Digital Quality Improvement Approach Reduces the Need for Rescue Antiemetics in High-Risk Patients: A Comparative Effectiveness Study Using Interrupted Time Series and Propensity Score Matching Analysis.

BACKGROUND: Affecting nearly 30% of all surgical patients, postoperative nausea and vomiting (PONV) can lead to patient dissatisfaction, prolonged recovery times, and unanticipated hospital admissions. There are well-established, evidence-based guidelines for the prevention of PONV; yet physicians inconsistently adhere to them. We hypothesized that an electronic medical record-based clinical decision support (CDS) approach that incorporates a new PONV pathway, education initiative, and personalized feedback reporting system can decrease the incidence of PONV. METHODS: Two years of data, from February 17, 2015 to February 16, 2016, was acquired from our customized University of California Los Angeles Anesthesiology perioperative data warehouse. We queried the entire subpopulation of surgical cases that received general anesthesia with volatile anesthetics, were ≥12 years of age, and spent time recovering in any of the postanesthesia care units (PACUs). We then defined PONV as the administration of an antiemetic medication during the aforementioned PACU recovery. Our CDS system incorporated additional PONV-specific questions to the preoperative evaluation form, creation of a real-time intraoperative pathway compliance indicator, initiation of preoperative PONV risk alerts, and individualized emailed reports sent weekly to clinical providers. The association between the intervention and PONV was assessed by comparing the slopes from the incidence of PONV pre/postintervention as well as comparing observed incidences in the postintervention period to what we expected if the preintervention slope would have continued using interrupted time series analysis regression models after matching the groups on PONV-specific risk factors. RESULTS: After executing the PONV risk-balancing algorithm, the final cohort contained 36,796 cases, down from the 40,831 that met inclusion criteria. The incidence of PONV before the intervention was estimated to be 19.1% (95% confidence interval [CI], 17.9%-20.2%) the week before the intervention. Directly after implementation of the CDS, the total incidence decreased to 16.9% (95% CI, 15.2%-18.5%; P = .007). Within the high-risk population, the decrease in the incidence of PONV went from 29.3% (95% CI, 27.6%-31.1%) to 23.5% (95% CI, 20.5%-26.5%; P < .001). There was no significant difference in the PONV incidence slopes over the entire pre/postintervention periods in the high- or low-risk groups, despite an abrupt decline in the PONV incidence for high-risk patients within the first month of the CDS implementation. CONCLUSIONS: We demonstrate an approach to reduce PONV using individualized emails and anesthesia-specific CDS tools integrated directly into a commercial electronic medical record. We found an associated decrease in the PACU administration of rescue antiemetics for our high-risk patient population.

Semaphorin-3A inhibits multiple myeloma progression in a mouse model.

Previous studies revealed that progression of multiple myeloma (MM) is associated with downregulation of semaphorin-3A (sema3A) expression in bone marrow endothelial cells. We therefore determined if serum sema3A concentrations are correlated with MM progression and if sema3A can affect MM progression. We find that the concentration of sema3A in sera of MM patients is strongly reduced and that the decrease is correlated with disease progression. A similar depletion is found in patients having acute myeloid leukemia and acute lymphoblastic leukemia but not in cancer forms that do not involve the bone marrow such as in colon cancer. Expression of a modified sema3A [furin-resistant sema3A (FR-sema3A)] stabilized against cleavage by furin-like proprotein convertases in CAG MM cells did not affect their behavior in-vitro. CAG cells injected into the tail vein of severe combined immunodeficient (SCID) mice home to the bone marrow and proliferate, mimicking MM disease progression. Disease progression in mice injected with CAG cells expressing FR-sema3A was inhibited, resulting in prolonged survival and a lower incidence of bone lesions. Histological examination and fluorescence-activated cell sorting analysis revealed that FR-sema3A expression reduced the infiltration of the CAG cells into the bone marrow, reduced bone marrow necrosis and reduced angiogenesis induced by the MM cells in the bone marrow. Our results suggest that measurement of sema3A serum concentrations may be of use for the diagnosis and for the monitoring of malignancies of the bone marrow such as MM. Furthermore, our results suggest that FR-sema3A may perhaps find use as an inhibitor of MM disease progression.

