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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
X-ray
Luminescence
Overlay
120 hours
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.
Post 24 hours
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