Paul R. Territo

The HMGB1-RAGE axis mediates traumatic brain injury–induced pulmonary dysfunction in lung transplantation

Traumatic brain injury induces acute lung injury that negatively impacts the physiology of the donor lung before and after lung transplantation. Sounding the Alarm for RAGE Only 20% of lungs are transplantable because traumatic brain injury, a major cause of death in organ doors, may induce acute lung injury. High-mobility group box-1 (HMGB1) release from the injured brain likely contributes to acute lung injury in donors by preferentially interacting with receptor for advanced glycation end products (RAGE) in the lung. Blocking the HMGB1-RAGE axis improves lung function in murine donors with traumatic brain injury and after transplant. In translational studies, lungs sourced from donors with high HMGB1 levels had worse pulmonary function after transplant. Targeting the HMGB1-RAGE axis may increase the number of lungs available for transplantation and improve patient outcomes. Traumatic brain injury (TBI) results in systemic inflammatory responses that affect the lung. This is especially critical in the setting of lung transplantation, where more than half of donor allografts are obtained postmortem from individuals with TBI. The mechanism by which TBI causes pulmonary dysfunction remains unclear but may involve the interaction of high-mobility group box-1 (HMGB1) protein with the receptor for advanced glycation end products (RAGE). To investigate the role of HMGB1 and RAGE in TBI-induced lung dysfunction, RAGE-sufficient (wild-type) or RAGE-deficient (RAGE−/−) C57BL/6 mice were subjected to TBI through controlled cortical impact and studied for cardiopulmonary injury. Compared to control animals, TBI induced systemic hypoxia, acute lung injury, pulmonary neutrophilia, and decreased compliance (a measure of the lungs’ ability to expand), all of which were attenuated in RAGE−/− mice. Neutralizing systemic HMGB1 induced by TBI reversed hypoxia and improved lung compliance. Compared to wild-type donors, lungs from RAGE−/− TBI donors did not develop acute lung injury after transplantation. In a study of clinical transplantation, elevated systemic HMGB1 in donors correlated with impaired systemic oxygenation of the donor lung before transplantation and predicted impaired oxygenation after transplantation. These data suggest that the HMGB1-RAGE axis plays a role in the mechanism by which TBI induces lung dysfunction and that targeting this pathway before transplant may improve recipient outcomes after lung transplantation.

Characterization of 11C-GSK1482160 for Targeting the P2X7 Receptor as a Biomarker for Neuroinflammation

The purinergic receptor subtype 7 (P2X7R) represents a novel molecular target for imaging neuroinflammation via PET. GSK1482160, a potent P2X7R antagonist, has high receptor affinity, high blood–brain barrier penetration, and the ability to be radiolabeled with 11C. We report the initial physical and biologic characterization of this novel ligand. Methods: 11C-GSK1482160 was synthesized according to published methods. Cell density studies were performed on human embryonic kidney cell lines expressing human P2X7R (HEK293-hP2X7R) and underwent Western blotting, an immunofluorescence assay, and radioimmunohistochemistry analysis using P2X7R polyclonal antibodies. Receptor density and binding potential were determined by saturation and association–disassociation kinetics, respectively. Peak immune response to lipopolysaccharide treatment in mice was determined in time course studies and analyzed via Iba1 and P2X7R Western blotting and Iba1 immunohistochemistry. Whole-animal biodistribution studies were performed on saline- or lipopolysaccharide-treated mice at 15, 30, and 60 min after radiotracer administration. Dynamic in vivo PET/CT was performed on the mice at 72 h after administration of saline, lipopolysaccharide, or lipopolysaccharide + blocking, and 2-compartment, 5-parameter tracer kinetic modeling of brain regions was performed. Results: P2X7R changed linearly with concentrations or cell numbers. For high-specific-activity 11C-GSK1482160, receptor density and Kd were 1.15 ± 0.12 nM and 3.03 ± 0.10 pmol/mg, respectively, in HEK293-hP2X7R membranes. Association constant kon, dissociation constant koff, and binding potential (kon/koff) in HEK293-hP2X7R cells were 0.2312 ± 0.01542 min−1⋅nM−1, 0.2547 ± 0.0155 min−1, and 1.0277 ± 0.207, respectively. Whole-brain Iba1 expression in lipopolysaccharide-treated mice peaked by 72 h on immunohistochemistry, and Western blot analysis of P2X7R for saline- and lipopolysaccharide-treated brain sections showed a respective 1.8- and 1.7-fold increase in signal enhancement at 72 h. Biodistribution of 11C-GSK1482160 in saline- and lipopolysaccharide-treated mice at 72 h was statistically significant across all tissues studied. In vivo dynamic 11C-GSK1482160 PET/CT of mice at 72 h after administration of saline, lipopolysaccharide, or lipopolysaccharide + blocking showed a 3.2-fold increase and 97% blocking by 30 min. The total distribution volumes for multiple cortical regions and the hippocampus showed statistically significant increases and were blocked by an excess of authentic standard GSK1482160. Conclusion: The current study provides compelling data that support the suitability of 11C-GSK1482160 as a radioligand targeting P2X7R, a biomarker of neuroinflammation.

