Dual-therapeutic reporter genes fusion for enhanced cancer gene therapy and imaging.

Two of the successful gene-directed enzyme prodrug therapies include herpes simplex virus-thymidine kinase (HSV1-TK) enzyme-ganciclovir prodrug and the Escherichia coli nitroreductase (NTR) enzyme-CB1954 prodrug strategies; these enzyme-prodrug combinations produce activated cytotoxic metabolites of the prodrugs capable of tumor cell death by inhibiting DNA synthesis and killing quiescent cells, respectively. Both these strategies also affect significant bystander cell killing of neighboring tumor cells that do not express these enzymes. We have developed a dual-combination gene strategy, where we identified HSV1-TK and NTR fused in a particular orientation can effectively kill tumor cells when the tumor cells are treated with a fusion HSV1-TK-NTR gene- along with a prodrug combination of GCV and CB1954. In order to determine whether the dual-system demonstrate superior therapeutic efficacy than either HSV1-TK or NTR systems alone, we conducted both in vitro and in vivo tumor xenograft studies using triple negative SUM159 breast cancer cells, by evaluating the efficacy of cell death by apoptosis and necrosis upon treatment with the dual HSV1-TK genes-GCV-CB1954 prodrugs system, and compared the efficiency to HSV1-TK-GCV and NTR-CB1954. Our cell-based studies, tumor regression studies in xenograft mice, histological analyses of treated tumors and bystander studies indicate that the dual HSV1-TK-NTR-prodrug system is two times more efficient even with half the doses of both prodrugs than the respective single gene-prodrug system, as evidenced by enhanced apoptosis and necrosis of tumor cells in vitro in culture and xenograft of tumor tissues in animals.

Molecular imaging: current status and emerging strategies.

In vivo molecular imaging has a great potential to impact medicine by detecting diseases in early stages (screening), identifying extent of disease, selecting disease- and patient-specific treatment (personalized medicine), applying a directed or targeted therapy, and measuring molecular-specific effects of treatment. Current clinical molecular imaging approaches primarily use positron-emission tomography (PET) or single photon-emission computed tomography (SPECT)-based techniques. In ongoing preclinical research, novel molecular targets of different diseases are identified and, sophisticated and multifunctional contrast agents for imaging these molecular targets are developed along with new technologies and instrumentation for multi-modality molecular imaging. Contrast-enhanced molecular ultrasound (US) with molecularly-targeted contrast microbubbles is explored as a clinically translatable molecular imaging strategy for screening, diagnosing, and monitoring diseases at the molecular level. Optical imaging with fluorescent molecular probes and US imaging with molecularly-targeted microbubbles are attractive strategies as they provide real-time imaging, are relatively inexpensive, produce images with high spatial resolution, and do not involve exposure to ionizing irradiation. Raman spectroscopy/microscopy has emerged as a molecular optical imaging strategy for ultrasensitive detection of multiple biomolecules/biochemicals with both in vivo and ex vivo versatility. Photoacoustic imaging is a hybrid of optical and US techniques involving optically-excitable molecularly-targeted contrast agents and quantitative detection of resulting oscillatory contrast agent movement with US. Current preclinical findings and advances in instrumentation, such as endoscopes and microcatheters, suggest that these molecular imaging methods have numerous potential clinical applications and will be translated into clinical use in the near future.

Molecular ultrasound imaging: current status and future directions.

Targeted contrast-enhanced ultrasound (molecular ultrasound) is an emerging imaging strategy that combines ultrasound technology with novel molecularly-targeted ultrasound contrast agents for assessing biological processes at the molecular level. Molecular ultrasound contrast agents are nano- or micro-sized particles that are targeted to specific molecular markers by adding high-affinity binding ligands onto the surface of the particles. Following intravenous administration, these targeted ultrasound contrast agents accumulate at tissue sites overexpressing specific molecular markers, thereby enhancing the ultrasound imaging signal. High spatial and temporal resolution, real-time imaging, non-invasiveness, relatively low costs, lack of ionising irradiation and wide availability of ultrasound systems are advantages compared to other molecular imaging modalities. In this article we review current concepts and future directions of molecular ultrasound imaging, including different classes of molecular ultrasound contrast agents, ongoing technical developments of pre-clinical and clinical ultrasound systems, the potential of molecular ultrasound for imaging different diseases at the molecular level, and the translation of molecular ultrasound into the clinic.

Indirect imaging of cardiac-specific transgene expression using a bidirectional two-step transcriptional amplification strategy.

