Rhino-orbito-cerebral mucormycosis(ROCM) is an invasive fungal infection that usually occurs in immunocompromised patients and sometimes presents as orbital apex syndrome(OAS) initially. It is rapidly fatal without an early diagnosis and treatment. We report the cases of invasive ROCM presenting with OAS initially in order to raise the attention of clinicians. We retrospectively investigated eleven cases of invasive ROCM presenting initially with OAS admitted between January 2006 and December 2013. We analyzed clinical features, results of laboratory and radiological examinations, nasal endoscopy, aggressive surgical excision and debridement, and medical management outcomes of each case. A total of eleven cases of invasive ROCM with OAS as an initial sign were presented. Mucormycosis was accompanied by type II diabetes mellitus in nine cases, renal transplant in one case, and injury caused by traffic accident in one case. Anterior rhinoscopy revealed palatine or nasal necrotic lesions in all patients, and transethmoidal optic nerve decompression was carried out in three patients at the same time. CT scan revealed rhino-orbital-cerebral involvement in every patient. All patients were given intravenous amphotericin B. Nine patients underwent surgical debridement of necrotic tissue. Three patients survived. ROCM is a severe, emergent and fatal infection requiring multidisciplinary management. It may often present with OAS initially. For ophthalmologist, mucormycosis must be considered in immunocompromised patients presenting with OAS initially, and anterior rhinoscopy is imperative before hormonotherapy, even in the cases absent of ketoacidosis induced by diabetes mellitus.

Purpose: The purpose of this study was to investigate the therapeutic effect of perillaldehyde (PAE) on Aspergillus fumigatus (A. fumigatus) keratitis. Methods: Human corneal epithelial cells (HCECs) were pretreated with PAE and stimulated with A. fumigatus mycelium. C57BL/6 mice were infected with A. fumigatus and treated with or without PAE 1 day after infection. Clinical scores, PCR, ELISA, and Western blotting were used to detect the expression of pro-inflammatory mediators, dendritic cell-associated c-type lectin-1 (Dectin-1), nuclear factor (erythroid-derived 2) like 2 (Nrf2), and heme oxygenase (HO-1). Nrf2 expression in HCECs pretreated with PAE was observed by immunofluorescence. NIMP-R14 protein expression and localization in mouse corneas were observed by immunofluorescence staining after treatment with PAE. Corneal colony counting, time-kill tests, and mycelial transformation inhibition tests were used to evaluate the antifungal effect of PAE. Results: C57BL/6 mice treated with PAE at 1 day after infection had a lower clinical score and decreased IL-1β, TNF-α, IL-6, Dectin-1, and MPO levels. PAE treatment significantly reduced neutrophil recruitments to the corneal stroma. Compared with the DMSO-treated group, PAE treatment significantly decreased mRNA and protein levels of pro-inflammatory cytokines and Dectin-1 in HCECs. PAE pretreatment before A. fumigatus stimulation obviously elevated the mRNA and protein levels of components of the Nrf2/HO-1 axis. HCECs pretreated with PAE before infection showed a weakened ability to inhibit inflammation in the presence of brusatol (BT; an Nrf2 inhibitor) or ZnPP (an HO-1 inhibitor). PAE treatment significantly reduced the fungal load of C57BL/6 mouse corneas and inhibited fungal growth in vitro. Conclusions: These data proved that PAE may ameliorate A. fumigatus keratitis by activating the Nrf2/HO-1 signaling pathway and inhibiting the Dectin-1 mediated inflammatory response and neutrophil recruitment. Furthermore, PAE exerts direct fungicidal activity on A. fumigatus.