The semaphorins and their receptors as modulators of tumor progression.

The semaphorins were initially characterized as repulsive axon guidance factors. However, they are currently also recognized as important regulators of diverse biological processes which include regulation of immune responses, angiogenesis, organogenesis, and a variety of additional physiological and developmental functions. The semaphorin family consists of more than 20 genes divided into seven subfamilies, all of which contain the sema domain signature. They usually transduce signals by activation of receptors belonging to the plexin family, either directly, or indirectly following the binding of some semaphorins to receptors of the neuropilin family which subsequently associate with plexins. Additional receptors which form complexes with these primary semaphorin receptors are also frequently involved in semaphorin signalling, and can strongly influence the nature of the biological responses of cells to semaphorins. Recent evidence suggests that semaphorins play important roles in the etiology of multiple forms of cancer. Some semaphorins such as some semaphorins belonging to the class-3 semaphorin subfamily, have been found to function as bona fide tumor suppressors and to inhibit tumor progression by various mechanisms. Because these class-3 semaphorins are secreted proteins, these semaphorins may potentially be used as anti-tumorigenic drugs. Other semaphorins, such as semaphorin-4D, function as inducers of tumor progression and represent targets for the development of novel anti-tumorigenic drugs. The mechanisms by which semaphorins affect tumor progression are diverse, ranging from direct effects on tumor cells to modulation of accessory processes such as modulation of immune responses and inhibition or promotion of tumor angiogenesis and tumor lymphangiogenesis. This review focuses on the diverse mechanisms by which semaphorins affect tumor progression.

Multimodality Molecular Imaging of Cardiac Cell Transplantation: Part II. In Vivo Imaging of Bone Marrow Stromal Cells in Swine with PET/CT and MR Imaging.

Purpose To quantitatively determine the limit of detection of marrow stromal cells (MSC) after cardiac cell therapy (CCT) in swine by using clinical positron emission tomography (PET) reporter gene imaging and magnetic resonance (MR) imaging with cell prelabeling. Materials and Methods Animal studies were approved by the institutional administrative panel on laboratory animal care. Seven swine received 23 intracardiac cell injections that contained control MSC and cell mixtures of MSC expressing a multimodality triple fusion (TF) reporter gene (MSC-TF) and bearing superparamagnetic iron oxide nanoparticles (NP) (MSC-TF-NP) or NP alone. Clinical MR imaging and PET reporter gene molecular imaging were performed after intravenous injection of the radiotracer fluorine 18-radiolabeled 9-[4-fluoro-3-(hydroxyl methyl) butyl] guanine ((18)F-FHBG). Linear regression analysis of both MR imaging and PET data and nonlinear regression analysis of PET data were performed, accounting for multiple injections per animal. Results MR imaging showed a positive correlation between MSC-TF-NP cell number and dephasing (dark) signal (R(2) = 0.72, P = .0001) and a lower detection limit of at least approximately 1.5 × 10(7) cells. PET reporter gene imaging demonstrated a significant positive correlation between MSC-TF and target-to-background ratio with the linear model (R(2) = 0.88, P = .0001, root mean square error = 0.523) and the nonlinear model (R(2) = 0.99, P = .0001, root mean square error = 0.273) and a lower detection limit of 2.5 × 10(8) cells. Conclusion The authors quantitatively determined the limit of detection of MSC after CCT in swine by using clinical PET reporter gene imaging and clinical MR imaging with cell prelabeling. (©) RSNA, 2016 Online supplemental material is available for this article.

Multimodality Molecular Imaging of Cardiac Cell Transplantation: Part I. Reporter Gene Design, Characterization, and Optical in Vivo Imaging of Bone Marrow Stromal Cells after Myocardial Infarction.

