Sequence-specific detection of different strains of LCMV in a single sample using tentacle probes.

BACKGROUND: Virus infections often result in quasispecies of viral strains that can have dramatic impacts on disease outcomes. However, sequencing of viruses to determine strain composition is time consuming and often cost-prohibitive. Rapid, cost-effective methods are needed for accurate measurement of virus diversity to understand virus evolution and can be useful for experimental systems. METHODS: We have developed a novel molecular method for sequence-specific detection of RNA virus genetic variants called Tentacle Probes. The probes are modified molecular beacons that have dramatically improved false positive rates and specificity in routine qPCR. To validate this approach, we have designed Tentacle Probes for two different strains of Lymphocytic Choriomeningitis Virus (LCMV) that differ by only 3 nucleotide substitutions, the parental Armstrong and the more virulent Clone-13 strain. One of these mutations is a missense mutation in the receptor protein GP1 that leads to the Armstrong strain to cause an acute infection and Clone-13 to cause a chronic infection instead. The probes were designed using thermodynamic calculations for hybridization between target or non-target sequences and the probe. RESULTS: Using this approach, we were able to distinguish these two strains of LCMV individually by a single nucleotide mutation. The assay showed high reproducibility among different concentrations of viral cDNA, as well as high specificity and sensitivity, especially for the Clone-13 Tentacle Probe. Furthermore, in virus mixing experiments we were able to detect less than 10% of Clone-13 cDNA diluted in Armstrong cDNA. CONCLUSIONS: Thus, we have developed a fast, cost-effective approach for identifying Clone-13 strain in a mix of other LCMV strains.

Heterobivalent ligands target cell-surface receptor combinations in vivo.

A challenge in tumor targeting is to deliver payloads to cancers while sparing normal tissues. A limited number of antibodies appear to meet this challenge as therapeutics themselves or as drug-antibody conjugates. However, antibodies suffer from their large size, which can lead to unfavorable pharmacokinetics for some therapeutic payloads, and that they are targeted against only a single epitope, which can reduce their selectivity and specificity. Here, we propose an alternative targeting approach based on patterns of cell surface proteins to rationally develop small, synthetic heteromultivalent ligands (htMVLs) that target multiple receptors simultaneously. To gain insight into the multivalent ligand strategy in vivo, we have generated synthetic htMVLs that contain melanocortin (MSH) and cholecystokinin (CCK) pharmacophores that are connected via a fluorescent labeled, rationally designed synthetic linker. These ligands were tested in an experimental animal model containing tumors that expressed only one (control) or both (target) MSH and CCK receptors. After systemic injection of the htMVL in tumor-bearing mice, label was highly retained in tumors that expressed both, compared with one, target receptors. Selectivity was quantified by using ex vivo measurement of Europium-labeled htMVL, which had up to 12-fold higher specificity for dual compared with single receptor expressing cells. This proof-of-principle study provides in vivo evidence that small, rationally designed bivalent htMVLs can be used to selectively target cells that express both, compared with single complimentary cell surface targets. These data open the possibility that specific combinations of targets on tumors can be identified and selectively targeted using htMVLs.

Antimicrobial distribution from local delivery depends on dose : a pilot study with MRI.

BACKGROUND: Tissue distribution after local delivery has been quantified over a period of 5 hours on 7-T MRI in a rabbit model using gadolinium-labeled diethylenetriamine pentaacetic acid (Gd-DTPA) as an antimicrobial surrogate; however, it is unknown how the Gd-DTPA load in a local depot will affect the duration of high-concentration Gd-DTPA in local tissues after surgical débridement. QUESTIONS/PURPOSES: We determined whether the Gd-DTPA load in bone cement affected its local tissue distribution over a period of 1 month after local delivery. METHODS: A 1-cm3 soft tissue dead space was created in the quadriceps of seven rabbits and filled with gadolinium-loaded bone cement. At 7, 14, and 33 days, the volume of tissue with a Gd-DTPA concentration of more than 14 µg/mL was calculated from T1-weighted images using 7-T MRI. Differences in volumes of distribution were analyzed with ANOVA. RESULTS: The volume of tissue with more than 14 µg/mL Gd-DTPA was much larger from higher gadolinium loads on Day 7 (p=0.02) (2121 mm3 for 10 g and 665 mm3 for 1 g) and smaller with time for the 10-g formulation (2121 mm3 on Day 7 and 1241 mm3 on Day 14). CONCLUSIONS: Volume of distribution and duration of Gd-DTPA after local delivery increased with increasing load in the cement and decreased with time. CLINICAL RELEVANCE: For local delivery, high antimicrobial concentrations would be expected in greater volumes of tissue, for longer durations, when higher antimicrobial loads are used.

