Novel Interactions of the Hormone Leptin Revealed by PET Imaging in Rodents and Rhesus Macaques

Leptin is a polypeptide hormone, secreted principally by adipose tissue, which functions as an afferent signal in a feedback loop to maintain body weight and energy homeostasis. In addition to its well documented effects on food intake and energy expenditure, leptin modulates the function of many other physiological systems in mammals, through actions in the central nervous system and periphery. Remarkably, despite extensive studies on leptin receptor expression, the physiological biodistribution of the hormone remains essentially unknown. In order to characterize the distribution leptin in mammals, we have developed methodologies to radiolabel the hormone and visualize its biodistribution using positron emission tomography (PET). Two complementary techniques were developed to label leptin using the positron emitting isotopes 68Ga and 18F. 68Ga labeling was accomplished by lysine-directed conjugation with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) followed by chelation of the isotope. 18F labeling was accomplished using a site-specific, two step labeling procedure in which an aminooxy moiety was introduced at the Cterminus of leptin using expressed protein ligation (EPL), which was subsequently derivitized with 18F-4-fluorobenzaldehyde in an aniline accelerated radiochemical oximation reaction. These probes were used for PET imaging in mice, rats, and in rhesus macaques. PET imaging in these organisms revealed that the hormone was rapidly taken up by the cortex of the kidney, bone marrow, and visceral organs. Uptake in the kidney was partially saturable with cold ligand, and was not mediated by leptin receptor (ObR). Subsequent analysis in with a kidney specific knockout of the multiligand endocytic transporter megalin revealed loss of leptin in the urine, which was confirmed using PET imaging. Thus, megalin is required for the uptake of leptin in the proximal convoluted tubule within the cortex of the kidney. Subsequent biodistribution experiments revealed that the hormone was taken up by the brain, spleen, liver, fat, and lungs in mice, and that this uptake was leptin receptor dependant. Furthermore, PET imaging in rhesus macaques revealed that leptin was absorbed by the bone marrow and liver. Thus, leptin may activate and modulate hematopoiesis by direct action on either hematopoietic precursors or the stromal support tissue. Thus, novel hysiologically significant interactions of the hormone leptin were revealed using PET

Effect of Time-of-Flight and Regularized Reconstructions on Quantitative Measurements and Qualitative Assessments in Newly Diagnosed Prostate Cancer With 18F-Fluorocholine Dual Time Point PET/MRI.

Recent technical advances in positron emission tomography/magnetic resonance imaging (PET/MRI) technology allow much improved time-of-flight (TOF) and regularized iterative PET reconstruction regularized iterative reconstruction (RIR) algorithms. We evaluated the effect of TOF and RIR on standardized uptake values (maximum and peak SUV [SUVmax and SUVpeak]) and their metabolic tumor volume dependencies and visual image quality for 18F-fluorocholine PET/MRI in patients with newly diagnosed prostate cancer. Fourteen patients were administered with 3 MBq/kg of 18F-fluorocholine and scanned dynamically for 30 minutes. Positron emission tomography images were divided to early and late time points (1-6 minutes summed and 7-30 minutes summed). The values of the different SUVs were documented for dominant PET-avid lesions, and metabolic tumor volume was estimated using a 50% isocontour and SUV threshold of 2.5. Image quality was assessed via visual acuity scoring (VAS). We found that incorporation of TOF or RIR increased lesion SUVs. The lesion to background ratio was not improved by TOF reconstruction, while RIR improved the lesion to background ratio significantly ( P < .05). The values of the different VAS were all significantly higher ( P < .05) for RIR images over TOF, RIR over non-TOF, and TOF over non-TOF. In conclusion, our data indicate that TOF or RIR should be incorporated into current protocols when available

Imaging glutathione depletion in the rat brain using ascorbate-derived hyperpolarized MR and PET probes.

