1. Mammalian phrenic nerve, in a trough filled with saline, was excited by magnetic coil (MC)-induced stimuli at defined stimulation sites, including the negative-going first spatial derivative of the induced electric field along a straight nerve, at a bend in the nerve, and at a cut nerve ending. At all such sites, the largest amplitude response for a given stimulator output setting was elicited by an induced damped polyphasic pulse consisting of an initial quarter-cycle hyperpolarization followed by a half-cycle depolarization compared with a predominantly 'monophasic' quarter-cycle depolarization. 2. Simulation studies demonstrated that the increased efficacy of the induced quarter-cycle hyperpolarizing-half-cycle depolarizing polyphasic pulse was mainly attributed to the greater duration of the outward membrane current phase, resulting in a greater outward charge transfer afforded by the half-cycle (i.e. quarter-cycles 2 and 3). The advantage of a fast rising initial quarter-cycle depolarization was more than offset by the slower rising, but longer duration depolarizing half-cycle. 3. Simulation further revealed that the quarter-cycle hyperpolarization-half-cycle depolarization showed only a 2·6% lowering of peak outward current and a 3·5% lowering of outward charge transfer at threshold, compared with a half-cycle depolarization alone. Presumably, this slight increase in efficacy reflects modest reversal of Na¤ inactivation by the very brief initial hyperpolarization. 4. In vitro, at low bath temperature, the nerve response to an initial quarter-cycle depolarization declined in amplitude as the second hyperpolarizing phase progressively increased in amplitude and duration. This 'pull-down' phenomenon nearly disappeared as the bath temperature approached 37°C. Possibly, at the reduced temperature, delay in generation of the action potential permitted the hyperpolarization phase to reduce excitation. 5. Pull-down was not observed in the thenar muscle responses to median nerve stimulation in a normal human at normal temperature. However, pull-down emerged when the median nerve was cooled by placing ice over the forearm. 6. In a nerve at subnormal temperature straddled with non-conducting inhomogeneities, polyphasic pulses of either polarity elicited the largest responses. This was also seen when stimulating distal median nerve at normal temperature. These results imply excitation by hyperpolarizing-depolarizing pulse sequences at two separate sites. Similarly, polyphasic pulses elicited the largest responses from nerve roots and motor cortex. 7. The pull-down phenomenon has a possible clinical application in detecting pathologically slowed activation of Na¤ channels. The current direction of the polyphasic waveform may become a significant factor with the increasing use of repetitive magnetic stimulators which, for technical reasons, induce a cosine-shaped half-cycle, preceded and followed by quartercycles of opposite polarity.

There remains an urgent need for the noninvasive tracking of transfused chimeric antigen receptor (CAR) T cells to determine their biodistribution, viability, expansion, and antitumor functionality. DOTA antibody reporter 1 (DAbR1) comprises a single-chain fragment of the antilanthanoid-DOTA antibody 2D12.5/G54C fused to the human CD4-transmembrane domain and binds irreversibly to lanthanoid ()-2-(4-acrylamidobenzyl)-DOTA (AABD). The aim of this study was to investigate whether DAbR1 can be expressed on lymphocytes and used as a reporter gene as well as a suicide gene for therapy of immune-related adverse effects. DAbR1 was subcloned together with green fluorescent protein into an SFG-retroviral vector and used to transduce CD3/CD28-activated primary human T cells and second-generation 1928z (CAR) T cells. Cell surface expression of DAbR1 was confirmed by cell uptake studies with radiolabeled AABD. In addition, the feasibility of imaging of DAbR1-positive T cells in vivo after intravenous injection ofY/Lu-AABD was studied and radiation doses determined. A panel of DAbR1-expressing T cells and CAR T cells exhibited greater than 8-fold increased uptake ofY-AABD in vitro when compared with nontransduced cells. Imaging studies showed Y-AABD was retained by DAbR1-positive T cells while it continuously cleared from normal tissues, allowing for in vivo tracking of intravenously administered CAR T cells. Normal-organ dose estimates were favorable for repeated PET/CT studies. Selective T cell ablation in vivo withLu-AABD seems feasible for clustered T-cell populations. We have demonstrated for the first time that T cells can be modified with DAbR1, enabling their in vivo tracking via PET and SPECT. The favorable biodistribution and high image contrast observed warrant further studies of this new reporter gene.

