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K DHARMALINGAM, SIVAGNANAM ANANTHI, PRAJNA LALITHA, N VENKATESH PRAJNA; Tear Proteome Profile of Patients with Fungal Keratitis. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4236.
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Mycotic keratitis is a major cause of corneal blindness in India. Analysis of differentially expressed proteins in tears from early, intermediate and late stage of fusarium keratitis infections will pave the way for a better understanding of the disease onset and progression.
Tear samples were collected from culture positive (Fusarium) fungal keratitis patients and categorized based on the duration of the infection as early (within 7 days), intermediate (7-14 days) & late (after 14 days) stage of infection. Tears from healthy individuals served as controls. Tear sample preparation for Fluorescence Difference Gel Electrophoresis (2D DIGE) was optimized and DeCyder software was used for spot comparison & quantitation of separated proteins. Selected protein spots were identified using nanoLC-MS/MS and Western analysis was performed for validation
TCA precipitation & ultrafiltration resulted in efficient sample concentration and desalting. Fluorescence Difference Gel Electrophoresis could detect more than 1500 protein spots, of which 105 protein spots were differentially expressed in infected tear. Among the proteins identified by mass spectrometry, seven showed increased expression while three others were decreased in expression in tears from fusarium keratitis patients. 2D western analysis showed that the expression of lipocalin and lacritin isoforms were reduced to negligible levels in the early stage of fungal infection and their expression increased marginally at intermediate and late stage of infection
Tear proteins, which showed altered expression, were involved in inflammatory process and local immune function. This is the first study to show the comparison of tear protein profile in three different stages of fungal keratitis. Based on our results it is evident that the host immune response significantly changes as the disease progresses. This gives a clue that the treatment option should take into consideration the infective disease stage. Taken together this study proves that tears can be used as a clinical source to study the host and pathogen specific responses in patients with fungal keratitis.
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