To observe Dectin-1 expression in fungal keratitis on rat models and to determine the role of Dectin-1 in innate immune response to Aspergillus fumigatus. Wistar rats were randomly divided into control, fungal keratitis and pretreatment (pretreated with Laminarin) groups. Samples were used for conducting immunohistochemical staining and real-time PCR to observe expression of cytokines like CCL2, CCL3, CXCL1, CXCL2, IL-1β, TNF-α, IL-6, IL-10. After fungal stimulations, all 7 inflammatory factors, except IL-10, increased with different levels. After 4 h of fungal stimulations, IL-1β, IL-6, CCL2, CXCL1 and CXCL2 of pretreatment groups were significantly (p < 0.05) lower than fungal groups, while the other 3 cytokines had no significant changes. After 8 h of fungal stimulations, IL-6 and CXCL1 of pretreatment groups were still significantly (p < 0.05) lower than fungal groups. With progress of fungus stimulation, expression of IL-1β,CXCL1 ,CXCL2,MCP-1 gradually increased, whilepretreated with Laminarin to block Dectin-1, these expression decreased, indicating that Dectin-1 maypromote immune reaction through them. IL-10 decreased in fungal group because of itsimmunosuppressive effect at 4h, and it began to increase at 8h to suppress Th1 inflammation response inorder to avoid excessive tissue damage. Dectin-1 in early period of innate immune responses in rat fungal keratitis might work through IL-1β, IL-6, CCL2, CXCL1, CXCL2 to recruit neutrophils and macrophages to participate anti-fungal immunity.

Hinokitiol (β-thujaplicin) is an important component of the essential oil extracted from Chamaecyparis obtuse, which prevents the decay and decomposition of temple and shrine buildings in Japan. Hinokiol has been shown to have a detrimental effect on various fungi such as Candida albicans and saprophytic fungi. However how hinokitiol works against Aspergillus fumigatus (A. fumigatus) has not been claimed. This study aims to investigate the adverse effects of hinokitiol on the disruption of the cell wall and cell membrane of A. fumigatus and to explore possible potential mechanisms or pathways. According to our results, hinokitiol negatively altered mycelium morphology, growth density, and cell plasma composition content. When incubated with human corneal epithelial cells (HCECs), hinokitiol saw a safe effect with concentrations below 12 μg/ml. Hinokitiol was shown to increase the cell membrane’s permeability by decreasing the cell membrane’s ergosterol content. The integrity of the cell wall was disrupted, as well as a significant increase in chitin degradation and chitinase activity. As determined by RNA-seq results, subsequent analysis, and qRT-PCR, altered transcript levels of cell walls and cell membranes-related genes (such as eglC) illustrated how hinokitiol affected the genetic profile of A. fumigatus. With this study, we recommend hinokitiol as an effective anti-A. fumigatus agent by reducing the amounts of key components in the cell wall and membrane by preventing production and accelerating breakdown.

Objective: To prepare a nanobody specific to dectin 1 and verify its specificity and anti-inflammatory effects on Aspergillus fumigatus keratitis. Methods: The nanobody was selected from a high-quality shark-antibody library constructed with phage-display technology. The nanobody was developed in the expression systems of Escherichia coli. Indirect ELISA was used to determine the specificity of the nanobody to recombinant dectin 1 protein. The potential of the nanobody to be recognized and expressed on the surfaces of cells and corneas was detected by immunofluorescence, and its anti-inflammatory effect on A. fumigatus keratitis was further verified. After infection with A. fumigatus, eyes of C57B L/6 mice were treated with nanobodies. Human corneal epithelial cells (HCECs) were pretreated with nanobodies and then incubated with A. fumigatus. Clinical scores and slit-lamp photography were used to assess disease response in mouse corneas. RT-PCR and ELISA were used to evaluate mRNA and protein expression of IL1β and IL6 in both mouse corneas and HCECs. Results: The nanobody was successfully expressed through microbial system and showed specific high-affinity binding to recombinant dectin 1. Furthermore, it exhibited specific binding to dectin 1 expressed on the surfaces of cells and recognized dectin 1 in mouse corneas. Importantly, it reduced clinical scores of A. fumigatus keratitis in mice compared with a PBS-treatment group. In addition, it decreased mRNA and protein expression of IL1β and IL6 in infected corneas and HCECs stimulated with A. fumigatus. Conclusion: These results suggest that this nanobody can bring about anti-inflammatory effects. This highlights the potential of these nanobodies as innovative therapeutic agents in A. fumigatus.