Distributable, metabolic PET reporting of tuberculosis.

Tuberculosis remains a large global disease burden for which treatment regimens are protracted and monitoring of disease activity difficult. Existing detection methods rely almost exclusively on bacterial culture from sputum which limits sampling to organisms on the pulmonary surface. Advances in monitoring tuberculous lesions have utilized the common glucoside [18F]FDG, yet lack specificity to the causative pathogen Mycobacterium tuberculosis (Mtb) and so do not directly correlate with pathogen viability. Here we show that a close mimic that is also positron-emitting of the non-mammalian Mtb disaccharide trehalose - 2-[18F]fluoro-2-deoxytrehalose ([18F]FDT) - is a mechanism-based reporter of Mycobacteria-selective enzyme activity in vivo. Use of [18F]FDT in the imaging of Mtb in diverse models of disease, including non-human primates, successfully co-opts Mtb-mediated processing of trehalose to allow the specific imaging of TB-associated lesions and to monitor the effects of treatment. A pyrogen-free, direct enzyme-catalyzed process for its radiochemical synthesis allows the ready production of [18F]FDT from the most globally-abundant organic 18F-containing molecule, [18F]FDG. The full, pre-clinical validation of both production method and [18F]FDT now creates a new, bacterium-selective candidate for clinical evaluation. We anticipate that this distributable technology to generate clinical-grade [18F]FDT directly from the widely-available clinical reagent [18F]FDG, without need for either custom-made radioisotope generation or specialist chemical methods and/or facilities, could now usher in global, democratized access to a TB-specific PET tracer.

Distributable, Metabolic PET Reporting of Tuberculosis.

Tuberculosis remains a large global disease burden for which treatment regimens are protracted and monitoring of disease activity difficult. Existing detection methods rely almost exclusively on bacterial culture from sputum which limits sampling to organisms on the pulmonary surface. Advances in monitoring tuberculous lesions have utilized the common glucoside [18F]FDG, yet lack specificity to the causative pathogen Mycobacterium tuberculosis (Mtb) and so do not directly correlate with pathogen viability. Here we show that a close mimic that is also positron-emitting of the non-mammalian Mtb disaccharide trehalose - 2-[18F]fluoro-2-deoxytrehalose ([18F]FDT) - can act as a mechanism-based enzyme reporter in vivo. Use of [18F]FDT in the imaging of Mtb in diverse models of disease, including non-human primates, successfully co-opts Mtb-specific processing of trehalose to allow the specific imaging of TB-associated lesions and to monitor the effects of treatment. A pyrogen-free, direct enzyme-catalyzed process for its radiochemical synthesis allows the ready production of [18F]FDT from the most globally-abundant organic 18F-containing molecule, [18F]FDG. The full, pre-clinical validation of both production method and [18F]FDT now creates a new, bacterium-specific, clinical diagnostic candidate. We anticipate that this distributable technology to generate clinical-grade [18F]FDT directly from the widely-available clinical reagent [18F]FDG, without need for either bespoke radioisotope generation or specialist chemical methods and/or facilities, could now usher in global, democratized access to a TB-specific PET tracer.

A sterilizing tuberculosis treatment regimen is associated with faster clearance of bacteria in cavitary lesions in marmosets.

Shortening the lengthy treatment duration for tuberculosis patients is a major goal of current drug development efforts. The common marmoset develops human-like disease pathology and offers an attractive model to better understand the basis for relapse and test regimens for effective shorter duration therapy. We treated Mycobacterium tuberculosis-infected marmosets with two drug regimens known to differ in their relapse rates in human clinical trials: the standard four-drug combination of isoniazid, rifampin, pyrazinamide, and ethambutol (HRZE) that has very low relapse rates and the combination of isoniazid and streptomycin that is associated with higher relapse rates. As early as 2 weeks, the more sterilizing regimen significantly reduced the volume of lung disease by computed tomography (P = 0.035) and also significantly reduced uptake of [(18)F]-2-fluoro-2-deoxyglucose by positron emission tomography (P = 0.049). After 6 weeks of therapy, both treatments caused similar reductions in granuloma bacterial load, but the more sterilizing, four-drug regimen caused greater reduction in bacterial load in cavitary lesions (P = 0.009). These findings, combined with the association in humans between cavitary disease and relapse, suggest that the basis for improved sterilizing activity of the four-drug combination is both its faster disease volume resolution and its stronger sterilizing effect on cavitary lesions. Definitive data from relapse experiments are needed to support this observation.