Jeannette Wilkins Award: Can locally delivered gadolinium be visualized on MRI? A pilot study.

BACKGROUND: Management of orthopaedic infections relies on débridement and local delivery of antimicrobials; however, the distribution and concentration of locally delivered antimicrobials in postdébridement surgical sites is unknown. Gadolinium-DTPA (Gd-DTPA) has been proposed as an imaging surrogate for antimicrobials because it is similar in size and diffusion coefficient to gentamicin. QUESTIONS/PURPOSES: Is in vivo distribution of locally delivered Gd-DTPA (1) visible on MRI; (2) reliably visualized by different observers; (3) affected by the anatomic delivery site; and (4) affected by the in vitro release rate from the delivery vehicle? METHODS: Twenty-four local delivery depots were imaged in nine rabbits using two anatomic sites (intramedullary canal, quadriceps) with Gd-DTPA in intermediate-porosity polymethylmethacrylate (PMMA) or high-porosity PMMA; six of the nine rabbits also had Gd-DTPA delivered in collagen at a third site (hamstring). A total of 45,000 fat-suppressed T1-weighted RARE scans were acquired using a 7-T Bruker Biospec MRI: nine rabbits, 2-mm slices over 10 cm, four TR values, 25 time periods (pre, every 15 minutes for 6 hours). T1 maps were constructed at every time period. Gd-DTPA distribution was observed qualitatively on the T1 maps. Interobserver reliability was determined. RESULTS: Locally delivered Gd-DTPA was visible. Interobserver agreement was excellent. Intramuscular delivery followed intermuscular planes; intramedullary delivery was contained within the canal by bone. Distribution from collagen decreased after 1 hour but from PMMA increased over 6 hours. CONCLUSIONS: Locally delivered Gd-DTPA can be visualized on MRI; distribution is affected by anatomical location and delivery vehicle. CLINICAL RELEVANCE: Contrast-based imaging using locally delivered Gd-DTPA may be useful as an antibiotic surrogate to determine antibiotic distribution in surgical sites.

Stromal Cell-Derived Factor-1a Autocrine/Paracrine Signaling Contributes to Spatiotemporal Gradients in the Brain.

INTRODUCTION: Stromal cell derived factor-1a (SDF-1a) and its receptor CXCR4 modulate stem cell recruitment to neural injury sites. SDF-1a gradients originating from injury sites contribute to chemotactic cellular recruitment. To capitalize on this injury-induced cell recruitment, further investigation of SDF-1a/CXCR4 signaling dynamics are warranted. Here, we studied how exogenous SDF-1a delivery strategies impact spatiotemporal SDF-1a levels and the role autocrine/paracrine signaling plays. METHODS: We first assessed total SDF-1a and CXCR4 levels over the course of 7 days following intracortical injection of either bolus SDF-1a or SDF-1a loaded nanoparticles in CXCR4-EGFP mice. We then investigated cellular contributors to SDF-1a autocrine/paracrine signaling via time course in vitro measurements of SDF-1a and CXCR4 gene expression following exogenous SDF-1a application. Lastly, we created mathematical models that could recapitulate our in vivo observations. RESULTS: In vivo, we found sustained total SDF-1a levels beyond 3 days post injection, indicating endogenous SDF-1a production. We confirmed in vitro that microglia, astrocytes, and brain endothelial cells significantly change SDF-1a and CXCR4 expression after exposure. We found that diffusion-only based mathematical models were unable to capture in vivo SDF-1a spatial distribution. Adding autocrine/paracrine mechanisms to the model allowed for SDF-1a temporal trends to be modeled accurately, indicating it plays an essential role in SDF-1a sustainment. CONCLUSIONS: We conclude that autocrine/paracrine dynamics play a role in endogenous SDF-1a levels in the brain following exogenous delivery. Implementation of these dynamics are necessary to improving SDF-1a delivery strategies. Further, mathematical models introduced here may be utilized in predicting future outcomes based upon new biomaterial designs.

