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The ability to study the microcirculation in real time is key to elucidating how the immune system and the associated microvasculature interact and influence one another within the lymph node (LN). Here, we present a method for near in-situ imaging of the inguinal LN. In particular, this method is ideal for the assessment of overall vascular health that influences immune functions and for the evaluation of T-cell motility. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, quantification of vessel magnitude and vessel integrity. Modified intravital microscopy (M-IVM) of the LNs allows direct evaluation of microvascular functions as well as real-time imaging of the direct interface between immune cells, the LN, and the microcirculation. Importantly, M-IVM technique can be readily combined with many other vascular and immunological techniques such as fluorescent cell labeling and assessment of sticking and rolling time as descripted. Furthermore, it can be adapted to study vasculature of other than the inguinal LN. Overall, this chapter provides a dependable method for fundamental vascular immunological assessment of LNs that is decidedly useful in a diverse range of investigations.

Objectives: This study investigated processes causing leaflet thickening and structural valve degeneration (SVD)., Background: Although transcatheter aortic valve replacement (TAVR) has changed the treatment of aortic stenosis, concerns remain regarding SVD, potentially related to valve thrombosis and thickening, based on studies using computed tomography (CT). Detailed histological analyses are provided to help attain insights into these processes., Methods: Explanted transcatheter heart valves (THVs) were evaluated for thrombosis, fibrosis, and calcification for quantification of leaflet thickness. Immunohistochemical and microscopy approaches were used to investigate SVD-associated mechanisms., Results: THVs (n = 23) were obtained from 22 patients (median 81 years of age; 50% male) from 0 to 2,583 days post TAVR. Maximal leaflet thickness increased relative to implant duration (ρ = 0.427; p = 0.027). THVs explanted after >2 years were thicker than those explanted after <2 years (p = 0.007). All THVs had adherent thrombus on both aortic and ventricular sides, which beyond 60 days was seen in combination with fibrosis and beyond 4 years had calcification. Early thrombus formation (<60 days) occurred despite rapid endothelialization with an abnormal hyperplastic phenotype. Fibrosis was observed in 6 patients on both the aortic and the ventricular THV surfaces, remodeled over time, and was associated with matrix metalloproteinase-1 expression. Five THVs showed overt calcification associated with adherent thrombus and fibrosis., Conclusions: There is a time-dependent degeneration of THVs consisting of thrombus formation, endothelial hyperplasia, fibrosis, tissue remodeling, proteinase expression, and calcification. Future investigation is needed to further understand these mechanisms contributing to leaflet thickening and SVD., (Copyright © 2019 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Introduction: Angina, myocardial ischemia, and coronary artery physiology in hypertrophic cardiomyopathy (HCM) are poorly understood. However, coronary computed tomography angiography (CCTA) with fractional flow reserve from CT (FFR CT ) analysis offers a non-invasive method for evaluation of coronary artery volume to myocardial mass ratio (V/M) that may provide insight into such mechanisms. Thus, we sought to investigate changes in V/M in HCM., Methods: A retrospective analysis was performed on 37 HCM patients and 37 controls matched for age, sex, and cardiovascular risk factors; CCTA-derived coronary artery lumen volume (V) and myocardial mass (M) were used to determine V/M. FFR CT values were calculated for the left anterior descending (LAD), left circumflex (LCx) and right coronary (RCA) arteries as well as the 3-vessel cumulative FFR CT values., Results: HCM patients had significantly increased myocardial mass (176 ± 84 vs. 119 ± 27 g, p < 0.0001) and total coronary artery luminal volume (4112 ± 1139 vs. 3290 ± 924 mm 3 , p < 0.0001) that resulted from increases in segmented luminal volumes of both the left and right coronary artery systems. However, HCM patients had significantly decreased V/M (23.8 ± 5.9 vs. 26.5 ± 5.3 mm 3 /g; p = 0.026) which was further decreased when restricting V/M analysis to those HCM patients with septal hypertrophy (22.4 mm 3 /g, p = 0.01) that was mild-moderately predictive of HCM (AUC = 0.68). HCM patients also showed significantly lower nadir FFR CT values in the LCx (0.87 ± 0.06 vs. 0.91 ± 0.06, p = 0.02), and cumulative 3-vessel FFR CT values (2.58 ± 0.18 vs. 2.63 ± 0.14, p = 0.006)., Conclusions: HCM patients demonstrate significantly greater coronary volume. Despite this, HCM patients suffer from decreased V/M. Further prospective studies evaluating the relationship between V/M, angina, and heart failure in HCM are needed., (Copyright © 2018 Society of Cardiovascular Computed Tomography. Published by Elsevier Inc. All rights reserved.)

