Highly sensitive and quantitative HiBiT-tagged Nipah virus-like particles: A platform for rapid antibody neutralization studies.

Biocontainment regulations restrict the research on NiV to BSL-4 laboratories, thus limiting the mechanistic studies related to viral entry and allied pathogenesis. Understanding the precise process of viral-particle production and host cell entry is critical for designing targeted therapies or particle-based vaccines. In this study, we have synthesized HiBiT-tagged-NiV-VLPs to ease in-vitro BSL-2 particle handling. We propose a simple yet effective approach of generating substantial amount of HiBiT-tagged NiV-VLPs in vitro by co-expressing viral structural proteins in HEK293T cells. Though homologous to parent virus, the incapacitated replication potential facilitates a BSL-2 handling of these particles. The inclusion of a highly sensitive HiBiT tag on these VLPs allows for a quick detection of viral binding and entry, as well as in assessing the efficiency of neutralizing antibodies in vitro using the NanoBiT technology. The HiBiT-tag binds in high affinity with LgBiT (Large BiT an 18 kDa fusion protein and complementary subunit of HiBiT peptide), and the resultant complex elicits high intensity luminescence in the presence of substrate. The VLPs produced were morphologically and functionally identical to the native virus, and the HiBiT-tag permitted their quick application in viral binding, entry, and antibody neutralization assays. "Thus, we report a simple setting for generating HiBiT-NiV VLPs which can be utilized in a BSL-2 laboratory, to concurrently quantify features of NiV assembly, binding and entry. This also offers an alternate-safe and effective platform for viral based antibody neutralization assays in vitro".

Epstein-Barr virus infection controls the concentration of the intracellular antioxidant glutathione by upregulation of the glutamate transporter EAAT3 in tumor cells.

Epstein-Barr virus or human herpesvirus 4 (EBV/HHV-4) is an omnipresent oncovirus etiologically associated with various B-cell lymphomas and epithelial cancers. The malignant transformation associated with the persistent expression of viral proteins often deregulates the host cellular machinery and EBV infection is coupled to elevated levels of reactive oxygen species. Here, we investigated the role that the glutamate transporter EAAT3 plays in regulating the antioxidant system as a protective mechanism of EBV-infected cells against the virus-induced oxidative stress. Our study demonstrated that the expression of EAAT3 was upregulated and localized to the plasma membrane in EBV latently infected and de novo EBV-infected cells. EAAT3 was regulated by the transcription factor NFAT5 in the infected cells. Membrane localized EAAT3 was found to be involved in the transportation of glutamate from the extracellular space into the cell, as EAAT3 and NFAT5 inhibitors markedly reduced the levels of intracellular glutamate levels in EBV latently infected cells. Additionally, our data demonstrated a notable decrease in the intracellular glutathione levels following treatment with an EAAT3 inhibitor. Collectively, our results suggest that upregulation of the glutamate transporter EAAT3 is an adaptation of EBV-infected cells to maintain cellular redox homeostasis against the virus-induced oxidative stress, and that this cellular balance could be therapeutically destroyed by targeting EAAT3 to impede EBV-associated cancers.

Tissue Engineering of Cartilage Using Collagen Scaffold Enriched with Plant Polysaccharides.

Degenerative diseases associated with articular cartilage pose a huge burden on health care economics. The nature of the tissue involved and the changes therein do not allow self-healing; and most of these problems are progressive. Tissue engineering offers some solutions provided we focus on the right kind of cells and the appropriate surrounding niches created for a particular tissue. The present study deals with the formation of polysaccharide rich stable scaffold of collagen after cross-linking with oxidized gum arabic. The scaffold was tested for its biocompatibility and ability to support cells. The in vitro cytotoxicity of the scaffolds toward induced pluripotent stem cells and chondrocytes was evaluated. Evaluation of expression of lineage specific markers indicates differentiation of induced pluripotent stem cells to chondrogenic lineage and maintenance of chondrocytes per se when grown in the scaffold. Animal studies were carried out to study the efficacy of the scaffold to repair the knee injuries. Cells along with the scaffold appeared to be the best filling, in repair of injured cartilage. These studies show that these scaffolds are potential candidates in applications such as tissue engineering of cartilage.

