Validation of RNA-based molecular clonotype analysis for virus-specific CD8+ T-cells in formaldehyde-fixed specimens isolated from peripheral blood

Recent advances in the field of molecular clonotype analysis have enabled detailed repertoire characterization of viably isolated antigen-specific T cell populations directly ex vivo. However, in the absence of a biologically contained FACS facility, peripheral blood mononuclear cell (PBMC) preparations derived from patients infected with agents such as HIV must be formaldehyde fixed to inactivate the pathogen; this procedure adversely affects nucleic acid template quality. Here, we developed and validated a method to amplify and sequence mRNA species derived from formaldehyde fixed PBMC specimens. Antigen-specific CD8+ cytotoxic T-lymphocyte populations were identified with standard fluorochrome-conjugated peptide-major histocompatibility complex class I tetramers refolded around synthetic peptides representing immunodominant epitopes from HIV p24 Gag (KRWII[M/L]GLNK/HLA B*2705) and CMV pp65 (NLVPMVATV/HLA A*0201 and TPRVTGGGAM/HLA B*0702), and acquired in separate laboratories with or without fixation. In the presence of proteinase K pre-treatment, the observed antigen-specific CD8+ T-cell repertoire determined by molecular clonotype analysis was statistically no different whether derived from fixed or unfixed PBMC. However, oligo-dT recovery methods were not suitable for use with fixed tissue as significant skewing of clonotypic representation was observed. Thus, we have developed a reliable RNA-based method for molecular clonotype analysis that is compatible with formaldehyde fixation and therefore suitable for use with primary human samples isolated by FACS outside the context of a biological safety level 3 containment facility.     

Rabbit ear model of injury-induced arterial smooth muscle cell proliferation. Kinetics, reproducibility, and implications

Recently, considerable interest has focused on the vascular smooth muscle cell (SMC) response to injury, particularly as it relates to restenosis after angioplasty. In an effort to find an optimal experimental model of arterial SMC proliferation after injury, we examined the effects of external injury to the central artery of the rabbit ear and assessed the reproducibility, morphological changes, and time course of cellular proliferation after such an injury. With rabbits under general anesthesia, direct pressure was applied at two sites along the central artery of the ears of 19 New Zealand White rabbits. Rabbits were maintained on a diet of 2.4% fat and 0.001% cholesterol throughout the experiment. In seven rabbits examined after 21 days, marked SMC proliferation with neointimal formation was observed at all 28 sites (100%). Mean neointimal area, expressed as a percentage of the area of the tunica media, was 82 +/- 40% (range, 21-203%). Compared with the uninvolved artery displaced 2 mm from the injury site, mechanical crush caused a 38% increase in total vessel area (p less than 0.001), a 40% decrease in luminal area (p less than 0.002), and no change in the area of the media. Serial histological studies were performed 1-42 days after injury, using light and electron microscopy and bromodeoxyuridine immunohistochemistry. Beginning at day 3, activated medial SMCs were noted to migrate through defects in the internal elastic membrane, with a gradual increase in neointimal area between days 5 and 12. Peak DNA synthesis was identified in the media 5 days after injury, with proliferative activity shifting almost exclusively to the neointima thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coronary artery lumen volume measurement using three-dimensional intravascular ultrasound: Validation of a new technique

Objective: To validate an automated algorithm for the measurement of lumen volumes of coronary arteries. Background: Current intravascular ultrasound systems use absolute measurements of and changes in areas and diameters for the assessment of coronary artery disease. However, the coronary artery is a three-dimensional structure of complex geometry and volume. Methods: We used a comprehensive imaging system designed to reconstruct planar intravascular ultrasound images in three dimensions. This system consisted of a 25 MHz transducer-tipped rigid probe (for in vitro studies) or a 25 MHz transducer-tipped catheter within a 3.9F monorail imaging sheath (for in vivo studies), a motorized catheter pullback device that withdrew the transducer at 0.5 mm/sec, and an image processing computer that stacked 15 image slices/mm of vessel axial length and then performed thresholding-based three-dimensional image rendering and lumen volume measurement. We imaged 13 human coronary vessels (6 RCA, 6 LAD, 1 LCX) in vitro and 16 vessels (8 LAD, 6 RCA, 2 SVG) in vivo. Results: In vitro studies: Lumen volumes derived by three-dimensional intravascular ultrasound were 171 ± 121 mm³ and compared very well with those derived by histology (160 ± 109 mm³, r = 0.97, SEE = 29 mm³, P < 0.001) and with those derived by manual planimetry of planar intravascular ultrasound images (150 ± 106 mm³, r = 0.97, SEE = 30 mm³, P < 0.001). In vivo studies: Lumen volumes derived by three-dimensional intravascular ultrasound were 74 ± 35 mm³ and compared well with those derived by quantitative angiography (52 ± 20 mm³, r = 0.71, SEE = 25 mm³, P < 0.002). Conclusions: Three-dimensional intravascular ultrasound is a new technique that can accurately measure coronary artery lumen volumes. Further technical improvements may help to establish this technique as the new standard for lumen volume measurement. © Wiley-Liss, Inc.     