Transcriptional targeting for cardiac gene therapy is limited by the relatively weak activity of most cardiac-specific promoters. We have developed a bidirectional plasmid vector, which uses a two-step transcriptional amplification (TSTA) strategy to enhance the expression of two optical reporter genes, firefly luciferase (fluc) and Renilla luciferase (hrluc), driven by the cardiac troponin T (cTnT) promoter. The vector was characterized in vitro and in living mice using luminometry and bioluminescence imaging to assess its ability to mediate strong, correlated reporter gene expression in a cardiac cell line and the myocardium, while minimizing expression in non-cardiac cell lines and the liver. In vitro, the TSTA system significantly enhanced cTnT-mediated reporter gene expression with moderate preservation of cardiac specificity. After intramyocardial and hydrodynamic tail vein delivery of an hrluc-enhanced variant of the vector, long-term fluc expression was observed in the heart, but not in the liver. In both the cardiac cell line and the myocardium, fluc expression correlated well with hrluc expression. These results show the vector's ability to effectively amplify and couple transgene expression in a cardiac-specific manner. Further replacement of either reporter gene with a therapeutic gene should allow non-invasive imaging of targeted gene therapy in living subjects.

[Dynamic MRI of the liver with parallel acquisition technique: characterization of focal liver lesions and analysis of the hepatic vasculature in a single MRI session]. / Dynamische MRT der leber mit paralleler akquisitionstechnik: charakterisierung fokaler leberläsionen und analyse des gefässstatus in einem untersuchungsgang.

PURPOSE: To retrospectively evaluate the performance of breath-hold contrast-enhanced 3D dynamic parallel gradient echo MRI (pMRT) for the characterization of focal liver lesions (standard of reference: histology) and for the analysis of hepatic vasculature (standard of reference: contrast-enhanced 64-detector row computed tomography; MSCT) in a single MRI session. MATERIALS AND METHOD: Two blinded readers independently analyzed preoperative pMRT data sets (1.5T-MRT) of 45 patients (23 men, 22 women; 28 - 77 years, average age, 48 years) with a total of 68 focal liver lesions with regard to image quality of hepatic arteries, portal and hepatic veins, presence of variant anatomy of the hepatic vasculature, as well as presence of portal vein thrombosis and hemodynamically significant arterial stenosis. In addition, both readers were asked to identify and characterize focal liver lesions. Imaging parameters of pMRT were: TR/TE/matrix/slice thickness/acquisition time: 3.1 ms/ 1.4 ms/ 384 x 224 / 4 mm/ 15 - 17 s. MSCT was performed with a pitch of 1.2, an effective slice thickness of 1 mm and a matrix of 512 x 512. RESULTS: Based on histology, the 68 liver lesions were found to be 42 hepatocellular carcinomas (HCC), 20 metastases, 3 cholangiocellular carcinomas (CCC) as well as 1 dysplastic nodule, 1 focal nodular hyperplasia (FNH) and 1 atypical hemangioma. Overall, the diagnostic accuracy was high for both readers (91 - 100 %) in the characterization of these focal liver lesions with an excellent interobserver agreement (kappa-values of 0.89 [metastases], 0.97 [HCC] and 1 [CCC]). On average, the image quality of all vessels under consideration was rated good or excellent in 89 % (reader 1) and 90 % (reader 2). Anatomical variants of the hepatic arteries, hepatic veins and portal vein as well as thrombosis of the portal vein were reliably detected by pMRT. Significant arterial stenosis was found with a sensitivity between 86 % and 100 % and an excellent interobserver agreement (kappa = 0.85). CONCLUSIONS: Diagnostic image quality remains good or excellent in most cases when the data acquisition time is accelerated by means of parallel imaging in dynamic MRI. It allows reliable detection and characterization of focal liver lesions as well as the depiction of hepatic vascular variants, portal vein thrombosis, and arterial stenosis. Introducing pMRT in routine liver MRI may be another step towards a simplified diagnostic work-up prior to liver surgery.

2-deoxy-2-[F-18]fluoro-D-glucose accumulation in ovarian carcinoma cell lines.