A murine model of neurofibromatosis type 2 that accurately phenocopies human schwannoma formation

Neurofibromatosis type 2 (NF2) is an autosomal dominant genetic disorder resulting from germline mutations in the NF2 gene. Bilateral vestibular schwannomas, tumors on cranial nerve VIII, are pathognomonic for NF2 disease. Furthermore, schwannomas also commonly develop in other cranial nerves, dorsal root ganglia and peripheral nerves. These tumors are a major cause of morbidity and mortality, and medical therapies to treat them are limited. Animal models that accurately recapitulate the full anatomical spectrum of human NF2-related schwannomas, including the characteristic functional deficits in hearing and balance associated with cranial nerve VIII tumors, would allow systematic evaluation of experimental therapeutics prior to clinical use. Here, we present a genetically engineered NF2 mouse model generated through excision of the Nf2 gene driven by Cre expression under control of a tissue-restricted 3.9kbPeriostin promoter element. By 10 months of age, 100% of Postn-Cre; Nf2(flox/flox) mice develop spinal, peripheral and cranial nerve tumors histologically identical to human schwannomas. In addition, the development of cranial nerve VIII tumors correlates with functional impairments in hearing and balance, as measured by auditory brainstem response and vestibular testing. Overall, the Postn-Cre; Nf2(flox/flox) tumor model provides a novel tool for future mechanistic and therapeutic studies of NF2-associated schwannomas.

Human Adipose‐Derived Stromal/Stem Cells Protect Against STZ‐Induced Hyperglycemia: Analysis of hASC‐Derived Paracrine Effectors

Adipose‐derived stromal/stem cells (ASCs) ameliorate hyperglycemia in rodent models of islet transplantation and autoimmune diabetes, yet the precise human ASC (hASC)‐derived factors responsible for these effects remain largely unexplored. Here, we show that systemic administration of hASCs improved glucose tolerance, preserved β cell mass, and increased β cell proliferation in streptozotocin‐treated nonobese diabetic/severe combined immunodeficient mice. Coculture experiments combining mouse or human islets with hASCs demonstrated that islet viability and function were improved by hASCs following prolonged culture or treatment with proinflammatory cytokines. Analysis of hASC‐derived factors revealed vascular endothelial growth factor and tissue inhibitor of metalloproteinase 1 (TIMP‐1) to be highly abundant factors secreted by hASCs. Notably, TIMP‐1 secretion increased in the presence of islet stress from cytokine treatment, while TIMP‐1 blockade was able to abrogate in vitro prosurvival effects of hASCs. Following systemic administration by tail vein injection, hASCs were detected in the pancreas and human TIMP‐1 was increased in the serum of injected mice, while recombinant TIMP‐1 increased viability in INS‐1 cells treated with interleukin‐1beta, interferon‐gamma, and tumor necrosis factor alpha. In aggregate, our data support a model whereby factors secreted by hASCs, such as TIMP‐1, are able to mitigate against β cell death in rodent and in vitro models of type 1 diabetes through a combination of local paracrine as well as systemic effects. Stem Cells 2014;32:1831–1842