Magnetic resonance spectroscopy (MRS) can be performed in vivo using commercial MRI systems to obtain biochemical information about tissues and cancers. Applications in brain, prostate and breast aid lesion detection and characterisation (differential diagnosis), treatment planning and response assessment. Multi-centre clinical trials have been performed in all these tissues. Single centre studies have been performed in many other tissues including cervix, uterus, musculoskeletal and liver. While generally MRS is used to study endogenous metabolites it has also been used in drug studies, for example those that include 19 F as part of their structure. Recently the hyperpolarisation of compounds enriched with 13 C such as [1- 13 C] pyruvate has been demonstrated in animal models and now in preliminary clinical studies, permitting the monitoring of biochemical processes with unprecedented sensitivity. This review briefly introduces the underlying methods and then discusses the current status of these applications.

Progressive limb and girdle muscle atrophy leading to loss of ambulation is a hallmark of dysferlinopathies, which include limb-girdle muscular dystrophy type 2B and Miyoshi myopathy. However, animal models fail to fully reproduce the disease severity observed in humans, with dysferlin-null (Dysf -/- ) mice exhibiting minor muscle damage and weakness without dramatic ambulatory dysfunction. As we have previously reported significant Dysf expression in blood vessels, we investigated the role of vascular function in development of muscle pathology by generating a Dysf-deficient mouse model with vascular disease. This was achieved by crossing Dysf -/- mice with ApoE -/- mice, which have high levels of nonHDL-associated cholesterol. Double-knockout Dysf -/- ApoE -/- mice exhibited severe ambulatory dysfunction by 11 months of age. In limb-girdle muscles, histology confirmed dramatic muscle wasting, fibrofatty replacement, and myofiber damage in Dysf -/- ApoE -/- mice without affecting the ratio of centrally nucleated myofibers. Although there were no major changes in ex vivo diaphragm and soleus muscle function, histological analyses revealed these muscles to be untouched by damage and remodelling. In all, these data suggest that cholesterol may be deleterious to dysferlinopathic muscle and lead to ambulatory dysfunction. Moreover, differences in plasma lipid handling between mice and humans could be a key factor affecting dysferlinopathy severity., (Copyright © 2018 by the American Society for Biochemistry and Molecular Biology, Inc.)

Wild mushrooms, especially from North America, have not been systematically explored for their medicinal properties. Here we report screening for the growth-inhibitory and immunomodulatory activities of 12 species collected from multiple locations in north-central British Columbia, Canada. Mushrooms were characterized using morphology and DNA sequencing, followed by chemical extraction into 4 fractions using 80% ethanol, 50% methanol, water, and 5% sodium hydroxide. Growth-inhibitory, immunostimulatory, and anti-inflammatory activities of 5 mushrooms (Leucocybe connata, Trichaptum abietinum, Hydnellum sp., Gyromitra esculenta, and Hericium coralloides) are reported here, to our knowledge for the first time. Growth-inhibitory effects were assessed using the cytotoxic MTT assay. Immunostimulatory activity was assessed by tumor necrosis factor-α production in Raw 264.7 macrophages, whereas anti-inflammatory activity was assessed based on the inhibition of lipopolysaccharide-induced tumor necrosis factor-α production. The ethanol and aqueous extracts of Hydnellum sp. were potent growth inhibitors, with a half-maximal inhibitory concentration of 0.6 mg/mL. All 5 fungi displayed strong immunostimulatory activity, whereas only L. connata and T. abietinum showed strong anti-inflammatory activity. For the 7 other fungi investigated, which included well-known medicinal species such as Inonotus obliquus, Phellinus igniarius, and Ganoderma applanatum, the remarkable similarities in the biological activities reported here, and by others for specimens collected elsewhere, suggest that mushrooms can produce similar metabolites regardless of their habitat or ecosystem. This is to our knowledge the first study to explore wild mushrooms from British Columbia for biological activities that are relevant to cancer, and the results provide an initial framework for the selection of mushroom species with the potential for discovery of novel anticancer compounds.