Oxidative stress is a critical feature of several common neurologic disorders. The brain is well adapted to neutralize oxidative injury by maintaining a high steady-state concentration of small-molecule intracellular antioxidants including glutathione in astrocytes and ascorbic acid in neurons. Ascorbate-derived imaging probes for hyperpolarized 13C magnetic resonance spectroscopy and positron emission tomography have been used to study redox changes (antioxidant depletion and reactive oxygen species accumulation) in vivo. In this study, we applied these imaging probes to the normal rat brain and a rat model of glutathione depletion. We first studied hyperpolarized [1-13C]dehydroascorbate in the normal rat brain, demonstrating its robust conversion to [1-13C]vitamin C, consistent with rapid transport of the oxidized form across the blood-brain barrier. We next showed that the kinetic rate of this conversion decreased by nearly 50% after glutathione depletion by diethyl maleate treatment. Finally, we showed that dehydroascorbate labeled for positron emission tomography, namely [1-11C]dehydroascorbate, showed no change in brain signal accumulation after diethyl maleate treatment. These results suggest that hyperpolarized [1-13C]dehydroascorbate may be used to non-invasively detect oxidative stress in common disorders of the brain

Regulation of Transgenic Class II Major Histocompatibility Genes in Murine Langerhans Cells

I-E is a class II major histocompatibility complex molecule normally expressed by Langerhans cells, A series of transgenic mice were developed previously that carry Eαd gene constructs with promoter-region deletions that cause expression of I-E by different cell types when maintained on a B6 (I-E[–]) genetic background. To study cis-acting gene sequences that regulate expression of class II proteins by Langerhans cells, we identified trans genie I-E expression by tissue immunoperoxidase staining and by epidermal cell suspension lmmunofluorescence cytometry. Mice with a transgene containing 1.4 kilobase pairs (kb) of flanking sequence 5' to the Eα initiation site expressed barely detectable levels of I-E on a tiny percentage of Langerhans cells, indicating that sequences promoting Langerhans cell expression of Eα exist between 2.0 and 1.4 kb 5' of the Eα initiation site. Removal of an additional 170 bp of 5' flanking sequence caused near-normal levels of expression by approximately one third of epidermal Langerhans cells, which contrasts with studies that showed minimal transgene expression by splenic dendritic cells in these animals, Thus, sequences between 1.4 and 1.23 kb 5' of the Eα initiation site decrease expression of I-E by epidermal Laugerhans cells, but enable I-E expression by splenic dendritic cells, These studies identify Langerhans cell-specific regulatory sequences and genetic regions controlling major histocompatibility complex class II gene expression in Langerhans cells and splenic dendritic cells. The genetic regions identified may be particularly important because differential regulation of class II major histocompatibility complex protein synthesis by Langerhans cells and dendritic cells may be crucial to immune functions of intact animals

Imaging Active Infection in vivo Using D-Amino Acid Derived PET Radiotracers.

Occult bacterial infections represent a worldwide health problem. Differentiating active bacterial infection from sterile inflammation can be difficult using current imaging tools. Present clinically viable methodologies either detect morphologic changes (CT/ MR), recruitment of immune cells (111In-WBC SPECT), or enhanced glycolytic flux seen in inflammatory cells (18F-FDG PET). However, these strategies are often inadequate to detect bacterial infection and are not specific for living bacteria. Recent approaches have taken advantage of key metabolic differences between prokaryotic and eukaryotic organisms, allowing easier distinction between bacteria and their host. In this report, we exploited one key difference, bacterial cell wall biosynthesis, to detect living bacteria using a positron-labeled D-amino acid. After screening several 14C D-amino acids for their incorporation into E. coli in culture, we identified D-methionine as a probe with outstanding radiopharmaceutical potential. Based on an analogous procedure to that used for L-[methyl-11C]methionine ([11C] L-Met), we developed an enhanced asymmetric synthesis of D-[methyl-11C]methionine ([11C] D-Met), and showed that it can rapidly and selectively differentiate both E. coli and S. aureus infections from sterile inflammation in vivo. We believe that the ease of [11C] D-Met radiosynthesis, coupled with its rapid and specific in vivo bacterial accumulation, make it an attractive radiotracer for infection imaging in clinical practice