18F-NaF PET Imaging of Early Coronary Artery Calcification

Studies to date have focused on 18F-sodium fluoride (18F-NaF) uptake in advanced lesions of coronary artery disease (CAD) patients [(1)][1]. However, others suggest that 18F-NaF soft-tissue uptake imaged by positron-emission tomography (PET) may occur in coronary arteries before the advanced stages

In Vivo UTE‐MRI Reveals Positive Effects of Raloxifene on Skeletal‐Bound Water in Skeletally Mature Beagle Dogs

Raloxifene positively affects mechanical properties of the bone matrix in part through modification of skeletal‐bound water. The goal of this study was to determine if raloxifene‐induced alterations in skeletal hydration could be measured in vivo using ultra‐short echotime magnetic resonance imaging (UTE‐MRI). Twelve skeletally mature female beagle dogs (n = 6/group) were treated for 6 months with oral doses of saline vehicle (VEH, 1 mL/kg/d) or raloxifene (RAL, 0.5 mg/kg/d). After 6 months of treatment, all animals underwent in vivo UTE‐MRI of the proximal tibial cortical bone. UTE‐MRI signal intensity versus echotime curves were analyzed by fitting a double exponential to determine the short and long relaxation times of water with the bone (dependent estimations of bound and free water, respectively). Raloxifene‐treated animals had significantly higher bound water (+14%; p = 0.05) and lower free water (–20%) compared with vehicle‐treated animals. These data provide the first evidence that drug‐induced changes in skeletal hydration can be noninvasively assessed using UTE‐MRI.

SOCS1 is a negative regulator of metabolic reprogramming during sepsis

Sepsis can induce an overwhelming systemic inflammatory response, resulting in organ damage and death. Suppressor of cytokine signaling 1 (SOCS1) negatively regulates signaling by cytokine receptors and Toll-like receptors (TLRs). However, the cellular targets and molecular mechanisms for SOCS1 activity during polymicrobial sepsis are unknown. To address this, we utilized a cecal ligation and puncture (CLP) model for sepsis; C57BL/6 mice subjected to CLP were then treated with a peptide (iKIR) that binds the SOCS1 kinase inhibitory region (KIR) and blocks its activity. Treatment with iKIR increased CLP-induced mortality, bacterial burden, and inflammatory cytokine production. Myeloid cell-specific SOCS1 deletion (Socs1Δmyel) mice were also more susceptible to sepsis, demonstrating increased mortality, higher bacterial loads, and elevated inflammatory cytokines, compared with Socs1fl littermate controls. These effects were accompanied by macrophage metabolic reprograming, as evidenced by increased lactic acid production and elevated expression of the glycolytic enzymes hexokinase, lactate dehydrogenase A, and glucose transporter 1 in septic Socs1Δmyel mice. Upregulation was dependent on the STAT3/HIF-1α/glycolysis axis, and blocking glycolysis ameliorated increased susceptibility to sepsis in iKIR-treated CLP mice. These results reveal a role of SOCS1 as a regulator of metabolic reprograming that prevents overwhelming inflammatory response and organ damage during sepsis.

