Introducing a new reporter gene, membrane-anchored luciferase, for multiplex bioluminescence imaging.

Bioluminescence reporter gene imaging is a robust, high-throughput imaging modality that is useful for tracking cells and monitoring biological processes, both in cell culture and in small animals. We introduced and characterized a novel bioluminescence reporter-membrane-anchored luciferase (maCLuc)-paired with a unique vargulin substrate. This luciferase-substrate pair has no cross-reactivity with established d-luciferin- or coelenterazine-based luciferase reporters.

Lactate Dehydrogenase A Depletion Alters MyC-CaP Tumor Metabolism, Microenvironment, and CAR T Cell Therapy.

To enhance human prostate-specific membrane antigen (hPSMA)-specific chimeric antigen receptor (CAR) T cell therapy in a hPSMA MyC-CaP tumor model, we studied and imaged the effect of lactate dehydrogenase A (LDH-A) depletion on the tumor microenvironment (TME) and tumor progression. Effective LDH-A short hairpin RNA (shRNA) knockdown (KD) was achieved in MyC-CaP:hPSMA Renilla luciferase (RLuc)-internal ribosome entry site (IRES)-GFP tumor cells, and changes in tumor cell metabolism and in the TME were monitored. LDH-A downregulation significantly inhibited cell proliferation and subcutaneous tumor growth compared to control cells and tumors.

Enhancement of PSMA-Directed CAR Adoptive Immunotherapy by PD-1/PD-L1 Blockade.

Chimeric antigen receptor (CAR) T cell therapy in hematologic malignancies has shown remarkable responses, but the same level of success has not been observed in solid tumors. A new prostate cancer model (Myc-CaP:PSMA(+)) and a second-generation anti-hPSMA human CAR T cells expressing a Click Beetle Red luciferase reporter) were used to study hPSMA targeting and assess CAR T cell trafficking and persistence by bioluminescence imaging (BLI). We investigated the antitumor efficacy of human CAR T cells targeting human prostate-specific membrane antigen (hPSMA), in the presence and absence of the target antigen; first alone and then combined with a monoclonal antibody targeting the human programmed death receptor 1 (anti-hPD1 mAb).

Comparative Analysis of Human Nucleoside Kinase-Based Reporter Systems for PET Imaging.

Purpose: Radionuclide-based reporter gene imaging has the sensitivity to monitor gene- and cell-based therapies in human subjects. Potential immunogenicity of current viral transgenes warrants development of human-based reporter systems. We compared human nucleoside kinase reporters to a panel of nucleoside analogs of FEAU, FMAU, and FIAU, including the first in vivo assessment of L-[F]FEAU.

Comparative Analysis of T Cell Imaging with Human Nuclear Reporter Genes.

Unlabelled: Monitoring genetically altered T cells is an important component of adoptive T cell therapy in patients, and the ability to visualize their trafficking/targeting, proliferation/expansion, and retention/death using highly sensitive reporter systems that do not induce an immunologic response would provide useful information. Therefore, we focused on human reporter gene systems that have the potential for translation to clinical studies. The objective of the in vivo imaging studies was to determine the minimum number of T cells that could be visualized with the different nuclear reporter systems.

Targeting the internal epitope of prostate-specific membrane antigen with 89Zr-7E11 immuno-PET.

Unlabelled: The potential of the positron-emitting (89)Zr has been recently investigated for the design of radioimmunoconjugates for immuno-PET. In this study, we report the preparation and in vivo evaluation of (89)Zr-desferrioxamine B (DFO)-7E11, a novel (89)Zr-labeled monoclonal antibody (mAb) construct for targeted imaging of prostate-specific membrane antigen (PSMA), a prototypical cell surface marker highly overexpressed in prostate cancer. The ability of (89)Zr-DFO-7E11 to delineate tumor response to therapy was also investigated, because it binds to the intracellular epitope of PSMA, which becomes available only on membrane disruption in dead or dying cells.

A new pyrimidine-specific reporter gene: a mutated human deoxycytidine kinase suitable for PET during treatment with acycloguanosine-based cytotoxic drugs.

