Technical Reports in 2010

A major problem in the clinical management of patients with brain tumors is distinguishing tumor recurrence from radiation-induced necrosis after brain tumor therapy. Zhou et al. use an MRI technique called amide proton transfer imaging to noninvasively differentiate between these two pathologies. The approach is successfully evaluated by comparing two orthotopic glioma models with a radiation necrosis model in rats.

Bhang and colleagues have developed a tumor-specific imaging strategy that uses the progression elevated gene-3 (PEG-3) promoter, known to be specifically associated with malignant transformation, to selectively drive the expression of luciferase or herpes simplex virus 1 thymidine kinase reporters. Systemic delivery of PEG-3 promoter–driven constructs using a nonviral gene delivery vehicle allowed detection of both primary tumors and micrometastatic disease in mouse models of human melanoma and breast cancer.

Deficiencies with current in vitro methods to assess cancer invasion prompted Todd Ridky and his colleagues to design a three-dimensional human organotypic epithelial cancer model using primary epithelial cells from multiple stratified epithelial tissues. The model recapitulates many of the features of tumor progression, including epithelial invasion through the intact basement membrane and supporting stroma. Studying epithelial tumor cell invasion in a more physiologic manner may help identify potential therapeutic targets for a range of epithelial tumors.

Grazyna Palczewska and her colleagues use the noninvasive imaging modality of two-photon microscropy to study the retinoid cycle in the mammalian eye at the subcellular level. They perform spectral analyses of endogenous fluorophores, including fluorescent retinyl esters in subcellular structures called retinosomes, as well as their potentially harmful condensation products. This may prove useful in assessing retinal changes in the human eye at the earliest stage and long before retinal diseases become apparent and result in loss of vision.

A lack of current methods for major histocompatibility class II epitope identification prompted Isamu Hartman and his colleagues to develop a cell-free system that they used to identify physiologically selected immunodominant epitopes of model antigens, as well as for de novo epitope identification.

From a therapeutic standpoint, one of the main drawbacks of artificial electrical stimulation of muscle is that large, fatigable motor units are recruited before smaller units, which is opposite to the normal physiological recruitment pattern. Researchers from Stanford University have circumvented this problem by stimulating muscle optically rather than electrically, providing enhanced functional performance and potential applications for the technique in neuromuscular physiology, neuroprosthetics and neurorehabilitation.

Standard methods of neutrophil isolation require skilled personnel, are time consuming and use large blood volumes. Kotz and his colleagues have developed a rapid microfluidic chip-based approach for rapidly isolating neutrophils directly from whole blood with 'on-chip' processing for mRNA and protein isolation. The device, which yields sufficient quantities and purities for downstream genomic or proteomic analysis, was validated in a multicenter clinical study of the immune response to severe trauma and burn injury.

One of the main shortcomings of cell therapy is the poor persistence and loss of functionality of donor cells after transfer. Using adjuvant drug–loaded nanoparticles conjugated directly to the surface of therapeutic donor cells, an approach designed to minimize the systemic side effects of adjuvant drugs, Matthias Stephan and his colleagues show enhanced functionality in a model of adoptive T cell therapy for cancer and of hematopoietic stem cell engraftment.

Our knowledge of the diversity of human T cell receptors (TCRs) is limited by tolerance mechanisms—most high-avidity autoreactive T cells are deleted. Li et al. have now transferred the entire TCR-αβ gene loci into mice, which will facilitate the study of T cell responses to human antigens.

Sullivan and his colleagues describe a novel microneedle patch-based system for vaccine delivery that targets the skin's antigen-presenting cells, providing improved immunogenicity and eliminating the hazards associated with using hypodermic needles. The group demonstrates the feasibility of this approach for influenza prophylaxis, whereby vaccine is encapsulated within microscopic polymeric needles that dissolve in the skin in minutes.

