Oxymatrine mitigates Aspergillus fumigatus keratitis by suppressing fungal activity and restricting pyroptosis.

Fungal keratitis (FK) is a refractory keratitis caused by excessive inflammation and fungal damage. Excessive inflammation can lead to tissue damage and corneal opacity, resulting in a poor prognosis for FK. Oxymatrine (OMT) is a natural alkaloid, which has rich pharmacological effects, such as antioxidant and anti-inflammation. However, its antifungal activity and the mechanism of action in FK have not been elucidated. This study confirmed that OMT suppressed Aspergillus fumigatus growth, biofilm formation, the integrity of fungal cell and conidial adherence. OMT not only effectively reduced corneal fungal load but also inflammation responses. OMT lessened the recruitment of neutrophils and macrophages in FK. In addition, OMT up-regulated the expression of Nrf2 and down-regulated the expression of IL-18, IL-1ß, caspase-1, NLRP3 and GSDMD. Pre-treatment with Nrf2 inhibitor up-regulated the expression of IL-1ß, IL-18, caspase-1, NLRP3 and GSDMD supressed by OMT. In conclusion, OMT has efficient anti-inflammatory and antifungal effects by suppressing fungal activity and restricting pyroptosis via Nrf2 pathway. OMT is considered as a potential option for the treatment of FK.

Kaempferol ameliorate the prognosis of Aspergillus fumigatus keratitis by reducing fungal load and inhibiting the Dectin-1 and p38 MAPK pathway.

Fungal keratitis is one of leading reasons for blindness in the world, which causes corneal blindness mainly due to excessive inflammatory responses. Kaempferol (KAE) is a natural flavonoid which has potent anti-inflammatory effects. However, whether KAE plays protective roles in fungal keratitis and the potentially protective mechanisms are unrevealed. Here we first investigated the anti-inflammatory and antifungal effects of KAE on Aspergillus fumigatus (A. fumigatus) keratitis in C57BL/6 mice. We found that treatment of KAE ameliorated the severity of keratitis, inhibited macrophages and neutrophils recruitment, depressed corneal fungal load, and declined the expression of TLR4 and Dectin-1 in A. fumigatus infected mice corneas. And in activated hyphae or Curdlan stimulated macrophages, pretreatment of KAE also significantly decreased the mRNA and protein expression of IL-1ß, TNF-α, MIP-2 and the phosphorylated-p38 (p-p38)/p38 MAPK ratio. In summary, KAE ameliorated the prognosis of fungal keratitis in C57BL/6 mice by reducing corneal fungal load, depressing the inflammatory cells recruitment, and downregulating the expression of inflammatory factors, and those effects depended on the inhibition of Dectin-1 and p38 MAPK pathway.

Resolvin D1 protects against Aspergillus fumigatus keratitis in diabetes by blocking the MAPK-NF-κB pathway.

Fungal keratitis (FK) is one of the main causes of blindness in China. People with diabetes are susceptible to corneal epithelial disease, even fungal keratitis. At present, there are few studies on this disease. Resolvins (Rv) has been reported as a mediators that exert crucial anti-inflammatory and immune regulation roles in serval diseases. In order to investigate the roles and underlying mechanism of Resolvins D1 (RvD1) on the Aspergillus fumigatus (A. fumigatus) keratitis in diabetes, we established in vivo and in vitro models of A. fumigatus keratitis, which were then exposed to high glucose. The expression levels of RvD1, 5-lipoxygenase (5-LOX), and 15-lipoxygenase (15-LOX) in A. fumigatus keratitis patients with diabetes were determined through Enzyme Linked Immunosorbent Assay (ELISA), Western blot and immunohistochemistry. Reactive Oxygen Species (ROS) production, ELISA, flow cytometry, Hematoxylin-Eosin (HE) staining and fungal loading determination were conducted to evaluate the severity of A. fumigatus infection. Lymphangiogenesis and angiogenesis were examined by immunofluorescence assay. Western blot was applied to detect the proteins of the MAPK-NF-κB pathway. The results showed that RvD1 diminished the high glucose-induced oxidative stress and inflammatory response, as evidenced by the reduction of ROS production, Interleukin-6 (IL-6), Interleukin-8 (IL-8), Heme Oxygenase-1 (HMOX-1), and the elevation of Cyclooxygenase-2 (COX2), Superoxide Dismutase (SOD-1), and Glutathione Peroxidase-2 (GPX2) levels in A. fumigatus-infected Human Corneal Endothelial Cells (HCECs). Additionally, lymphangiogenesis and angiogenesis prominently decreased after intervention with RvD1. Furthermore, RvD1 significantly reduced the levels of p-MEK1/2 and p-ERK1/2, and restrained the NF-κB and GPR32 activation. The above results showed that RvD1 protects against A. fumigatus keratitis in diabetes by suppressing oxidative stress, inflammatory response, fungal growth, and immunoreaction via modulating MAPK-NF-κB pathway. RvD1 provides clues for the therapeutic targets of Fungal keratitis complicated with diabetes.