Multi-parametric magnetic resonance imaging (mpMRI) offers a unique insight into tumor biology by combining functional MRI techniques that inform on cellularity (diffusion-weighted MRI), vascular properties (dynamic contrast-enhanced MRI), and metabolites (magnetic resonance spectroscopy) and has scope to provide valuable information for prognostication and response assessment. Challenges in the application of mpMRI in the clinic include the technical considerations in acquiring good quality functional MRI data, development of robust techniques for analysis, and clinical interpretation of the results. This article summarizes the technical challenges in acquisition and analysis of multi-parametric MRI data before reviewing the key applications of multi-parametric MRI in clinical research and practice., Competing Interests: The authors report no conflicts of interest.

Purpose: To estimate the exchange rates of labile (1) H in citrate and spermine, metabolites present in prostatic secretions, to predict the size of the citrate and spermine CEST effects in vivo., Methods: CEST z-spectra were acquired at high-field [11.7 Tesla (T)] from citrate and spermine solutions at physiological pH (6.5) using saturation power 6 μT. CEST was performed at different temperatures to determine exchange regimes (slow, intermediate or fast). For low pH solutions of spermine, exchange rates were estimated from resonance line width, fitting z-spectra using the Bloch equations incorporating exchange, and using quantifying exchange using saturation time experiments (QUEST). These rates were extrapolated to physiological pH., Results: Citrate showed little CEST effect at pH 6.5 and temperature (T) = 310 K (maximum 0.001% mM(-1) ), indicating fast exchange, whereas spermine showed greater CEST effects (maximum 0.2% mM(-1) ) indicating intermediate-to-fast exchange. Extrapolating data acquired from low pH spermine solutions predicts exchange rates at pH 6.5 and T of 310 K of at least 2 × 10(4) s(-1) ., Conclusion: Citrate and spermine show minimal CEST effects at 11.7T even using high saturation power. These effects would be much less than 2% at clinical field-strengths due to relatively faster exchange and would be masked by CEST from proteins. Magn Reson Med 76:742-746, 2016. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited., (© 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine.)

Purpose: To investigate the clinical utility of the reverse gradient algorithm in correcting distortions in diffusion-weighted images of the cervix and for increasing diagnostic performance., Materials and Methods: Forty-one patients ages 25-72 years (mean 40 ± 11 years) with suspected or early stage cervical cancer were imaged at 3T using an endovaginal coil. T2 -weighted (W) and diffusion-weighted images with right and left phase-encode gradient directions were obtained coronal to the cervix (b = 0, 100, 300, 500, 800 s mm(-2) ). Differences in angle of the endocervical canal to the x-axis between T2 W and right-gradient, left-gradient, and corrected images were measured. Uncorrected and corrected images were assessed for diagnostic performance when viewed together with T2 W images by two independent observers against subsequent histology., Results: The angles of the endocervical canal relative to the x-axis were significantly different between the T2 W images and the right-gradient images (P = 0.007), approached significance for left-gradient images (P = 0.055), and were not significantly different after correction (P = 0.95). Corrected images enabled a definitive diagnosis in 34% (n = 14) of patients classified as equivocal on uncorrected images. Tumor volume in this subset was 0.18 ± 0.44 cm(3) (mean ± SD; sensitivity of detection 100% [8/8], specificity 50% [3/6] for an experienced observer). Correction did not improve diagnostic performance for the less-experienced observer., Conclusion: Distortion-corrected diffusion-weighted images improved correspondence with T2 W images and diagnostic performance in a third of cases., (© 2015 The Authors Journal of Magnetic Resonance Imaging published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine.)

The use of magnetic resonance imaging (MRI) in radiotherapy (RT) planning is rapidly expanding. We review the wide range of image contrast mechanisms available to MRI and the way they are exploited for RT planning. However a number of challenges are also considered: the requirements that MR images are acquired in the RT treatment position, that they are geometrically accurate, that effects of patient motion during the scan are minimized, that tissue markers are clearly demonstrated, that an estimate of electron density can be obtained. These issues are discussed in detail, prior to the consideration of a number of specific clinical applications. This is followed by a brief discussion on the development of real-time MRI-guided RT.