Tarik Massoud and colleagues offer a new, noninvasive molecular imaging technique based on split reporter complementation for quantifying and imaging protein-protein interactions—cytoplasmic and nuclear—in vivo using positron emission tomography. They use a split reporter system based on the enzyme herpes simplex virus type 1 thymidine kinase, an approach designed to significantly improve the sensitivity and dynamic range of imaging protein-protein interactions.

Harald Ott and his colleagues build on their earlier work, based on reconstruction of a decellularized heart, to develop a new way to bioengineer a functioning lung. Through a process of decellularization, seeding with endothelial and epithelial cells, and maturation in an innovative bioreactor system, followed by transplantation into rats of the regenerated lungs in orthotopic position, the group was able to demonstrate adequate ventilation, blood flow and gas exchange in vivo for short periods of time.

A critical donor organ shortage currently limits the treatment of patients with severe liver failure. Building on earlier work with decellularized hearts, Basak Uygun et al. have developed a transplantable liver graft using a decellularized liver matrix. This approach preserves the structural and functional characteristics of the native microvascular network, supports efficient recellularization and, when transplanted into rats, allows the viability and metabolic function of hepatocytes to be maintained.

Using the skin epidermis as a model for studies of epithelial biology and tumorigenesis and lentiviruses carrying RNAi or Cre recombinase, Beronja and colleagues describe a noninvasive, in vivo method for cell type–specific loss-of-function studies in the surface epithelia of mouse embryos. This approach can be used for the rapid functional assay of genes, the dissection of genetic interactions within complex regulatory pathways, and the study of the role of putative tumor suppressors and oncogenes in regulating growth control.

Fan et al. present a method to dissect the cellular immune response to transplanted tissue by tracking various T cell populations in both the circulatory compartment by in vivo flow cytometry and at the graft site by endoscopic confocal microscopy. The allograft response was followed for two weeks in a mouse model in which pancreatic islet transplants were placed beneath the renal capsule. This approach may help to develop treatment options that improve transplantation outcomes.

Most cases of esophageal cancer can be prevented if detected early at the precancerous high-grade dysplasia stage in patients presenting with Barrett's esophagus, a condition that almost always precedes this form of cancer. Here, Qiu and colleagues have developed a multispectral imaging system that uses endoscopic polarized scanning spectroscopy to perform rapid optical scanning and imaging of the entire esophageal surface, providing a diagnosis in near real time.

A technique for expanding hematopoietic stem cell numbers could have many clinical applications, including improving bone marrow transplantation and recovery from myelotoxic chemotherapy. In this report, Heather Himburg et al. suggest a new strategy to accomplish this by identifying pleiotrophin, a growth factor not previously known to affect hematopoiesis, as an inducer of both mouse and human hematopoietic stem cell expansion ex vivo and of hematopoietic stem cell regeneration in vivo.

McMillin et al. describe a drug screening platform that takes into account the tumor microenvironment, in particular tumor-stromal interactions, enabling the screening of the antitumor activity of candidate anticancer agents in the context of such interactions. The in vitro tumor cell–specific bioluminescence imaging assay is both high throughput and scalable.

Lukas Flatz et al. have exploited the characteristics of the lymphocytic choriomeningitis virus (LCMV) to create a vaccine vector platform that elicits potent CD8⁺ T cell immunity. Using a recombinant, replication-defective LCMV, they show that these modified viral vectors target dendritic cells in vivo and trigger cytotoxic T lymphocyte responses that compare favorably with existing vectors. Other benefits include low global seroprevalence to LCMV and minimal interference of preexisting antibodies with vaccine efficacy.

Colleen Delaney and colleagues have developed a new culture system for umbilical cord blood progenitors that improves myeloid engraftment in cord blood transplants using a Notch ligand–mediated ex vivo expansion strategy. The study demonstrates rapid hematopoietic engraftment in a NOD/SCID mouse model and a phase 1 myeloablative cord blood transplantation trial. The strategy could lead to better clinical outcomes as a result of reduced morbidity and mortality.