Tissue engineering of collagen scaffolds crosslinked with plant based polysaccharides.

Ideally, a bioscaffold should mimic the characteristics of an extracellular matrix of a living organ of interest. The present study deals with the formation of composite scaffolds of collagen with gum arabic. Collagen was cross-linked with oxidized gum arabic having aldehyde groups to form a porous block. By changing the oxidation level of gum arabic, incorporation of the polysaccharides into the scaffold could be varied resulting in scaffolds with variable polysaccharide to protein content. A series of scaffolds were made by altering collagen concentration and oxidation level of gum arabic. The scaffolds were tested for their physical properties, stability, biocompatibility and ability to support the cell growth. Results implied that variable polysaccharide incorporation into the scaffolds was possible depending on the oxidation level of gum arabic which could influence the swelling behavior. The scaffolds showed non-toxic behavior towards the mesenchymal stem cells and nucleus pulposa cells using viability assay in culture conditions up to 30 days; the growth of cells was seen at all combinations of gels. Nucleus pulposa cells were able to maintain their phenotype in the GACO gels. The studies show that these scaffolds are potential candidates in applications, such as tissue engineering, and can be designed to match the requirement of different cell/tissues as per their ECM.

The Role of Virtual Histology Intravascular Ultrasound in the Identification of Coronary Artery Plaque Vulnerability in Acute Coronary Syndromes.

Markers of coronary plaque vulnerability, such as a high lipid burden, increased inflammatory activity, and a thin fibrous cap, have been identified in histological studies. In vivo, grayscale intravascular ultrasound (IVUS) provides more in-depth information on coronary artery plaque burden than conventional angiography but is unable to accurately distinguish between noncalcific tissue types within the plaque. An analysis of IVUS radiofrequency backscatter based on spectral pattern recognition, such as virtual histology IVUS, allows detailed scrutiny of plaque composition and classification of coronary lesions. This review discusses the virtual histology IVUS technology and its accuracy in identifying vulnerable plaque features, focusing on its use in predicting patient outcomes after acute coronary syndrome, and its limitations in clinical practice.

Ex vivo validation of 45 MHz intravascular ultrasound backscatter tissue characterization.

AIMS: The objectives of the present study are to describe the algorithm for VH(®) IVUS using the 45-MHz rotational IVUS catheter and the associated ex vivo validation in comparison to the gold standard histology. METHODS AND RESULTS: The first phase of the present study was to construct the 45 MHz VH IVUS algorithm by using a total of 55 human coronary artery specimens [111 independent coronary lesions and 510 homogenous regions of interest (ROIs)], obtained at autopsy. Regions were selected from histology and matched with their corresponding IVUS data to build the plaque classification system using spectral analysis and statistical random forests. In the second phase, the ex vivo validation of the VH IVUS algorithm assessed a total of 1060 ROIs (120 lesions from 60 coronary arteries) in comparison with histology. In an independent manner, two interventional cardiologists also classified a randomly selected subset of the ROIs for assessment of inter- and intra-observer reproducibility of VH IVUS image interpretation.When including all ROIs, the predictive accuracies were 90.8% for fibrous tissue, 85.8% for fibro fatty tissue, 88.3% for necrotic core, and 88.0% for dense calcium. The exclusion of ROIs in the acoustically attenuated areas improved the predictive accuracies, ranging from 91.9 to 96.8%. The independent analysis of randomly selected 253 ROIs showed substantial agreement for inter-observer (k = 0.66) and intra-observer (k = 0.88) reproducibility. CONCLUSION: Tissue classification by 45 MHz VH IVUS technology, when not influenced by calcium-induced acoustic attenuation, provided combined tissue accuracy >88% to identify tissue types compared with the gold standard histologic assessment, with high inter- and intra-observer reproducibility.

Reproducibility of intravascular ultrasound radiofrequency data analysis (virtual histology) with a 45-MHz rotational imaging catheter in ex vivo human coronary arteries.