Multimodality Imaging Methods for Assessing Retinoblastoma Orthotopic Xenograft Growth and Development

Genomic studies of the pediatric ocular tumor retinoblastoma are paving the way for development of targeted therapies. Robust model systems such as orthotopic xenografts are necessary for testing such therapeutics. One system involves bioluminescence imaging of luciferase-expressing human retinoblastoma cells injected into the vitreous of newborn rat eyes. Although used for several drug studies, the spatial and temporal development of tumors in this model has not been documented. Here, we present a new model to allow analysis of average luciferin flux () through the tumor, a more biologically relevant parameter than peak bioluminescence as traditionally measured. Moreover, we monitored the spatial development of xenografts in the living eye. We engineered Y79 retinoblastoma cells to express a lentivirally-delivered enhanced green fluorescent protein-luciferase fusion protein. In intravitreal xenografts, we assayed bioluminescence and computed , as well as documented tumor growth by intraocular optical coherence tomography (OCT), brightfield, and fluorescence imaging. In vivo bioluminescence, ex vivo tumor size, and ex vivo fluorescent signal were all highly correlated in orthotopic xenografts. By OCT, xenografts were dense and highly vascularized, with well-defined edges. Small tumors preferentially sat atop the optic nerve head; this morphology was confirmed on histological examination. In vivo, in xenografts showed a plateau effect as tumors became bounded by the dimensions of the eye. The combination of modeling and in vivo intraocular imaging allows both quantitative and high-resolution, non-invasive spatial analysis of this retinoblastoma model. This technique will be applied to other cell lines and experimental therapeutic trials in the future.

Comparison of standardized uptake values with volume of distribution for quantitation of [11C]PBR28 brain uptake

INTRODUCTION [(11)C]PBR28 is a high-affinity ligand for the Translocator Protein 18 kDa (TSPO), which is considered to be a marker for microglial activation. Volume of distribution (VT) estimated with an arterial plasma input function is the gold standard for quantitation of [(11)C]PBR28 binding. However, arterial sampling is impractical at many PET sites for multiple reasons. Reference region modeling approaches are not ideal for TSPO tracers, as the existence of a true reference region cannot be assumed. Given that it would be desirable to have a non-invasive index of [(11)C]PBR28 binding, we elected to study the utility of the semi-quantitative metric, standardized uptake value (SUV) for use in brain [(11)C]PBR PET studies. The primary goal of this study was to determine the relationship between SUV and VT. METHODS We performed a retrospective analysis of data from sixteen [(11)C]PBR28 PET scans acquired in baboons at baseline and at multiple time points after IV injection of lipopolysaccharide, an endotoxin that transiently induces neuroinflammation. For each scan, data from 14 brain regions of interest were studied. VT was estimated with the Logan plot, using metabolite-corrected input functions. SUV was calculated with data from 30 to 60 minutes after [(11)C]PBR28 injection. RESULTS Within individual PET studies, SUV tended to correlate well with VT. Across studies, the relationship between SUV and VT was variable. CONCLUSIONS From study to study, there was variability in the degree of correlation between [(11)C]PBR28 VT and SUV. There are multiple physiological factors that may contribute to this variance. ADVANCES IN KNOWLEDGE As currently applied, the non-invasive measurement of SUV does not appear to be a reliable outcome variable for [(11)C]PBR28. Additional work is needed to discover the source of the discrepancy in SUV between [(11)C]PBR28 scans. IMPLICATIONS FOR PATIENT CARE There is a need to develop alternatives to arterial plasma input functions for TSPO ligands in order to facilitate multi-center trials.

Preclinical Evidence for the Use of Sunitinib Malate in the Treatment of Plexiform Neurofibromas.

Plexiform neurofibromas (pNF) are pathognomonic nerve and soft tissue tumors of neurofibromatosis type I (NF1), which are highly resistant to conventional chemotherapy and associated with significant morbidity/mortality. Disruption of aberrant SCF/c‐Kit signaling emanating from the pNF microenvironment induced the first ever objective therapeutic responses in a recent phase 2 trial. Sunitinib malate is a potent, highly selective RTK inhibitor with activity against c‐Kit, PDGFR, and VEGFR, which have also been implicated in the pathogenesis of these lesions. Here, we evaluate the efficacy of sunitinib malate in a preclinical Krox20;Nf1flox/− pNF murine model.