The authors wish to acknowledge Mark Rodgers for outlining the infection procedures and L. Eoin Carney and Brian Lopresti for guidance in establishing these imaging procedures. Funding for this work has been provided by The Bill and Melinda Gates Foundation (J.L.F., P.L.L.), National Institutes of Health, National Institutes of Allergy and Infectious Diseases R01 AI111871 (P.L.L.), National Heart Lung and Blood Institute R01 HL106804 (J.L.F.), R01 HL110811.

<ol>
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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4+T+cell+depletion+exacerbates+acute+Mycobacterium+tuberculosis+while+reactivation+of+latent+infection+is+dependent+on+severity+of+tissue+depletion+in+cynomolgus+macaques.">CD4 T cell depletion exacerbates acute Mycobacterium tuberculosis while reactivation of latent infection is dependent on severity of tissue depletion in cynomolgus macaques.</a> AIDS Res Hum Retroviruses. 28 (12), 1693-1702 (2012).</li><li>Mattila, J. T., Diedrich, C. R., Lin, P. L., Phuah, J., Flynn, J. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digitally+Barcoding+Mycobacterium+tuberculosis+Reveals+In+Vivo+Infection+Dynamics+in+the+Macaque+Model+of+Tuberculosis.">Digitally Barcoding Mycobacterium tuberculosis Reveals In Vivo Infection Dynamics in the Macaque Model of Tuberculosis.</a> MBio. 8 (3), (2017).</li><li>Lin, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metronidazole+prevents+reactivation+of+latent+Mycobacterium+tuberculosis+infection+in+macaques.">Metronidazole prevents reactivation of latent Mycobacterium tuberculosis infection in macaques.</a> Proc Natl Acad Sci U S A. 109 (35), 14188-14193 (2012).</li><li>Lin, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+necrosis+factor+neutralization+results+in+disseminated+disease+in+acute+and+latent+Mycobacterium+tuberculosis+infection+with+normal+granuloma+structure+in+a+cynomolgus+macaque+model.">Tumor necrosis factor neutralization results in disseminated disease in acute and latent Mycobacterium tuberculosis infection with normal granuloma structure in a cynomolgus macaque model.</a> Arthritis Rheum. 62 (2), 340-350 (2010).</li><li>Phuah, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+B+Cell+Depletion+on+Early+Mycobacterium+tuberculosis+Infection+in+Cynomolgus+Macaques.">Effects of B Cell Depletion on Early Mycobacterium tuberculosis Infection in Cynomolgus Macaques.</a> Infect Immun. 84 (5), 1301-1311 (2016).</li><li>Martinez, V., Castilla-Lievre, M. A., Guillet-Caruba, C., Grenier, G., Fior, R., Desarnaud, S., Doucet-Populaire, F., Boue, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=(18)F-FDG+PET/CT+in+tuberculosis:+an+early+non-invasive+marker+of+therapeutic+response.">(18)F-FDG PET/CT in tuberculosis: an early non-invasive marker of therapeutic response.</a> Int J Tuberc Lung Dis. 16 (9), 1180-1185 (2012).</li><li>Malherbe, S. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persisting+positron+emission+tomography+lesion+activity+and+Mycobacterium+tuberculosis+mRNA+after+tuberculosis+cure.">Persisting positron emission tomography lesion activity and Mycobacterium tuberculosis mRNA after tuberculosis cure.</a> Nat Med. 22 (10), 1094-1100 (2016).</li><li>Chen, R. 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The authors have nothing to disclose.

