Experimental model of Fusarium solani keratitis in rats

AIM: To establish a repeatable rat model of Fusarium solani keratitis (F. solani keratitis) that mimicked fungal keratitis in humans. METHODS: Wistar rats’ corneas were scratched on the superficial stroma after scraping the unilateral corneal epithelia. Then, the corneal surface was inoculated with different inoculum dose of F. solani spore suspension. Doses ranged from 106 to 109 colony-forming unit per milliliter (CFU/mL). The treated corneas were covered by contact lenses that were made of Parafilm M membrane. Negative controls were inoculated with sterile phosphate-buffered saline (PBS). For statistical analysis, corneas were evaluated daily on a 12-point scale to check the state of corneal inflammation. Furthermore, the pathological characteristics of this model were investigated. RESULTS: The rat model of F. solani keratitis was established by the combination methods of corneal trauma and parafilm-made contact lens and inoculation of fungus spore suspension. 106 and 107CFU/mL of F. solani induced mild corneal infection, while 108CFU/mL of F. solani was sufficient to induce moderate infection that was consistent with human keratomycosis. Dose of 109CFU/mL of F. solani was excessive and led to perforated corneas. CONCLUSION: The rat model of F. solani keratitis, established by the combinational methods of corneal trauma, parafilm-made contact lens and the appropriate dose of inoculum, that imitates the developing processes of F. solani keratitis in human beings and provides a repeatable method of creating a rat model.     

Prophylactic Knockdown of the miR-183/96/182 Cluster Ameliorates Pseudomonas aeruginosa–Induced Keratitis

PurposePreviously, we demonstrated that miR-183/96/182 cluster (miR-183C) knockout mice exhibit decreased severity of Pseudomonas aeruginosa (PA)-induced keratitis. This study tests the hypothesis that prophylactic knockdown of miR-183C ameliorates PA keratitis indicative of a therapeutic potential.MethodsEight-week-old miR-183C wild-type and C57BL/6J inbred mice were used. Locked nucleic acid–modified anti-miR-183C or negative control oligoribonucleotides with scrambled sequences (NC ORNs) were injected subconjunctivally 1 day before and then topically applied once daily for 5 days post-infection (dpi) (strain 19660). Corneal disease was graded at 1, 3, and 5 dpi. Corneas were harvested for RT-PCR, ELISA, immunofluorescence (IF), myeloperoxidase and plate count assays, and flow cytometry. Corneal nerve density was evaluated in flatmounted corneas by IF staining with anti-β-III tubulin antibody.ResultsAnti-miR-183C downregulated miR-183C in the cornea. It resulted in an increase in IL-1β at 1 dpi, which was decreased at 5 dpi; fewer polymorphonuclear leukocytes (PMNs) at 5 dpi; lower viable bacterial plate count at both 1 and 5 dpi; increased percentages of MHCII⁺ macrophages (Mϕ) and dendritic cells (DCs), consistent with enhanced activation/maturation; and decreased severity of PA keratitis. Anti-miR-183C treatment in the cornea of naïve mice resulted in a transient reduction of corneal nerve density, which was fully recovered one week after the last anti-miR application. miR-183C targets repulsive axon-guidance receptor molecule Neuropilin 1, which may mediate the effect of anti-miR-183C on corneal nerve regression.ConclusionsProphylactic miR-183C knockdown is protective against PA keratitis through its regulation of innate immunity, corneal innervation, and neuroimmune interactions.     

Isorhamnetin Ameliorates Aspergillus fumigatus Keratitis by Reducing Fungal Load,Inhibiting Pattern-Recognition Receptors and Inflammatory Cytokines

