Manipulation of gene expression by an ecdysone-inducible gene switch in tumor xenografts

BACKGROUND: Rapid, robust and reversible induction of transgene expression would significantly facilitate cancer gene therapy as well as allow the in vivo functional study of newly discovered genes in tumor formation and progression. The popularity of the ecdysone inducible gene switch system has led us to investigate whether such a system can successfully regulate gene expression in a syngeneic tumor system in vivo. RESULTS: MBT-2 and Panc02 carcinoma cells were transfected with components of a modification of the ecdysone switch system driving firefly luciferase (F-Luc). In vitro luciferase expression ± ecdysone analog GS-E indicated a robust induction with minimal baseline activity and complete decay after 24 hours without drug. In vitro selection of MBT-2 transfected cell clones which had complete absence of F-Luc expression in the absence of stimulation but which expressed this gene at high levels in response to GS-E were chosen for in vivo evaluation. Tumors from engineered MBT-2 cells were grown to 5 mm in diameter prior to GS-E administration, animals euthanized and tumors removed at 6, 12 and 24 hours after GS-E administration and assayed for F-Luc activity. GS-E resulted in a maximal induction of F-Luc activity at 6 hours in tumor tissue with almost complete reversion to control levels by 12 hours. CONCLUSIONS: This study is the first demonstration that robust and reversible transgene expression in tumors is feasible using the ecdysone system, allowing future rapid in vivo functional characterization of gene function or gene therapy applications

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Efficacy of Flotetuzumab in Combination with Cytarabine in Patient-Derived Xenograft Models of Pediatric Acute Myeloid Leukemia

Children with acute myeloid leukemia (AML) have a poor prognosis despite the intensification of chemotherapy. Future efforts to improve outcomes should focus on more precise targeting of leukemia cells. CD123, or IL3RA, is expressed on the surface of nearly all pediatric AML samples and is a high-priority target for immunotherapy. The efficacy of an investigational dual-affinity retargeting antibody (DART) molecule (CD123 × CD3; MGD006 or flotetuzumab) was assessed in two distinct patient-derived xenograft (PDX) models of pediatric AML. MGD006 simultaneously binds to CD123 on target cells and CD3 on effector T cells, thereby activating T cells and redirecting them to induce cytotoxicity in target cells. The concurrent treatment of cytarabine and MGD006 was performed to determine the effect of cytarabine on T-cell counts and MGD006 activity. Treatment with MGD006 along with an allogeneic human T-cell infusion to act as effector cells induced durable responses in both PDX models, with CD123 positivity. This effect was sustained in mice treated with a combination of MGD006 and cytarabine in the presence of T cells. MGD006 enhanced T-cell proliferation and decreased the burden of AML blasts in the peripheral blood with or without cytarabine treatment. These data demonstrate the efficacy of MGD006 in prolonging survival in pediatric AML PDX models in the presence of effector T cells and show that the inclusion of cytarabine in the treatment regimen does not interfere with MGD006 activity

A Transcriptome-Wide Screen for mRNAs Enriched in Fetal Leydig Cells: CRHR1 Agonism Stimulates Rat and Mouse Fetal Testis Steroidogenesis

<div><p>Fetal testis steroidogenesis plays an important role in the reproductive development of the male fetus. While regulators of certain aspects of steroidogenesis are known, the initial driver of steroidogenesis in the human and rodent fetal testis is unclear. Through comparative analysis of rodent fetal testis microarray datasets, 54 candidate fetal Leydig cell-specific genes were identified. Fetal mouse testis interstitial expression of a subset of these genes with unknown expression (<em>Crhr1</em>, <em>Gramd1b</em>, <em>Itih5</em>, <em>Vgll3</em>, and <em>Vsnl1</em>) was verified by whole-mount <em>in situ</em> hybridization. Among the candidate fetal Leydig cell-specific factors, three receptors (CRHR1, PRLR, and PROKR2) were tested for a steroidogenic function using <em>ex vivo</em> fetal testes treated with receptor agonists (CRH, PRL, and PROK2). While PRL and PROK2 had no effect, CRH, at low (approximately 1 to 10) nM concentration, increased expression of the steroidogenic genes <em>Cyp11a1, Cyp17a1, Scarb1, and Star</em> in GD15 mouse and GD17 rat testes, and in conjunction, testosterone production was increased. Exposure of GD15 fetal mouse testis to a specific CRHR1 antagonist blunted the CRH-induced steroidogenic gene expression and testosterone responses. Similar to <em>ex vivo</em> rodent fetal testes, ≥10 nM CRH exposure of MA-10 Leydig cells increased steroidogenic pathway mRNA and progesterone levels, showing CRH can enhance steroidogenesis by directly targeting Leydig cells. <em>Crh</em> mRNA expression was observed in rodent fetal hypothalamus, and CRH peptide was detected in rodent amniotic fluid. Together, these data provide a resource for discovering factors controlling fetal Leydig cell biology and suggest that CRHR1 activation by CRH stimulates rat and mouse fetal Leydig cell steroidogenesis <em>in vivo</em>.</p> </div

GD17 rat testis steroidogenic gene expression after treatment with CRH, PRL, or PROK2.

<p>Testes were exposed for 24 hours <i>in vitro</i> to varying concentrations of CRH, PRL, or PROK2. Taqman-based qRT-PCR was used for determination of mRNA levels. Three to four samples per group. Mean ±SD are shown for all data. C: control. Asterisk indicates significance for p-value of <0.05 when compared with controls.</p

List of genes highly enriched in fetal Leydig Cells.

<p>Genes in bold are known fetal Leydig cell-specific genes. Fold change values are the increase in mRNA levels from GD11 to GD13 in <i>Mafb+</i> cells.</p

Effects of CRH on steroidogenesis in GD17 rat testis.

<p>A) Treatment with CRH increased steroidogenesis in GD17 rat testis. Seven to nineteen testes per group were exposed for 24 hours <i>ex vivo.</i> mRNA levels were determined using Taqman-based qRT-PCR. Mean ±SD are shown for all data. Asterisk indicates significance for p-value of <0.05 when compared with controls. B) Testosterone secretion increased in GD17 rat testis treated with CRH. Media were collected from eight to twenty samples per group for testosterone radioimmunoassay analysis. Mean ±SD is shown for all data. An asterisk indicates significance of a p-value <0.05.</p

Cycle threshold (Ct) values of <i>Crh</i> and <i>Ucn1</i> mRNA in rat fetal tissues.

<p>ND = Not detectable; Ct value over 34. Applied Biosystems does not recommend using any data with a Ct value over 34 due to poor accuracy.</p

Effect of CRH on MA-10 cell steroidogenesis.

<p>A) CRH exposure of MA-10 cells increased steroidogenic gene expression at different time points. MA-10 cells were exposed to 10 nM CRH for 1, 3, 6 or 24 hours. Three to six replicates were analyzed at each time point. Taqman-based qRT-PCR was used to determine mRNA levels. Mean ±SD are shown for all data. Asterisk indicates significance for p-value of <0.05 when compared with controls. B) CRH concentration-response of MA-10 cell steroidogenic mRNA expression. MA-10 cells, five replicates/group, were exposed to varying concentrations of CRH for 6 hours. Taqman-based qRT-PCR was used to determine mRNA levels. Mean ±SD shown for all data. Asterisk indicates significance for p-value of <0.05 when compared with controls. C) CRH increased MA-10 cell progesterone secretion. Eight replicates/group were treated for 6 hours, and progesterone levels in media were quantified by radioimmunoassay. Mean ±SD shown for all data. C: Control. Asterisk indicates significance of a p-value <0.05.</p