Data acquired from PET/CT can be used as surrogate measurements for many aspects of M. tuberculosis infection that would be unobservable without such technology. PET/CT is much more sensitive than X-ray technology, which is often used in macaques studies. PET/CT provides structural, spatial and functional information. The analyses described above have many practical applications such as monitoring disease progression, assessing effectiveness of drug treatment, and providing risk factors for reactivation6,10,11,13.
Tracking the spread of granulomas and FDG uptake of individual lesions can be compared between control and experimental groups to not only provide specific location of the infection, but also follow dissemination of disease25. For example, in work from Coleman et al. describing early infection in cynomolgus macaques, following the infection progression can determine whether the infection within an animal is going to remain active and worsen or be contained by the immune system (i.e. LTBI)11. This is just one example of the power that PET/CT imaging has in studying the progression of disease with respect to granulomas. The same method can be used to study a wide variety of experimental parameters over time. For example, enumeration of granulomas that establish by 4 weeks post-infection can provide a powerful outcome measure for a vaccine, since the best vaccines would prevent or limit granuloma establishment following challenge. Another outcome measure for vaccines could be in limiting dissemination. These quantifiable outcome measures provide important data without the need to perform early necropsies on the animals. A limitation of assessing individual granulomas is the sensitivity of the CT scanner; visualizing granulomas &#60;1 mm in size is often not possible.
Evaluation of mediastinal lymph nodes (MLNs) is important when studying M. tuberculosis infection as well. MLNs are important for T-cell priming and trafficking of immune cells during infection. However, in nearly all macaques, at least one, and sometimes several MLNs can become infected. Thus, MLNs are an additional site of bacterial persistence during active TB and LTBI, and can serve as a reservoir for bacteria, possibly contributing to reactivation26,27. In cases of severe MLN involvement, airways can be compressed. Large necrotic MLNs can erode into the airways, leading to dissemination of the infection. Analyzing PET/CT data on lymph nodes is more complex than granulomas because the structural components of the nodes are not readily visible on CT scans. Additionally, only the lymph nodes that are metabolically active can be analyzed due to FDG uptake. Because of this, when analyzing hot lymph nodes, it is important to ensure that the PET image is scaled to the same maximum and minimum intensity scale for each image analysis to ensure consistency. Since they can be large structures, the necrotic center can be negative for FDG avidity, and thus FDG avidity can appear to be reduced over time, even when disease is increasing in the MLNs.
Total lung FDG avidity represents the total inflammation within the lungs. The amount of lung inflammation is an indication of disease severity and is correlated with bacterial burden6,10,28 therefore this quantitative and objective assessment has numerous applications. To measure total FDG avidity, all voxels within a PET image that show an SUV of greater than 2.3 are combined in a single volume-of-interest (VOI) and the total SUV value of the entire VOI is the final avidity value. This value was selected from literature that compared SUV values of FDG uptake in lung tumors to various infectious pathologies in humans24. It is important to note that this total FDG avidity value is limited only to disease in the lung space and all FDG uptake non-disease related or located in close proximity to the lungs must not be considered. In addition, total lung FDG avidity does not include MLNs. While the avidity of individual granulomas and lymph nodes allows us to see the variability of TB outcomes within the host, FDG avidity of the total lung is integral to evaluating the host as a whole. These methods also function as analytical tools to measure drug response to TB disease. Previous work has shown that TB drug treatment can reduce the size and FDG avidity of individual granulomas over time12 and these changes were associated with reduced bacterial burden. Changes in inflammation over the full course of drug regimens can also be used to assess drug efficacy or failure.
Due to the highly detailed nature of these procedures, a fair amount of troubleshooting may be necessary in order to obtain the most consistent data during studies. It is the goal of this paper to outline the procedures to enable individuals throughout the world to use these techniques while keeping in mind that precise attention to detail is essential. Persons evaluating images should be very familiar with anatomy and physiology in order to recognize abnormality within particular scans. Image readers need to recognize non-normal probe uptake throughout the body because TB can spread beyond the thoracic cavity. Additionally, PET and CT registration in imaging is not a perfect process and occasional deviations on image registration can occur; recognizing this can be vitally important when evaluating very small disease features (i.e. 1 - 2 mm granulomas). A pre-infection scan can be particularly useful as a comparator to identify normal lung (and other organ) structures and PET patterns and recognize those that are new or changed post-infection. Another important component to this analysis is background measurement. All PET data are normalized to muscle uptake as a physiological baseline because FDG uptake is based on metabolism. A combination of rhomboid and serratus muscles in the back are used for background measurements because of proximity to the thoracic cavity and relative consistency in FDG uptake in fasted animals. In the case of M. tuberculosis-infected macaques, this is preferable to using another organ, such as the liver, for background measurements as M. tuberculosis can infect the liver and liver metabolism can be affected when treating animals with various anti-TB drugs. Taking the above factors into account as well as ensuring that all image regions-of-interest are saved at the end of analysis should yield highly reproducible results.
In summary, PET/CT offers a unique and powerful method for investigating M. tuberculosis infection in non-human primates, providing quantitative outcome measures that relate to initial infection, dissemination, and bacterial burden. This allows for tracking the variable outcomes of infection across individual animals without the need for necropsies at various time points, thus saving resources and reducing animal use. This technology is directly translatable to humans, as PET/CT has been used in several studies to assess drug treatment in TB, as well as LTBI in HIV- and HIV+ subjects10,29,30,31. Finally, this technology and quantitative tool set for analyzing PET/CT data are likely to be useful in the future for vaccine efficacy studies and can likely be used as a template for analysis of other infectious diseases in animal models and human subjects.