Unlabelled: In this article, we describe a series of new human-derived reporter genes based on human deoxycytidine kinase (dCK) suitable for clinical PET.

Methods: Native dCK and its mutant reporter genes were tested in vitro and in vivo for their phosphorylation of pyrimidine- and acycloguanosine-based radiotracers including 2'-deoxy-2'-fluoroarabinofuranosylcytosine, 2'-fluoro-2'-deoxyarabinofuranosyl-5-ethyluracil (FEAU), penciclovir, and 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine (FHBG) and clinically applied antiviral and anticancer drugs.

Results: Cells transduced with dCK mutant reporter genes showed high in vitro and in vivo uptake of pyrimidine-based radiopharmaceuticals ((18)F-FEAU) comparable to that of herpes simplex virus type-1 thymidine kinase (HSV1-tk)-transduced cells.

PET imaging of HSV1-tk mutants with acquired specificity toward pyrimidine- and acycloguanosine-based radiotracers.

Purpose: The aim of this study was to create an alternative mutant of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene with reduced phosphorylation capacity for acycloguanosine derivatives, but not pyrimidine-based compounds that will allow for successful PET imaging.

Methods: A new mutant of HSV1-tk reporter gene, suitable for PET imaging using pyrimidine-based radiotracers, was developed. The HSV1-tk mutant contains an arginine-to-glutamine substitution at position 176 (HSV1-R176Qtk) of the nucleoside binding region of the enzyme.

Monitoring the efficacy of adoptively transferred prostate cancer-targeted human T lymphocytes with PET and bioluminescence imaging.

Unlabelled: Noninvasive imaging technologies have the potential to enhance the monitoring and improvement of adoptive therapy with tumor-targeted T lymphocytes. We established an imaging methodology for the assessment of spatial and temporal distributions of adoptively transferred genetically modified human T cells in vivo for treatment monitoring and prediction of tumor response in a systemic prostate cancer model.

Methods: RM1 murine prostate carcinoma tumors transduced with human prostate-specific membrane antigen (hPSMA) and a Renilla luciferase reporter gene were established in SCID/beige mice.

Absorption of iron in rats with experimental enteritis.

Inflammatory conditions of the gastrointestinal tract and iron-deficiency anemia are very common in humans. Acute intestinal inflammation was pathologically established in rats by intraluminal administration of acetic acid into the duodenum and the proximal jejunum. The study included two control groups of intact (untreated) rats and sham-operated (saline-treated) rats for each intestinal segment.

Paraquat-induced formation of leukotriene B4 in rat lungs: modulation by N-acetylcysteine.

The present work is focused on the formation of the inflammatory mediator leukotriene B4 (LTB4) in the lungs of paraquat (PQ)-intoxicated rats. The levels of LTB4 and the number of neutrophils in lung lavages of PQ-intoxicated rats, measured 12 h after 30 mg/kg PQ, increased significantly compared with those of control animals; administration of 50 mg/kg IP N-acetylcysteine (NAC), 8 h after PQ, inhibited this effect. The release of LTB4 from alveolar macrophages (AM) or alveolar epithelial type II cells from healthy animals incubated with PQ and/or NAC did not offer' an explanation for the effect of these chemicals on LTB4 in the bronchoalveolar lavage fluid (BALF).

N-acetylcysteine delays the infiltration of inflammatory cells into the lungs of paraquat-intoxicated rats.

We investigated a possible role for N-acetylcysteine (NAC), a well-known antioxidant and free radical scavenger, against oxidative lung damage as observed in the in vivo model of paraquat-intoxicated rats. The administration of two ip doses of 50 mg/kg NAC to paraquat-intoxicated animals did not change the glutathione status of the lungs, as determined by the measurement of nonprotein sulfhydryl (NP-SH) groups. The administration of NAC did however suppress the paraquat-induced release of chemoattractants for neutrophils in the bronchoalveolar fluid when the lavage was carried out 12 hr after the administration of 30 mg/kg paraquat.

Phenotypic and functional immaturity of human umbilical cord blood T lymphocytes.