Purpose To use multimodality reporter-gene imaging to assess the serial survival of marrow stromal cells (MSC) after therapy for myocardial infarction (MI) and to determine if the requisite preclinical imaging end point was met prior to a follow-up large-animal MSC imaging study. Materials and Methods Animal studies were approved by the Institutional Administrative Panel on Laboratory Animal Care. Mice (n = 19) that had experienced MI were injected with bone marrow-derived MSC that expressed a multimodality triple fusion (TF) reporter gene. The TF reporter gene (fluc2-egfp-sr39ttk) consisted of a human promoter, ubiquitin, driving firefly luciferase 2 (fluc2), enhanced green fluorescent protein (egfp), and the sr39tk positron emission tomography reporter gene. Serial bioluminescence imaging of MSC-TF and ex vivo luciferase assays were performed. Correlations were analyzed with the Pearson product-moment correlation, and serial imaging results were analyzed with a mixed-effects regression model. Results Analysis of the MSC-TF after cardiac cell therapy showed significantly lower signal on days 8 and 14 than on day 2 (P = .011 and P = .001, respectively). MSC-TF with MI demonstrated significantly higher signal than MSC-TF without MI at days 4, 8, and 14 (P = .016). Ex vivo luciferase activity assay confirmed the presence of MSC-TF on days 8 and 14 after MI. Conclusion Multimodality reporter-gene imaging was successfully used to assess serial MSC survival after therapy for MI, and it was determined that the requisite preclinical imaging end point, 14 days of MSC survival, was met prior to a follow-up large-animal MSC study. (©) RSNA, 2016 Online supplemental material is available for this article.

MRI detection of transcriptional regulation of gene expression in transgenic mice.

Ferritin, the iron storage protein, was recently suggested to be a candidate reporter for the detection of gene expression by magnetic resonance imaging (MRI). Here we report the generation of TET:EGFP-HAferritin (tet-hfer) transgenic mice, in which tissue-specific inducible transcriptional regulation of expression of the heavy chain of ferritin could be detected in vivo by MRI. We show organ specificity by mating the tet-hfer mice with transgenic mice expressing tetracycline transactivator (tTA) in liver hepatocytes and in vascular endothelial cells. Tetracycline-regulated overexpression of ferritin resulted in specific alterations of the transverse relaxation rate (R(2)) of water. Transgene-dependent changes in R(2) were detectable by MRI in adult mice, and we also found fetal developmental induction of transgene expression in utero. Thus, the tet-hfer MRI reporter mice provide a new transgenic mouse platform for in vivo molecular imaging of reporter gene expression by MRI during both embryonic and adult life.

Pathological angiogenesis is induced by sustained Akt signaling and inhibited by rapamycin.

Endothelial cells in growing tumors express activated Akt, which when modeled by transgenic endothelial expression of myrAkt1 was sufficient to recapitulate the abnormal structural and functional features of tumor blood vessels in nontumor tissues. Sustained endothelial Akt activation caused increased blood vessel size and generalized edema from chronic vascular permeability, while acute permeability in response to VEGF-A was unaffected. These changes were reversible, demonstrating an ongoing requirement for Akt signaling for the maintenance of these phenotypes. Furthermore, rapamycin inhibited endothelial Akt signaling, vascular changes from myrAkt1, tumor growth, and tumor vascular permeability. Akt signaling in the tumor vascular stroma was sensitive to rapamycin, suggesting that rapamycin may affect tumor growth in part by acting as a vascular Akt inhibitor.

Department of Anesthesiology Skilled Peer Support Program Outcomes: Second Victim Perceptions.