Mycobacterium tuberculosis has plagued humans for millennia and causes more mortality than any other single infectious agent in the world today. In 2015, there were 10.5 million reported new cases of tuberculosis (TB) globally1 with the majority of cases emanating from India, Indonesia, China, Nigeria, Pakistan, and South Africa. Estimates place the global death toll from TB at 1.4 million people during that same time period. This value is nearly 25% lower than the death rate 100 years ago. Although drug sensitive TB is treatable, the regimen is lengthy requiring multiple medications and compliance is a concern. The emergence of multi drug-resistant (MDR) strains accounted for ~580,000 of the new TB cases in 2015. The successful treatment rate of patients with MDR strains of M. tuberculosis is only estimated to be around 50%. Even more alarming is the emergence of extensively resistant (XDR) strains of M. tuberculosis, which are resistant to nearly every available drug. Thus, new techniques are needed within the TB research field that enhance the ability to diagnose TB, increase the immunological understanding of the disease process, and allow for the screening of novel treatments and prevention strategies including antibiotic regimens and vaccine efficacy studies.
M. tuberculosis is an aerobic acid-fast bacillus that is physically characterized by its very complex outer cell wall and slow growth kinetics. Infection generally occurs through inhalation of individual bacteria contained in aerosolized droplets that are expelled from a symptomatic, infected individual while coughing, sneezing, or singing. Of the exposed individuals that develop infection, only 5 - 10% of people develop active clinical TB. The remaining 90% have a varying spectrum of asymptomatic infections that ranges from subclinical infection to no disease at all, all of which is classified clinically as latent TB infection (LTBI)2,3. Of the population that has this asymptomatic infection, approximately 10% will develop active TB by reactivation of the contained infection in their lifetime. The risk of reactivation dramatically increases if a person with asymptomatic infection contracts HIV or undergoes treatment with an immunosuppressive drug, such as TNF inhibitors4,5,6. Active TB disease also presents as a spectrum, with most people having pulmonary TB, which affects the lungs and thoracic lymph nodes. However, M. tuberculosis can infect any organ, so that the infection can also present in extrapulmonary sites of involvement.
The pathologic hallmark of M. tuberculosis infection is an organized spherical structure of host cells, called the granuloma. Macrophages, T cells and B cells are major components of the granuloma, with variable numbers of neutrophils7. The center of the granuloma is often necrotic. Thus, granulomas function as an immune microenvironment to kill or contain the bacilli, preventing spread to other parts of the lungs. However, M. tuberculosis can subvert killing by the granuloma, and persist within these structures for decades. Consistent and regular monitoring for the development of active TB disease after new infection or reactivation of LTBI is impractical, scientifically challenging and time consuming. Techniques that study these processes longitudinally, in humans and human-like animal models, are extremely useful to the scientific community in furthering the understanding of the many complexities of M. tuberculosis infection and disease.
PET/CT is an extremely useful imaging technique that has been employed to study a vast range of disease states in humans and animal models8. PET is a functional technique that uses positron-emitting radioactive compounds as a reporter. These radioisotopes are typically functionalized to a metabolic compound, such as glucose, or to a targeting group that is designed to bind to a receptor of interest. Since the radiation emitted from PET isotopes is powerful enough to penetrate tissue, very low concentrations can be used which allows for study below saturation levels in receptor-targeting compounds and at a low enough concentration to have no impact on metabolic processes when using agents such as 2-deoxy-2-(18F)Fluoro-D-glucose (FDG). CT is a three-dimensional x-ray imaging technique that uses varying levels of x-ray attenuation to identify physical characteristics of organs within the body9. When paired with PET, CT is used as a map to determine specific locations and structures that show uptake of a PET radiotracer. PET/CT is a powerful tool for in vivo imaging of both humans and animal models infected with M. tuberculosis infection that has led to many important insights into pathogenesis, response to drug treatment, disease spectrum, etc6,10,11,12. This work describes specific PET/CT analytical methods to study TB in non-human primate models longitudinally using parameters such as granuloma size, FDG uptake in individual lesions, whole lung and lymph node FDG avidity, and detection of extrapulmonary disease6,10,11,12.
This manuscript describes methodologies of imaging analysis in non-human primates (NHPs), specifically cynomolgus macaques, which are used to longitudinally evaluate disease progression and drug treatment following infection with M. tuberculosis. NHPs are a valuable animal model because when inoculated with a low dose of M. tuberculosis Erdman strain, animals show a variety of disease outcomes with ~50% developing active TB and the remaining animals having asymptomatic infection (i.e. controlling the infection, LTBI), providing the closest model to the clinical disease spectrum seen in humans3,13,14,15,16. Reactivation of LTBI in macaques is triggered by the same agents that cause reactivation in humans, examples of which include human immunodeficiency virus (HIV, using simian immunodeficiency virus (SIV) as the macaque version of HIV), CD4 depletion or tumor necrosis factor (TNF) neutralization13,16. In addition, macaques present with pathology that is extremely similar to that seen in humans, including the organized granulomas that form in lungs or other organs17. Thus, this model has provided important insights into the basic host-pathogen interactions in M. tuberculosis infection, as well as valuable knowledge about drug regimens and vaccines for tuberculosis14,18,19,20,21.
PET/CT imaging provides the ability to follow the appearance, distribution, and progression of individual granulomas. This work has primarily used FDG as a probe, which, as a glucose analogue, incorporates into metabolically active host cells, such as macrophages, neutrophils, and lymphocytes8, all of which are in granulomas. Thus, FDG is a proxy for host inflammation. The analysis procedures detailed herein uses OsiriX, a widely used DICOM viewer available for purchase and use. The image analysis methods described track the shape, size, and metabolic activity (via FDG uptake) of individual granulomas over time and uses imaging as a map for identifying specific lesions upon animal necropsy. Additionally, a separate method has been developed that quantifies the summation of FDG uptake in the lung above a specific threshold (SUV &#8805; 2.3) and uses this value to evaluate differences between control and experimental groups across studies ranging from vaccine trials to co-infection models. These data support that this overall measure of FDG uptake in lungs is correlated with bacterial burden, thus providing information about the disease status. Similar analyses can be performed on the FDG uptake of thoracic lymph nodes to study progression of disease as well. The following protocol describes the experimental process from animal infection through image analysis.