Anti-vascular endothelial growth factor treatment normalizes tuberculosis granuloma vasculature and improves small molecule delivery.

Tuberculosis (TB) causes almost 2 million deaths annually, and an increasing number of patients are resistant to existing therapies. Patients who have TB require lengthy chemotherapy, possibly because of poor penetration of antibiotics into granulomas where the bacilli reside. Granulomas are morphologically similar to solid cancerous tumors in that they contain hypoxic microenvironments and can be highly fibrotic. Here, we show that TB-infected rabbits have impaired small molecule distribution into these disease sites due to a functionally abnormal vasculature, with a low-molecular-weight tracer accumulating only in peripheral regions of granulomatous lesions. Granuloma-associated vessels are morphologically and spatially heterogeneous, with poor vessel pericyte coverage in both human and experimental rabbit TB granulomas. Moreover, we found enhanced VEGF expression in both species. In tumors, antiangiogenic, specifically anti-VEGF, treatments can "normalize" their vasculature, reducing hypoxia and creating a window of opportunity for concurrent chemotherapy; thus, we investigated vessel normalization in rabbit TB granulomas. Treatment of TB-infected rabbits with the anti-VEGF antibody bevacizumab significantly decreased the total number of vessels while normalizing those vessels that remained. As a result, hypoxic fractions of these granulomas were reduced and small molecule tracer delivery was increased. These findings demonstrate that bevacizumab treatment promotes vascular normalization, improves small molecule delivery, and decreases hypoxia in TB granulomas, thereby providing a potential avenue to improve delivery and efficacy of current treatment regimens.

Differential virulence and disease progression following Mycobacterium tuberculosis complex infection of the common marmoset (Callithrix jacchus).

Existing small-animal models of tuberculosis (TB) rarely develop cavitary disease, limiting their value for assessing the biology and dynamics of this highly important feature of human disease. To develop a smaller primate model with pathology similar to that seen in humans, we experimentally infected the common marmoset (Callithrix jacchus) with diverse strains of Mycobacterium tuberculosis of various pathogenic potentials. These included recent isolates of the modern Beijing lineage, the Euro-American X lineage, and M. africanum. All three strains produced fulminant disease in this animal with a spectrum of progression rates and clinical sequelae that could be monitored in real time using 2-deoxy-2-[(18)F]fluoro-d-glucose (FDG) positron emission tomography (PET)/computed tomography (CT). Lesion pathology at sacrifice revealed the entire spectrum of lesions observed in human TB patients. The three strains produced different rates of progression to disease, various extents of extrapulmonary dissemination, and various degrees of cavitation. The majority of live births in this species are twins, and comparison of results from siblings with different infecting strains allowed us to establish that the infection was highly reproducible and that the differential virulence of strains was not simply host variation. Quantitative assessment of disease burden by FDG-PET/CT provided an accurate reflection of the pathology findings at necropsy. These results suggest that the marmoset offers an attractive small-animal model of human disease that recapitulates both the complex pathology and spectrum of disease observed in humans infected with various M. tuberculosis strain clades.

Meropenem-clavulanic acid shows activity against Mycobacterium tuberculosis in vivo.