Simplified synthesis and relaxometry of magnetoferritin for magnetic resonance imaging.

Magnetoferritin nanoparticles have been developed as high-relaxivity, functional contrast agents for MRI. Several previous techniques have relied on unloading native ferritin and re-incorporation of iron into the core, often resulting in a polydisperse sample. Here, a simplified technique is developed using commercially available horse spleen apoferritin to create monodisperse magnetoferritin. Iron oxide atoms were incorporated into the protein core via a step-wise Fe(II)Chloride addition to the protein solution under low O(2) conditions; subsequent filtration steps allow for separation of completely filled and superparamagnetic magnetoferritin from the partially filled ferritin. This method yields a monodisperse and homogenous solution of spherical particles with magnetic properties that can be used for molecular magnetic resonance imaging. With a transverse per-iron and per-particle relaxivity of 78 mM(-1) sec(-1) and 404,045 mM(-1) sec(-1), respectively, it is possible to detect ∼ 10 nM nanoparticle concentrations in vivo.

Two-step synthesis of multivalent cancer-targeting constructs.

Selective targeting of constructs to pathological cells by conjugating one or more ligands for an overexpressed receptor has been proposed to enhance the delivery of therapeutics to and imaging of specific cells of interest. Previous work in our lab has demonstrated the efficacy of targeting glioblastoma cells with a multivalent, biomacromolecular construct targeted to the alpha(6)beta(1)-integrin. However, solid-phase synthesis of this construct was inefficient in terms of cost and number of steps. Here we show proof-of-concept of a two-step synthesis that can be used to create similar constructs targeted to glioblastoma cells. Specifically, a well-defined aldehyde side chain polymer was synthesized and oxime chemistry was employed to conjugate ligands specific for the alpha(6)beta(1)-integrin. These constructs were then tested in competitive binding, fluorescence binding, and toxicity assays, through which we demonstrate that constructs are multivalent, preferentially target glioblastoma cells, and are nontoxic. Rapid, potentially low-cost synthesis of targeting constructs will enable their use in the clinic and for personalized medicine.

Tentacle probe sandwich assay in porous polymer monolith improves specificity, sensitivity and kinetics.

Nucleic acid sandwich assays improve low-density array analysis through the addition of a capture probe and a specific label, increasing specificity and sensitivity. Here, we employ photo-initiated porous polymer monolith (PPM) as a high-surface area substrate for sandwich assay analysis. PPMs are shown to enhance extraction efficiency by 20-fold from 2 microl of sample. We further compare the performance of labeled linear probes, quantum dot labeled probes, molecular beacons (MBs) and tentacle probes (TPs). Each probe technology was compared and contrasted with traditional hybridization methods using labeled sample. All probes demonstrated similar sensitivity and greater specificity than traditional hybridization techniques. MBs and TPs were able to bypass a wash step due to their 'on-off' signaling mechanism. TPs demonstrated reaction kinetics 37.6 times faster than MBs, resulting in the fastest assay time of 5 min. Our data further indicate TPs had the most sensitive detection limit (<1 nM) as well as the highest specificity (>1 x 10(4) improvement) among all tested probes in these experiments. By matching the enhanced extraction efficiencies of PPM with the selectivity of TPs, we have created a format for improved sandwich assays.

Glioblastoma targeting via integrins is concentration dependent.

A novel approach to treat cancer more selectively is achieved by targeting drugs to cells via conjugating the drug or imaging agent to an antibody or ligand for a cell surface receptor that is over-expressed by the target cell population. Previous work by us has suggested that enhanced specificity can be obtained by multivalency of binding moieties. In this study we investigated the binding specificity of a multivalent construct including three peptides segments (TWYKIAFQRNRK), which bind the alpha(6)beta(1)-integrin, linked by poly(ethylene glycol) spacers. The binding specificity of the constructs was calculated by quantifying their binding to target cells (glioma cells, SF 767) relative to non-targeted cells (normal human astrocytes, NHA). Dodecapeptide constructs (monovalent) exhibit specificity equal to the ratio of receptor expression at all concentrations. However, trivalent constructs demonstrated a sharp increase in specificity at concentrations less than the affinity of the receptor-ligand bond (4.28 microM). These experiments (conducted at 4 degrees C) were consistent with the theoretical prediction and indicate that the biophysical model captures the basic trend of the data in the absence of receptor internalization, although the concentration at which increased specificity is observed is greater than predicted. The biophysical model does not predict the results of 37 degrees C experiments, and this is shown to be due to internalization which occurs at 37 degrees C but not at 4 degrees C.