<table><tbody><tr><td>Ketamine</td><td>Henry Schein</td><td>23061</td><td>Henry Schein</td></tr><tr><td>Telazol</td><td>Zoetis</td><td>4866</td><td>Henry Schein</td></tr><tr><td>Cetacaine</td><td>Patterson Vet Generics</td><td>07-892-6862</td><td>Patterson</td></tr><tr><td>Sterile saline</td><td>Hospira</td><td>07-800-9721</td><td>Patterson</td></tr><tr><td>7H11 agar</td><td>BD</td><td>283810</td><td>BD Biosciences</td></tr><tr><td>IV catheter</td><td>Surflash</td><td>07-806-7659</td><td>Patterson</td></tr><tr><td>18F-FDG</td><td>Zevacor</td><td>N/A</td><td></td></tr><tr><td>Endotracheal tube</td><td>Jorgensen Labs Inc</td><td>07-887-0284</td><td>Patterson</td></tr><tr><td>Artificial tears</td><td>Patterson Vet Generics</td><td>07-888-1663</td><td>Patterson</td></tr><tr><td>Isoflurane</td><td>Zoetis</td><td>07-806-3204</td><td>Patterson</td></tr><tr><td>Neurologica Ceretom CT</td><td>Samsung Neurologica</td><td>N/A</td><td></td></tr><tr><td>Siemens Focus 220 microPET</td><td>Siemens Molecular Imaging Systems</td><td>N/A</td><td></td></tr><tr><td>Inveon Research Software</td><td>Siemens Molecular Imaging Systems</td><td>N/A</td><td></td></tr><tr><td>OsiriX</td><td>Pixmeo</td><td>N/A</td><td></td></tr></tbody></table>