PURPOSE: To evaluate 2-deoxy-2-[F-18]fluoro-D-glucose (FDG) accumulation in human ovarian carcinoma cell lines compared with control tumor cell lines known to accumulate FDG. PROCEDURES: FDG accumulation assays were performed in 15 different ovarian carcinoma cell lines at 1, 2, and 3 hours after incubation with 1 microCi of FDG. Results were compared with FDG accumulation in six different control tumor cell lines. 2-deoxy-2-[F-18]fluoro-D-glucose accumulation was expressed as counts per minute (cpm) in cells and normalized to initial cpm in medium and total protein content of cell lysates. RESULTS: FDG accumulation in all 15 ovarian carcinoma cell lines was equal to or higher than 0.0005 +/- 8.6 10(-5) cpm in cells/cpm in medium/mug protein at all three different time points. In two ovarian carcinoma cell lines (ES-2, poorly differentiated clear cell carcinoma, and OVCAR-3, poorly differentiated papillary adenocarcinoma), FDG accumulation was not statistically, significantly different compared to the control cell line with the highest FDG accumulation (LS 174T human colorectal adenocarcinoma) at two or more time points (P > or = 0.07). In 2 of 15 (13%) ovarian carcinoma cell lines (OVCAR5 epithelial carcinoma and SKOV3 clear cell carcinoma), FDG accumulation was lower than that in the control cell line with the lowest FDG accumulation (HT-29 human colorectal adenocarcinoma) at one or more time points (P < 0.05). CONCLUSIONS: Most human ovarian carcinoma cell lines showed comparable FDG accumulations with control cell lines known to accumulate FDG. This study lays the foundations for further comparisons with other ovarian cancer cell lines and for other positron emission tomography tracers.

Retrospectively ECG-gated multi-detector row CT of the chest: does ECG-gating improve three-dimensional visualization of the bronchial tree?

PURPOSE: To determine the impact of retrospectively ECG-gated multi-detector row CT (MDCT) on three-dimensional (3D) visualization of the bronchial tree and virtual bronchoscopy (VB) as compared to non-ECG-gated data acquisition. MATERIALS AND METHODS: Contrast-enhanced retrospectively ECG-gated and non-ECG-gated MDCT of the chest was performed in 25 consecutive patients referred for assessment of coronary artery bypass grafts and pathology of the ascending aorta. ECG-gated MDCT data were reconstructed in diastole using an absolute reverse delay of - 400 msec in all patients. In 10 patients additional reconstructions at - 200 msec, - 300 msec, and - 500 msec prior to the R-wave were performed. Shaded surface display (SSD) and virtual bronchoscopy (VB) for visualization of the bronchial segments was performed with ECG-gated and non-ECG-gated MDCT data. The visualization of the bronchial tree underwent blinded scoring. Effective radiation dose and signal-to-noise ratio (SNR) for both techniques were compared. RESULTS: There was no significant difference in visualizing single bronchial segments using ECG-gated compared to non-ECG-gated MDCT data. However, the total sum of scores for all bronchial segments visualized with non-ECG-gated MDCT was significantly higher compared to ECG-gated MDCT (P < 0.05). The summary scores for visualization of bronchial segments for different diastolic reconstructions did not differ significantly. The effective radiation dose and the SNR were significantly higher with the ECG-gated acquisition technique (P < 0.05). CONCLUSION: The bronchial tree is significantly better visualized when using non-ECG-gated MDCT compared to ECG-gated MDCT. Additionally, non-ECG-gated techniques require less radiation exposure. Thus, the current retrospective ECG-gating technique does not provide any additional benefit for 3D visualization of the bronchial tree and VB.

Time-effectiveness, observer-dependence, and accuracy of measurements of left ventricular ejection fraction using 4-channel MDCT.

PURPOSE: To evaluate the time-effectiveness, inter-observer variance, and accuracy of left ventricular ejection fraction (EF) measurements using retrospectively ECG-gated four-channel multi-detector row CT (MDCT) angiography in comparison with biplane cine-ventriculography. MATERIALS AND METHODS: Twenty consecutive patients underwent retrospectively ECG-gated MDCT angiography and conventional coronary angiography with biplane ventriculography. Raw MDCT data were reconstructed at 0 % - 90 % of the cardiac cycle in increments of 10 %. Ten geometrically identical multiplanar reformations parallel to the short axis of the heart were reconstructed in each patient. Three blinded readers segmented the left ventricle in the end-systolic and end-diastolic phase using standardized window settings in order to determine the EF. The EF was measured with biplane cine-ventriculography by two blinded readers and was compared with MDCT. The time needed for post-processing was recorded and the inter-observer agreement for both imaging techniques was assessed. RESULTS: Mean post-processing time was 63 +/- 3 min per patient for MDCT and 5.5 +/- 1.2 min for ventriculography. MDCT and ventriculography showed a good correlation (r = 0.83, p < 0.0001) for measurement of the EF. Mean errors of EF measurements for the three MDCT readers compared with the mean of the ventriculography were - 6.3 +/- 6.6 %, - 4.7 +/- 7.1 % and - 4.6 +/- 5.7 %, respectively. The mean differences between the three readers assessing MDCT were - 1.6 +/- 3.2 % (reader 1 versus 2, r = 0.96), - 1.6 +/- 5.6 % (1 versus 3, r = 0.95) and - 0.011 +/- 2.9 % (2 versus 3, r = 0.97, p < 0.0001). The mean differences between the two readers assessing ventriculography was 0.32 +/- 5.1 % (r = 0.88, p < 0.0001). CONCLUSIONS: MDCT correlates well with biplane cine-ventriculography but has the tendency to underestimate the left ventricular EF. Measurements using MDCT have a high inter-observer agreement, however, the time needed for additional MDCT data post-processing is still unacceptably long.

