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Victor Llorens, Blanca Molins, Marina Mesquida, Maite Sainz De La Maza, Javier Zarranz-Ventura, Anna Sala-Puigdollers, Julian Gonzalez-Martin, Alfredo Adan Civera; Infection model of the human retinal pigment epithelium cell line ARPE-19 by Mycobacterium tuberculosis complex bacillus Calmette-Guérin. . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3103.
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© ARVO (1962-2015); The Authors (2016-present)
To establish and characterize a novel infection model of human retinal pigment epithelium (RPE) by M. bovis bacillus Calmette-Guérin (BCG).
BCG strain Pasteur 1173 P2 was cultured in Lowenstein-Jensen medium. Isolated colonies were suspended in culture media and disaggregated. Suspensions were adjusted to Mc Farland 1.0. ARPE-19 cells were grown until 90% confluence and bacterial inoculum was dispensed at a MOI 100:1 for 3 h. Control and infected ARPE-19 cells were trypsinized and seeded at a concentration of 100 cells/µl. Cytotoxicity and cytoproliferation assays were determined by WST-1 reagent and crystal violet dye elution (CVDE), respectively 3, 24, 48, 72 and 96 h post infection. To determine infection rates, ARPE-19 cells were trypsinized and counted by trypan blue. A cell suspension of 50 μl was lysed, serially diluted and cultured. Colony forming units (CFU) were counted and the infection rate was determined as CFU/cells. In parallel, 50µl of cell suspension were stained with modified Ziehl-Neelsen and infection rate determined by modified Crowle method. Control and infected epithelium were cultured for 2 months and analyzed for cyto-bacteriological interaction by optical microscopy.
Infection rate peaked at 48 h. We observed a substantial difference in infection rate between the two methods (16%±5.7% by CFU count and 40%±7.7% by microscopy, p=0.058). Correlation between both methods was moderate (r=0.538). Microscopy examination of infected cells showed adhesion, phagocytosis, intracellular proliferation and cytolysis with extracellular invasion by BCG bacteria (Fig.1, a to e, respectively). Interestingly, some giant infected cells were also observed at early infection stages. In late infected epithelium (1 month), we observed exosome release in those cells around BCG colonies (Fig.2). Cytotoxicity in infected cells was minimal and did not differ significantly from uninfected cells. However, cytoproliferation was significantly enhanced by BCG infection at 48 h (p=0.042).
BCG can infect ARPE-19 cells with little toxicity. Exosome release from infected cells could be on the bases of a sustained intraocular immune response in tuberculosis-related uveitis (TRU). This reproducible biosafe model provides new opportunities for the understanding of the pathogenesis of TRU.
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