PurposeIsorhamnetin is a natural flavonoid with both antimicrobial and anti-inflammatory properties, but its effect on fungal keratitis (FK) remains unknown. The current study aims to investigate the antifungal and anti-inflammatory effects of isorhamnetin against mouse Aspergillus fumigatus keratitis.MethodsIn vitro, the lowest effective concentration of isorhamnetin was assessed by minimum inhibitory concentration and cytotoxicity tests in human corneal epithelial cells (HCECs) and RAW264.7 cells. The antifungal property was investigated by scanning electron microscopy and propidium iodide uptake test. The anti-inflammatory effect of isorhamnetin in HCECs and RAW264.7 cells was observed by quantitative real-time polymerase chain reaction (qRT-PCR). In the eyes of mice with A. fumigatus keratitis, FK severity was evaluated using clinical score, plate counting, histological staining and periodic acid Schiff staining. In vivo, the anti-inflammatory effect of isorhamnetin was examined by immunofluorescence staining, myeloperoxidase assay, Western blot, enzyme-linked immunosorbent assay, and qRT-PCR.ResultsIn HCECs and RAW264.7 cells, isorhamnetin significantly inhibited A. fumigatus conidia growth and hyphae viability at 80 µg/mL without affecting cell viability. In vitro, isorhamnetin altered A. fumigatus hyphal morphology and membrane integrity. In A. fumigatus keratitis mouse model, isorhamnetin treatment alleviated the severity of FK by reducing corneal fungal load and inhibiting neutrophil recruitment. In addition, the mRNA and protein expression levels of TLR-2, TLR-4, Dectin-1, IL-1β, and tumor necrosis factor-α were significantly decreased in isorhamnetin-treated groups in vivo and in vitro.ConclusionsIsorhamnetin improves the prognosis of A. fumigatus keratitis in mice by inhibiting the growth of A. fumigatus, reducing the recruitment of neutrophils and downregulating inflammatory factors.     

Corneal nerve alterations in acute Acanthamoeba and fungal keratitis: an in vivo confocal microscopy study

Purpose

To study sub-basal corneal nerve alterations in patients with acute Acanthamoeba keratitis (AK) and fungal keratitis (FK), using laser in vivo confocal microscopy (IVCM).

Methods

A retrospective analysis of IVCM (Heidelberg Retina Tomograph 3/Rostock Cornea Module) images of 10 AK corneas and 4 FK corneas was performed, and the results compared with those of 10 normal and 12 acute herpetic keratitis (HK) corneas. Sub-basal corneal nerves were analyzed with respect to total number of nerves, main nerve trunks, branching pattern and total length of nerves per image, as well as tortuosity. For each variable, results for three frames were averaged and analyzed using analysis of variance.

Results

Total corneal nerve length was significantly (P<0.0001) reduced in patients with AK (193.4±124.5 μm) and FK (268.6±257.4 μm) when compared with normal controls (3811.84±911.4 μm). Total nerve counts in patients with AK (3.9±1.2) and FK (3.6±3.2) were significantly (P<0.0001) decreased in comparison with normal controls (24.7±5.5). The number of main nerve trunks and nerve branching was found to be significantly lower in AK and FK corneas, when compared with controls. There was a statistically significant decrease in the above parameters when compared with HK controls.

Conclusions

The sub-basal corneal nerve plexus is significantly diminished in eyes with AK and FK, as demonstrated by IVCM. These results are more profound than previously reported findings of a diminished nerve plexus in HK.     

The Role of SREC-Ⅰ in Innate Immunity to Aspergillus fumigatus Keratitis

PurposeTo determine the role of scavenger receptor expressed by endothelial cell-1 (SREC-Ⅰ) in vitro and in a mouse model of Aspergillus fumigatus keratitis.MethodsSREC-Ⅰ mRNA and protein expression were tested in both normal and A fumigatus stimulated human corneal epithelial cells (HCECs). Immunofluorescence was used to detect SREC-Ⅰ expression in human corneas with or without A fumigatus infection. HCECs were incubated with SREC-Ⅰ small interfering RNA, then the mRNA levels of LOX-1, IL-1β, and TNF-α were detected after A fumigatus stimulation. A mouse fungal keratitis (FK) model was established and SREC-Ⅰ mRNA and protein expression were detected by RT-PCR, Western blot and immunofluorescence. The severity of FK was evaluated by clinical score. CLCX1, LOX-1, IL-1β, and TNF-α mRNA expression levels were tested before and after anti–SREC-Ⅰ treatment.ResultsSREC-Ⅰ expressed in normal and A fumigatus treated HCECs and human corneal epithelium. In vitro experiment showed that SREC-Ⅰ mRNA and protein levels were significantly increased after A fumigatus stimulation. SREC-Ⅰ small interfering RNA treatment inhibited the expressions of LOX-1, IL-1β, and TNF-α in HCECs. The expressions of CLCX1, LOX-1, IL-1β, and TNF-α were elevated in mice with A fumigatus keratitis, which could be decreased by SREC-Ⅰ–neutralizing antibody treatment.ConclusionsSREC-Ⅰ is a key mediator in inflammatory response induced by A fumigatus keratitis. SREC-Ⅰ blockade could be a potential therapeutic approach for FK.     