Cellular communication network factor 1 (CCN1) is a dynamically expressed, matricellular protein required for vascular development and tissue repair. The CCN1 gene is a presumed target of Yes-associated protein (YAP), a transcriptional coactivator that regulates cell growth and organ size. Herein, we demonstrate that the CCN1 promoter is indeed a direct genomic target of YAP in endothelial cells (ECs) of new blood vessel sprouts and that YAP deficiency in mice downregulates CCN1 and alters cytoskeletal and mitogenic gene expression. Interestingly, CCN1 overexpression in cultured ECs inactivates YAP in a negative feedback and causes its nuclear exclusion. Accordingly, EC-specific deletion of the CCN1 gene in mice mimics a YAP gain-of-function phenotype, characterized by EC hyperproliferation and blood vessel enlargement. CCN1 brings about its effect by providing cells with a soft compliant matrix that creates YAP-repressive cytoskeletal states. Concordantly, pharmacological inhibition of cell stiffness recapitulates the CCN1 deletion vascular phenotype. Furthermore, adeno-associated virus-mediated expression of CCN1 reversed the pathology of YAP hyperactivation and the subsequent aberrant growth of blood vessels in mice with ischemic retinopathy. Our studies unravel a new paradigm of functional interaction between CCN1 and YAP and underscore the significance of their interplay in the pathogenesis of neovascular diseases.

This is the initial report of an α-based pre-targeted radioimmunotherapy (PRIT) using 225 Ac and its theranostic pair, 111 In. We call our novel tumor-targeting DOTA-hapten PRIT system “proteus-DOTA” or “Pr.” Herein we report the first results of radiochemistry development, radiopharmacology, and stoichiometry of tumor antigen binding, including the role of specific activity, anti-tumor efficacy, and normal tissue toxicity with the Pr-PRIT approach (as α-DOTA-PRIT). A series of α-DOTA-PRIT therapy studies were performed in three solid human cancer xenograft models of colorectal cancer (GPA33), breast cancer (HER2), and neuroblastoma (GD2), including evaluation of chronic toxicity at ~20 weeks of select survivors. Methods: Preliminary biodistribution experiments in SW1222 tumor-bearing mice revealed that 225 Ac could not be efficiently pretargeted with current DOTA-Bn hapten utilized for 177 Lu or 90 Y, leading to poor tumor uptake in vivo . Therefore, we synthesized Pr consisting of an empty DOTA-chelate for 225 Ac, tethered via a short polyethylene glycol linker to a lutetium-complexed DOTA for picomolar anti-DOTA chelate single-chain variable fragment (scFv) binding. Pr was radiolabeled with 225 Ac and its imaging surrogate, 111 In. In vitro studies verified anti-DOTA scFv recognition of [ 225 Ac]Pr, and in vivo biodistribution and clearance studies were performed to evaluate hapten suitability and in vivo targeting efficiency. Results: Intravenously (i.v.) administered 225 Ac- or 111 In-radiolabeled Pr in mice showed rapid renal clearance and minimal normal tissue retention. In vivo pretargeting studies show high tumor accumulation of Pr (16.71 ± 5.11 %IA/g or 13.19 ± 3.88 %IA/g at 24 h p.i. for [ 225 Ac]Pr and [ 111 In]Pr, respectively) and relatively low uptake in normal tissues (all average ≤ 1.4 %IA/g at 24 h p.i.). Maximum tolerated dose (MTD) was not reached for either [ 225 Ac]Pr alone or pretargeted [ 225 Ac]Pr at administered activities up to 296 kBq/mouse. Single-cycle treatment consisting of α-DOTA-PRIT with either huA33-C825 bispecific anti-tumor/anti-DOTA-hapten antibody (BsAb), anti-HER2-C825 BsAb, or hu3F8-C825 BsAb for targeting GPA33, HER2, or GD2, respectively, was highly effective. In the GPA33 model, no complete responses (CRs) were observed but prolonged overall survival of treated animals was 42 d for α-DOTA-PRIT vs. 25 d for [ 225 Ac]Pr only ( P < 0.0001); for GD2, CRs (7/7, 100%) and histologic cures (4/7, 57%); and for HER2, CRs (7/19, ...

The retina is a complex neurovascular structure that conveys light/visual image through the optic nerve to the visual cortex of the brain. Neuronal and vascular activities in the retina are physically and functionally intertwined, and vascular alterations are consequential to the proper function of the entire visual system. In particular, alteration of the structure and barrier function of the retinal vasculature is commonly associated with the development of vasoproliferative ischemic retinopathy, a set of clinically well-defined chronic ocular microvascular complications causing blindness in all age groups. Experimentally, the retinal tissue provides researchers with a convenient, easily accessible, and directly observable model suitable to investigate whether and how newly identified genes regulate vascular development and regeneration. The six mammalian CCN gene-encoded proteins are part of an extracellular network of bioactive molecules that regulate various aspects of organ system development and diseases. Whether and how these molecules regulate the fundamental aspects of blood vessel development and pathology and subsequently the neurovascular link in the retina are open-ended questions. Sophisticated methods have been developed to gain insight into the pathogenesis of retinal vasculopathy. This chapter describes several useful methodologies and animal models to investigate the regulation and potential relevance of the CCN proteins in vasoproliferative diseases of the retina.