T cells that cannot respond to TGF-β escape control by CD4+CD25+ regulatory T cells

CD4+CD25+ regulatory T (T reg) cells play a pivotal role in control of the immune response. Transforming growth factor-β (TGF-β) has been shown to be required for T reg cell activity; however, precisely how it is involved in the mechanism of suppression is poorly understood. Using the T cell transfer model of colitis, we show here that CD4+CD45RBhigh T cells that express a dominant negative TGF-β receptor type II (dnTβRII) and therefore cannot respond to TGF-β, escape control by T reg cells in vivo. CD4+CD25+ T reg cells from the thymus of dnTβRII mice retain the ability to inhibit colitis, suggesting that T cell responsiveness to TGF-β is not required for the development or peripheral function of thymic-derived T reg cells. In contrast, T reg cell activity among the peripheral dnTβRII CD4+CD25+ population is masked by the presence of colitogenic effector cells that cannot be suppressed. Finally, we show that CD4+CD25+ T reg cells develop normally in the absence of TGF-β1 and retain the ability to suppress colitis in vivo. Importantly, the function of TGF-β1−/− T reg cells was abrogated by anti–TGF-β monoclonal antibody, indicating that functional TGF-β can be provided by a non–T reg cell source

Coded Aperture and Compton Imaging for the Development of 225^{225}Ac-based Radiopharmaceuticals

Targeted alpha-particle therapy (TAT) has great promise as a cancer treatment. Arguably the most promising TAT radionuclide that has been proposed is $^{225}$Ac. The development of $^{225}$Ac-based radiopharmaceuticals has been hampered due to the lack of effective means to study the daughter redistribution of these agents in small animals at the preclinical stage. The ability to directly image the daughters, namely $^{221}$Fr and $^{213}$Bi, via their gamma-ray emissions would be a boon for preclinical studies. That said, conventional medical imaging modalities, including single photon emission computed tomography (SPECT) based on pinhole collimation, cannot be employed due to sensitivity limitations. As an alternative, we propose the use of both coded aperture and Compton imaging with the former modality suited to the 218-keV gamma-ray emission of $^{221}$Fr and the latter suited to the 440-keV gamma-ray emission of $^{213}$Bi. This work includes coded aperture images of $^{221}$Fr and Compton images of $^{213}$Bi in tumor-bearing mice injected with $^{225}$Ac-based radiopharmaceuticals. These results are the first demonstration of visualizing and quantifying the $^{225}$Ac daughters in small animals via coded aperture and Compton imaging and serve as a stepping stone for future radiopharmaceutical studies

NF-κB-inducing kinase regulates selected gene expression in the Nod2 signaling pathway

The innate immune system surveys the extra- and intracellular environment for the presence of microbes. Among the intracellular sensors is a protein known as Nod2, a cytosolic protein containing a leucine-rich repeat domain. Nod2 is believed to play a role in determining host responses to invasive bacteria. A key element in upregulating host defense involves activation of the NF-κB pathway. It has been suggested through indirect studies that NF-κB-inducing kinase, or NIK, may be involved in Nod2 signaling. Here we have used macrophages derived from primary explants of bone marrow from wild-type mice and mice that either bear a mutation in NIK, rendering it inactive, or are derived from NIK(−/−) mice, in which the NIK gene has been deleted. We show that NIK binds to Nod2 and mediates induction of specific changes induced by the specific Nod2 activator, muramyl dipeptide, and that the role of NIK occurs in settings where both the Nod2 and TLR4 pathways are activated by their respective agonists. Specifically, we have linked NIK to the induction of the B-cell chemoattractant known as BLC and suggest that this chemokine may play a role in processes initiated by Nod2 activation that lead to improved host defense