A novel 3D culture model of fungal keratitis to explore host-pathogen interactions within the stromal environment.

Fungal keratitis (FK) pathology is driven by both fungal growth and inflammation within the corneal stroma. Standard in vitro infection models ̶ involving co-culture of the pathogen and the corneal cells in tissue culture medium ̶ are sufficient to probe host responses to the fungus; however, they lack the physiological structure and nutrient composition of the stroma to accurately study fungal invasiveness and metabolic processes. We therefore sought to develop a culture model of FK that would allow for both host and fungal cell biology to be evaluated in parallel. Towards this end, we employed a previously described system in which primary human cornea fibroblasts (HCFs) are cultured on transwell membranes, whereupon they secrete a three-dimensional (3D) collagen matrix that resembles the human stroma. We demonstrated that two common mold agents of FK, Fusarium petroliphilum and Aspergillus fumigatus, penetrated into these constructs and caused a disruption of the collagen matrix that is characteristic of infection. HCF morphology appeared altered in the presence of fungus and electron microscopy revealed a clear internalization of fungal spores into these cells. Consistent with this apparent phagocyte-like activity of the HCFs, mRNA and protein levels for several pro-inflammatory cytokines/chemokines (including TNFα, IL-1ß, IL-6, and IL-8) were significantly upregulated compared to uninfected samples. We similarly found an upregulation of several HCF metalloproteases (MMPs), which are enzymes that breakdown collagen during wound healing and may further activate pro-inflammatory signaling molecules. Finally, several fungal collagenase genes were upregulated during growth in the constructs relative to growth in tissue culture media alone, suggesting a fungal metabolic shift towards protein catabolism. Taken together, our results indicate that this 3D-stromal model provides a physiologically relevant system to study host and fungal cell pathobiology during FK.

GSDMD, an executor of pyroptosis, is involved in IL-1ß secretion in Aspergillus fumigatus keratitis.

The protein GSDMD is an important performer of pyroptosis and a universal substrate for the inflammatory caspase. However, the role and regulatory mechanism of GSDMD in Aspergillus fumigatus keratitis is remains unknown. Here we detected GSDMD protein in the cornea of normal and fungal-infected C57BL/6 mice. Human corneal epithelial cell (HCECs) were preincubated with a hydrochloride solution (IFNR inhibitor), ruxolitinib (JAK/STAT inhibitor), belnacasan (caspase-1 inhibitor) before infection with A. fumigatus conidia. Mice corneas were infected with Aspergillus fumigatus after pretreatment of GSDMD siRNA via subconjunctival injection. After, samples were harvested at specific time points and the expression of GSDMD and IL-1ß was assessed by PCR, Western blot and immunofluorescence staining. Compared with the control group, we observed that the expression of GSDMD in fungal-infected mice cornea was significantly increased. After pretreatment with IFNR, JAK/STAT and caspase-1 inhibitors before fungal infection, the expression of GSDMD was significantly inhibited compared to the DMSO control in HCECs. Moreover, the GSDMD siRNA treatment have significantly weaken corneal inflammatory response, decreasing the proinflammatory factor IL-1ß secretion and reducing neutrophils and macrophages recruitment in mice infected corneas. In summary, the data here provided evidences that GSDMD, an executor of pyroptosis, is involved in the early immune response of A. fumigatus keratitis. Additionally, the inhibition of GSDMD expression can affect the secretion of IL-1ß and the recruitment of neutrophil and macrophages by blocking IFNR, JAK/STAT and caspase-1 signaling pathway. The protein GSDMD may emerge as a potential therapeutic target for A. fumigatus keratitis.

