Technical Reports in 2013

One of the most likely substrates for metabolic imaging of response to treatment in cancer is glucose, but until now, using hyperpolarized 13C-labelled glucose has been problematic because of the short lifetime of the hyperpolarization in this molecule. Using [U-¹³C, U-²H]glucose, Tiago Rodrigues et al. now show that they are able to image its glycolytic conversion to lactate in two mouse tumor models in vivo, and that in one model, flux is markedly reduced after treatment with the chemotherapeutic drug etoposide.

There are currently a paucity of approaches for the direct in vivo assessment of rates of hepatic mitochondrial oxidation and anaplerotic flux in humans. With this in mind, Douglas Befroy and colleagues have developed a new ¹³C-labeling strategy that they use in combination with ¹³C magnetic resonance spectroscopy, which should prove useful in determining the potential role of changes in hepatic mitochondrial fat oxidation in diseases such as nonalcoholic fatty liver disease and type 2 diabetes.

Matthias Hackl and his colleagues developed a new serial multiphoton microscopy approach to track migrating podocytes and parietal epithelial cells over time in vivo in the intact kidney. Use of this new approach is demonstrated in several mouse models and should help answer questions surrounding podocyte proliferation and migration to other (peri)glomerular regions, thus shedding light on the mechanisms of glomerular injury and regeneration.

There is an urgent need for quantitative magnetic resonance approaches for assessing brain development, as well as for studying the effects of drugs on neural tissue inflammation. Aviv Mezer and colleagues have developed a neuroimaging method for the quantification of local tissue volume and tissue-surface interaction, producing reliable quantitative measurements across a range of scanners. They apply their method to both the healthy brain and individuals with relapsing-remitting multiple sclerosis.

Progress in T cell receptor (TCR) gene therapy has been hampered by the lack of a rapid and efficient screening system for antigen-specific TCRs. Here, Kobayashi et al. have developed a direct single-cell TCR cloning system for cloning antigen-specific TCRs from peripheral blood in 10 d. The approach is used to clone and analyze Epstein-Barr virus–specific TCRs from healthy donors with latent Epstein-Barr virus infection, as well as TCRs from peptide-vaccinated patients with hepatocellular carcinoma.

The lack of robust and high-throughput technologies to analyze the human TCR repertoire has been a bottleneck in the analysis of human T cell responses. Linnemann and colleagues have addressed this issue by using a TCR gene capture technology that, because of its quantitative nature, allows the rapid identification of TCRab pairs from bulk populations of cells without the need for single-cell cloning. Such an approach should be useful in obtaining defined antigen-reactive TCRs for therapeutic purposes.

Thorek et al. describe an activatable imaging agent based on a radioactive decay signal through the Cerenkov luminescence effect, which is light produced by β-particle–emitting radionuclides, such as clinically available PET tracers. The approach offers a means of simultaneously investigating multiple disease-relevant biological activities in vivo—the radioluminescent readout providing a quantitative measure of enzymatic activation with reduced background.

Qiong Wang and colleagues introduce the AdipoChaser mouse, an in vivo tool to track the formation and turnover of adipocytes. They use this inducible mature adipocyte lineage-tracing system to monitor adipogenesis and follow the formation of white and beige adipocytes under different conditions: high-fat diet, cold exposure and β-adrenergic stimulation. The system produced some interesting findings on in vivo adipogenesis, including that beige adipocytes differentiate de novo from specialized precursors rather than by transdifferentiation of mature white adipocytes.

Hans Wehrl et al. use a multimodal approach to demonstrate the feasibility of measuring functional brain responses in activated and resting states with PET and MRI simultaneously. Using this method, which allows brain function studies to be conducted on multiple time and metabolic scales, they could tease apart the complex relationships between neural activity, blood flow and oxygenation that form the basis of the blood oxygen level–dependent (BOLD) effect.

