.2 years (range, 23-76 years) in controls (P = 0.914). The ratio of male to female was 1.1 in schizophrenic patients and 1.0 in controls (P = 0.914). Ten women (52.6%) with schizophrenia and 2 (20%) in the control group (P = 0.096) were menopausal or post-menopausal. The OSDI [0.0 (0.0-4.2) vs. 7.3 (2.1-14.6)] and TBUT [4.5 (3.0-6.0) vs. 10.0 (3.5-11.0)] were significantly lower in patients with schizophrenia than in controls (P = 0.003 and P = 0.009, respectively). The rate of MGL [36.5 (17.5-47.5) vs. 8.5 (0.0-17.5)] increased in schizophrenic patients (P < 0.001). Among pro-inflammatory cytokines, the levels of interleukin (IL)-1α, IL-6, IL-11, IL-12A, IL-15, IL-17A, and granulocyte colony-stimulating factor (G-CSF) in tears were elevated in the schizophrenia group (all P < 0.01). Most of the chemokines examined were at increased levels in the tears of schizophrenics (all P < 0.05). The levels of matrix metalloproteinases-9 (MMP-9) and intercellular adhesion molecule-1 (ICAM-1) were also higher in the schizophrenic patients (all P < 0.001). The concentrations of IL-1Ra, tissue-inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-2 in the schizophrenia group were decreased (all P < 0.001). In schizophrenic patients, the level of CCL2 in tears was positively correlated with OSDI (R = 0.34, P = 0.03). The increasing TIMP-1 and decreasing IL-5 were correlated with increasing LLT (R = 0.33, P = 0.035; R = -0.35, P = 0.027, respectively). The level of ICAM-1 was then positively correlated with partial blink rate (PBR) (R = 0.33, P = 0.035). There was a negative correlation between IL-8 and the Schirmer I-test (R = -0.41, P = 0.009). CONCLUSIONS: Patients with schizophrenia were more likely to experience asymptomatic DED, with mild symptoms and obvious signs. The inflammatory cytokines in the tears of schizophrenic patients differed greatly from that of non-schizophrenic patients.

A multi-center study evaluating the correlation between meibomian gland dysfunction and depressive symptoms.

Increased prevalence of depression has been found in patients with meibomian gland dysfunction (MGD); however, specific conditions of patients suffered from MGD and depression remains unclear. Our aim was to investigate the prevalence of depression in patients with MGD and analyze the risk factors of depression in MGD patients. In this multi-center cross-sectional study, we enrolled 830 MGD patients and 114 normal controls. Demographic information was collected by designed questionnaires about lifestyle habits, systemic and ocular medical history. Ophthalmic examinations were performed in a formal order. Depression symptoms were evaluated with a questionnaire survey using a modified self-rating depression scale (M-SDS). The correlations between the M-SDS score and demographic and clinical information were analyzed. The prevalence of hyperlipidemia and obstructive sleep apnea-hypopnea syndrome (OSAHS) was higher in the MGD group. There were 78 cases (9.4%) with depressive symptoms in the MGD group whereas there were 4 cases (3.5%) in controls. Similarly, M-SDS was higher in the MGD group. The characteristics of depression in the MGD group included: crying spells, sleep disturbance and depressed appetite. Some systemic diseases (OSAHS, allergy, skin disease) and the use of contact lenses was correlated with an increased risk of depressive symptoms in MGD patients.

Cytokeratin 13 Is a New Biomarker for the Diagnosis of Limbal Stem Cell Deficiency.

PURPOSE: The purpose of this study was to evaluate the expression of cytokeratin (K) 13 on the corneal surface and to validate its application in the diagnosis of limbal stem cell deficiency (LSCD). METHODS: This prospective comparative study included 26 corneal impression cytology (IC) specimens from patients diagnosed with LSCD. Twenty-three IC specimens from normal donors served as controls. K12 and K13 expression were detected on the IC specimens by immunohistochemistry study. The number of K12 + or K13 + cells in all areas of the IC was quantified using ImageJ software. RESULTS: The epithelial cells harvested from IC specimens from control corneas were all K12 + . In eyes with LSCD, K13 + and K12 + /K13 + cells accounted for 93.8% and 2.6%, respectively, in the cornea. In eyes with sectoral LSCD, the median number of K13 + cells in the clinically affected area was higher than that in the unaffected area (810.0 vs. 115.0 cells/mm 2 ; P < 0.001). No significant correlation was found between the LSCD severity and the number of K12 + cells (r = -0.284, P = 0.16) or K13 + cells (r = -0.011, P = 0.95). The presence of at least 16 K13 + cells/mm 2 was suggestive of LSCD. CONCLUSIONS: Identification of K13 + cells on IC specimens provides a simple and reliable method to detect conjunctival epithelial cells on the cornea. K13 is a marker for diagnosing LSCD and localizing the involved area in sectoral LSCD.