The cGAS-STING signaling pathway contributes to the inflammatory response and autophagy in Aspergillus fumigatus keratitis.

Fungal keratitis is a serious corneal infection, which can lead to significant visual impairment and blindness. The cGAS-STING signaling pathway has emerged as a key player in innate immunity by sensing of invading pathogens. However, the role of the cGAS-STING pathway in Aspergillus fumigatus (A. fumigatus) keratitis is still unknown. In this study, we showed that the cGAS-STING signaling pathway was activated in human corneal epithelial cells (HCECs) and in mouse corneas infected with A. fumigatus. Knockdown of cGAS reduced A. fumigatus-induced production of pro-inflammatory cytokines, including TNF-α, IL-1ß, IL-6, and IFN-ß. However, reconstruction of cGAS activity restored the inflammatory response in HCECs infected with A. fumigatus. A specific cGAS inhibitor, RU.521, could also significantly inhibit A. fumigatus-induced inflammatory cytokine expression. In addition, we found that cGAS was indispensable for the autophagy flux evoked by A. fumigatus infection. Moreover, inhibition of cGAS using siRNA or RU.521 alleviated the severity of A. fumigatus keratitis in the mouse cornea. Therefore, the cGAS-STING signaling pathway contributes to the progression of A. fumigatus keratitis and targeting this pathway may provide therapeutic potential.

Loss of Down Syndrome Critical Region-1 Mediated-Hypercholesterolemia Accelerates Corneal Opacity Via Pathological Neovessel Formation.

OBJECTIVE: The calcineurin-NFAT (nuclear factor for activated T cells)-DSCR (Down syndrome critical region)-1 pathway plays a crucial role as the downstream effector of VEGF (vascular endothelial growth factor)-mediated tumor angiogenesis in endothelial cells. A role for DSCR-1 in different organ microenvironment such as the cornea and its role in ocular diseases is not well understood. Corneal changes can be indicators of various disease states and are easily detected through ocular examinations. Approach and Results: The presentation of a corneal arcus or a corneal opacity due to lipid deposition in the cornea often indicates hyperlipidemia and in most cases, hypercholesterolemia. Although the loss of Apo (apolipoprotein) E has been well characterized and is known to lead to elevated serum cholesterol levels, there are few corneal changes observed in ApoE-/- mice. In this study, we show that the combined loss of ApoE and DSCR-1 leads to a dramatic increase in serum cholesterol levels and severe corneal opacity with complete penetrance. The cornea is normally maintained in an avascular state; however, loss of Dscr-1 is sufficient to induce hyper-inflammatory and -oxidative condition, increased corneal neovascularization, and lymphangiogenesis. Furthermore, immunohistological analysis and genome-wide screening revealed that loss of Dscr-1 in mice triggers increased immune cell infiltration and upregulation of SDF (stromal derived factor)-1 and its receptor, CXCR4 (C-X-C motif chemokine ligand receptor-4), potentiating this signaling axis in the cornea, thereby contributing to pathological corneal angiogenesis and opacity. CONCLUSIONS: This study is the first demonstration of the critical role for the endogenous inhibitor of calcineurin, DSCR-1, and pathological corneal angiogenesis in hypercholesterolemia induced corneal opacity.

Fungal keratitis: Pathogenesis, diagnosis and prevention.

As a kind of serious, potentially sight-threatening corneal infections with poor prognosis, fungal keratitis can bring a heavy economic burden to patients and seriously affect the quality of life, especially those in developing countries where fungal keratitis is more prevalent. Typical clinical features include immune rings, satellite lesions, pseudopods, hypha moss, hypopyon and endothelial plaques. The ideal therapeutic effects could not be achieved by current treatments for many reasons. Therefore, under the current status, understanding the pathogenesis, early diagnosis and prevention strategies might be of great importance. Here, in this review, we discuss the recent progresses that may advance our understanding of pathogenesis, early diagnosis and prevention of fungal keratitis.

Fusarium infection alters the m6A-modified transcript landscape in the cornea.

