Development of a novel ex vivo model of corneal fungal adherence

Zhou, Qingjun; Chen, Hao; Qu, Mingli; Wang, Qian; Yang, Lingling; Xie, Liping

doi:10.1007/s00417-010-1601-9

Cited by 8 publications

(9 citation statements)

References 39 publications

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“…Recently, there has been some interest in the use of ex vivo corneal models to study keratitis [ 12 , 13 ]. Although these models lack immune elements, the 3D architecture remains, as do the intracellular innate immune molecules and cellular–stromal components.…”

Section: Introductionmentioning

confidence: 99%

“…Although these models lack immune elements, the 3D architecture remains, as do the intracellular innate immune molecules and cellular–stromal components. These models have been used for studying wound healing [ 14 ], microbial adherence [ 12 ] and molecular microbial pathogenicity [ 13 ]. In our laboratory, we have used ex-vivo corneal models to study corneal epithelial regeneration [ 15 , 16 ] and to develop models of inflammation.…”

Section: Introductionmentioning

confidence: 99%

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Ex vivo rabbit and human corneas as models for bacterial and fungal keratitis

Pinnock

Shivshetty

Roy

et al. 2016

Graefes Arch Clin Exp Ophthalmol

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PurposeIn the study of microbial keratitis, in vivo animal models often require a large number of animals, and in vitro monolayer cell culture does not maintain the three-dimensional structure of the tissues or cell-to-cell communication of in vivo models. Here, we propose reproducible ex vivo models of single- and dual-infection keratitis as an alternative to in vivo and in vitro models.MethodsExcised rabbit and human corneoscleral rims maintained in organ culture were infected using 108 cells of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans or Fusarium solani. The infection was introduced by wounding with a scalpel and exposing corneas to the microbial suspension or by intrastromal injection. Post-inoculation, corneas were maintained for 24 and 48 h at 37 °C. After incubation, corneas were either homogenised to determine colony-forming units (CFU)/cornea or processed for histological examination using routine staining methods. Single- and mixed-species infections were compared.ResultsWe observed a significant increase in CFU after 48 h compared to 24 h with S. aureus and P. aeruginosa. However, no such increase was observed in corneas infected with C. albicans or F. solani. The injection method yielded an approximately two- to 100-fold increase (p < 0.05) in the majority of organisms from infected corneas. Histology of the scalpel-wounded and injection models indicated extensive infiltration of P. aeruginosa throughout the entire cornea, with less infiltration observed for S. aureus, C. albicans and F. solani. The models also supported dual infections.ConclusionsBoth scalpel wounding and injection methods are suitable for inducing infection of ex vivo rabbit and human cornea models. These simple and reproducible models will be useful as an alternative to in vitro and in vivo models for investigating the detection and treatment of microbial keratitis, particularly when this might be due to two infective organisms.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Ex vivo rabbit and human corneas as models for bacterial and fungal keratitis

Pinnock

Shivshetty

Roy

et al. 2016

Graefes Arch Clin Exp Ophthalmol

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“…The advantages of using goat corneas are that no ethical approval is required, the cornea is large and comparable to that of humans, and they can be procured easily from abattoirs. Ex vivo models have been used to study keratitis caused by Candida ,[ 11 ] Fusarium ,[ 12 ] herpes simplex virus,[ 5 ] S. aureus , and P. aeruginosa . [ 7 ] The mouse corneal organ culture developed for Candida infection was used to study fungal adhesion mechanisms and drug screening.…”

Section: Discussionmentioning

confidence: 99%

“…[ 7 ] The mouse corneal organ culture developed for Candida infection was used to study fungal adhesion mechanisms and drug screening. [ 11 ] Fusarium infection was modeled using human corneal buttons stabilized in an artificial anterior chamber (Refractive Technologies, Cleveland, OH). [ 12 ] Recently, rabbit corneas were used in an ex vivo model for Pseudomonas and Staphylococcus keratitis.…”