analysis of 18fdg pet/ct imaging as a tool for studying mycobacterium tuberculosis infection and treatment in non-human primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Identification and Analysis of Individual Lesions
Individual granulomas can be visualized for number, size, and FDG uptake qualitatively to understand the general scope of the infection process (Figure 1). Using these images, counting granulomas over time is a quantitative measure of disease spread. Figure 2 depicts individual granuloma counts over time in a group of 10 animals. Of the 10 animals, three developed active disease and six developed latent infection. One animal showed no signs of active disease but was occasionally culture positive (in gastric aspirate and/or bronchoalveolar lavage samples) for M. tuberculosis, placing it within the spectrum of disease between active and latent and was thus removed from the analysis for this particular experiment. Of the three animals with active disease, one animal developed miliary disease by 12 weeks post-infection and was euthanized (this is identified in Figure 2 as TNTC [Too Numerous To Count]). From 6 weeks after infection and thereafter, animals that would later develop active disease showed statistically higher numbers of granulomas than animals that would develop latent infection.
To better characterize and distinguish granulomas between active and latent animals, individual lesions on PET scans were analyzed to determine whether there was a difference in FDG uptake pattern between the two groups. In all of the active infection animals, there was an increase in FDG uptake in every granuloma from three to six weeks post infection (Figure 3A). Conversely, granulomas in animals that developed latent infection showed a variation in FDG uptake with some lesions increasing, decreasing, or showing the same uptake from three to six weeks (Figure 3B). These results are compared in groups showing the difference in change between latent and active animals at three weeks vs. six weeks (Figure 3C) and three weeks vs. 24 weeks (Figure 3D). In both cases, the granulomas of active animals showed a positive and significantly different change in SUV (within each individual animal (Figure 3A and 3B) and when compared by groups of animals (Figure 3C and 3D).
Analyzing FDG Uptake in &#34;Hot&#34; Lymph Nodes
As mediastinal lymph nodes are not readily visualized on CT scans unless greatly enlarged, PET images must be used to identify these diseased tissues. When analyzing lymph nodes, it is critical that the image is always scaled to the same maximum and minimum PET scale to maintain consistency throughout the process. When comparing the MLNs of animals that developed active or latent disease, Lin et al. showed through ROI analysis of MLNs that while FDG uptake in lymph nodes were similar between active and latent animals at 3 weeks, MLNs from active animals showed significantly higher uptake at 6 weeks11. Differences were seen to a greater degree at 8 and 12 weeks (Figure 4). Thus, PET/CT data can be utilized to assess significant differences in lymph nodes in addition to studying granulomas in infected animals.
Total Lung FDG Avidity
As an example of the power of evaluating total lung FDG avidity, Lin et al. showed that elevated lung inflammation in animals clinically classified as LTBI correlates with risk of reactivation6. In this study, LTBI cynomolgus macaques (infected with low-dose M. tuberculosis) were PET/CT imaged (6 months post-infection) prior to tumor necrosis factor (TNF) neutralization to assess the spectrum of lesions seen in clinically defined LTBI and determine risk of reactivation. Animals with higher total lung FDG avidity were more likely to reactivate (Figure 5). Over 90% of the animals with more than 103 lung FDG avidity or with visible (by scan) at least one extra-pulmonary site of infection reactivated after TNF neutralization. Only one animal that did not reactivate exceeded this total lung FDG avidity threshold. Thus, PET/CT parameters can be a powerful tool to predicting clinical outcome though the specific parameters must be scientifically identified.
As an example to show utility in drug treatment scenarios, Coleman et al. conducted a study testing oxazolidinones in humans and cynomolgus macaques where total lung FDG avidity was measured pre-drug treatment, and one and two months post-treatment10. Fold changes were calculated to show drug response one month (Figure 6A) and two months post-treatment (Figure 6B). At both time points, control animals showed significantly higher FDG avidity in the entire lung space than drug-treated animals. In all of the drug-treated animals, total lung inflammation decreased over the two-month treatment regimen, while in most of the control animals, total lung inflammation increased over time or was unchanged.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56375/56375fig1.jpg" /> 
