Monitoring the Granulomal Micro-environment in a Monkey Model of Tuberculosis Using a Novel Fluorescence Bronchoscope

Abstract

Tuberculosis is a disease that infects one in three humans today. The long, expensive drug course required to cure the active form, combined with its increasing resistance to conventional antibiotic treatment, necessitates the development of a new class of TB drugs. To evaluate the efficacy of these drugs, as well as to gain increased basic knowledge of the disease's progression, we are building a novel 0.8 mm diameter bronchoscope as part of a multi-institutional initiative to develop a primate-based tuberculosis model system. In order to monitor the in vivo microenvironment of the tuberculosis granuloma, monkeys will be inoculated with transgenic pH reporting tuberculosis bacilli and GFP expression measured to quantify the local pH and other micro-environmental parameters. CT scans will be used to reveal the induced nodules/lesions and guide the bronchoscope to the granulomas. The lung tissue itself contains many 488 nm excitable endogenous fluorophores (e.g. elastin, collagen) and autofluorescence limits the level of reporter quantification. To overcome this problem we are employing a photoactivatable protein (Dronpa) as the reporter expressed by the bacteria. Using a novel pulsed UV/Blue non-laser light source, the protein's fluorescence can be modulated to distinguish reporter signal from the constant autofluorescence background and therefore produce highly quantitative measurements of changes in the granuloma microenvironment during the progression of the disease and during drug treatment.(Supported by the Gates Foundation and NIH/NIBIB P41 RR04224 to WRZ.)