BACKGROUND: Despite the frequent use of spectral analysis of intravascular ultrasound radiofrequency data (VH(®) IVUS) in clinical studies, the assessment for reproducibility using this with high frequency IVUS remains unexplored. PURPOSE: The aim of this study was to examine the reproducibility of VH IVUS using 45-MHz rotational IVUS in ex vivo human coronary arteries. METHODS: Data were collected using 45-MHz VH IVUS (Revolution(®), Volcano Corporation, San Diego, CA, USA) via a series of pullbacks from eight human coronary artery specimens. Imaging data were analyzed by two independent observers. Intraobserver and interobserver reproducibility were assessed using five pullbacks from five vessels. The intercatheter reproducibility was assessed using three different catheters in each of the five vessels. The intracatheter reproducibility was assessed between the two sequential pullbacks from each of the 15 catheters used in the intercatheter assessment. RESULTS: Geometrical measurements consistently showed low variability (relative difference <10%) and excellent intraclass correlation coefficients (ICCs), ranging from 0.88 to 1.00. With respect to the compositional measurements, the relative differences were predominantly higher than those of geometrical measurements. In particular, fibrous-fatty area showed a higher relative difference (17.5% in intercatheter assessment) compared to fibrous, necrotic core, and dense calcium areas (6.5%, 8.4%, and 6.4%, respectively). However, each compositional measurement also showed acceptable reproducibility (ICCs ranging from 0.82 to 1.00). CONCLUSIONS: The 45-MHz rotational VH IVUS technology had acceptable reproducibility with respect to geometrical and compositional assessments in ex vivo human coronary arteries. These data are crucial when designing future longitudinal studies addressing geometrical measurements and plaque characterization by 45-MHz VH IVUS.

Layer-dependent variation in the anisotropy of apparent integrated backscatter from human coronary arteries.

Clinical imaging of the coronary arteries in the cardiac catheterization laboratory using intravascular ultrasound (IVUS) is known to display a three-layered appearance, corresponding to the intima/plaque, media and adventitia. It is not known whether ultrasonic anisotropy arising from these tissues may alter this pattern in future IVUS systems that insonify in the forward direction or obliquely. In anticipation of such devices, the current study was carried out by imaging fresh human coronary arteries in two orthogonal directions in vitro. Twenty-six sites from 12 arteries were imaged with a side-looking IVUS system, and with an acoustic microscope both radially and axially. Side-looking IVUS and radial acoustic microscopy scans demonstrated the typical "bright-dark-bright" pattern of the backscatter, with the media being significantly darker than the other two layers. Images obtained in the axial orientation exhibited a markedly different pattern, with the relative brightness of the media significantly larger than that of the intima/plaque.

Tissue characterisation using intravascular radiofrequency data analysis: recommendations for acquisition, analysis, interpretation and reporting.

This document suggests standards for the acquisition, measurement, and reporting of radiofrequency data analysis (virtual histology - VH) intravascular ultrasound (IVUS) studies. Readers should view this document as the authors' best attempt in an area of rapidly evolving investigation, an area where rigorous evidence is not yet available or widely accepted. Nevertheless, this document is based on known pathologic data as well as previously reported imaging data; where practical, this data is summarised in the current document, a document which will also include recommendations for future evolution of the technology.

Intravascular ultrasound measures of coronary atherosclerosis are associated with the Framingham risk score: an analysis from a global IVUS registry.

AIMS: In addition to an adjunctive imaging platform during coronary angiography, intravascular ultrasound (IVUS) with Virtual Histology (VH) is increasingly being used to quantify coronary atherosclerosis. The relationship between VH-IVUS measures of coronary atherosclerosis and traditional cardiovascular risk factors has not been completely described. The objective of this study was to determine if an association exists between VH-IVUS measures of coronary atherosclerosis and the Framingham risk score in a prospective, multinational registry. METHODS AND RESULTS: Patients enrolled from 2004-2006 at 37 multinational centres in the prospective VHIVUS Global Registry were analysed. All subjects underwent diagnostic coronary angiography followed by IVUS. A Framingham risk score (FRS) was calculated for each subject, then stratified into three exclusive estimates (<10%, 10-19%, or >or= 20%) for future coronary heart disease (CHD) event risk over 10 years. Among 531 patients, plaque volume of the most diseased 10 mm segment increased with increasing FRS (P=0.006, adjusted for multiple comparisons). Patients with higher FRS estimates of CHD risk had a higher proportion of plaque classified as thin cap fibroatheroma compared with patients in the middle and lower risk score categories (21.4% vs 15.2% and 11.3%, respectively, P=0.008, adjusted for multiple comparisons). CONCLUSIONS: Using data from a large, multinational VH-IVUS registry we describe an association between the Framingham risk score and VH-IVUS measures of atherosclerosis within the most diseased 10 mm segment, namely plaque volume and the proportion of thin cap fibroatheroma.

