Whole‐Body Imaging of Infection Using Fluorescence

Kong, Ying; Akin, Ali R.; Francis, Kevin P.; Zhang, Ning; Troy, Tamara L.; Xie, Hexin; Rao, Jianghong; Cirillo, Suat L. G.; Cirillo, Jeffrey D.

doi:10.1002/9780471729259.mc02c03s21

Cited by 14 publications

(12 citation statements)

References 32 publications

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“…In our own laboratory, we have targeted diagnostic strategies, since they have the potential to change the TB management landscape due to the fact that current diagnostic methods often cannot make a diagnosis prior to transmission [1,15]. Recent optical imaging technological developments have created more sensitive ways to visualize and quantify mycobacteria within cells and in vivo [2,3,6–9,12,21]. Despite these advances, reduction in signal due to tissue depth make application to diagnosis in humans extremely difficult.…”

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New directions using reporter enzyme fluorescence (REF) as a tuberculosis diagnostic platform

et al. 2016

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SUMMARY Although tuberculosis (TB) is one of the most common causes of morbidity and mortality in humans worldwide and diagnostic methods have been in place for more than 100 years, diagnosis remains a challenge. The main problems with diagnosis relate to the time needed to obtain a definitive result, difficulty in obtaining sputum, the primary clinical material used, and the ability of the causative agent, Mycobacterium tuberculosis, to cause disease in nearly any tissue within the body. In order to decrease incidence of TB, discovery of a novel interventions will be required, since current technologies have only been able to control numbers of infections, not reduce them. Diagnostic innovation is particularly needed because there are no effective pediatric or extrapulmonary TB diagnostic methods and multiple-drug resistance is only identified in less than 25% of those patients that are thought to have it. The most common diagnostic method worldwide remains acid-fast stain on sputum, with a threshold of ~10,000 bacteria/ml that is only reached ~5–6 months after development of symptoms. In order to obtain definitive diagnostic results earlier during the disease process, we have developed a diagnostic method designated reporter enzyme fluorescence (REF) that utilizes BlaC produced by M. tuberculosis and custom substrates to produce a specific fluorescent signal with as few as 10 bacteria/ml in clinical samples. We believe that the unique biology of the REF technique will allow it to contribute new diagnostic information that is complementary to all existing diagnostic tests as well as those currently known to be in development.

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confidence: 99%

New directions using reporter enzyme fluorescence (REF) as a tuberculosis diagnostic platform

et al. 2016

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“…The IVIS ® Lumina II optical imaging system, used predominantly for non-invasive biophotonic imaging in rodent animal models [32,33], proved to be an ideal tool for the quantitative analysis and spatial distribution of GFP expression in leaves of N. benthamiana plants. Utilizing the standard GFP filter settings and software of the system we were able to greatly reduce the background fluorescence from chlorophyll in the plant tissue, which has been reported to be especially problematic when imaging GFP reporters [14].…”

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Visualization of plant viral suppressor silencing activity in intact leaf lamina by quantitative fluorescent imaging

et al. 2011

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BackgroundTransient expression of proteins in plants has become a favoured method over the production of stably transformed plants because, in addition to enabling high protein yields, it is both fast and easy to apply. An enhancement of transient protein expression can be achieved by plant virus-encoded RNA silencing suppressor proteins. Since viral suppressor proteins differ in their efficiency to enhance transient protein expression in plants, we developed a whole-leaf green fluorescent protein (GFP)-based imaging assay to quantitatively assess suppressor protein activity.ResultsIn a transient GFP-expression assay using wild-type and GFP-transgenic N. benthamiana, addition of the plant viral suppressors Beet mild yellowing virus (BMYV-IPP) P0 or Plum pox virus (PPV) HC-Pro was shown to increase fluorescent protein expression 3-4-fold, 7 days post inoculation (dpi) when compared to control plants. In contrast, in agroinfiltrated patches without suppressor activity, near complete silencing of the GFP transgene was observed in the transgenic N. benthamiana at 21 dpi. Both co-infiltrated suppressors significantly enhanced GFP expression over time, with HC-Pro co-infiltrations leading to higher short term GFP fluorescence (at 7 dpi) and P0 giving higher long term GFP fluorescence (at 21 dpi). Additionally, in contrast to HC-Pro co-infiltrations, an area of complete GFP silencing was observed at the edge of P0 co-infiltrated areas.ConclusionsFluorescence imaging of whole intact leaves proved to be an easy and effective method for spatially and quantitatively observing viral suppressor efficiency in plants. This suppressor assay demonstrates that plant viral suppressors greatly enhanced transient GFP expression, with P0 showing a more prolonged suppressor activity over time than HC-Pro. Both suppressors could prove to be ideal candidates for enhancing target protein expression in plants.

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“…[30] A detailed protocol for the method of infection was described previously. [31] 24 hours after inoculation, the mice were imaged using the whole-body imaging system with intravital excitation. Following imaging, the lungs were excised, homogenized, and 10-fold serial dilutions of lung homogenates were plated on 7H11 selective media for culturing and CFU counting.…”

Section: Methodsmentioning

confidence: 99%

Intravital excitation increases detection sensitivity for pulmonary tuberculosis by whole‐body imaging with β‐lactamase reporter enzyme fluorescence

Nooshabadi

Yang

Cheng

et al. 2016

Journal of Biophotonics

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Tuberculosis is a pulmonary disease with an especially high mortality rate in immuno-compromised populations, specifically children and HIV positive individuals. The causative agent, Mycobacterium tuberculosis (Mtb), is a very slow growing and difficult organism to work with, making both diagnosis and development of effective treatments cumbersome. We utilize a fiber-optic fluorescence microendoscope integrated with a whole-body imaging system for in vivo Mtb detection. The system exploits an endogenous enzyme of Mtb (β-lactamase, or BlaC) using a BlaC-specific NIR fluorogenic substrate. In the presence of BlaC, this substrate is cleaved and becomes fluorescent. Using intravital illumination of the lung to excite this probe, sensitivity of the optical system increases over trans- and epi-illumination methods of whole-body fluorescence imaging. We demonstrate that integration of these imaging technologies with BlaC-specific fluorescent reporter probe improves the level of detection to ~100 colony forming units, a 100× increase in sensitivity in comparison to epi-illumination and a 10× increase in sensitivity in comparison to previous work in intravital excitation of tdTomato-expressing Mtb. This lower detection threshold enables the study of early stage bacterial infections with clinical strains of Mtb and longitudinal studies of disease pathogenesis and therapeutic efficacy with multiple time points in a single animal.

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Whole‐Body Imaging of Infection Using Fluorescence

Cited by 14 publications

References 32 publications

New directions using reporter enzyme fluorescence (REF) as a tuberculosis diagnostic platform

New directions using reporter enzyme fluorescence (REF) as a tuberculosis diagnostic platform

Visualization of plant viral suppressor silencing activity in intact leaf lamina by quantitative fluorescent imaging

Intravital excitation increases detection sensitivity for pulmonary tuberculosis by whole‐body imaging with β‐lactamase reporter enzyme fluorescence

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