The carbapenems imipenem and meropenem in combination with clavulanic acid reduced the bacterial burden in Mycobacterium tuberculosis-infected macrophages by 2 logs over 6 days. Despite poor stability in solution and a short half-life in rodents, treatment of chronically infected mice revealed significant reductions of bacterial burden in the lungs and spleens. Our results show that meropenem has activity in two in vivo systems, but stability and pharmacokinetics of long-term administration will offer significant challenges to clinical evaluation.

Disrupted erythropoietin signalling promotes obesity and alters hypothalamus proopiomelanocortin production.

Although erythropoietin (Epo) is the cytokine known to regulate erythropoiesis, erythropoietin receptor (EpoR) expression and associated activity beyond haematopoietic tissue remain uncertain. Here we show that mice with EpoR expression restricted to haematopoietic tissues (Tg) develop obesity and insulin resistance. Tg-mice exhibit a decrease in energy expenditure and an increase in white fat mass and adipocyte number. Conversely, Epo treatment of wild-type (WT)-mice increases energy expenditure and reduces food intake and fat mass accumulation but shows no effect in body weight of Tg-mice. EpoR is expressed at a high level in white adipose tissue and in the proopiomelanocortin (POMC) neurons of the hypothalamus. Although Epo treatment in WT-mice induces the expression of the polypeptide hormone precursor, POMC, mice lacking EpoR show reduced levels of POMC in the hypothalamus. This study provides the first evidence that mice lacking EpoR in non-haematopoietic tissue become obese and insulin resistant with loss of Epo regulation of energy homeostasis.

In vivo micro-CT imaging of liver lesions in small animal models.

Three-dimensional micro computed tomography (microCT) offers the opportunity to capture images liver structures and lesions in mice with a high spatial resolution. Non-invasive microCT allows for accurate calculation of vessel tortuosity and density, as well as liver lesion volume and distribution. Longitudinal monitoring of liver lesions is also possible. However, distinguishing liver lesions from variations within a normal liver is impossible by microCT without the use of liver- or tumor-specific contrast-enhancing agents. The combination of microCT for morphologic imaging with functional imaging, such as positron emission tomography (PET) or single photon emission tomography (SPECT), offers the opportunity for better abdominal imaging and assessment of structure discrepancies visible by functional imaging. This paper describes methods of current microCT imaging options for imaging of liver lesions compared to other imaging techniques in small animals.

Noninvasive monitoring of a murine model of metastatic pheochromocytoma: a comparison of contrast-enhanced microCT and nonenhanced MRI.

PURPOSE: To compare contrast-enhanced micro-computed tomography (microCT) and nonenhanced respiratory-triggered magnetic resonance imaging (MRI) in an animal model of metastatic pheochromocytoma. Animal models are becoming important in the study of cancer treatment and imaging is useful in minimizing the number of animals needed and reducing costs associated with autopsies. However, the choice of imaging modality is still evolving. MATERIALS AND METHODS: Adult female nude mice were injected by tail vein with a mouse pheochromocytoma (MPC) cell line (MPC 4/30PRR) to create a metastatic model. After optimizing imaging techniques, eight mice were imaged with both respiratory triggered MRI and microCT and the findings were verified histologically. RESULTS: MicroCT and MRI were approximately equal in their ability to detect hepatic metastases at a size threshold of 350 microm. In the lungs, MRI was more sensitive than microCT, detecting lesions 0.6 mm in diameter versus 1 mm for microCT. Additionally, MRI was more sensitive for lesions in the kidneys, bone, ovaries, and adrenal glands. MRI demonstrated a higher contrast-to-noise ratio (CNR) than microCT. CONCLUSION: In addition to the advantage of not exposing the animal to ionizing radiation, MRI provided a more complete assessment of the extent of metastases in this model compared to microCT.

Comparison of Fenestra VC Contrast-enhanced computed tomography imaging with gadopentetate dimeglumine and ferucarbotran magnetic resonance imaging for the in vivo evaluation of murine liver damage after ischemia and reperfusion.