Potential of dual-energy computed tomography to characterize atherosclerotic plaque: ex vivo assessment of human coronary arteries in comparison to histology.

BACKGROUND: Noninvasive characterization of coronary atherosclerotic plaque is limited with current computed tomography (CT) techniques. Dual-energy CT (DECT) has the potential to provide additional attenuation data for better differentiation of plaque components. OBJECTIVE: We attempted to characterize coronary atherosclerotic plaque with DECT. METHODS: Seven human coronary arteries acquired at autopsy were scanned consecutively at 80 and 140 kVp with CT. Vessels were perfused with saline, and data were acquired before and after contrast agent injection. Lesions were identified, and attenuation measurements were made from CT image quadrants. CT quadrants were classified as densely calcified, fibrocalcific, fibrous, lipid-rich, or normal vessel wall, corresponding to matched histology images. Attenuation values at each peak tube voltage were compared within plaque types for both noncontrast and contrast scans. Further, dual-energy index (DEI) values computed from attenuation were analyzed for classification of plaque. RESULTS: In 14 lesions, a total of 56 quadrants were identified. Histology results classified 8 (14%) as densely calcified, 8 (14%) as fibrocalcific, 9 (16%) as fibrous, 5 (9%) as lipid-rich, and 25 (45%) as normal vessel wall. Calcified lesions attenuated significantly more at 80 kVp in both contrast and noncontrast scans, whereas fibrous plaque attenuated more at 80 kVp only for contrast-enhanced scans. No differences were found for lipid-rich plaques. Using DEI values, only densely calcified plaques could be distinguished from other plaque types except fibrocalcific plaques in contrast images. CONCLUSIONS: Only densely calcified and fibrocalcific plaques showed a true change in attenuation at 80 versus 140 kVp. Therefore, calcified plaques could be distinguished from noncalcified plaques with DECT, but further classification of plaque types was not possible.

Normalization and backscatter spectral analysis of human carotid arterial data acquired using a clinical linear array ultrasound imaging system.

The risk of plaque rupture in carotid atherosclerotic disease is associated more closely with the composition of plaque rather than the severity of stenosis. The constituents of plaque can be determined from ultrasonic spectral parameters obtained from normalized backscatter tissue data. Calibration of the data is done using echoes off a specular reflector which removes the system response of an ultrasound transducer, Terason (Teratech Corporation), from the backscatter data. A reference spectrum study is used to compare specular reflectors based on time domain (echo) and frequency domain (power spectrum, centroid and parabola test) analysis. Nylon and a tissue-mimicking phantom (velocity = 1560 m/s, slope of attenuation = 0.7 dB/cm MHz) have an intermediate acoustic impedance with respect to water and appear good choices as specular reflectors for clinical ultrasound imaging scanners compared to Plexiglas and other higher reflecting materials. A tissue-mimicking phantom is used to correct for attenuation in plaque, diffraction and saturation of electronics of the ultrasound scanner. Autoregressive power spectrum estimation methods are used to extract spectral parameters (spectral slope, y-intercept, midband fit, maximum and minimum power with corresponding frequencies, and integrated backscatter) from calibrated tissue data and linear and quadratic discriminant rules developed for classification of carotid arterial plaque. Regions of interest (n = 64; 64 samples x 8 scan lines with 30 MHz sampling frequency) consisting of 48 fibrous-fibrofatty (Class 1), 11 thrombus-necrotic core (Class 2), and 5 dense calcium (Class 3) areas selected for analysis show that fibrosis can be differentiated from necrosis and calcification. The quadratic discriminant rule identified necrosis with a lower misclassification rate (9.1%) than the linear discriminant rule (18.2%).

Automated coronary plaque characterisation with intravascular ultrasound backscatter: ex vivo validation.