Tentacle probes: eliminating false positives without sacrificing sensitivity.

The majority of efforts to increase specificity or sensitivity in biosensors result in trade-offs with little to no gain in overall accuracy. This is because a biosensor cannot be more accurate than the affinity interaction it is based on. Accordingly, we have developed a new class of reagents based on mathematical principles of cooperativity to enhance the accuracy of the affinity interaction. Tentacle probes (TPs) have a hairpin structure similar to molecular beacons (MBs) for enhanced specificity, but are modified by the addition of a capture probe for increased kinetics and affinity. They produce kinetic rate constants up to 200-fold faster than MB with corresponding stem strengths. Concentration-independent specificity was observed with no false positives at up to 1 mM concentrations of variant analyte. In contrast, MBs were concentration dependent and experienced false positives above 3.88 muM of variant analyte. The fast kinetics of this label-free reagent may prove important for extraction efficiency, hence sensitivity and detection time, in microfluidic assays. The concentration-independent specificity of TPs may prove extremely useful in assays where starting concentrations and purities are unknown as would be the case in bioterror or clinical point of care diagnostics.

Effects of linker length and flexibility on multivalent targeting.

Increasing valence can enhance the ability of molecular targeting constructs to bind specifically to targeted cells for drug delivery. Here, we mathematically model the length and flexibility of a linker used to conjoin two peptide ligands of a divalent targeting construct and investigate the influence both on binding avidity and specificity. Four different models are used to approximate varying degrees of linker flexibility (random coil, rigid rod, jointed rods, and combined rod-random coil) and for each linker a binding enhancement factor (VR) is derived that quantifies the increased rate of each construct's second binding event over the first. Results indicate that the moderately flexible models can best reproduce experimentally measured avidities. Also, the magnitude of VR, in conjunction with receptor density and ligand concentration, significantly influences the achievable specificity. Thus, the model elucidates important considerations in designing multivalent targeting constructs for use in delivery of targeted therapy or imaging.

Temporal and spatial control of neural effects following intracerebral microinfusion.

Spatial and temporal control of neural drug delivery is critical for many therapeutic applications and analyses of brain patterns and behavior. Specifically, for localized injections that serve to deliver drug or inactivate an isolated tissue region in order to observe changes in neural activity at that site, excess distribution into surrounding regions may confound analysis or adversely affect healthy tissue. Here, we develop a mass transport model that simulates a short period of initial infusion of inactivating drug, followed by a successive convective wash with artificial cerebrospinal fluid (aCSF), while tracking the regions of tissue that are above a certain threshold concentration of inactivating agent. We analyze the effect of parameters such as effective diffusion coefficient, extracellular volume fraction, and injectate concentration upon spatiotemporal distribution profiles. Further, we observe the effects of following the initial injection with a wash-out period with aCSF upon the breadth of the volume affected by the injectate. These simulations indicate that, by injecting small volumes of drug at low concentrations and following them with an aCSF flush, a well-delineated region of tissue can be altered for a controlled duration.

In Vitro Validation of Targeting and Comparison to Mathematical Modeling.

Nanoparticle and other drug delivery platforms have demonstrated promising potential for the delivery of therapeutics or imaging agents in a specific and targeted manner. While a variety of drug delivery platforms have been applied to medicine, in vitro and in silico optimization and validation of these targeting constructs needs to be conducted to maximize in vivo delivery and efficacy. Here, we describe the mathematical and experimental models to predict and validate the transport of a peptide targeting construct through a mock tissue environment to specifically target tumor cells, relative to non-tumor cells. We provide methods to visualize and analyze fluorescence microscopy images, and also describe the methods for creating a finite element model (FEM) that validates important parameters of this experimental system. By comparing and contrasting mathematical modeling results with experimental results, important information can be imparted to the design and functionality of the targeting construct. This information will help to optimize construct design for future therapeutic delivery applications.

Tandem Histone-Binding Domains Enhance the Activity of a Synthetic Chromatin Effector.