Figure 1. Serial FDG PET/CT Images Showing a Disseminating and Stable Pattern of Granuloma Evolution During the Course of Early Infection. 
(Top row) Primary granulomas (white arrows) were first established at 3 weeks post-infection, while new granulomas developed adjacent to existing lesions (green arrows) or in new sites (yellow arrows). Animals that would later develop active TB developed more lesions during the course of infection. (Bottom row) Primary granulomas (white arrows) of latent animals usually remained stable, with few new granulomas developing through the course of infection. wks PI, weeks post-infection. Figure taken from Coleman et al.11 <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56375/56375fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56375/56375fig2v2.jpg" /> 
Figure 2. Representative Data of Depicting the Median and Range of Granuloma Counting from CT Scans Comparing Animals with Active Infection (Red Symbols) to Latent Infection (Green Symbols). 
Active infection animals had more granulomas than latently infected animals as early as six weeks post-infection. P &#60; 0.05 (*) by Mann-Whitney test. Weeks PI, weeks post-infection. TNTC, too numerous to count. Figure adapted from Coleman et al.11 <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56375/56375fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56375/56375fig3.jpg" /> 
Figure 3. Utilization of ROI Analysis on PET Images to Show that Metabolic Activity of Lung Granulomas Differs Between Active and Latently Infected Animals During Early Infection. 
Individual granulomas in active animals [A] have a significant increase in metabolic activity (measured as standard uptake value normalized to muscle uptake [SUVCMR]) between 3 and 6 weeks after infection while latently infected animals [B] do not. The change in metabolic activity in lesions between latent and active animals as groups were compared at 3 vs. 6 weeks [C] and 3 vs. 24 weeks [D] showing that actively infected animals (red squares) have a significantly larger change in uptake than latent animals (green circles) at both time points. Solid black lines represent the median. The Wilcoxon rank-sum test was used to analyze data in panels A and B. For panels C and D, values were analyzed by the Mann-Whitney test. P &#60; 0.0001 (****) for all panels. Figure adapted from Coleman et al.11 <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56375/56375fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56375/56375fig4.jpg" /> 
Figure 4. ROI Analysis of PET Images to Show Differences in FDG Uptake in Mediastinal Lymph Nodes Between Animals with Active Disease (Red Squares) and Latent Disease (Green Circles). 
Uptake was higher in actively infected animals at 6, 8, and 12 weeks post infection. Each dot represents an individual lymph node. Solid black lines represent the median. P &#60; 0.05 (*), P &#60; 0.01 (**), and P &#60; 0.001 (***) by Mann-Whitney test. Figure adapted from Coleman et al.11 <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56375/56375fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56375/56375fig5.jpg" /> 
Figure 5. ROI Analysis of Total Lung FDG Avidity Measurements at Six Months Post Infection Highlighting Differences in FDG Uptake in Animals Which Reactivate from Latent Infection (Pink Squares) in Comparison to Animals Which Remain Latent (Green Circles). 
Three of the four &#34;reactivated&#34; animals that lie below the dotted line had extra-pulmonary disease prior to TNF (tumor necrosis factor) neutralization. Each dot represents one animal. Dotted line represents threshold value for likely reactivation risk. Solid black lines represent the median. P &#60; 0.01 (**) from Mann-Whitney Test. Data adapted from Lin et al.6 <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56375/56375fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56375/56375fig6.jpg" /> 
Figure 6. Representative Total Lung FDG Avidity ROI Measurements Highlighting Overall Change in Inflammation Measured in Lungs Comparing Untreated (Red Circles) and Linezolid Treated (Blue Circles) Monkeys. 
FDG avidity was measured before linezolid treatment (30 mg/kg daily) and the fold change in total FDG uptake was measured at 1 month [A] and 2 months [B] post treatment. Each monkey is represented by an individual circle. Solid black lines represent the median. P &#60; 0.01 (**) per Mann-Whitney Test. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56375/56375fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates at JoVE.com