N6-methyladenosine (m6A) is the most common post-transcriptional modification of RNA in eukaryotes that regulates the post-transcriptional expression level of genes without changing the base sequence. The role of m6A in fungal keratitis has not yet been elucidated. Here, we aimed to identify m6A modification changes and their potential roles in fungal keratitis. The murine model of fungal keratitis was established by inoculating mice with Fusarium solani (F. solani). The overall m6A level was detected via an m6A RNA methylation assay kit. The expression levels of key m6A modification-related genes were estimated by quantitative real-time polymerase chain reaction (PCR). The expression and localization of METTL (methyltransferase like)3, the key component of the m6A methyltransferase complex, was determined by immunostaining and Western blotting (WB). Immunoprecipitation methylation microarray was used to describe the changes in m6A modification in F. solani-infected corneal tissue. The overall m6A level in corneal tissue on the 5th day in the F. solani-treated group was upregulated compared with that in the control group. The demethylase levels were unaltered, but the level of the methylase METTL3 was increased significantly after fungal infection. Additionally, differences were found in m6A modifications in 1137 mRNAs, of which 780 were hypermethylated and 357 were hypomethylated. To the best of our knowledge, the present work is the first investigation on the m6A modification profiles in experimental fungal keratitis, and it may provide a potential therapeutic target.

Quantitative profiling of tear proteome reveals down regulation of zinc alpha-2 glycoprotein in Aspergillus flavus keratitis patients.

Corneal mycotic ulceration is predominantly due to Aspergillus and Fusarium solani infection in tropical countries. In this study, we examined the proteome profile of tear samples from A. flavus keratitis patients at various stages of infection. The proteome was profiled using 2D PAGE and the protein levels were quantified using 2D DIGE. Alpha-1-antitrypsin, apolipoprotein, haptoglobin, lactoferrin and albumin were up regulated while cystatin SA III precursor, lacrimal lipocalin precursor, lacritin precursor and Zinc alpha-2 glycoprotein (ZAG) were down regulated in tear fluid. In the case of ZAG all proteoforms were down regulated as the disease progressed from early to late stage of infection. Western blot analysis confirmed the results observed using DIGE. Further, there were no gender specific differences in the levels of ZAG expression in keratitis patient tear film. Published results show up regulation of ZAG in Fusarium keratitis patient tear indicating subtle changes in the early events of host response to these two fungal pathogens. We conclude that ZAG level could be used as an indicator of A. flavus or F. solani infection, even during the early stage of the disease.

Interactions of thymic stromal lymphopoietin with TLR2 and TLR4 regulate anti-fungal innate immunity in Aspergillus fumigatus-induced corneal infection.

Thymic stromal lymphopoietin (TSLP) is an interleukin 7 (IL-7)-like four helix bundle cytokine that plays diverse roles in the regulation of immune responses. In fungal infection, pattern recognition receptors (PRRs), including the cell surface Toll-like receptors (TLRs) and cytoplasmic NOD-like receptors, recognize pathogen-associated molecular patterns to initiate downstream signal cascades to active immune responses. Our previous studies reported that, in vitro human cornea epithelium cells represented a novel target of TSLP and that TSLP/TSLPR/STAT5 signaling played an important role in the response to Aspergillus fumigatus challenge. TSLP downstream signaling molecules upregulated TLR2 and MyD88/NF kappa B-p65 signaling. This phenomenon suggested that TSLP had an impact on PRRs in antifungal immunity. In mouse fungal keratitis induced by A. fumigatus, TSLP was mainly expressed in the epithelium as well as in some infiltrated immune cells in a time-dependent manner. Exogenous TSLP with Aspergillus led to severe keratitis and worse corneal recovery with higher levels of TLR2, TLR4, IL-6, and IL-8 as well as increased neutrophil infiltration. By contrast, when TSLP was suppressed by siRNA, fungal keratitis was mild with higher levels of antimicrobial peptides such as human beta-defensin (hBD9). Taken together, our data revealed an unreported function of TSLP in mediating an anti-fungal inflammatory response and serving as a target to control tissue injury and infection in A. fumigatus keratitis.

Expression of cytokines in aqueous humor from fungal keratitis patients.