AIMS: Atherosclerosis is considered both a systemic and focal disease. Current diagnostic tools do not allow adequate in vivo identification and characterisation of lesions. Advanced spectral analysis of IVUS backscatter has displayed the potential for real-time plaque characterisation. The aim of this study is to determine the ex vivo accuracy of automated plaque characterisation by spectral analysis of intravascular ultrasound (IVUS) backscatter. METHODS AND RESULTS: Plaques (n=184) from 51 coronary arteries were imaged by IVUS. The arteries were then pressure fixed and matching histology collected. Regions were selected from histology and corresponding IVUS data were used to build the plaque classification system using spectral analysis and classi-fication trees. Tissue-maps were validated ex vivo by comparison with histology via 899 selected regions (n=94 plaques) that comprised 471 fibrous tissue (FT), 130 fibro-fatty (FF), 132 necrotic-core (NC) and 156 dense-calcium (DC) regions. The overall predictive accuracies were 93.5% for FT, 94.1% for FF, 95.8% for NC, and 96.7% for DC with sensitivities and specificities ranging from 72% to 99%. The Kappa statistic was calculated to be 0.845 indicating very high agreement with histology. CONCLUSIONS: Automated spectral analysis of IVUS backscatter provides accurate ex vivo information on plaque composition, with considerable potential for assessment of plaque vulnerability in real-time.

Contrast enhancement of coronary atherosclerotic plaque: a high-resolution, multidetector-row computed tomography study of pressure-perfused, human ex-vivo coronary arteries.

OBJECTIVES: The objective of this study was to investigate the effect of contrast injection on atherosclerotic coronary plaque attenuation measured using multidetector-row computed tomography. BACKGROUND: Recent multidetector-row computed tomography studies have described the characterization of coronary atherosclerotic plaque on the basis of Hounsfield unit values. The influence of contrast injection on the attenuation of individual plaque components, however, is unknown. METHODS: Using a pressurized perfusion system, 10 human coronary arteries were examined postmortem with multidetector-row computed tomography and histology. Pre-enhanced, peak-enhanced, and delayed enhanced multidetector-row computed tomography images were acquired during continuous perfusion of the vessel. A total of 37 focal atherosclerotic plaques were identified. Vessel wall attenuation was measured from multidetector-row computed tomography images during all three enhancement phases. On the basis of the histology, plaques were categorized as noncalcified (predominantly fibrous or predominantly fibrofatty), mixed calcified (calcified fibrous or calcified necrotic core), or densely calcified. The mean Hounsfield unit was compared among contrast phases for all plaques and in plaque subgroups. RESULTS: We observed contrast enhancement of atherosclerotic plaques within the vessel wall. For noncalcified plaques including both fibrous and fibrofatty plaques, the mean Hounsfield unit of the vessel wall during and after contrast injection exceeded the mean value before injection (t-test, P<0.002). CONCLUSION: The present study demonstrates that intra-arterial injection of iodinated contrast agent results not only in luminal enhancement but also in atherosclerotic plaque enhancement in pressure-perfused coronary arteries imaged ex vivo. Plaque enhancement should be considered when characterizing plaque components on the basis of Hounsfield unit with multidetector-row computed tomography.

Real-time plaque characterization and visualization with spectral analysis of intravascular ultrasound data.