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

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JoVE Journal

Immunology and Infection

18.7K Views.  University of Pittsburgh School of Medicine. Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Video: Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) imaging. Co-mutations in the KRAS proto-oncogene and the LKB1 tumor suppressor gene are frequent events in lung cancer that drive hypermetabolic, glycolytic tumor growth. A critical pathway regulating the growth and metabolism of these tumors is the mechanistic target of the rapamycin (mTOR) pathway, which can be effectively targeted using selective catalytic mTOR kinase inhibitors. The mTOR inhibitor MLN0128 suppresses glycolysis in mice bearing tumors with Kras and Lkb1 co-mutations, referred to as KL mice. The therapy response in KL mice is first measured by 18F-FDG PET and computed tomography (CT) imaging before and after the delivery of MLN0128. By utilizing 18F-FDG PET/CT, researchers are able to measure dynamic changes in the glucose metabolism in genetically engineered mouse models (GEMMs) of lung cancer following a therapeutic intervention with targeted therapies. This is followed by ex vivo autoradiography and a quantitative immunohistochemical (qIHC) analysis using morphometric software. The use of qIHC enables the detection and quantification of distinct changes in the biomarker profiles following treatment as well as the characterization of distinct tumor pathologies. The coupling of PET imaging to quantitative histology is an effective strategy to identify metabolic and therapeutic responses in vivo in mouse models of disease.

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

In this protocol, we describe how to utilize [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography (18F-FDG PET/CT) imaging to measure the tumor metabolic response to the targeted therapy MLN0128 in a Kras/Lkb1 mutant mouse model of lung cancer and coupled imaging with high resolution ex vivo autoradiography and quantitative histology.

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography ...

Cancer Research

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular interactions that exist within this microenvironment. Yet the BM microenvironment cannot be easily replicated in vitro, which potentially limits the physiologic relevance of many in vitro and ex vivo experimental models. These issues can be overcome by utilizing a xenograft model in which luciferase (LUC)-transfected 8226 MM cells will specifically engraft in the mouse skeleton. When these mice are given the appropriate substrate, D-luciferin, the effects of therapy on tumor growth and survival can be analyzed by measuring changes in the bioluminescent images (BLI) produced by the tumors in vivo. This BLI data combined with positronic-emission tomography/computational tomography (PET/CT) analysis using the metabolic marker 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) is used to monitor changes in tumor metabolism over time. These imaging platforms allow for multiple noninvasive measurements within the tumor/BM microenvironment.

Here we use bioluminescent, X-ray, and positron-emission tomography/computed tomography imaging to study how inhibiting mTOR activity impacts bone marrow-engrafted myeloma tumors in a xenograft model. This allows for physiologically relevant, non-invasive, and multimodal analyses of the anti-myeloma effect of therapies targeting bone marrow-engrafted myeloma tumors in vivo.

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular ...

Hybrid &#181;CT-FMT imaging and image analysis

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to assess the biodistribution of novel probes and to assess disease progression using established molecular probes or reporter genes. The combination with an anatomical modality, e.g., micro computed tomography (&#181;CT), is beneficial for image analysis and for fluorescence reconstruction. We describe a protocol for multimodal &#181;CT-FMT imaging including the image processing steps necessary to extract quantitative measurements. After preparing the mice and performing the imaging, the multimodal data sets are registered. Subsequently, an improved fluorescence reconstruction is performed, which takes into account the shape of the mouse. For quantitative analysis, organ segmentations are generated based on the anatomical data using our interactive segmentation tool. Finally, the biodistribution curves are generated using a batch-processing feature. We show the applicability of the method by assessing the biodistribution of a well-known probe that binds to bones and joints.

Hybrid  &#181;CT-FMT imaging and image analysis

We describe a protocol for hybrid imaging, combining fluorescence-mediated tomography (FMT) with micro computed tomography (&#181;CT). After fusion and reconstruction, we perform interactive organ segmentation to extract quantitative measurements of the fluorescence distribution.

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to ...