Comparative study on costs incurred for treatment of patients with bacterial and fungal keratitis - A retrospective analysis

Purpose:To compare the costs associated with medications and travel of patients with smear-proven bacterial keratitis and fungal keratitis in a tertiary care center in India.Methods:Retrospective analysis of case records of a cohort of patients who presented between April 2017 and March 2018 to a tertiary care center in India, with infectious keratitis who were smear-positive for bacteria or fungi, and whose costs of treatment and travel were supported by a philanthropic program.Results:In total, 672 case records of 177 smear-positive bacterial keratitis (BK) and 495 smear-positive fungal keratitis (FK) were included in the study. Further, 62% of BK and 75% of FK received more than one antimicrobial drug (P < 0.001). The mean total medication cost (INR) was significantly more in FK (959.1 ± 675.2) compared to BK (674.9 ± 463.7) (P < 0.0001). The mean medication cost (INR) per visit was also more for FK (201.1 ± 109.4) compared to BK (155.2 ± 84.1) (P < 0.0001). The mean total medication cost was significantly more for FK for both patients who healed with medical treatment (611.6 ± 395.6 for BK, 801.5 ± 599.9 for FK, P = 0.0005) and for patients who required TPK (953.7 ± 653.1 for BK, 1374.6 ± 701.5 for FK, P = 0.0023) compared to their respective counterparts in BKConclusion:Patients with fungal keratitis incurred significantly more on medications compared to patients with bacterial keratitis irrespective of whether they had healed with successful medical treatment or required therapeutic keratoplasty. Prolonged duration of treatment and the high costs of antifungal medications account for the significant economic burden of fungal keratitis.     

Analysis of MicroRNA Expression in Tears of Patients with Herpes Epithelial Keratitis: A Preliminary Study

PurposeHerpes epithelial keratitis (HEK) is the most common form of herpes simplex virus (HSV) eye involvement, and understanding the molecular mechanisms underlying HEK is important. We investigated the expression of microRNAs (miRNAs) in the tears of patients with HEK.MethodsTear samples from eight patients with HEK and seven age-matched controls were evaluated. Clinical ophthalmologic evaluation was performed, and an anterior segment photograph was obtained after fluorescence staining. Dendritic or geographic ulcer areas were measured using ImageJ software. The expression of 43 different miRNAs in tears was measured using real-time polymerase chain reaction and compared between patients with HEK and controls. Differences in miRNA expression between the dendritic and geographic ulcer groups and correlations involving miRNA expression and ulcer area were evaluated.ResultsOf the 43 miRNAs, 23 were upregulated in patients with HEK compared to normal controls. MiR-15b-5p, miR-16-5p, miR-20b-5p, miR-21-5p, miR-23b-3p, miR-25-3p, miR-29a-3p, miR-30a-3p, miR-30d-5p, miR-92a-3p, miR-124-3p, miR-127-3p, miR-132-3p, miR-142-3p, miR-145-5p, miR-146a-5p, miR-146b-5p, miR-155-5p, miR-182-5p, miR-183-5p, miR-221-3p, miR-223-3p, and miR-338-5p were significantly upregulated in patients with HEK. MiR-29a-3p exhibited significant differences between the dendritic and geographic ulcer groups. All 23 miRNAs with significant differences between patients with HEK and the control group were not significantly correlated with ulcer area.ConclusionsTwenty-three miRNAs were significantly upregulated in the tears of patients with HEK, and the expression of miRNAs may play important roles in herpes infection in relation to host immunity.     