MR angiography: noninvasive vascular imaging of the abdomen

Magnetic resonance angiography (MRA) has been used to image abdominal vessels less frequently than renal arteries. Until the use of fast contrast-enhanced (CE) techniques, an important limitation was the acquisition time of phase-contrast or time-of-flight imaging and, consequently, the creation of motion artifacts. Recent advances in MRA technology have shortened acquisition times, so it is now possible to obtain successive images in the arterial and then the portal phase. MRA can be used as an adjunct to any MR examination to assess, e.g., the arterial feeding of hepatocellular carcinoma, the encasement of arteries, and segmental portal thrombosis in pancreatic carcinoma. However, MRA has been used mainly to study chronic mesenteric ischemia, portal vein diseases, and complications from liver transplantation. The portal venous system is exquisitely portrayed with this method; MRA is as accurate as digital subtraction angiography (DSA) in the diagnosis of portal vein diseases. Acute mesenteric ischemia is an emergency in which computed tomography is the most appropriate imaging modality. Conversely, chronic mesenteric ischemia is best examined with CE-MRA, which is almost as accurate as DSA. CE-MRA is superior to DSA for the simultaneous exploration of the aorta, renal arteries, and iliac arteries, thereby providing a panoramic view of abdominal vascular involvement. MRA can be coupled with measurements of flow. With this functional approach, MRA is the only modality that can completely assess vascular diseases of the abdomen.     

High-resolution magnetic resonance imaging at 2 Tesla: potential for atherosclerotic lesions exploration in the apolipoprotein E knockout mouse

INTRODUCTION: The aim of the present study was to evaluate the potential of high-resolution MRI at 2 Tesla (T) for direct noninvasive imaging of the aortic wall in a mouse model of atherosclerosis. MATERIAL AND METHODS: A specific mouse antenna was developed and sequence parameters were adjusted. T(1)- and T2-weighted images of abdominal aorta were obtained at 2 T with a spatial resolution of 86 x 86 x 800 microm3 in vivo. With a dedicated small coil, ex vivo MRI of the aorta was performed with a spatial resolution of 54 x 54 x 520 microm3. RESULTS: In vivo, the aortic wall was clearly defined on T(2)-weighted images in 15 of 16 mice: along the aorta the lumen circumference ranged from 1.07 to 3.61 mm and mean wall thickness from 0.11 to 0.67 mm. In vivo measurements of plaque distribution were confirmed by ex vivo MR imaging and by histology, with a good correlation with histology regarding lumen circumference (r = 0.94) and wall thickness (r = 0.97). CONCLUSION: Magnetic resonance imaging at 2 T to analyze in vivo atherosclerotic lesions in mice is possible with a spatial resolution of 86 x 86 x 800 microm3 and thus can be used for noninvasive follow-up in evaluation of new drugs.     

Pilot MR evaluation of pharmacokinetics and relaxivity of specific blood pool agents for MR angiography