Purpose: To compare the depiction of metabolite signals in short and long echo time (TE) prostate cancer spectra at 3T, and to quantify their concentrations in tumors of different stage and grade, and tissues adjacent to tumor., Materials and Methods: First, single-voxel magnetic resonance imaging (MRI) spectra were acquired from voxels consisting entirely of tumor, as defined on T2-weighted and diffusion-weighted (DW)-MRI and from a biopsy-positive octant, at TEs of 32 msec and 100 msec in 26 prostate cancer patients. Then, in a separate cohort of 26 patients, single-voxel TE = 32 msec MR spectroscopy (MRS) was performed over a partial-tumor region and a matching, contralateral normal-appearing region, defined similarly. Metabolite depiction was compared between TEs using Cramér-Rao lower bounds (CRLB), and absolute metabolite concentrations were calculated from TE = 32 msec spectra referenced to unsuppressed water spectra., Results: Citrate and spermine resonances in tumor were better depicted (had significantly lower CRLB) at TE = 32 msec, while the choline resonance was better depicted at TE = 100 msec. Citrate and spermine concentrations were significantly lower in patients of more advanced stage, significantly lower in Gleason grade 3+4 than 3+3 tumors, and significantly lower than expected from the tumor fraction in partial-tumor voxels (by 14 mM and 4 mM, respectively, P < 0.05)., Conclusion: Citrate and spermine resonances are better depicted at short TE than long TE in tumors. Reduction in these concentrations is related to increasing tumor stage and grade in vivo, while reductions in the normal-appearing tissues immediately adjacent to tumor likely reflect tumor field effects., (© 2015 Wiley Periodicals, Inc.)

Measurement of tissue lactate using (1) H MRS is often confounded by overlap with intense lipid signals at 1.3 ppm. Single-voxel localization using PRESS is also compromised by the large chemical shift displacement between voxels for the 4.1 ppm (-CH) resonance and the 1.3 ppm -CH3 resonance, leading to subvoxels with signals of opposite phase and hence partial signal cancellation. To reduce the chemical shift displacement to negligible proportions, a modified semi-LASER sequence was written ("FOCI-LASER", abbreviated as fLASER) using FOCI pulses to permit high RF bandwidth even with the limited RF amplitude characteristic of clinical MRI scanners. A further modification, MQF-fLASER, includes a selective multiple-quantum filter to detect lactate and reject lipid signals. The sequences were implemented on a Philips 3 T Achieva TX system. In a solution of brain metabolites fLASER lactate signals were 2.7 times those of PRESS. MQF-fLASER lactate was 47% of fLASER (the theoretical maximum is 50%) but still larger than PRESS lactate. In oil, the main 1.3 ppm lipid peak was suppressed to less than 1%. Enhanced suppression was possible using increased gradient durations. The minimum detectable lactate concentration was approximately 0.5 mM. Coherence selection gradients needed to be at the magic angle to avoid large water signals derived from intermolecular multiple-quantum coherences. In pilot patient measurements, lactate peaks were often observed in brain tumours, but not in cervix tumours; lipids were effectively suppressed. In summary, compared with PRESS, the fLASER sequence yields greatly superior sensitivity for direct detection of lactate (and equivalent sensitivity for other metabolites), while the single-voxel single-shot MQF-fLASER sequence surpasses PRESS for lactate detection while eliminating substantial signals from lipids. This sequence will increase the potential for in vivo lactate measurement as a biomarker in targeted anti-cancer treatments as well as in measurements of tissue hypoxia., (© 2015 The Authors. NMR in Biomedicine published by John Wiley & Sons, Ltd.)

Back-to-back (1)H MRSI scans, using an endorectal and phased-array coil combination, were performed on 18 low-risk patients with prostate cancer at 3 T, employing TEs of 32 and 100 ms in order to compare metabolite visualization at each TE. Outer-volume suppression of lipid signals was performed using regional saturation (REST) slabs and the quantification of spectra at both TEs was achieved with the quantitation using quantum estimation (QUEST) routine. Metabolite nulling experiments in an additional five patients found that there were negligible macromolecule background signals in prostate spectra at TE = 32 ms. Metabolite visibility was judged using the criterion Cramér-Rao lower bound (CRLB)/amplitude < 20%, and metabolite concentrations were corrected for relaxation effects and referenced to the data acquired in corresponding water-unsuppressed MRSI scans. For the first time, the prostate metabolites spermine and myo-inositol were quantified individually in vivo, together with citrate, choline and creatine. All five metabolite visibilities were higher in TE = 32 ms MRSI than in TE = 100 ms MRSI. At TE = 32 ms, citrate was visible in 99.0% of lipid-free spectra, whereas, at TE = 100 ms, no metabolite simulation of citrate matched the in vivo peaks. Spermine, choline and creatine were visualised separately in 30.4% more spectra at TE = 32 ms than at TE = 100 ms, and myo-inositol in 72.5% more spectra. T2 values were calculated for spermine (53 ± 16 ms), choline (62 ± 17 ms) and myo-inositol (90 ± 48 ms). Data from the TE = 32 ms spectra showed that the concentrations of citrate and spermine secretions were positively correlated in both the peripheral zone and central gland (R(2)  = 0.73 and R(2)  = 0.43, respectively), and that the citrate content was significantly higher in the former at 64 ± 22 mm than in the latter at 32 ± 16 mm (p = 0.01). However, lipid contamination at TE = 32 ms was substantial; therefore, to make clinical use of the greater visualisation of prostate metabolites at TE = 32 ms rather than at TE = 100 ms, three-dimensional MRSI at TE = 32 ms with effective lipid suppression must be implemented., (© 2014 The Authors. NMR in Biomedicine published by John Wiley & Sons, Ltd.)