OBJECTIVES: Comparison of intravenous Fenestra VC-enhanced computed tomography (CT) with gadopentetate dimeglumine and Ferucarbotran contrast-enhanced magnetic resonance imaging (MRI) for the in vivo imaging of hepatic ischemia/reperfusion injury (IRI) in a murine model. MATERIAL AND METHODS: After induction of hepatic IRI by left liver lobe (LLL) ischemia (30, 45, and 75 minutes) and reperfusion (4 hours and 24 hours), a total of 130 mice were imaged either by Fenestra VC-enhanced 3-D CT or by dynamic, T1-weighed gadopentetate dimeglumine or static, T2*-weighed Ferucarbotran 2-D MRI (4.7 T). RESULTS: Detection of liver tissue damage as a consequence of IRI was not possible by CT or MRI without the use of contrast media. (1) Mice subjected to liver IRI (45 minutes of ischemia) and injected with Fenestra VC showed a distinct liver enhancement of the viable liver tissue or a nonenhancement of the necrotic tissue. The Fenestra VC CT-unenhanced liver volume increased as a function of time of ischemia and reperfusion. The unenhanced liver volume also correlated positively with serum liver enzyme activities and damage scores from liver histology. (2) The signal intensities (SI) between normal liver tissue and livers subjected to 30 minutes of ischemia were not different on dynamic gadopentetate dimeglumine-enhanced magnetic resonance images. More severe IRI as induced by 45 or 75 minutes of ischemia was characterized by (a) early hyperenhancement of regions in the LLL with rapid increase of SI higher than that observed in the undamaged liver within the first few minutes and (b) delayed hyperenhancement in the later course after gadopentetate dimeglumine injection, respectively. (3) Ferucarbotran MRI detected signs of IRI after only 30 minutes of liver ischemia and hence detected IRI earlier than Fenestra VC or gadopentetate dimeglumine. With longer duration of ischemia, Ferucarbotran SI increased in the LLL, but viable and necrotic tissues were not clearly distinguishable. CONCLUSIONS: MicroCT with Fenestra VC enhancement and MRI using either gadopentetate dimeglumine or Ferucarbotran enhancement of the liver revealed that all techniques allow in vivo determination of hepatic IRI as a function of the duration of ischemia and reperfusion of the liver. However, Fenestra VC-enhanced CT of the murine liver is superior to gadopentetate dimeglumine and Ferucarbotran for localization, quantification, and differentiation of viable from metabolically inactive/damaged liver tissue after hepatic ischemia/reperfusion but Fenestra VC is less sensitive than Ferucarbotran to detect the early onset of subtle consequences of hepatic IRI.

Skeletal abnormalities and extra-skeletal ossification in mice with restricted Gsalpha deletion caused by a renin promoter-Cre transgene.

We have recently generated a transgenic mouse line (termed hRen-Cre) that expresses Cre-recombinase under the control of a 12.2-kb fragment of the human renin promoter. In the present study, we have crossed hRen-Cre mice with a mouse strain in which exon 1 of the Gnas gene is flanked by loxP sites. Gnas encodes the alpha-subunit of the stimulatory G protein (Gs alpha). Our aim has been to generate a mouse model with locally restricted inactivation of Gs alpha to extend studies of the role of Gs alpha function in vivo. Mice with local Cre-mediated inactivation of Gs alpha (rCre-Gs alpha) are viable and fertile. Their most obvious phenotype consists of marked skeletal malformations of the forelimbs in which computer-tomography scans reveal shortened and fused extremity bones. Extraskeletal ossifications occur in the subcutis and in skeletal muscles associated with the affected long bones. Plasma calcium, phosphate and parathyroid hormone are normal. Skin histology has demonstrated diffuse mineralization and ossification associated with the basal cells of hair follicles. This phenotype in part resembles syndromes in humans associated with loss-of-function of Gs alpha, such as Albright hereditary osteodystrophy and progressive osseous heteroplasia. The renal phenotype of rCre-Gs alpha mice is inconspicuous. Plasma renin concentration, ambient urine osmolarity, and the glomerular filtration rate of rCre-Gs alpha mice do not differ from controls. The absence of measurable functional changes in the renin-angiotensin system indicates insufficient Cre expression in juxtaglomerular granular cells in this strain of mice. Nevertheless, the present report reaffirms the importance of Gs alpha signaling for bone development and the suppression of ectopic ossification.