BACKGROUND: Although a series of reports on corneal fungal infection have been published, studies on pathogenic mechanisms and inflammation-associated cytokines remain limited. In this study, aqueous humor samples from fungal keratitis patients were collected to examine cytokine patterns and cellular profile for the pathogenesis of fungal keratitis. METHODS: The aqueous humor samples were collected from ten patients with advanced stage fungal keratitis. Eight aqueous humor samples from patients with keratoconus or corneal dystrophy were taken as control. Approximately 100 µl to 300 µl of aqueous humor in each case were obtained for examination. The aqueous humor samples were centrifuged and the cells were stained and examined under optical microscope. Bacterial and fungal cultures were performed on the aqueous humor and corneal buttons of all patients. Cytokines related to inflammation including IL-1ß, IL-6, IL-8, IL-10, TNF-α, and IFN-γ were examined using multiplex bead-based Luminex liquid protein array systems. RESULTS: Fungus infection was confirmed in these ten patients by smear stains and/or fungal cultures. Bacterial and fungal cultures revealed negative results in all aqueous humor specimens. Polymorphonuclear leukocytes were the predominant infiltrating cells in the aqueous humor of fungal keratitis. At the advanced stages of fungal keratitis, the levels of IL-1ß, IL-6, IL-8, and IFN-γ in the aqueous humor were significantly increased when compared with control (p<0.01). The levels of IL-10 and TNF-α also showed an ascending trend but with no statistical significance. CONCLUSIONS: High concentration of IL-1ß, IL-6, IL-8, and IFN-γ in the aqueous humor was associated with fungal keratitis.

Phospholipase Cγ2 is critical for Ca2+ flux and cytokine production in anti-fungal innate immunity of human corneal epithelial cells.

BACKGROUND: Fungal keratitis (FK) is a sight-threatening disease, accounting for a significant portion with its complex presentation, suboptimal efficacy of the existing therapies and uncontrollable excessive innate inflammation. Phospholipase C-γ2 (PLCγ2) is a non-receptor tyrosine kinase that plays an important role at the early period of innate immunity. This study aimed to identify the role of PLCγ2 in Dectin-1-mediated Ca2+ Flux and its effect on the expression of proinflammatory mediators at the exposure to Aspergillus fumigatus (A. fumigatus) hyphae antigens in human corneal epithelial cells (HCECs). METHODS: The HCECs were preincubated with or without different inhibitors respectively before A. fumigatus hyphae stimulation. Intracellular calcium flux in HCECs and levels of PLCγ2 and spleen-tyrosine kinase (Syk) were detected by fluorescence imaging and Western Blotting. The expression of proinflammatory mediators was determined by reverse transcriptase polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). RESULTS: We demonstrated that an intracellular Ca2+ flux in HCECs was triggered by A. fumigatus hyphae and could be reduced by pre-treatment with PLCγ2-inhibitor U73122. A. fumigatus hyphae induced PLCγ2 phosphorylation was regulated by Dectin-1 via Syk. Furthermore, PLCγ2-deficient HCECs showed a drastic impairment in the Ca2+ signaling and the secretion of IL-6, CXCL1 and TNF-α. CONCLUSIONS: PLCγ2 plays a critical role for Ca2+ Flux in HCECs stimulated by A. fumigatus hyphae. Syk acts upstream of PLCγ2 in the Dectin-1 signaling pathway. The expressions of proinflammatory mediators induced by A. fumigatus are regulated by the activation of Dectin-1-mediated PLCγ2 signaling pathway in HCECs.

Antimicrobial efficacy of corneal cross-linking in vitro and in vivo for Fusarium solani: a potential new treatment for fungal keratitis.