Ciliogenesis and autophagy are coordinately regulated by EphA2 in the cornea to maintain proper epithelial architecture

PurposeTo understand the relationship between ciliogenesis and autophagy in the corneal epithelium.MethodssiRNAs for EphA2 or PLD1 were used to inhibit protein expression in vitro. Morpholino-anti-EphA2 was used to knockdown EphA2 in Xenopus skin. An EphA2 knockout mouse was used to conduct loss of function studies. Autophagic vacuoles were visualized by contrast light microscopy. Autophagy flux, was measured by LC3 turnover and p62 protein levels. Immunostaining and confocal microscopy were conducted to visualize cilia in cultured cells and in vivo.ResultsLoss of EphA2 (i) increased corneal epithelial thickness by elevating proliferative potential in wing cells, (ii) reduced the number of ciliated cells, (iii) increased large hollow vacuoles, that could be rescued by BafA1; (iv) inhibited autophagy flux and (v) increased GFP-LC3 puncta in the mouse corneal epithelium. This indicated a role for EphA2 in stratified epithelial assembly via regulation of proliferation as well as a positive role in both ciliogenesis and end-stage autophagy. Inhibition of PLD1, an EphA2 interacting protein that is a critical regulator of end-stage autophagy, reversed the accumulation of vacuoles, and the reduction in the number of ciliated cells due to EphA2 depletion, suggesting EphA2 regulation of both end-stage autophagy and ciliogenesis via PLD1. PLD1 mediated rescue of ciliogenesis by EphA2 depletion was blocked by BafA1, placing autophagy between EphA2 signaling and regulation of ciliogenesis.ConclusionOur findings demonstrate a novel role for EphA2 in regulating both autophagy and ciliogenesis, processes that are essential for proper corneal epithelial homeostasis.     

Foldscope: A smartphone based diagnostic tool for fungal keratitis

Purpose:Smartphone-based microscopy tool like foldscope (FS) may serve the purpose of a low-cost diagnostic alternative to the compound light microscope especially in areas with limited resources. The purpose of this study was to detect fungal pathogens causing keratitis on direct smear by smartphone-mounted FS and to evaluate the efficacy of FS against routine compound light microscope (CLM).Methods:The prospective study was conducted at a tertiary eye care center from September 2019 to March 2020. The study included 60 smear examinations (Gram stain [GM] n = 30, Lactophenol Cotton Blue [LCB] n = 30) to detect fungal pathogens from corneal scraping material of clinically suspected fungal keratitis (FK) cases. The diagnostic utility of FS was compared with CLM for both GM and LCB wet mount. Data collected were used to quantify the agreement using Cohen’s kappa between CLM and FS imaging.Results:Forty-six samples out of 60 were positive for fungi using CLM. GM stain and LCB showed 22/30 (73.33%) and 24/30 (80%) positive results with CLM, respectively. Moderate agreement (0.49) was observed between CLM and FS with the smartphone method. LCB mount showed high specificity of 1.00 over 0.87 of GM stain for FS with the smartphone.Conclusion:Direct smear can be an early and sensitive measure to diagnose FK other than clinical suspicion. The smartphone-mounted FS has limited sensitivity as an alternative to CLM, but excellent specificity in the present study for FK. The FS as a smartphone-based diagnostic tool is simple, portable, and inexpensive in resource-constrained rural or remote clinical and public health settings in the absence of CLM and other higher diagnostic modalities.     

Repressed miR-34a Expression Dictates the Cell Fate to Corneal Endothelium Failure

PurposeTo reveal the mechanism triggering the functional disparity between degenerated and non-degenerated corneal endothelium cells in the water efflux from corneal stroma to the anterior chamber.MethodsThe varied levels of the microRNA (miR)-34, miR-378, and miR-146 family in human corneal endothelium and cultured cells thereof were investigated using 3D-Gene Human miRNA Oligo Chips. Concomitantly, CD44, p53, c-Myc, matrix metalloprotease (MMP)-2 expression, and Ras homolog gene family member A (Rho A) activity was correlated to the expression intensities of these microRNAs, partly complemented with their altered expression levels with the transfection of the corresponding mimics and inhibitors. The levels of miRs were further associated with intracellular pH (pHi) and mitochondrial energy homeostasis.ResultsP53-inducible miR-34a/b repressed CD44 expression, and CD44 was repressed with the elevated c-Myc. The repressed miR-34a activated the CD44 downstream factors Rho A and MMP-2. MiR-34a mimics downregulated pHi, inducing the skewing of mitochondrial respiration to oxidative phosphorylation. The oxidative stress (H₂O₂) induced on human corneal endothelial cells, which repressed miR-34a/b expression, may account for the impaired signaling cascade to mitochondrial metabolic homeostasis necessary for an efficient water efflux from the corneal stroma.ConclusionsThe upregulated expression of CD44, through repressed miR-34a/b by reactive oxygen species and elevated c-Myc by oxidative stress, may impair mitochondrial metabolic homeostasis, leading to human corneal endothelial failure.     