BACKGROUND: Most anesthesia providers experience an adverse event during their training or career. Limited evidence suggests skilled peer support programs (SPSPs) reduce initial distress and support adaptive functioning and coping. This study evaluated second victim perceptions of a voluntary SPSP. METHODS: An SPSP was developed and implemented for all clinical and administrative personnel in the Department of Anesthesiology and Perioperative Medicine in three hospitals and six outpatient surgery centers in December 2017. The program incorporated the Scott Three-Tiered Interventional Model of Second Victim Support. Surveys were offered to clinicians in the department prior to implementation of the SPSP and again 18 months after implementation. Among the subset of respondents who experienced a serious adverse patient event, the authors used multiple logistic regression models that adjusted for role and number of night shifts per month to examine differences in perceived resource availability and post-event support received following implementation of the program. RESULTS: There were 94 surveys (83 complete; 11 partially complete) collected prior to implementation and 84 surveys (67 complete; 17 partially complete) collected after implementation. A total of 25 individuals took the survey at both pre and post (19 complete). After implementation, 62.5% of respondents indicated that institutional support had improved since the occurrence of their serious adverse patient event. Statistical models identified a significant improvement in the probability that a clinician agreed with the statement "I think that the organization learned from the event and took appropriate steps to reduce the chance of it happening again" at post vs. pre (adjusted odds ratio [aOR] 3.9, 95% confidence interval [CI] 1.01-15.1. A statistically significant increase from pre to post in the perceived availability of formal emotional support was identified (aOR 5.2, 95% CI 1.9-22.5). CONCLUSION: Implementation of a skilled peer support program within a large department of anesthesiology can improve institutional-based emotional support.

Encephaloduroarteriosynangiosis Operative Technique and Intraoperative Anesthesia Management: Treatment From Both Sides of the Curtain.

BACKGROUND: Encephaloduroarteriosynangiosis (EDAS) is a form of indirect revascularization for cerebral arterial steno-occlusive disorders. EDAS has gained growing interest as a technique applicable to pediatric and adult populations for several types of ischemic cerebral steno-occlusive conditions. OBJECTIVE: To present a team-oriented, multidisciplinary update of the EDAS technique for application in challenging adult cases of cerebrovascular stenosis/occlusion, successfully implemented in more than 200 cases. METHODS: We describe and demonstrate step-by-step a multidisciplinary-modified EDAS technique, adapted to maintain uninterrupted intensive medical management of patients' stroke risk factors and anesthesia protocols to maintain strict hemodynamic control. RESULTS: A total of 216 EDAS surgeries were performed in 164 adult patients, including 65 surgeries for patients with intracranial atherosclerotic disease and 151 operations in 99 patients with moyamoya disease. Five patients with intracranial atherosclerotic disease had recurrent strokes (3%), and there was one perioperative death. The mean clinical follow-up was 32.9 mo with a standard deviation of 31.1. There was one deviation from the surgical protocol. There were deviations from the anesthesia protocol in 3 patients (0.01%), which were promptly corrected and did not have any clinical impact on the patients' condition. CONCLUSION: The EDAS protocol described here implements a team-oriented, multidisciplinary adaptation of the EDAS technique. This adaptation resides mainly in 3 points: (1) uninterrupted administration of intensive medical management, (2) strict hemodynamic control during anesthesia, and (3) meticulous standardized surgical technique.

Ferritin as a reporter gene for MRI: chronic liver over expression of H-ferritin during dietary iron supplementation and aging.

The iron storage protein, ferritin, provides an important endogenous MRI contrast that can be used to determine the level of tissue iron. In recent years the impact of modulating ferritin expression on MRI contrast and relaxation rates was evaluated by several groups, using genetically modified cells, viral gene transfer and transgenic animals. This paper reports the follow-up of transgenic mice that chronically over-expressed the heavy chain of ferritin (h-ferritin) in liver hepatocytes (liver-hfer mice) over a period of 2 years, with the aim of investigating the long-term effects of elevated level of h-ferritin on MR signal and on the well-being of the mice. Analysis revealed that aging liver-hfer mice, exposed to chronic elevated expression of h-ferritin, have increased R(2) values compared to WT. As expected for ferritin, R(2) difference was strongly enhanced at high magnetic field. Histological analysis of these mice did not reveal liver changes with prolonged over expression of ferritin, and no differences could be detected in other organs. Furthermore, dietary iron supplementation significantly affected MRI contrast, without affecting animal wellbeing, for both wildtype and ferritin over expressing transgenic mice. These results suggest the safety of ferritin over-expression, and support the use of h-ferritin as a reporter gene for MRI.