Detection of submucosal gastric fundal varices with multi-detector row CT angiography.

BACKGROUND AND AIM: The diagnosis of submucosal fundal varices is challenging. Currently, endoscopy and endoscopic ultrasound (EUS) are considered most useful for this purpose. The aim of this study was to evaluate if multi-detector row CT (MDCT) angiography contributes to the diagnosis of submucosal fundal varices. PATIENTS AND METHODS: Twenty two patients with endoscopically suspected fundal varices were prospectively included in the study. All patients underwent EUS and MDCT angiography. Levels of agreement between EUS and MDCT angiography for the detection of submucosal and perigastric fundal varices were evaluated by three blinded independent readers. In addition, variceal size and location, as well as afferent and efferent vessels of the submucosal varices, were determined. RESULTS: Good or excellent image quality of MDCT angiography was obtained in 21/22 patients (95%). Based on EUS, submucosal varices were detected in 16 of 22 patients (73%) and perigastric varices in 22/22 patients (100%). Using MDCT angiography, the presence of submucosal varices was confirmed in all of these 16 patients by all three readers. Perigastric varices were also confirmed in all 22 patients by all three readers. In addition, all three readers noted the presence of a submucosal varix in an additional patient which was not detected on initial EUS. MDCT angiography showed an excellent interobserver reliability with regard to variceal diameter (kappa=0.90) and variceal location (kappa=0.94). Based on MDCT angiography, afferent and efferent vessels of submucosal varices included the left gastric vein in 11 (65%), the posterior/short gastric veins in 15 (88%), gastrorenal shunts in 10 (59%), the left inferior phrenic vein in six (35%), and the left pericardiophrenic vein in six (35%) of 17 patients. CONCLUSIONS: MDCT angiography is equivalent to EUS in terms of detection and characterisation of fundal varices, in particular with regard to the distinction between submucosal and perigastric fundal varices.

[Contrast-enhanced MR angiography for differentiation between perigastric and submucosal gastric fundal varices]. / Kontrastmittelverstärkte MR-Angiographie zur Differenzierung zwischen perigastrischen und submukösen Fundusvarizen des Magens.

PURPOSE: To evaluate contrast-enhanced MR angiography for the distinction between perigastric and submucosal fundal varices. MATERIALS AND METHODS: Nineteen consecutive patients with clinically suspected fundal varices underwent contrast-enhanced MR angiography and endoscopic ultrasound (EUS) within one week. Diagnostic confidence for the detection of perigastric and submucosal fundal varices was compared between MR angiography (two radiologists) and EUS (one gastroenterologist), and the agreement of size and location was evaluated. RESULTS: Both MR angiography and EUS detected perigastric varices in all 19 patients and submucosal fundal varices in 14 of the 19 patients. The interobserver reliability of MR angiography was good for measuring the variceal diameter (kappa = 0.76) and excellent for localizing the varices (kappa = 1.0). EUS and MR angiography agreed in 12 of 14 patients (86 %) in determining variceal diameter and location. CONCLUSIONS: Contrast-enhanced MR angiography is comparable to endoscopic ultrasound in the detection and characterization of gastric fundal varices.

Spiral-CT angiography to assess feasibility of endovascular aneurysm repair in patients with ruptured aortoiliac aneurysm.