Fusion proteins that specifically interact with biochemical marks on chromosomes represent a new class of synthetic transcriptional regulators that decode cell state information rather than DNA sequences. In multicellular organisms, information relevant to cell state, tissue identity, and oncogenesis is often encoded as biochemical modifications of histones, which are bound to DNA in eukaryotic nuclei and regulate gene expression states. We have previously reported the development and validation of the "polycomb-based transcription factor" (PcTF), a fusion protein that recognizes histone modifications through a protein-protein interaction between its polycomb chromodomain (PCD) motif and trimethylated lysine 27 of histone H3 (H3K27me3) at genomic sites. We demonstrated that PcTF activates genes at methyl-histone-enriched loci in cancer-derived cell lines. However, PcTF induces modest activation of a methyl-histone associated reporter compared to a DNA-binding activator. Therefore, we modified PcTF to enhance its binding avidity. Here, we demonstrate the activity of a modified regulator called Pc2TF, which has two tandem copies of the H3K27me3-binding PCD at the N-terminus. Pc2TF has a smaller apparent dissociation constant value in vitro and shows enhanced gene activation in HEK293 cells compared to PcTF. These results provide compelling evidence that the intrinsic histone-binding activity of the PCD motif can be used to tune the activity of synthetic histone-binding transcriptional regulators.

Specificity and mobility of biomacromolecular, multivalent constructs for cellular targeting.

Effective targeting of drugs to cells requires that the drug reach the target cell and interact specifically with it. In this study, we synthesized a biomacromolecular, multivalent construct intended to target glioblastoma tumors. The construct was created by linking three dodecapeptides, reported to bind the alpha 6beta1 integrin, with poly(ethylene glycol) linkers. The construct is intended to be delivered locally, and it demonstrates a more homogeneous and more rapid perfusion profile in comparison with quantum dots. The binding specificity of the construct was investigated by using glioblastoma cells and normal human astrocyte cells. The results reveal qualitative differences in binding between glioma and normal human astrocyte cells, with a moderate increase in binding avidity due to multivalency (0.79 microM for the trivalent construct versus 4.28 microM for the dodecapeptide). Overall, biomacromolecular constructs appear to be a promising approach for targeting with high biocompatibility, good perfusion abilities, and specificity.

Bioactive surface for neural electrodes: decreasing astrocyte proliferation via transforming growth factor-beta1.

Implantation of deep-brain recording devices is a traumatic event, which inevitably elicits reactive gliosis. The ensuing glial scar encapsulating the implanted device impedes the long-term functional recording capability of the microelectrode. In this work, a bioactive surface is prepared by conjugation of transforming growth factor-beta one (TGF-beta1) and laminin to dextran, which is in turn conjugated to a biomaterial substrate. Poly-L-lysine coated surfaces are treated with oxidized dextran, and the dextran is re-oxidized with sodium metaperiodate to generate hemiacetal structures to which TGF-beta1 and laminin are covalently bound. Covalent conjugation of the ligand is confirmed by enzyme-linked immunosorbent assay. A primary cell line of astrocytes is incubated on a surface conjugated with laminin and TGF-beta1 and a surface only conjugated with laminin. Proliferation on the laminin plus TGF-beta1 surface is 57% less (p < 0.002) than the control surface (laminin alone). The results demonstrate that conjugated TGF-beta1 retains its efficacy toward astrocyte proliferation and represents a potential strategy for reducing glial scar formation in vivo.

Control of self-assembling oligopeptide matrix formation through systematic variation of amino acid sequence.

In order to elucidate design principles for biocompatible materials that can be created by in situ transformation from self-assembling oligopeptides, we investigate a class of oligopeptides that can self-assemble in salt solutions to form three-dimensional matrices. This class of peptides possesses a repeated sequence of amino acid residues with the type: hydrophobic/negatively-charged/hydrophobic/positively-charged. We systematically vary three chief aspects of this sequence type: (1) the hydrophobic side chains: (2) the charged side-chains: and (3) the number of repeats. Employing a rheometric assay to judge matrix formation, we determine the critical concentration of NaCl salt solution required to drive transformation from viscous state to gel state. We find that increasing side-chain hydrophobicity decreases the critical salt concentration in accord with our previous validation of DLVO theory for explaining this self-assembly phenomenon Caplan et al. (Biomacromolecules 1 (2000) 627). Further, we find that increasing the number of repeats yields a biphasic dependence-first decreasing, then increasing, the critical salt concentration. We believe that this result is likely due to an unequal competition between a greater hydrophobic (favorable) effect and a greater entropic (unfavorable) effect as the peptide length is increased. Finally, we find that we can use this understanding to rationally alter the charged side-chains to create a self-assembling oligopeptide sequence that at pH 7 remains viscous in the absence of salt but gels in the presence of physiological salt concentrations, a highly useful property for technological applications.