MRI reporter genes.

Noninvasive molecular imaging of dynamic processes has benefited tremendously from the use of reporter genes. These genes encode for proteins that emit light, bind radiolabeled probes, or, as covered in this review, modulate MRI contrast. Reporter genes play a pivotal role in monitoring cell trafficking, gene replacement therapy, protein-protein interactions, neuronal plasticity, and embryonic development. Several strategies exist for generating MRI contrast: using enzyme-catalyzed chemical modification of metal-based contrast agents or (phosphorus) metabolites, iron-binding and iron-storage proteins to accumulate iron as a contrast agent, and artificial proteins for imaging based on chemical exchange saturation transfer. MRI reporter genes have the advantage that the specific signal can be coregistered with soft-tissue anatomy and functional tissue information and have, therefore, become an active and growing area of scientific interest.

Transient Intraoperative Central Diabetes Insipidus in Moyamoya Patients Undergoing Revascularization Surgery: A Mere Coincidence?

We present 2 patients with Moyamoya disease undergoing revascularization surgery who developed transient intraoperative central diabetes insipidus with spontaneous resolution in the immediate postoperative period. We speculate that patients with Moyamoya disease may be predisposed to a transient acute-on-chronic insult to the arginine vasopressin-producing portion of their hypothalamus mediated by anesthetic agents. We describe our management, discuss pertinent literature, and offer possible mechanisms of this transient insult. We hope to improve patient safety by raising awareness of this potentially catastrophic complication.

A Sema3C Mutant Resistant to Cleavage by Furin (FR-Sema3C) Inhibits Choroidal Neovascularization.

In age-related macular degeneration (AMD), abnormal sub retinal choroidal neovascularization (CNV) is a major cause of blindness. FR-sema3C is a point mutated form of semaphorin-3C that is resistant to cleavage by furin like pro-protein convertases (FPPC). We have found in previous work that FR-sema3C functions as an anti-angiogenic factor. In this study we investigated the possible use of FR-sema3C as an inhibitor of CNV. FR-sema3C inhibits VEGF as well as PDGF-BB signal transduction in endothelial cells and to less extent bFGF induced signal transduction using a mechanism that does not depend upon the binding of VEGF like the drugs that are currently the mainstay treatment for AMD. CNV was induced in eyes of C57 black mice by laser photocoagulation. Intravitreal injection of FR-Sema3C or aflibercept (VEGF-trap) was then used to inhibit CNV formation. Invading choroidal vessels were visualized a week later by injection of FITC-dextran into the circulation, followed by the measurement of the area of the invading blood vessels. Injection of 0.1 µg FR-Sema3C inhibited CNV by 55% (P<0.01) and was as effective as 5 µg aflibercept. FR-sema3C did not display any adverse effects on retinal function following its injection into eyes of healthy mice as assessed by optokinetic reflex (OKR) and Electro-retinogram (ERG) criteria. Furthermore, FR-sema3C did not induce apoptosis in the retina as determined by TUNEL nor was there any discernable structural damage to the retina as assessed by several immuno-histochemical criteria. Our results suggest that FR-sema3C could perhaps be used for the treatment of AMD, and that it may perhaps be of benefit to patients that do not respond well to current treatments relying on VEGF sequestering agents.

The role of the semaphorins in cancer.