A controlled study of amniotic membrane transplantation for acute Pseudomonas keratitis

ObjectiveTo evaluate the efficacy of amniotic membrane transplantation (AMT) to improve the outcomes of acute Pseudomonas keratitis as compared with a control group.DesignProspective interventional case series with retrospective controls.ParticipantsWe studied 14 eyes with Pseudomonas keratitis as the AMT group and 11 eyes with Pseudomonas keratitis as the control group.MethodsEyes in the AMT group were treated with antibiotic therapy followed by single-layer AMT at 2 to 3 days. Eyes in the control group received only antibiotic therapy. Patients were followed for 11.1 ± 2.4 months.ResultsIn the AMT group, pain significantly decreased from a mean score of 2.4 ± 0.5 preoperatively to 1.1 ± 0.9 at day 2 postoperatively (p < 0.001). Corneal epithelial defects healed completely within 13.2 ± 2.6 days in the AMT group compared with 15.5 ± 3.4 days in the control group (p = 0.07). At final follow-up visits, the sizes of corneal opacity and deep neovascularization were not different between the 2 groups. However, the mean score for density of the corneal opacity was significantly less in the AMT group compared with the control group (2.1 ± 0.4 vs 2.5 ± 0.7, respectively, p = 0.04). Although the best corrected visual acuity using hard contact lenses was not different between the 2 groups, uncorrected visual acuity was better in the AMT group (0.45 ± 0.22 logMAR) than in the control group (0.71 ± 0.32 logMAR, p = 0.03). No patient in either group developed significant corneal thinning or perforation.ConclusionsAMT in acute Pseudomonas keratitis was associated with immediate pain relief, less density of the final corneal opacity, and better uncorrected visual acuity at the final follow-up visit.     

Expression of dectin-1 during fungus infection in human corneal epithelial cells

AIM:To evaluate the expression of dendritic cell-associated C-type lectin-1 (dectin-1) in human corneal epithelial (HCE) cells infected by fungus.METHODS:A total of 20 cases of healthy donor corneas were group A, and 20 patients (20 eyes) suffered from fungal keratitis (FK) composed group B. Real-time qPCR and immunohistochemistry were applied to detect dectin-1 expression in corneal epithelium of both groups. HCE cells were cultured with aspergillus fumigatus (AF) antigens in vitro. The expression of dectin-1 mRNA was measured by real-time qPCR at the stimulation of 0, 4, 8 and 24h separately. Dectin-1 protein was detected by immunocytochemistry at 0 and 24h separately.RESULTS: Dectin-1 expressed in corneal epithelium of normal persons and FK patients. Vitro cellular experiment showed that the expression of dectin-1 mRNA in HCE cells began to increase after stimulation of AF antigens at 4h, and dectin-1 protein expression increased after stimulation at 24h.CONCLUSION: Dectin-1 expressed in corneal epithelium of normal persons. AF antigens stimulation can elevate the expression of dectin-1 in HCE cells in vitro.     

TSLP Produced by Aspergillus fumigatus-Stimulated DCs Promotes a Th17 Response Through the JAK/STAT Signaling Pathway in Fungal Keratitis