The role of SDF-1α-ECM crosstalk in determining neural stem cell fate.

The consequences of central nervous system injury are far-reaching and debilitating and, while an endogenous repair response to neural injury has been observed in recent years, the mechanisms behind this response remain unclear. Neural progenitor/stem cell (NPSC) migration to the site of injury from the neural stem cell niches (e.g. subventricular zone and hippocampus) has been observed to be vasophilic in nature. While the chemotactic stimuli directing NPSC homing to injury is not well established, it is thought to be due in part to an increasing gradient of chemotactic cytokines, such as stromal cell-derived factor 1α (SDF-1α). Based on these recent findings, we hypothesize that critical crosstalk between SDF-1α and the extracellular matrix (ECM) drives injury-induced NPSC behavior. In this study, we investigated the effect of SDF-1α and ECM substrates (Matrigel, laminin, and vitronectin) on the migration, differentiation, and proliferation of NPSCs in vitro using standard assays. The results demonstrated that SDF-1α and laminin-based ECM (Matrigel and laminin) significantly and synergistically enhanced NPSC migration and acute neuronal differentiation. These effects were significantly attenuated with the addition of AMD3100 (an antagonist against the SDF-1α receptor, CXCR4). SDF-1α alone significantly increased NPSC proliferation regardless of ECM substrate, however no synergy was observed between SDF-1α and the ECM. These results serve to elucidate the relationship between adhesive and soluble signaling factors of interest and their effect on NPSC behavior following neural injury. Furthermore, these results better inform the next generation of biomaterials aimed at stimulating endogenous neural regeneration for neural injury and neurodegenerative diseases.

Distribution of molecules locally delivered from bone cement.

Revision of infected orthopedic implants is successful in most cases when antimicrobials are delivered locally (mixed with bone cement or bone graft which is placed in the site from which the infected tissue was removed); however, there is still a substantial rate of recurrence most likely due to the antimicrobials not achieving a therapeutic dose at all locations in the tissue. To study transport within this environment, gadolinium chelated in diethylene triamine pentaacetic acid (Gd-DTPA), a MRI contrast agent with size and solubility similar to two common antimicrobials (gentamicin and vancomycin), was mixed with bone cement, implanted in vivo into two models of orthopedic surgical wounds, and imaged using MRI 5.5 h after implantation. Image thresholding was used to create two-dimensional and three-dimensional representations of areas/volumes containing detectable concentrations of Gd-DTPA. Distribution is found to be anisotropic with Gd-DTPA transporting preferentially anterior of the implant toward the skin. When fascia is not closed over the implant site, Gd-DTPA transports to the skin and along the subcutaneous plane. The distance transported indicates that transport is likely driven by convection. Finally, the tissue concentration of Gd-DTPA is much less than the concentration loaded into the bone cement.

Spatiotemporal quantification of local drug delivery using MRI.

Controlled release formulations for local, in vivo drug delivery are of growing interest to device manufacturers, research scientists, and clinicians; however, most research characterizing controlled release formulations occurs in vitro because the spatial and temporal distribution of drug delivery is difficult to measure in vivo. In this work, in vivo magnetic resonance imaging (MRI) of local drug delivery was performed to visualize and quantify the time resolved distribution of MRI contrast agents. Three-dimensional T1 maps (generated from T1-weighted images with varied TR) were processed using noise-reducing filtering. A segmented region of contrast, from a thresholded image, was converted to concentration maps using the equation 1/T1=1/T1,0+R1C, where T1,0 and T1 are the precontrast and postcontrast T1 map values, respectively. In this technique, a uniform estimated value for T 1,0 was used. Error estimations were performed for each step. The practical usefulness of this method was assessed using comparisons between devices located in different locations both with and without contrast. The method using a uniform T1,0, requiring no registration of pre- and postcontrast image volumes, was compared to a method using either affine or deformation registrations.