Follow-up on the therapeutic effects of a budesonide, azithromycin, montelukast, and acetylcysteine (BAMA) regimen in children with post-infectious bronchiolitis obliterans.

BACKGROUND: Post-infectious bronchiolitis obliterans (PIBO) is a rare, severe chronic lung disease without optimal treatment. Currently, treatment in children mainly relies on systemic corticosteroids, but long-term use of these drugs may lead to adverse reactions. This study aimed to evaluate the short-term efficacy of the budesonide, azithromycin, montelukast, and acetylcysteine (BAMA) regimen in paediatric PIBO patients and whether it can reduce systemic corticosteroid use. METHODS: This was a prospective study. From June 2017 to July 2020, patients diagnosed with PIBO at Yuying Children's Hospital of Wenzhou Medical University were treated with the BAMA regimen for 3 months. Methylprednisolone was added only when the clinical manifestations did not improve or deteriorated. All patients were followed up every 2 to 4 weeks, and changes in clinical symptoms were recorded. Pulmonary function tests and high-resolution computed tomography (HRCT) were performed, and systemic corticosteroid use was recorded after the 3-month follow-up. RESULTS: A total of 75 patients with PIBO were included; overall, 54 patients completed the course of treatment. After treatment, the respiratory manifestations were improved in 37 patients (68.5%), but 4 patients (7.4%) showed decreased exercise tolerance, and 2 patients (3.7%) were readmitted to the hospital. Additionally, 17 (56.7%) of the 30 patients whose pulmonary function was re-examined showed improvement, and 28 (77.8%) of the 36 patients who underwent HRCT showed marked improvement. Importantly, 20 patients (54.1%) received systemic corticosteroids for 2 weeks or less, while 31.5% of patients used no corticosteroids. CONCLUSIONS: The BAMA regimen effectively relieved clinical symptoms and signs of PIBO in children, improved pulmonary function and HRCT manifestations, and reduced the use of systemic corticosteroids.

Characteristics of Toxic Keratopathy, an In Vivo Confocal Microscopy Study.

Purpose: Toxic keratopathy (TK) involves complex clinical manifestations and is difficult to differentiate from other ocular surface diseases by conventional slit-lamp examination. The challenge faced by clinicians in confidently diagnosing TK cannot be underestimated. This study aimed to explore the microstructural characteristics and diagnostic parameters by in vivo confocal microscopy (IVCM) in TK. Methods: In this prospective, cross-sectional, comparative study, slit-lamp and IVCM examinations were performed on 20 normal eyes and 54 eyes with TK. Based on slit-lamp imaging, TK subjects were divided into four groups: superficial punctate keratitis (n = 10 eyes), pseudodendritic keratitis (n = 14 eyes), ulcerative keratitis (UK; n = 16 eyes), and ring keratitis (RK; n = 14 eyes). The microstructural characteristics of TK were described according to the following IVCM parameters: basal cell (BC) density, dendritiform cell (DC) density, DC size, corneal nerve fiber (CNF) length, nerve tortuosity, and keratocyte reflectivity. A receiver operating characteristic (ROC) curve model was also formulated to compare the predictive power of BC density, DC density, and CNF length. Results: TK eyes showed significantly higher values for DC density (45.8 cells/mm2; range, 25.0-100.0) compared with normal eyes (24.0 cells/mm2; range, 20.8-32.3; P = 0.013; DC size (111.0 µm2; range, 92.0-137.8) compared with normal eyes (63.7 µm2; range, 47.7-70.3; P < 0.001); nerve tortuosity (0.08; range, 0.05-0.09) compared with normal eyes (0.04; range, 0.02-0.04; P < 0.001); and keratocyte reflectivity. BC density and CNF length values were found to be significantly less than those for normal controls (both P < 0.001). In all subgroups, CNF length appeared to be significantly lower than that of controls (all P < 0.001), and DC density was only statistically significantly higher in the UK (P = 0.003) and RK (P < 0.001) groups. Corneal fluorescein staining had no correlation with the analyzed IVCM parameters (all P ˃ 0.05). However, the increase in DC density and DC size showed negative correlations to CNF length (density: r = -0.325, P < 0.005; size: r = -0.493, P < 0.005), as well as positive correlations to duration and frequency of topical eye drops and DC size (density: r = 0.361, P < 0.05; size: r = 0.581, P < 0.05). A ROC curve showed that CNF length had the strongest predictive power, with the estimated area under the curve being 0.992 ± 0.008. Conclusions: Lower BC density and CNF length, greater DC density and DC size, and greater keratocyte reflectivity were the microstructural characteristics of TK. The role of subbasal nerve, inflammatory response, and limbal stem cells in the progression of TK and the appropriate treatment of different TK stages are future research directions. Translational Relevance: The evaluation of basal cells, subbasal nerve, and dendritiform cells is helpful to our understanding of the pathological process of TK.