PurposeThe purpose of this study was to explore the role of thymic stromal lymphopoietin (TSLP) secreted by Aspergillus fumigatus–stimulated dendritic cells (DCs) during the T helper 17 (Th17) immune response, and further clarify the mechanisms contributing to the Th17 immune response of fungal keratitis (FK).MethodsA carboxyfluorescein diacetate succinimidyl ester assay, PCR, and flow cytometry were performed to detect Th17 differentiation of CD4⁺ T cells; PCR, ELISA, and Western blot were used to detect the expression of TSLP and JAK/STAT–related proteins; Signaling pathways involved in Th17 response was evaluated using RNA sequence; C57BL/6 mice were infected with A. fumigatus and treated with ruxolitinib or BBI608. Slit-lamp examination, fluorescein staining, and clinical scores were used to assess the clinical manifestation.ResultsA. fumigatus–infected DCs could drive naïve CD4⁺ T-cell proliferation and promote the production of Th17 cytokines IL-17A, IL-17F, and IL-22. A. fumigatus stimulation increased the expression of TSLP in DCs. DC-derived TSLP contributed to a Th17-type inflammatory response via the JAK/STAT signaling pathway. TSLP small interfering RNA, TSLPR small interfering RNA, or JAK/STAT inhibitors inhibited the Th17 immune response induced by A. fumigatus–infected DCs. Moreover, TSLP promoted A. fumigatus keratitis disease progression in a mouse model. However, inhibition of the JAK/STAT signaling pathway using a specific inhibitor reversed the development of FK by A. fumigatus infection.ConclusionsTSLP secreted by A. fumigatus–stimulated DCs played a significant role in the Th17-dominant immune response of FK through its JAK/STAT activation. Our findings may contribute to the elucidation of the molecular mechanisms of FK and to the development of novel therapeutic approaches.     

Queratitis fúngica por Colletotrichum spp. A propósito de un caso

Case reportA 23 years old male with an unremarkable past medical history suffered an injury with a branch of a lemon tree in the right eye two days prior to presentation. The slit-lamp examination showed a central corneal erosion with a white tree-shaped stromal infiltrate and Tyndall +/++ in anterior chamber. Cultivation of corneal scraping was positive for Colletotrichum spp. The patient responded favourably to topical amphotericin.DiscussionColletotrichum spp. is an uncommon cause of keratitis, usually secondary to corneal erosion caused by plant material and should be included in the differential diagnosis of fungal keratitis.     

Multilayer amniotic membrane transplantation for reconstruction of deep corneal ulcers

PurposeTo evaluate the efficacy of multilayer amniotic membrane transplantation for reconstruction of corneal epithelium and stroma in the context of deep corneal ulcers.DesignProspective, noncomparative, interventional case series.ParticipantsEleven consecutive patients with deep corneal ulcers refractory to conventional treatment; six patients had herpetic keratitis and five had other forms of neurotrophic keratitis.InterventionMultilayer amniotic membrane transplantation with kryopreserved human amniotic membrane.Main outcome measuresIntegrity of corneal epithelium and stroma, opacification, and appearance of grafted membrane during 12 months follow-up.ResultsAmniotic membrane transplantation markedly reduced ocular inflammation in all patients. Epithelium healed above all corneal ulcers within 4 weeks and remained stable in 9 of 11 patients for 1 year. Two patients with recurrent epithelial defect suffered from severe neurotrophic keratitis. Following transplantation the amniotic membranes gradually dissolved over a period of 12 months, but stromal thickness remained stable.ConclusionAmniotic membrane transplantation allows corneal surface reconstruction in patients with persistent epithelial defects. The multilayer technique is useful for treating deep corneal ulcers and even descemetoceles. Because the procedure results in stability of the ocular surface over a period of more than 12 months in most patients, it may be considered an alternative to conventional surgical techniques for ocular surface reconstruction.     

Retinal Neuroprotective Effect of Mesenchymal Stem Cells Secretome Through Modulation of Oxidative Stress,Autophagy, and Programmed Cell Death