BACKGROUND: To evaluate spiral computed tomography (SCT) angiography for assessment of feasibility of endovascular aneurysm repair (EVAR) in patients with ruptured aortoiliac aneurysm (AAA). PATIENTS AND METHODS: 24 patients (mean age 74 years; range, 69 to 82 years) with suspicion of ruptured AAA and stable hemodynamics were preoperatively examined by using a SCT scanner in the emergency room. SCT angiography was performed from the suprarenal aorta to the femoral bifurcation after a fixed injection delay time of 30 seconds. After that a venous phase SCT scan, beginning at the last image position and ending at the upper thoracic aperture, was performed. RESULTS: The mean acquisition time of the SCT scan was 80 seconds (range 70 to 100 seconds), the mean overall procedure time, including image reconstruction, 5 minutes (range, 4 to 6 minutes). 2D images were directly evaluated during CT data acquisition, and 3D image reconstructions within 10 minutes (range, 8 to 11 minutes) after the SCT scan. AAA rupture was assessed in 14/24 patients (58%): in 10/14 patients (71%) rupture was contained to the retroperitoneum, and in 4/14 patients (29%) intraperitoneal rupture was observed. Successful EVAR was performed in 6/14 patients (43%) with ruptured AAA, and in 8/10 patients (80%) without ruptured AAA. Open surgery was exclusively performed in 6/24 patients (25%) with inappropriate anatomy for EVAR and in 4/24 patients (17%) with intraperitoneal rupture. CONCLUSIONS: Spiral computed tomography angiography is a reliable technique to assess feasibility of endovascular aneurysm repair in patients with ruptured aortic aneurysm. However, it can only be recommended for patients with stable hemodynamics, despite of the short acquisition time.

Worsening enterocolitis in neonates: diagnosis by CT examination of urine after enteral administration of iohexol.

Perforation, a severe complication of necrotizing enterocolitis (NEC), has a high mortality rate. Recently, we presented a new technique for evaluation of NEC: measuring the CT attenuation coefficient of urine after oral administration of iohexol. We present three cases of neonates with NEC who demonstrated serial increases in urine CT attenuation coefficients, all of whom subsequently deteriorated clinically and radiographically. Surgery in all three cases confirmed severe necrosis and/or perforation. These three cases suggest that the CT attenuation coefficient of urine after oral administration of iohexol may be a more sensitive indicator of NEC severity, progression, and perforation than clinical evaluation and radiography. More investigation is necessary, but eventually, this noninvasive technique may be able to decrease morbidity and mortality by predicting the need for surgical intervention or more aggressive medical management of NEC before perforation occurs.

Systemic spread of meconium peritonitis.

Meconium peritonitis is a chemical peritonitis which occurs following bowel perforation during fetal life. It is generally looked upon as benign, resulting in no long-term sequelae. We present a case of a newborn infant with meconium peritonitis who developed infarcts in several organs. At autopsy the infarcts proved to be caused by emboli as a result of intravascular dissemination of meconium. To our knowledge, this is the first reported case of systemic spread of meconium peritonitis in the literature and suggests that meconium peritonitis may have more serious implications than generally thought.

Amiloride-inhibitable Na+ conductance in rat proximal tubule.

Previous single-channel recordings from the luminal membrane of the rabbit proximal tubule have revealed amiloride-inhibitable Na+ channels of a characteristic conductance range. The present study aimed to pursue this issue in rat proximal tubule. Control rats were compared to those put on a low-Na+ diet or pretreated by triamcinolone injections (s.c.). Stimulation of Na+ absorption by glucocorticoids was verified by examining the transepithelial voltage in Ussing chamber studies of the distal colon. The membrane voltage (Vm) of isolated, in-vitro-perfused proximal tubule segments was measured in patch-clamp and impalement studies. It was found that amiloride hyperpolarized Mv significantly by 2.1 +/- 0.9 mV (n = 26) in tubules of control rats, by 3.9 +/- 0.7 mV (n = 12) in rats put on a low-Na+ diet and by 3.7 +/- 1.0 mV (n = 17) in rats treated with glucocorticoids. The effect of amiloride was concentration dependent with a half-maximal effect at < 1 micromol/l. RT-PCR techniques were used to search for the presence of the alpha-, beta- and gamma-subunits of the epithelial Na+ channel in isolated proximal tubule segments. The presence of the respective mRNAs was verified. These data indicate that: (1) amiloride-inhibitable Na+ channels are present in rate proximal tubules; (2) the Na+ conductance may be up-regulated by Na+ deprivation but is still very limited when compared to total cell conductance; (3) therefore, the contribution of Na+-channel-mediated absorption to total proximal Na+ absorption is probably small.