There is a pressing need for techniques that can be used for the noninvasive assessment of response to therapy and staging of disease. As many pathological conditions are associated with disordered glucose metabolism, such as diabetes, stroke and cancer, Simon Walker-Samuel and his colleagues have developed a noninvasive MRI-based method for imaging glucose uptake in vivo termed glucose chemical exchange saturation transfer (glucoCEST). This potentially cost-effective approach does not require the use of radiolabeled glucose analogs or ionizing radiation and allows nonlabeled glucose to be imaged at physiological quantities.

Building on earlier work, Dekkers et al. describe the first application of their intestinal organoid culture technology to the study of human disease, in this case cystic fibrosis. These so called 'mini-guts', which recapitulate the essential in vivo intestinal tissue architecture in vitro, are used to develop a rapid and quantitative assay to measure mutant cystic fibrosis transmembrane conductance regulator (CFTR) function, as well as test the efficacy of correctors and potentiators of mutant CFTR.

Marsilius Mues et al. have overcome previous obstacles precluding the expression of genetically encoded calcium indicators (GECIs) in immune cells by developing a new fluorescence resonance energy transfer–based GECI for the functional calcium imaging of T cells in vivo. Using two-photon imaging, the group traced the real-time activation of T cells in peripheral lymph nodes after antigen application, as well as in the CNS during experimental autoimmune encephalomyelitis.

The work of Dmitri Lodygin and his colleagues focuses on the important issue of when and where autoaggressive T cells are activated within their target organ to trigger autoimmune disease. Using intravital two-photon imaging and a new molecular sensor—a genetically encoded fluorescent sensor of nuclear factor of activated T cells (NFAT) combined with a fluorescently tagged version of nuclear histone protein H2B—the group was able to pinpoint key T cell activation events in experimental autoimmune encephalomyelitis, a model for multiple sclerosis.

Building on their earlier work on heart and lung organ engineering, Jeremy Song and his colleagues have now adapted the technology of using decellularized scaffolds to develop bioengineered kidneys for transplantation. When reseeded with endothelial and epithelial cells, and after orthotopic transplantation in rats, the grafts became perfused by the recipient's circulation, produced urine and provided clearance of metabolites.

The noninvasive detection of myeloperoxidase (MPO)-mediated oxidative stress in deep tissue inflammatory foci has been hampered by poor penetration of luminol-emitted short wavelength light due to tissue absorption and scattering. To circumvent this, Daniel Ansaldi and his colleagues have adopted a chemiluminescence resonance energy transfer approach whereby near-infrared (NIR) nanoparticles are used to red-shift luminol-emitted blue light to the NIR. Improved in vivo detectability of MPO is demonstrated in a lipopolysaccharide-induced pulmonary inflammation model, as well as in deep tissue tumor metastases.

A limited T cell receptor (TCR) repertoire after allogeneic hematopoietic stem cell transplantation can be associated with poor clinical outcomes and greater susceptibility to infections. Jeroen van Heijst and colleagues have combined 5′ RACE PCR with deep sequencing technology to provide a reproducible and quantitative measure of the recovery of TCR diversity during the first year of transplantation with three commonly used stem cell sources in patients with various hematologic malignancies.

Michalina Gora and her colleagues have developed a tethered capsule endoscope in the form of a swallowable pill that does not require sedation and is the size of a one-cent coin. Once swallowed, the device was well tolerated and used to capture three-dimensional microstructural images of the digestive tract, particularly the esophagus, using optical frequency domain imaging. Feasibility was demonstrated in patients with Barrett’s esophagus, including high-grade dysplasia.

Much of the current understanding of oxygen transport at the capillary level comes from mathematical models. Building on earlier work, Alexandre Parpaleix and his colleagues use two-photon phosphorescence lifetime microscopy to show how brain activity can be noninvasively imaged from measurements of oxygen dynamics in capillaries. They demonstrate the presence of an oxygen partial pressure (PO₂) initial dip at the level of capillaries and show that tissue PO₂ can be inferred from erythrocyte-associated transient values.