PurposeDegenerative mechanisms of retinal neurodegenerative diseases (RND) share common cellular and molecular signalization pathways. Curative treatment does not exist and cell-based therapy, through the paracrine properties of mesenchymal stem cells (MSC), is a potential unspecific treatment for RND. This study aimed to evaluate the neuroprotective capability of human bone marrow (bm) MSC secretome and its potential to modulate retinal responses to neurodegeneration.MethodsAn in vitro model of spontaneous retinal neurodegeneration was used to compare three days of monocultured neuroretina (NR), NR cocultured with bmMSC, and NR cultured with bmMSC secretome. We evaluated retinal morphology markers (Lectin peanut agglutinin, rhodopsin, protein kinase C α isoform, neuronal-specific nuclear protein, glial fibrillary acidic protein, TdT-mediated dUTP nick-end labeling, and vimentin) and proteins involved in apoptosis (apoptosis-inductor factor, caspase-3), necroptosis (MLKL), and autophagy (p62). Besides, we analyzed the relative mRNA expression through qPCR of genes involved in apoptosis (BAX, BCL2, CASP3, CASP8, CASP9), necroptosis (MLKL, RIPK1, RIPK3), autophagy (ATG7, BCLIN1, LC3B, mTOR, SQSTM1), oxidative stress (COX2, CYBA, CYBB, GPX6, SOD1, TXN2, TXNRD1) and inflammation (IL1, IL6, IL10, TGFb1, TNFa).ResultsThe bmMSC secretome preserves retinal morphology, limits pro-apoptotic– and pro-necroptotic–related gene and protein expression, modulates autophagy-related genes and proteins, and stimulates the activation of antioxidant-associated genes.ConclusionsThe neuroprotective ability of the bmMSC secretome is associated with activation of antioxidant machinery, modulation of autophagy, and inhibition of apoptosis and necroptosis during retinal degeneration. The neuroprotective effect of bmMSC secretomes in the presence/absence of MSC looks similar. Our current results reinforce the hypothesis that the human bmMSC secretome slows retinal neurodegeneration and may be a therapeutic option for treating RND.     

The use of in vivo confocal microscopy in fungal keratitis – Progress and challenges

Fungal keratitis (FK) is a serious and sight-threatening corneal infection with global reach. The need for prompt diagnosis is paramount, as a delay in initiation of treatment could lead to irreversible vision loss. Current “gold standard” diagnostic methods, namely corneal smear and culture, have limitations due to diagnostic insensitivity and their time-consuming nature. PCR is a newer, complementary method used in the diagnosis of fungal keratitis, whose results are also sample-dependent. In vivo confocal microscopy (IVCM) is a promising complementary diagnostic method of increasing importance as it allows non-invasive real-time direct visualization of potential fungal pathogens and manifesting infection directly in the patient's cornea. In numerous articles and case reports, FK diagnosis by IVCM has been evaluated, and different features, approaches, sensitivity/specificity, and limitations have been noted. Here, we provide an up-to-date, comprehensive review of the current literature and present the authors' combined recommendations for fungal identification in IVCM images, while also looking to the future of FK assessment by IVCM using artificial intelligence methods.     

Baicalein Protects Against Aspergillus fumigatus Keratitis by Reducing Fungal Load and Inhibiting TSLP-Induced Inflammatory Response

PurposeTo investigate the antifungal and anti-inflammatory effects of baicalein on Aspergillus fumigatus (A. fumigatus) keratitis and the underlying mechanisms.MethodsThe noncytotoxic antifungal concentration of baicalein was determined using CCK8, cell scratch assay, minimum inhibitory concentration, biofilm formation, scanning electron microscopy, propidium iodide uptake test and adherence assay in vitro and Draize test in vivo. In fungal keratitis (FK) mouse models, clinical score and plate count were used to evaluate FK severity, and myeloperoxidase assay and immunofluorescence staining were performed to examine neutrophil infiltration and activity. Real-time PCR, ELISA, and Western blot were performed to explore the anti-inflammatory activity of baicalein and the underlying mechanisms in vivo and in vitro.ResultsBaicalein at 0.25 mM (noncytotoxic) significantly inhibited A. fumigatus growth, biofilm formation, and adhesion in vitro. In A. fumigatus keratitis mice, baicalein mitigated FK severity, reduced fungal load, and inhibited neutrophil infiltration and activity. Baicalein not only suppressed mRNA and protein levels of proinflammatory factors IL-1β, IL-6, and TNF-α, but also inhibited the expression of thymic stromal lymphopoietin (TSLP) and TSLP receptor (TSLPR) in vivo and in vitro. In HCECs, mRNA and protein levels of IL-1β, IL-6, and TNF-α were significantly lower in the TSLP siRNA–treated group, while higher in the rTSLP-treated group than in the corresponding control. Baicalein treatment significantly inhibited rTSLP induced the expression of IL-1β, IL-6, and TNF-α.ConclusionsBaicalein plays a protective role in mouse A. fumigatus keratitis by inhibiting fungal growth, biofilm formation, and adhesion, and suppressing inflammatory response via downregulation of the TSLP/TSLPR pathway.