Successful implementation of bone marrow transplantation for hematopoietic reconstitution is limited by the lack of suitably HLA-matched donors and by the occurrence of graft-versus-host disease that frequently accompanies this procedure. Recent clinical reports have implied that the use of umbilical cord blood as a source of transplantable stem cells may solve these problems. To date, definitive experiments have not been performed to assess the immunological potential of T cells found in umbilical cord blood, which could mediate graft-versus-host disease.

Doppler flow velocities in single umbilical arteries.

Fetal compromise has been associated with an umbilical artery waveform pattern of low or absent diastolic velocity relative to systolic velocity. Fetuses with single umbilical arteries have an increased risk of major malformations, mortality, retarded fetal growth, and prematurity. In this study Doppler flow velocities were obtained in 13 fetuses (four twin fetuses and nine singletons) with a single umbilical artery.

Significance of oligohydramnios complicating pregnancy.

Oligohydramnios is a serious complication of pregnancy that is associated with a poor perinatal outcome. Eighty pregnancies complicated by oligohydramnios constitute the basis for this retrospective study. Forty patients had premature rupture of the membranes; of these, outcomes were good in 25.

Doppler studies of vena cava flows in human fetuses. Insights into normal and abnormal cardiac physiology.

We examined vena cava Doppler flow velocity tracings from 69 fetuses between 22 and 40 weeks' gestation. Twenty-three fetuses had arrhythmias. Fifteen fetuses had absent end-diastolic Doppler velocities in the umbilical artery, a condition associated with intrauterine growth retardation, and 15 normal fetuses with normal umbilical artery Doppler velocity ratios were matched by gestational age.

Fetal pericardial effusion.

Forty-four fetuses with pericardial effusions have been identified by ultrasonographic examinations. The clinical histories and courses of these patients were reviewed. At least eight different clinical features accompanied and were probably responsible for the pericardial effusions.

Prenatal diagnosis of congestive heart failure in a fetus with a sacrococcygeal teratoma.

Two-dimensional Doppler echocardiography was used to diagnose congestive heart failure in a fetus with a large sacrococcygeal teratoma. Ultrasound performed for size-date inconsistency revealed a 27.5-week fetus with hydrops and a large solid and cystic mass in the sacral region.

Fetal cardiac Doppler flow studies in prenatal diagnosis of heart disease.

The prenatal diagnosis of fetal cardiac disease has become increasingly accurate as the technology of ultrasound has improved. Although two-dimensional real-time ultrasound remains the primary method of diagnosis, Doppler blood flow velocity estimates can provide valuable pathophysiologic information to support the anatomic diagnosis. We present six cases in which Doppler studies contributed to the accuracy of the diagnosis of fetal heart disease, including tetralogy of Fallot, right and left ventricular hypoplasia, atrioventricular canal defect, double-outlet right ventricle, and pulmonic stenosis.

Changes in intracardiac Doppler blood flow velocities in fetuses with absent umbilical artery diastolic flow.

Umbilical artery Doppler blood flow velocity studies were used to identify 14 fetuses with absent flow during diastole to determine the significance of absent umbilical artery diastolic flow. Outcomes of these fetuses were recorded, and the associated intracardiac Doppler changes were identified in 12 of them. Maximal and mean intracardiac flow velocities were measured, and volume flows through the right (tricuspid valve, pulmonary valve) and left (mitral valve, aortic valve) sides of the heart were compared.

Congenital heart block and cardiac anomalies in the absence of maternal connective tissue disease.

The association of congenital heart block with maternal connective tissue disease and autoimmunity has been recently reported. Two cases of heart block were diagnosed in utero at 23 and 24 weeks' gestation. Both mothers had extensive workups for connective tissue disease that were negative.

Cardiac Doppler flows during fetal arrhythmias: physiologic consequences.

Eighty-six fetuses of 21-41 weeks' gestation with arrhythmias were studied with ultrasound and heart rate monitoring. The type of arrhythmia was identified by M-mode studies and was confirmed by postnatal electrocardiogram in 70 infants. The most common arrhythmia was premature atrial contractions (76), followed by premature ventricular contractions (five), paroxysmal supraventricular tachycardia (four), and atrial fibrillation/flutter (one).