Augmented Wnt signaling in a mammalian model of accelerated aging.

The contribution of stem and progenitor cell dysfunction and depletion in normal aging remains incompletely understood. We explored this concept in the Klotho mouse model of accelerated aging. Analysis of various tissues and organs from young Klotho mice revealed a decrease in stem cell number and an increase in progenitor cell senescence. Because klotho is a secreted protein, we postulated that klotho might interact with other soluble mediators of stem cells. We found that klotho bound to various Wnt family members. In a cell culture model, the Wnt-klotho interaction resulted in the suppression of Wnt biological activity. Tissues and organs from klotho-deficient animals showed evidence of increased Wnt signaling, and ectopic expression of klotho antagonized the activity of endogenous and exogenous Wnt. Both in vitro and in vivo, continuous Wnt exposure triggered accelerated cellular senescence. Thus, klotho appears to be a secreted Wnt antagonist and Wnt proteins have an unexpected role in mammalian aging.

Hemolysis in sickle cell mice causes pulmonary hypertension due to global impairment in nitric oxide bioavailability.

Pulmonary hypertension is a highly prevalent complication of sickle cell disease and is a strong risk factor for early mortality. However, the pathophysiologic mechanisms leading to pulmonary vasculopathy remain unclear. Transgenic mice provide opportunities for mechanistic studies of vascular pathophysiology in an animal model. By microcardiac catheterization, all mice expressing exclusively human sickle hemoglobin had pulmonary hypertension, profound pulmonary and systemic endothelial dysfunction, and vascular instability characterized by diminished responses to authentic nitric oxide (NO), NO donors, and endothelium-dependent vasodilators and enhanced responses to vasoconstrictors. However, endothelium-independent vasodilation in sickle mice was normal. Mechanisms of vasculopathy in sickle mice involve global dysregulation of the NO axis: impaired constitutive nitric oxide synthase activity (NOS) with loss of endothelial NOS (eNOS) dimerization, increased NO scavenging by plasma hemoglobin and superoxide, increased arginase activity, and depleted intravascular nitrite reserves. Light microscopy and computed tomography revealed no plexogenic arterial remodeling or thrombi/ emboli. Transplanting sickle marrow into wild-type mice conferred the same phenotype, and similar pathobiology was observed in a nonsickle mouse model of acute alloimmune hemolysis. Although the time course is shorter than typical pulmonary hypertension in human sickle cell disease, these results demonstrate that hemolytic anemia is sufficient to produce endothelial dysfunction and global dysregulation of NO.

Anatomical and functional imaging of tumors in animal models: focus on pheochromocytoma.

This review focuses on anatomical (computed tomography, magnetic resonance imaging) and functional (positron emission tomography) imaging methods for tumor localization and identification of experimentally induced tumors in animal models, especially pheochromocytoma. Although anatomical imaging can precisely locate primary and metastatic tumors, functional imaging has high specificity for some tumors, especially those of endocrine origin. This is due to the fact that endocrine tumor cells take up hormone precursors, express hormone receptors and transporters, and synthesize, store, and release hormones. These characteristic properties of endocrine tumors enable investigators to create highly specific radiopharmaceuticals, particularly for positron emission tomography. For example, localization of pheochromocytoma involves [18F]-6F-dopamine. It is a highly specific radiopharmaceutical since it uses the norepinephrine transporter system expressed in most pheochromocytoma cells. Here we review both anatomical and functional imaging methods that are used conjointly in order to localize and identify specific characteristics of tumors.