RATIONALE AND OBJECTIVES: To evaluate the use of two new blood pool contrast agents (P760, P775) compared with a low-molecular-weight gadolinium chelate in MR angiography. METHODS: The r1 efficiency of P760 was evaluated in vitro at 1.5 T; 3D abdominal contrast-enhanced MR angiography with qualitative analysis was compared in four rabbits after injection of incremental doses of P760 and in one rabbit after Gd-DOTA. A dynamic MR study was performed using a 2D T1-weighted turbo-flash MR sequence after injection of P760, P775, and Gd-DOTA. Each compound was tested at equivalent doses in three rabbits to assess r1 efficiency. Quantitative analysis of signal intensity in the aorta, the inferior vena cava, the renal cortex, and the medulla was performed. RESULTS: In vitro, the r1 efficiency of P760 was 23.3 mmol(-1) x L x sec(-1) at 1.5 T. Injection of a dose of P760 10 times less than Gd-DOTA allowed similar vessel visualization. The signal intensity peak and first-pass contrast kinetics in the aorta and the inferior vena cava were similar with the three products. Compared with P760 and Gd-DOTA, P775 allowed a greater renal cortex signal intensity at the first pass and a faster decrease on delayed images. CONCLUSIONS: The superior r1 efficiency of P760 and P775 was confirmed in vitro and in vivo at 1.5 T compared with Gd-DOTA, and P775 proved to be a rapid-clearance blood pool agent.     

Influence of bolus volume and dose of gadolinium chelate for first-pass myocardial perfusion MR imaging studies

First-pass MR myocardial perfusion measurements require a well-defined left ventricular (LV) blood pool input function. We used a peripheral intravenous (IV) injection of a gadolinium (Gd) chelate to obtain a well-characterized LV time-intensity curve. Using a strongly T1-weighted subsecond MR sequence, we performed cardiac MR imaging after administering an IV bolus injection of one of three different doses of the Gd chelate: a standard dose (0.1 mmol/kg, group I, n = 8); a low dose with two bolus volumes (0.01 mmol/kg, l/10e bolus volume, group n, n = 7, and 0.01 mmol/kg diluted in saline, same bolus volume as group I, group III, n = 3); and an intermediate dose (0.05 mmol/kg, group IV, n = 5). Unlike in group I (high dose), in groups n and m (low dose), the LV curve had a well-defined first peak, followed by a downslope and a recirculation peak. With the intermediate dose (group IV), a saturation effect still remained on the LV curve. The signal intensity (SI) enhancement of the myocardium was respectively 580 ± 77% at 0.1 mmol/kg, 362 ± 95% at 0.05 mmol/kg, and at 0.01 mmol/kg, it was 184 ± 33% in group II and 272 ± 8% in group m. In conclusion, with subsecond T1-weighted MR imaging and a low dose of Gd chelate (i.e., 0.01 mmol/kg). the LV input function is a well-defined first step for MR perfusion modeling.     

Prospective comparison of MR lung perfusion and lung scintigraphy

This study attempted to assess the accuracy and potential of lung magnetic resonance (MR) perfusion imaging compared with perfusion scintigraphy in the evaluation of patients with suspected lung perfusion defects. The technique, which uses an inversion recovery turbo-FLASH sequence with ultra-short TE (1.4 msec), was tested in 24 patients suspected clinically of having acute pulmonary embolism (n = 19) and in patients with severe pulmonary emphysema (n = 5). Perfusion lung scintigraphy was performed within 48 hours prior to the MRI examination in both groups of patients. The dynamic study was acquired in the coronal plane and consisted of 10 images of 6 slices (a total of 60 images per series). Gadopentetate dimeglumine (0.1 mmol/kg) was manually injected as a compact bolus during the acquisition of the first image. Three senior radiologists reviewed all unprocessed two-dimensional coronal sections. They were blinded to clinical data and other imaging modalities. For the three observers, the average sensitivity and specificity of MR were 69% and 91%, respectively. The overall agreement between MR and scintigraphy appears to be good, with a good correlation between the two modalities (kappa = 0.63). However, the data showed variability depending on the location of the perfusion defect, with higher accuracy in the upper lobes. The agreement between MR perfusion and scintigraphy appears to be moderate in the left inferior lobe (kappa = 0.48). The data showed an overall good interobserver agreement (kappa = 0.66). MR perfusion of the lung is a promising technique in detecting lung perfusion defects.     

Plaque density on CT, a potential marker of ischemic stroke

The authors sought to determine in a retrospective analysis whether carotid plaque soft TD on CT is associated with recent ischemic neurologic events. Among 141 patients (99 asymptomatic), 106 plaques with more than 50% stenosis were selected for density measurements. They found an odds ratio for neurologic events associated with a 10-point decrease in density of 1.54 (p = 0.002), showing an association between plaque density and neurologic events.