BACKGROUND: Fungal keratitis is one of the major causes of visual impairment worldwide. However, the effectiveness of corneal collagen cross-linking (CXL) for fungal keratitis remains controversial. In this study, we developed an in vitro and an in vivo models to assess the efficacy of CXL for Fusarium keratitis. METHODS: The effect of in vitro CXL fungicidal was evaluated on the cultures of Fusarium solani which were exposed to irradiation for different durations. Viability of fungal was appraised under four conditions: no treatment (control); CXL: UVA (365 nm)/riboflavin; riboflavin and UVA (365 nm). Each batch of sterile plate culture was irradiated for different CXL durations. The in vivo Therapeutic effect was studied on a mouse keratitis model. The animals were divided randomly into three groups: group A with no treatment (control); Group B with CXL treatment for two minutes and group C with CXL treatment for three minutes. The CXL procedure was performed 24 h post inoculation in each group. All mice with corneal involvement were scored daily for 7 days and 10 days after infection. Corneals were extracted at various time points for quantitative fungal recovery. Histological evaluations were conducted to calculate the number of polymorphonuclear cells. RESULTS: Viability of fungal decreased significantly in CXL group with 30-min irradiation compared with that in control, riboflavin and UVA groups (P < 0.01). The colony-forming units (CFUs) of fungal solutions in culture significantly decreased with CXL treatment (P < 0.05). Clinical scores, corneal lesion, corneal opacity, neovascularization and the depth of ulceration scores in group B and group C were remarkably lower than that in group A (P < 0.05, P = 0.001, P = 0.001, P = 0.034 and P = 0.025 respectively). Scores of group C were much lower than that in group B. Histological revealed that destruction of corneal collagen fibers and infiltration of inflammatory cells into corneal tissue in group B and group C were much lower than that in group A. CONCLUSIONS: We believe that CXL treatment may be applied to fungal keratitis, therapeutic efficacy will improve with longer treatment duration.

Ocular amphotericin B delivery by chitosan-modified nanostructured lipid carriers for fungal keratitis-targeted therapy.

CONTEXT: Fungal keratitis, a corneal fungal infection of the eye caused mainly by Candida species, has become the leading cause of blindness resulting from corneal disease in China. Present limitations in the management of ophthalmic fungal infections include the inability to provide long-term extraocular drug delivery without compromising intraocular structures and/or systemic drug exposure. OBJECTIVE: The aim of this study was to construct amphotericin B (AmB) loaded, chitosan-modified, nanostructured lipid carriers (AmB-CH-NLC) for prolonged ocular application and for the improvement of the targeted delivery of AmB to the ocular mucosa. MATERIALS AND METHODS: The AmB-CH-NLC was produced by the method of emulsion evaporation-solidification at low temperature. The particle size, zeta potential, and encapsulation efficiency, drug-release behavior, and corneal penetration ability were performed in vitro and in vivo. RESULTS AND DISCUSSION: The prepared AmB-CH-NLC nanoparticles exhibited a measured size of 185.4 nm, a zeta potential of 27.1 mV, and an entrapment efficiency of 90.9%. Sustained drug release behavior was observed in vitro. The in vivo ocular pharmacokinetic study indicated improved bioavailability of AmB-CH-NLC. The corneal penetration study showed that the AmB-CH-NLC could successfully penetrate into the cornea with no obvious irritation to the rabbits' eyes. CONCLUSION: The results support that this novel nanomedicine could be a promising system for effective ocular delivery of amphotericin B for fungal keratitis-targeted therapy.

Neutrophil extracellular traps involvement in corneal fungal infection.

PURPOSE: Neutrophils release neutrophil extracellular traps (NETs) when defending against invading microorganisms. We investigated the existence of NETs in fungal keratitis. METHODS: Fourteen patients with unilateral fungal keratitis were included. Detailed information about each patient was recorded, including (1) patient history (onset of symptoms and previous therapy), (2) ocular examination findings by slit-lamp biomicroscopy, (3) laboratory findings from direct smear examination and culture of corneal scrapings, (4) NET formation, and (5) treatment strategy and prognosis. Immunofluorescence staining was used to evaluate the existence of NETs on corneal scrapings. The relationship between the quantification of NETs and the clinical character of the fungal keratitis was identified. RESULTS: NETs were identified in all 14 patients. Patients with a higher grade of NET formation and fewer fungal hyphae always showed a good treatment response and a short course of infection. NETs were consistently found mixed with fungal hyphae in the corneal scrapings from infected patients. No statistical significance was found between the grade of NETs formed and the course of infection before presentation, and no relationship between the quantification of NETs and the size of the ulcer was found. CONCLUSIONS: The results suggest that NETs are involved in fungal keratitis. The number of NETs in infected corneas may provide a tool for evaluating the prognosis for fungal keratitis.

Histopathological clues in the diagnosis of fungal infection by Scedosporium in a case of endophthalmitis starting as conjunctivitis.