MicroCT for high-resolution imaging of ectopic pheochromocytoma tumors in the liver of nude mice.

Successful outcomes for patients with cancer often depend on the early detection of tumor and the prompt initiation of active therapy. Despite major advances in the treatment of many cancers, early-stage lesions often go undetected due to the suboptimal resolution of current anatomical and functional imaging modalities. This limitation also applies to preclinical animal tumor models that are crucial for the evaluation and development of new therapeutic approaches to cancer. We report a new mouse model of metastatic pheochromocytoma, generated using tail vein injection of the mouse pheochromocytoma cell (MPC) line that reproducibly generated multiple liver tumors in the animals. Furthermore, we show that in vivo microCT imaging enhanced using a hepatobiliary-specific contrast agent, glyceryl-2-oleyl-1,3-di-7-(3-amino-2,4,6-triiodophenyl)-heptanoate (DHOG), detected tumors as small as 0.35 mm as early as 4 weeks after the injection of the tumor cells. This model may be useful for in vivo studies of tumor biology and for development of new strategies to treat metastatic pheochromocytoma.

Evaluation of human fetal bone implants in SCID mice as a model of prostate cancer bone metastasis.

The metastasis of prostate cancer cells to the bone marrow constitutes the major source of morbidity and mortality in prostate cancer. Studying this process has been hampered by the lack of preclinical models to evaluate novel therapeutics and to study the biology of the disease. One proposed model utilizes human fetal bone implants to serve as the target for prostate cancer cells injected via the tail vein. We employed this model to test the ability of zoledronic acid to prophylax and to treat bone metastases. To improve the rate of bone metastasis, we used two bone implants instead of one to evaluate the cell lines PC3 and PC3M, a more metastatic subline. For this purpose we generated the novel cell line PC3EGFPLuc, which can be used for luminescence and/or fluorescence imaging in vivo. We did not observe bone implant metastases in 52 mice, with 90 bone implants following tail vein injection of 1x10(6) PC3 or PC3M cells. Soft tissue lesions in the buttocks and hind limbs as well as cellular growth in the hindlimbs were observed via bioluminescence imaging. This evidence together with literature findings suggests that this model produces artifactual 'bone metastasis' lesions.

Lymphatic dysfunction in transgenic mice expressing KSHV k-cyclin under the control of the VEGFR-3 promoter.

Kaposi sarcoma-associated herpesvirus (KSHV) infects endothelial cells within KS tumors, and these cells express the KSHV latent-cycle gene k-cyclin (kCYC) as well as vascular endothelial growth factor receptor 3 (VEGFR-3), a marker for lymphatic endothelium. To further understand KSHV-mediated pathogenesis, we generated transgenic mice expressing kCYC under the control of the VEGFR-3 promoter. kCYC mRNA and functional protein expression within tissue correlated with VEGFR-3 expression and were most abundantly detected within lung tissue. Clinically, most transgenic mice died within 6 months of age secondary to progressive accumulation of chylous pleural fluid. In skin, edema was detected by magnetic resonance imaging and mice demonstrated persistent erythema of the ears following trauma. Histologically, erythematous skin showed extravasation of erythrocytes and accumulation of erythrocytes within lymphatic lumens. In addition, lymphatic drainage of injected contrast dyes was markedly impaired in transgenic mice. Karyomegaly, a feature observed in kCYC-expressing cells in vitro, was detected in many tissues, and selectively occurred within lymphatic endothelial cells expressing kCYC mRNA by in situ hybridization. In summary, kCYC expression within VEGFR-3+ cells of mice causes marked impairment of lymphatic function. kCYC may contribute to the development of certain clinical and histologic features of KS, including localized edema and retention of extravasated erythrocytes within KS tumors.