The semaphorins were initially characterized as axon guidance factors, but have subsequently been implicated also in the regulation of immune responses, angiogenesis, organ formation, and a variety of additional physiological and developmental functions. The semaphorin family contains more then 20 genes divided into 7 subfamilies, all of which contain the signature sema domain. The semaphorins transduce signals by binding to receptors belonging to the neuropilin or plexin families. Additional receptors which form complexes with these primary semaphorin receptors are also frequently involved in semaphorin signaling. Recent evidence suggests that semaphorins also fulfill important roles in the etiology of multiple forms of cancer. Some semaphorins have been found to function as bona-fide tumor suppressors and to inhibit tumor progression by various mechanisms while other semaphorins function as inducers and promoters of tumor progression.

Rigorous anaesthesia management protocol for patients with intracranial arterial stenosis: a prospective controlled-cohort study.

OBJECTIVES: Reducing variability is integral in quality management. As part of the ongoing Encephaloduroarteriosynangiosis Revascularisation for Symptomatic Intracranial Arterial Stenosis (ERSIAS) trial, we developed a strict anaesthesia protocol to minimise fluctuations in patient parameters affecting cerebral perfusion. We hypothesise that this protocol reduces the intraoperative variability of targeted monitored parameters compared to standard management. DESIGN: Prospective cohort study of patients undergoing encephaloduroarteriosynangiosis surgery versus standard neurovascular interventions. Patients with ERSIAS had strict perioperative management that included normocapnia and intentional hypertension. Control patients received regular anaesthetic standard of care. Minute-by-minute intraoperative vitals were electronically collected. Heterogeneity of variance tests were used to compare variance across groups. Mixed-model regression analysis was performed to establish the effects of treatment group on the monitored parameters. SETTING: Tertiary care centre. PARTICIPANTS: 24 participants: 12 cases (53.8 years ± 16.7 years; 10 females) and 12 controls (51.3 years ± 15.2 years; 10 females). Adults aged 30-80 years, with transient ischaemic attack or non-disabling stroke (modified Rankin Scale <3) attributed to 70-99% intracranial stenosis of the carotid or middle cerebral artery, were considered for enrolment. Controls were matched according to age, gender and history of neurovascular intervention. MAIN OUTCOME MEASURES: Variability of heart rate, mean arterial blood pressure (MAP), systolic blood pressure and end tidal CO2 (ETCO2) throughout surgical duration. RESULTS: There were significant reductions in the intraoperative MAP SD (4.26 vs 10.23 mm Hg; p=0.007) and ETCO2 SD (0.94 vs 1.26 mm Hg; p=0.05) between the ERSIAS and control groups. Median MAP and ETCO2 in the ERSIAS group were higher (98 mm Hg, IQR 23 vs 75 mm Hg, IQR 15; p<0.001, and 38 mm Hg, IQR 4 vs 32 mm Hg, IQR 3; p<0.001, respectively). CONCLUSIONS: The ERSIAS anaesthesia protocol successfully reduced intraoperative fluctuations of MAP and ETCO2. The protocol also achieved normocarbia and the intended hypertension. TRIAL REGISTRATION NUMBER: NCT01819597; Pre-results.

The role of heparanase in lymph node metastatic dissemination: dynamic contrast-enhanced MRI of Eb lymphoma in mice.

Heparanase expression has been linked to increased tumor invasion, metastasis, and angiogenesis and with poor prognosis. The aim of the study was to monitor the effect of heparanase expression on lymph node metastasis, in heparanase-overexpressing subcutaneous Eb mouse T-lymphoma tumors, and their draining lymph node. Dynamic contrast-enhanced magnetic resonance imaging (MRI) using biotin-BSA-GdDTPA-FAM/ROX was applied for analysis of blood volume, vascular permeability, and interstitial convection, and for detection of very early stages of such metastatic dissemination. Eb tumors increased extravasation, interstitial convection, and lymphatic drain of the contrast material. Interstitial flow directions were mapped by showing radial outflow interrupted in some tumors by directional flow toward the popliteal lymph node. Heparanase expression significantly increased contrast enhancement of the popliteal lymph node but not of the primary tumor. Changes in MR contrast enhancement preceded the formation of pathologically detectable metastases, and were detectable when only a few enhanced green fluorescent protein (EGFP)-expressing Eb cells were found near and within the nodes. These results demonstrate very early, heparanase-dependent vascular changes in lymph nodes that were visible by MRI following administration of biotin-BSA-GdDTPA-FAM/ROX, and can be used for studying the initial stages of lymph node infiltration.