Section: Discussionmentioning

confidence: 99%

Ex vivo caprine model to study virulence factors in keratitis

Madhu¹,

Jha²,

Karthyayani³

et al. 2018

J Ophthalmic Vis Res

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Purpose:To develop an infectious keratitis model using caprine (goat) corneas and to investigate the expression of virulence factors during infection.Methods:Goat eyes were surface-sterilized and dissected, and the corneas were placed on an agarose-gelatin solid support (0.5% in phosphate-buffered saline) in a 12-well culture plate containing 10% fetal bovine serum-supplemented culture medium for 3 weeks. Cell viability tests (trypan blue and MTT) were performed on the cultured corneas. Corneas were infected with Pseudomonas aeruginosa and Fusarium solani separately. Infection progression was observed via histological analysis and hematoxylin and eosin (H-E) staining. For Pseudomonas-infected corneas, expression of eight virulence genes (exoS, exoT, exoY, alpR, prpL, lasA, lasB, and algD) was determined via quantitative real-time PCR (qRT-PCR) at 48-h and 72-h time-points. For Fusarium-infected corneas, expression of five proteases (C7Z0E6, C7ZFW9, C7Z7U2, C7ZNV5, and C7YY94) was quantified via qRT-PCR at 2, 4, and 8 days after infection. Protease from infected corneas was detected via gelatin zymography.Results:Goat corneas with a viable epithelium could be maintained for 15 days. Pseudomonas infection progressed rapidly, and complete corneal degradation was observed on day 4 after infection. Fusarium infection progressed more slowly. Histological analysis and H-E staining of Fusarium-infected cornea revealed mycelia penetrating all layers of the cornea. qRT-PCR revealed expression of all eight virulence factors, and statistically significant difference in expression of prpL and alpR in Pseudomonas-infected corneas. Expression of C7ZNV5 was highest in Fusarium-infected corneas.Conclusion:Goat corneas can be used to evaluate the expression of virulence factors involved in Pseudomonas and Fusarium infection.

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“…Inhibition assays at the eye organ model level were performed as described previously [48]. In brief, Balb/C mice were killed after anesthesia, their corneal epithelia were scarified with a 26-G syringe needle for “+” to mimic the situation of wounds occurred to the corneas.…”

Section: Methodsmentioning

confidence: 99%

Phage Display against Corneal Epithelial Cells Produced Bioactive Peptides That Inhibit Aspergillus Adhesion to the Corneas

et al. 2012

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Dissection of host-pathogen interactions is important for both understanding the pathogenesis of infectious diseases and developing therapeutics for the infectious diseases like various infectious keratitis. To enhance the knowledge about pathogenesis infectious keratitis, a random 12-mer peptide phage display library was screened against cultured human corneal epithelial cells (HCEC). Fourteen sequences were obtained and BLASTp analysis showed that most of their homologue counterparts in GenBank were for defined or putative proteins in various pathogens. Based on known or predicted functions of the homologue proteins, ten synthetic peptides (Pc-A to Pc-J) were measured for their affinity to bind cells and their potential efficacy to interfere with pathogen adhesion to the cells. Besides binding to HCEC, most of them also bound to human corneal stromal cells and umbilical endothelial cells to different extents. When added to HCEC culture, the peptides induced expression of MyD88 and IL-17 in HCEC, and the stimulated cell culture medium showed fungicidal potency to various extents. While peptides Pc-C and Pc-E inhibited Aspergillus fumigatus (A.f) adhesion to HCEC in a dose-dependent manner, the similar inhibition ability of peptides Pc-A and Pc-B required presence of their homologue ligand Alb1p on A.f. When utilized in an eyeball organ culture model and an in vivo A.f keratitis model established in mouse, Pc-C and Pc-E inhibited fungal adhesion to corneas, hence decreased corneal disruption caused by inflammatory infiltration. Affinity pull-down of HCEC membrane proteins with peptide Pc-C revealed several molecules as potential receptors for this peptide. In conclusion, besides proving that phage display-selected peptides could be utilized to interfere with adhesion of pathogens to host cells, hence could be exploited for managing infectious diseases including infectious keratitis, we also proposed that the phage display technique and the resultant peptides could be used to explore host-pathogen interactions at molecular levels.

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Development of a novel ex vivo model of corneal fungal adherence

Abstract: The corneal organ culture was a suitable ex vivo model for the research of fungal adhesion mechanisms and drug screening.

Cited by 8 publications

References 39 publications

Ex vivo rabbit and human corneas as models for bacterial and fungal keratitis

Ex vivo rabbit and human corneas as models for bacterial and fungal keratitis

Ex vivo caprine model to study virulence factors in keratitis

Phage Display against Corneal Epithelial Cells Produced Bioactive Peptides That Inhibit Aspergillus Adhesion to the Corneas

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