4288

in vivo Imaging of Early Inflammation Response in Bacterial Infection.

Rao V. L. Papineni*, Sean Orton, William McLaughlin and Benjamin Geldhof
Carestream Molecular Imaging, 4 Research Dr., Woodbridge, CT, 06525, USA.
* rao.papineni1@carestreamhealth.com

Abstract
X-ray Staphylococcus aureus (S. aureus) causes significant morbidity and mortality worldwide and a prime cause in hospital infections. Staphylococcus aureus bactereremia is prevalent in neutropenic cancer patients, and malignancies form a sizeable risk factor for methycillinresistant S. aureus (MRSA) infections. Targeting the virulence products is a promising approach for developing novel therapeutics. A detailed understanding of the virulence factors and the resultant immune response is quite essential for such development. Here, we established an athymic mice model to assess the initial immune response to S.aureus inoculation. As the polymorphonuclear neutrophils (PMN) and macrophages are a part of the early response, determination of its myeloperoxidase (MPO) activity in vivo is established. MPO is an inflammatory heme protein and utilizes hydrogen peroxide in the process of reactive oxygen species generation. 24 hrs after the intra muscular injection of the S.aureus bacterial lysate, the MPO activity was detected by i.p injection of luminol in mice. The blue luminescence resulting from the MPO activity was imaged with a commercially available multimodal imaging system. The luminescence images were overlaid on planar X-ray images for anatomical coregistration. The results show a robust MPO activity in the mouse bladder. This increase in MPO activity as a result of neutrophil activation at the bladder region was further confirmed using non-invasive X-ray contrast imaging of the bladder and co-registering the luminescence and the X-ray density signals at the bladder. The initial response shown here has similarities with the mechanism suggested recently for myobacterium bovis bacillus Calmette-Guerin (BCG) treatment of bladder cancer. Bacterial components, its structures that are involved in eliciting the robust PMN infiltration in bladder will be very valuable in the developing better bladder cancer anti-tumor treatments. Luminescence Overlay

24 hours

Non Invasive Luminescence Imaging of Myeloperoxidase Activity at the Urinary Bladder

Figure-3: Left Panels: Multimodal non invasive imaging of staphylococcus aureus inoculated athymic mouse. Representative mouse images obtained after 24 hours of injection with s.aureus. Luminescence image (center) was obtained few minutes after the luminol administration, and was followed by taking a 2.5 min exposure X-ray image (left). The overlay of the luminescence on X-ray image is shown (right) for anatomical co registration of the myeloperoxidase (MPO) signals. The MPO activity was detected at the site of injection (near the lower left thigh) along with a substantial amount at the bladder region. The increase in MPO activity as a result of neutrophil activation at the bladder region was further confirmed using radiopaque contrast agents, coregistering the luminescence and the X-ray density signals at the bladder (see legend- right) Figure-4: Lower Middle Panels: Robust myeloperoxidase activity observed at the bladder. Luminescence (center panel below) signals were relatively stronger at the bladder region of the mice (A and B; below) imaged after 120 hours of bacterial inoculation. The localization of MPO signals to the bladder was confirmed (lower right) using X-ray contrast imaging with iodinated compound (iodixanol). To obtain non invasive X-ray contrast at the bladder (lower left), the mice were earlier injected with a single bolus (lateral tail vein) of 100 l of Visipaque, an iodinated X-ray contrast agent (GE Healthcare Ltd., Piscataway, NY, USA). The X-ray images were captured using a 0.2 mm Aluminum X-ray filter; 2.5 min exposure; 2 X 2 binning. The MPO activity was negligible at the bacterial inoculation site (left thigh region) as observed from the lack of luminescence signals.

X-ray

Luminescence

Overlay

120 hours

in vivo Imaging of Myeloperoxidase Activity

Visualizing Early Inflammation Response
50 l bolus of staphylococcus aureus was injected (i.m) at a single site shown in the schematic diagram ( Fig-1: right panel). The bacterial colony obtained from the agar plate was subjected to robust mechanical homogenization using a 27 G syringe in PBS (pH 7.4). The cloudy components were injected directly to determine the inflammatory response. The in vivo myeloperoxidase (MPO) activity in response to the bacterial inoculation was determined by injecting luminol substrate (i.p. 100 l of 5 mg/ml Luminol, Sigma-Aldrich MO, USA). The MPO activity responsive luminescence was imaged using the development phase imaging system with a cooled CCD Camera along with the X-ray contrast images of the subject.

Luminol

Iodixanol

Inoculation site

Mouse A
4 min 14 min 24 min

Mouse B
40 min

Mouse A

Mouse B

Mouse A

Mouse B

in vivo luminescence from luminol (5amino-2,3-dihydro-1,4-phthalazinedione) was shown earlier to be a product of myeloperoxidase (MPO) activity (Gross et al. Nature Medicine 2009). The MPO system in host defense mechanisms is represented by the schematic diagram (Fig-2: left panel). MPO catalyzes the production of hypochlorous acid (HOCL) utilizing H2O2 and chloride ions.

Bladder ROI

Figure 5: Myeloperoxidase activity at urinary bladder peaks at later stages of infection. Luminescence signals (Bottom left panels) obtained after 24 hours post-bacterial infection show low MPO activity at the bladder compared to that at the bacterial inoculation site. By 120 hours (above), the neutrophil activation at bladder (MPO activity) is at its maximal. Four contiguous images (4, 14, 24, 40 min time points) of luminescence captured after a single bolus of luminol administration display gradual increase in the appearance of bladder signal (ROI encircledorange) compared to that of the inoculation site (ROI encircled -yellow)

Conclusions
An inflammation model for non invasive imaging of polymorphonuclear neutrophils (PMN) action at the urinary bladder during initial phase of host-pathogen defense is established.
Dr .Rao Papineni

Initial inflammation response in this bacterial infection model show similarities with the mechanism observed in myobacterium bovis bacillus Calmette-Guerin (BCG) treatment of cancer of bladder. The following methodology can be adapted (in vivo drug screening) for developing novel therapeutics - targeting the virulence factor of bacterial infection and in better bladder cancer treatments.

Courtesy: Toxins 2011 ( 2072-6651).

Post 24 hours

Inoculation Site ROI

Disclaimer. Carestreams pre-clinical imaging systems are not licensed to perform certain optical imaging applications that involve the in vivo imaging in mammals of (i) genetically expressed bioluminescent or fluorescent protein or (ii) conjugates of cells and light generating molecules, such applications are covered by patents owned or controlled by Caliper Life Sciences, Inc. Such patents include the following: U.S. Patents Nos. 5,650,135; 6,217,847; 7,198,774; 6,649,143; 6,939,533; 6,916,462; 6,923,951; 6,890,515; 6,908,605; 5,824,468; 6,638,752; 6,737,245 and 6,867,348; U.S. Patent Application No. 11/818,208; European Patent No. 0861093 and European Patent Application No. 991246406; Japanese Patent Nos. 3786704 and 3786903; Canadian Patent No. 2237983; Singapore Patent No. 53708; Hong Kong Patent No. 1018747; and Chinese Patent No. 951980068.

"Molecular Imaging - Wisdom To See For Maladies To Flee" Dr. Rao V. L. Papineni

Carestream Health, Inc.