Cutaneous fungal infections can result in disastrous episodes if improperly diagnosed and treated, especially in immunosuppressed patients. Although dermatopathologists are highly familiar with some filamentous fungi - such as Aspergillus and Zygomycetes - they are not so aware of other less common species. We report a case of ocular infection by Scedosporium apiospermum that started as conjunctivitis and resulted in Phthisis bulbi and subsequent exeresis of the left eye. We describe some of the main morphological features of the fungus as well as the important morphological clues for the differential diagnosis with some similar species, such as Aspergillus, Scopulariopsis, Fusarium, Paecilomyces and Zygomycetes.

Targeting iron acquisition blocks infection with the fungal pathogens Aspergillus fumigatus and Fusarium oxysporum.

Filamentous fungi are an important cause of pulmonary and systemic morbidity and mortality, and also cause corneal blindness and visual impairment worldwide. Utilizing in vitro neutrophil killing assays and a model of fungal infection of the cornea, we demonstrated that Dectin-1 dependent IL-6 production regulates expression of iron chelators, heme and siderophore binding proteins and hepcidin in infected mice. In addition, we show that human neutrophils synthesize lipocalin-1, which sequesters fungal siderophores, and that topical lipocalin-1 or lactoferrin restricts fungal growth in vivo. Conversely, we show that exogenous iron or the xenosiderophore deferroxamine enhances fungal growth in infected mice. By examining mutant Aspergillus and Fusarium strains, we found that fungal transcriptional responses to low iron levels and extracellular siderophores are essential for fungal growth during infection. Further, we showed that targeting fungal iron acquisition or siderophore biosynthesis by topical application of iron chelators or statins reduces fungal growth in the cornea by 60% and that dual therapy with the iron chelator deferiprone and statins further restricts fungal growth by 75%. Together, these studies identify specific host iron-chelating and fungal iron-acquisition mediators that regulate fungal growth, and demonstrate that therapeutic inhibition of fungal iron acquisition can be utilized to treat topical fungal infections.

γδ T cells regulate the expression of cytokines but not the manifestation of fungal keratitis.

As an important immunoregulatory cell type, the role of γδ T cells in fungal keratitis (FK) is unclear. We observed the distribution of γδ T cells in infected corneas in vivo by two-photon microscopy. The γδ T cells were depleted by neutralizing antibodies. The cytokine expression profile was obtained by protein arrays to determine the cytokines regulated by γδ T cells. ICAM-1, MIP-2 and IL-17A were evaluated by ELISA assays to confirm the role of γδ T cells in FK. We counted the number of neutrophils, evaluated the volume of fungal hyphae and analyzed the manifestation of the disease. The γδ T cells increased significantly at 36 h and 72 h post fungal infection (P < 0.05) and migrated from the limbus to the infection site. The neutralizing antibodies completely depleted the γδ T cells in 24 h. The depletion of γδ T cells led to up regulation of 25 cytokines and down regulation of 3 cytokines. ICAM-1, MIP-2 and IL-17A changed significantly because of the depletion of γδ T cells (P < 0.05). However, the number of neutrophils, volume of fungal hyphae and manifestation of the disease was not affected by the depletion of γδ T cells. Our results demonstrated that γδ T cells have a role in FK via regulation of some cytokines but did not affect the manifestation of this disease, suggesting that γδ T cells are not the key regulator cells in this disease.

Cell penetrating peptides as efficient nanocarriers for delivery of antifungal compound, natamycin for the treatment of fungal keratitis.

PURPOSE: Enhancing the penetration ability of the antifungal drug natamycin, known to possess poor penetration ability through the corneal epithelium, by complexing with cell penetrating peptides. METHODS: The drug, natamycin was conjugated to a cell penetrating peptide, Tat-dimer (Tat2). The uptake ability of the conjugate in human corneal epithelial cells and its antifungal activity against filamentous fungi, F.solani has been elucidated. RESULTS: The cellular penetration ability of natamycin increased upon conjugation with Tat2. The conjugation between natamycin and Tat2 also lead to enhanced solubility of the drug in aqueous medium. The antifungal activity of the conjugate increased two- folds in comparison to unconjugated natamycin against clinical isolates of F.solani. CONCLUSION: The formation of CPP-natamycin complex is clinically significant as it may enhance the bioavailability of natamycin in corneal tissues and aid in efficient management of fungal keratitis.