Corneal Changes in Acanthamoeba Keratitis at Various Levels of Severity: An In Vivo Confocal Microscopic Study.

Purpose: To investigate the relationship between Acanthamoeba cysts and inflammatory cells in Acanthamoeba keratitis (AK) by in vivo confocal microscopy (IVCM). Methods: A case-control study included 30 patients with AK and 20 normal controls. The severity of the AK was divided into mild, moderate, and severe. The central cornea and four standard quadrants of the peripheral cornea were imaged by IVCM. The cyst infiltration and dendritic cell (DC) density and maturity (size, length, field, and number of dendrites) were quantified. The relationship between clinical severity, cyst density, and DC alterations was characterized by Spearman correlation analysis. Results: The maximum cyst density in the mild, moderate, and severe groups was 31.3 cysts/mm2 (17.2-32.8), 62.5 cysts/mm2 (59.3-103.1), and 162.5 cysts/mm2 (65.6-215.6), respectively. Compared to normal participants, a significant increase in the mean corneal DC density was detected in patients with AK (290.2 ± 97.0 vs. 25.3 ± 8.3 cells/mm2; P < 0.001). Patients with AK presented an increase in median DC size (178.3 vs. 63.6 µm2/cell, P < 0.001), median DC field (518.1 vs. 256.6 µm2/cell, P = 0.008), and median DC dendrite length (42.3 vs. 14.7 µm/cell, P < 0.001). Increased AK severity was correlated with an increase in cyst density, DC size, and dendrite length (all P < 0.05). An increase in cyst density was significantly correlated with an increase in DC density (ß = 0.484, P = 0.026) and DC size (ß = 0.557, P = 0.009). Conclusions: Cyst density and depth of infiltration as well as maturity of the surrounding DC increased significantly with the severity of AK. Translational Relevance: Quantitative analysis of cyst density and DC maturity may provide a new method of evaluating the severity of AK.

Evaluation of Toluidine Blue-Mediated Photodynamic Therapy for Experimental Bacterial Keratitis in Rabbits.

Objective: The objective of this study was to evaluate the effect of toluidine blue-mediated photodynamic therapy on experimental bacterial keratitis in rabbits. Methods: Bacterial keratitis was induced in rabbits by the injection of 200 µl Staphylococcus aureus (S. aureus) solution into the anterior stroma of the right cornea. Rabbits were randomly divided into four groups: toluidine blue O and red light (TBOR), levofloxacin eye drops (LEV), the combination of TBOR and LEV (TBOR + LEV), and a control group. Clinical manifestations, histopathology, and transmission electron microscopy were analyzed at various time points. Results: Conjunctival injection and surface area of the corneal ulcer in the TBOR group and the TBOR + LEV group showed significant improvement from baseline after 7 days of treatment. Compared to baseline, the depth of corneal infiltration was decreased at day 14 in the TBOR and TBOR + LEV groups. Microscopic analysis of the TBOR and TBOR + LEV groups showed that the structure of each layer was intact, and there were no inflammatory cells in the corneal stroma. Additionally, IL-1ß and TNF-α were highly expressed in the LEV and control groups but were lower in the TBOR and TBOR + LEV groups. Under transmission electron microscopy, the corneas in the TBOR and TBOR + LEV groups were intact, whereas in the LEV and control groups, double-walled structures corresponding to S.aureus were found in the superficial stromal layer. Conclusions: TBOR demonstrated in vivo antibacterial efficacy against S.aureus. Translational Relevance: This study found in vivo antibacterial efficacy of toluidine blue-mediated photodynamic therapy on rabbit experimental bacterial keratitis, thus providing an additional new option for clinical treatment of bacterial keratitis.

Progress in the basic and clinical research on the Schlemm's canal.

Glaucoma is a leading cause of irreversible blindness in the world. Intraocular pressure (IOP) plays a key role in glaucoma development and progression. Schlemm's canal (SC), an important structure of the anterior chamber angle, regulates the flow of aqueous humor and maintains IOP. Because of its special function of aqueous outflow, the SC has been intensive investigated recently. Several characteristics of SC in anatomy, physiology and pathophysiology have been revealed. Compare to normal, glaucomatous SC cells are more sensitive to substrate stiffness, have higher stiffness and and lower porosity leading to higher outflow resistance. And SC collapse caused by acute IOP increase is partially or totally reversal. With advanced inspection techniques, high-quality images of the SC can be obtained in vivo, which facilitates SC quantitative measurements clinically and allows us to investigate a new therapy paradigm for glaucoma. In this review, we summarize the basic and clinical research that focused on mechanisms of aqueous outflow resistance and SC changes in physiological, pathological, and post-treatment states.