Receptor-mediated targeting of a photosensitizer by its conjugation to gonadotropin-releasing hormone analogues.

Photodynamic therapy uses a combination of light, oxygen, and a photosensitizer to induce the death of malignant cells. To improve the selectivity of a photosensitizer toward cancerous cells that express gonadotropin-releasing hormone (GnRH) receptors, protoporphyrin IX (PpIX) was conjugated to a GnRH agonist, [d-Lys6]GnRH, or to a GnRH antagonist, [d-pGlu1, d-Phe2, d-Trp3, d-Lys6]GnRH. The condensation of the peptide with PpIX was carried out in a homogeneous solution using benzotriazole-1-yloxytris(pyrrolidinophosphonium) hexafluorophosphate as a coupling reagent. Although these conjugates had lower binding affinity to rat pituitary GnRH receptors than their parent analogues, they fully preserved their agonistic or antagonistic activity in vitro and in vivo. The GnRH agonist conjugate proved to be long-acting in vivo. Thus, 24 h after its administration to rats (2 nmol/rat), serum LH concentrations were significantly higher than in rats treated with the same amount of the parent peptide. The conjugates, notably the agonist, were more phototoxic toward pituitary gonadotrope alphaT3-1 cell line than was unconjugated PpIX. In contrast to PpIX, the phototoxicity of the conjugates toward alphaT3-1 cells or to human breast cancer cells (MCF-7 cells that were transfected with human GnRH receptors) was alleviated by co-incubation with the parent peptide, indicating that phototoxicity is receptor-mediated. The selectivity of the GnRH antagonist conjugate to gonadotrope cells in a primary pituitary culture was approximately 10 times higher than that of the unconjugated PpIX. Thus, GnRH-based conjugates may affect cancer cells not only by acting as classic GnRH analogues to reduce the plasma levels of steroids by desensitization of the pituitary gland but also by selective photodamage of cells that express GnRH receptors.

Noninvasive reporter gene imaging of human Oct4 (pluripotency) dynamics during the differentiation of embryonic stem cells in living subjects.

PURPOSE: Human pluripotency gene networks (PGNs), controlled in part by Oct4, are central to understanding pluripotent stem cells, but current fluorescent reporter genes (RGs) preclude noninvasive assessment of Oct4 dynamics in living subjects. PROCEDURES: To assess Oc4 activity noninvasively, we engineered a mouse embryonic stem cell line which encoded both a pOct4-hrluc (humanized renilla luciferase) reporter and a pUbi-hfluc2-gfp (humanized firefly luciferase 2 fused to green fluorescent protein) reporter. RESULTS: In cell culture, pOct4-hRLUC activity demonstrated a peak at 48 h (day 2) and significant downregulation by 72 h (day 3) (p=0.0001). Studies in living subjects demonstrated significant downregulation in pOct4-hRLUC activity between 12 and 144 h (p = 0.001) and between 12 and 168 h (p = 0.0003). pOct4-hRLUC signal dynamics after implantation was complex, characterized by transient upregulation after initial downregulation in all experiments (n = 10, p = 0.01). As expected, cell culture differentiation of the engineered mouse embryonic stem cell line demonstrated activation of mesendodermal, mesodermal, endodermal, and ectodermal master regulators of differentiation, indicating potency to form all three germ layers. CONCLUSIONS: We conclude that the Oct4-hrluc RG system enables noninvasive Oct4 imaging in cell culture and in living subjects.