Bioengineering

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

PET Imaging and Biodistribution Analysis in Mice Using BCAMs and SaaS Tools

Preclinical Positron Emission Tomography with Body Conforming Animal Molds for Cloud-Based Automated Image Analysis in Mice

Positron Emission Tomography (PET) is a molecular imaging modality that can be used to investigate a multitude of pharmacological questions, such as biomarker modulation, receptor occupancy, and biodistribution of compounds of interest. In biodistribution studies, experimental subjects are often longitudinally imaged after receiving the test article. The images are then analyzed to derive the compound&#39;s distribution profile in various organs at different timepoints. This constitutes a crucial step in drug development to understand the distribution and potentially binding profile of an investigative compound. Standard/manual methods of PET imaging-based biodistribution analyses, however, are labor-intensive and time-consuming and are often associated with high inter-operator variability. Further, it is challenging to keep the animals&#39; positions consistent across different timepoints. To address these shortcomings, a series of mouse Body Conforming Animal Molds (BCAMs) were used to enable rigid and consistent positioning of animals during PET/CT imaging acquisition. Further, a Software-as-a-Service (SaaS) platform consisting of a cloud-based Organ Probability Map (OPM) and an artificial intelligence-powered segmentation tool were employed to enable reliable and automated quantitation of in vivo PET imaging data. The workflow presented here includes (1) prepping mice for imaging with the BCAMs, including the proper implantation of subcutaneous tumors to be compatible with the molds, (2) acquiring PET/CT images with BCAMs using the G8 scanner, and 3) performing automated organ segmentation and biodistribution analysis using the cloud-based SaaS. [18F]FDG was used as an exemplar tracer here, but other biomarkers and/or radio-labeled compounds can be readily adapted into the workflow. This procedure can be executed accurately and effectively with minimal training, and the automated PET data analysis yielded satisfactory results consistent with the manual method.

Author Spotlight: Standardizing Mouse In Vivo PET Imaging with Body Conforming Molds and Automated Analysis

This protocol describes the procedure for performing in vivo PET imaging on mice using Body Conforming Animal Molds (BCAMs) in the G8 PET/CT scanner. Technical details on mouse preparation, including proper tumor implantation, optimal positioning, BCAM-assisted PET/CT image acquisition, and data analysis, are provided.

Positron Emission Tomography (PET) is a molecular imaging modality that can be used to investigate a multitude of pharmacological questions, such as ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). Impaired uptake and metabolism of glucose in multiple organ systems due to insulin resistance is a hallmark of type 2 diabetes. The ability of this technique to extract an image-derived input function from the vena cava using an iterative deconvolution method eliminates the requirement of the collection of arterial blood samples. Fitting of tissue and vena cava time activity curves to a two-tissue, three compartment model permits the estimation of kinetic micro-parameters related to the 18F-FDG uptake from the plasma to the intracellular space, the rate of transport from intracellular space to plasma and the rate of 18F-FDG phosphorylation. This methodology allows for multiple measures of glucose uptake and metabolism kinetics in the context of longitudinal studies and also provides insights into the efficacy of therapeutic interventions.

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This study describes a protocol that uses 18F-FDG and positron emission tomography/computed tomography (PET/CT) imaging, together with kinetic modelling, to quantify the in vivo, real-time uptake of 18F-FDG into tissues.

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). ...

Medicine

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Biology

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9&#8211;18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...

FDG Based PET/CT Scan: A Method of Using Radiotracer for Non-invasive Imaging of Tumor Metabolism

Source: Momcilovic M. et al., <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/57167/utilizing-18f-fdg-petct-imaging-quantitative-histology-to-measure">Utilizing&#160;18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer.</a> J. Vis. Exp. (2018)
This article describes multimodality imaging of cancerous tissues with PET and CT diagnostic imaging by using a radiotracer 18F-FDG. Cancerous tissue with high glycolytic activity uptakes more 18F-FDG.

Source: Momcilovic M. et al., Utilizing&#160;18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in ...

Analysis of ¹⁸FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

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Chapters in this video

Utilizing ¹⁸F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

Hybrid µCT-FMT imaging and image analysis

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Preclinical Positron Emission Tomography with Body Conforming Animal Molds for Cloud-Based Automated Image Analysis in Mice

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Determining Glucose Metabolism Kinetics Using ¹⁸F-FDG Micro-PET/CT

Functional Imaging of Brown Fat in Mice with ¹⁸F-FDG micro-PET/CT

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

FDG Based PET/CT Scan: A Method of Using Radiotracer for Non-invasive Imaging of Tumor Metabolism