Coronary artery disease is the number one cause of death in the United States and the Western world, and approximately 250,000 affected people die per year without ever being admitted to a hospital. One of the main reasons of such a high death-rate without any diagnosis is that more than 50 or heart-attacks) occur in patients with no prior history of known heart disease or symptoms. Coronary artery disease leads to the occlusion of arteries that are vital in providing nutrients to the heart muscles. The disease develops by progressive accumulation or formation of "plaque" within an artery. Certain types of plaques could occlude blood flow and yet might be "stable". These plaques usually have a high fibrous content, and are known as hard plaques. On the other hand, "unstable" or "soft" plaques might not cause much occlusion but could be vulnerable to rupture. Rupture of such plaques could lead to total or partial occlusion in arteries resulting in sudden cardiac death or heart-attack. In fact, 68 coronary arteries are less than 50.Intravascular ultrasound (IVUS) is a minimally invasive imaging modality that provides cross-section images of arteries in real-time, allowing visualization of atherosclerotic plaques in vivo. In standard IVUS gray-scale images, calcified regions of plaque and dense fibrous components generally reflect ultrasound energy well and thus appear bright and homogeneous on IVUS images. Conversely, regions of low echo reflectance in IVUS images are usually labeled "soft" or "mixed" plaque. However, this visual interpretation has been demonstrated to be very inconsistent in accurately determining plaque composition and does not allow real-time assessment of quantitative plaque constituents.Spectral analysis of the backscattered radiofrequency (RF) ultrasound signals allows detailed assessment of plaque composition. Advanced mathematical techniques can be employed to extract spectral information from these RF data to determine composition. The spectral content or signature of RF data reflected from tissue depends on density, compressibility, concentration, size, etc. A combination of spectral parameters were used to develop statistical classification schemes for analysis of in vivo IVUS data in real-time. The clinical data acquisition system is ECG gated and the analysis software developed by our group reconstructs IVUS gray-scale images from the acquired RF data. A combination of spectral parameters and active contour models is used for real-time 3D plaque segmentation followed by computation of color-coded tissue maps for each image cross-section and longitudinal views of the entire vessel. The "fly-through" mode allows one to visualize the complete length of the artery internally with the histology components at the lumen surface. In addition, vessel and plaque metrics such as areas and volumes of individual plaque components (collagen, fibro-lipid, calcium, lipid-core) are also available.

Regularized autoregressive analysis of intravascular ultrasound backscatter: improvement in spatial accuracy of tissue maps.

Autoregressive (AR) models are qualified for analysis of stochastic, short-time data, such as intravascular ultrasound (IVUS) backscatter. Regularization is required for AR analysis of short data lengths with an aim to increase spatial accuracy of predicted plaque composition and was achieved by determining suitable AR orders for short data records. Conventional methods of determining order were compared to the use of trend in the mean square error for determining order. Radio-frequency data from 101 fibrous, 56 fibro-lipidic, 50 calcified, and 70 lipid-core regions of interest (ROIs) were collected ex vivo from 51 human coronary arteries with 30 MHz unfocused IVUS transducers. Spectra were computed for AR model orders between 3-20 for data representing ROIs of two sizes (32 and 16 samples at 100 MHz sampling frequency) and were analyzed in the 17-42 MHz bandwidth. These spectra were characterized based on eight previously identified parameters. Statistical classification schemes were computed from 75% of the data and cross-validated with the remaining 25% using matched histology. The results determined the suitable AR order numbers for the two ROI sizes. Conventional methods of determining order did not perform well. Trend in the mean square error was identified as the most suitable factor for regularization of short record lengths.

Coronary plaque classification with intravascular ultrasound radiofrequency data analysis.

BACKGROUND: Atherosclerotic plaque stability is related to histological composition. However, current diagnostic tools do not allow adequate in vivo identification and characterization of plaques. Spectral analysis of backscattered intravascular ultrasound (IVUS) data has potential for real-time in vivo plaque classification. METHODS AND RESULTS: Eighty-eight plaques from 51 left anterior descending coronary arteries were imaged ex vivo at physiological pressure with the use of 30-MHz IVUS transducers. After IVUS imaging, the arteries were pressure-fixed and corresponding histology was collected in matched images. Regions of interest, selected from histology, were 101 fibrous, 56 fibrolipidic, 50 calcified, and 70 calcified-necrotic regions. Classification schemes for model building were computed for autoregressive and classic Fourier spectra by using 75% of the data. The remaining data were used for validation. Autoregressive classification schemes performed better than those from classic Fourier spectra with accuracies of 90.4% for fibrous, 92.8% for fibrolipidic, 90.9% for calcified, and 89.5% for calcified-necrotic regions in the training data set and 79.7%, 81.2%, 92.8%, and 85.5% in the test data, respectively. Tissue maps were reconstructed with the use of accurate predictions of plaque composition from the autoregressive classification scheme. CONCLUSIONS: Coronary plaque composition can be predicted through the use of IVUS radiofrequency data analysis. Autoregressive classification schemes performed better than classic Fourier methods. These techniques allow real-time analysis of IVUS data, enabling in vivo plaque characterization.