31P magnetic resonance spectroscopy (31P MRS) can measure intracellular pH (pHi) using the chemical shift difference between pH-dependent inorganic phosphate (Pi) and a pH-independent reference peak. This study compared three different frequency reference peaks [phosphocreatine (PCr), α resonance of adenosine triphosphate (αATP) and water (using 1H MRS)] in a cohort of 10 volunteers and eight patients with non-Hodgkin's lymphoma (NHL). Well-resolved chemical shift imaging (CSI) spectra were acquired on a 1.5T scanner for muscle, liver and tumour. The pH was calculated for all volunteers and patients using the available methods. The consistency of the resulting pH was evaluated. The direct Pi–PCr method was best for those spectra with a very well-defined PCr, such as muscle (pH=7.05 ± 0.02). In liver, the Pi–αATP method gave more consistent results (pH=7.30 ± 0.06) than the calibrated water-based method (pH=7.27 ± 0.11). In NHL nodes, the measured pH using the Pi–αATP method was 7.25 ± 0.12. Given that the measured range includes some biological variation in individual patients, treatment-related changes of the order of 0.1 pH units should be detectable., (2013 John Wiley & Sons, Ltd.)

Rationale and Objectives: Based on their association with malignant proliferation, using noninvasive phosphorus MR spectroscopic imaging ((31)P MRSI), we measured the tumor content of the phospholipid-related phosphomonoesters (PME), phosphoethanolamine and phospholcholine, and its correlation with treatment outcome in newly diagnosed patients with diffuse large B-cell lymphoma (DLBCL) receiving standard first-line chemotherapy., Experimental Design: The PME value normalized to nucleoside triphosphates (PME/NTP) was measured using (31)P MRSI in tumor masses of 20 patients with DLBCL before receiving standard first-line chemotherapy. Response at 6 months was complete in 13 patients and partial in seven. Time to treatment failure (TTF) was ≤11 months in eight patients, from 18 to 30 months in three, and ≥60 months in nine., Results: On a t test, the pretreatment tumor PME/NTP mean value (SD, n) of patients with a complete response at 6 months was 1.42 (0.41, 13), which was significantly different from the value of 2.46 (0.40, 7) in patients with partial response (P < .00001). A Fisher test significantly correlated the PME/NTP values with response at 6 months (sensitivity and specificity at 0.85, P < .004) while a Cox proportional hazards regression significantly correlated the PME/NTP values with TTF (hazard ratio = 5.21, P < .02). A Kaplan-Meier test set apart a group entirely composed of patients with TTF ≤ 11 months (hazard ratio = 8.66, P < .00001)., Conclusions: The pretreatment tumor PME/NTP values correlated with response to treatment at 6 months and time to treatment failure in newly diagnosed patients with DLBCL treated with first-line chemotherapy, and therefore they could be used to predict treatment outcome in these patients., (Copyright © 2013 AUR. Published by Elsevier Inc. All rights reserved.)

This study aimed to characterize changes in lipid saturation using magnetic resonance spectroscopy of sensitive (HeLa) and resistant (C33A; Me180) cervical cancer cell lines following exposure to paclitaxel to explore lipid profiles as biomarkers of drug resistance. Spectra were acquired at 11.74 T. Flow cytometry, electron, and confocal microscopy assessed cellular morphology. Western blots assessed cytoplasmic phospholipase A(2) , fatty acid synthase, and acyl-CoA synthetase1 expression. After 24 h of paclitaxel exposure, >60% of cells showed mitotic arrest. At 48 h, HeLa cells showed apoptosis while C33A/Me180 cells showed normal morphology indicating resistance. MR-visible lipids increased significantly in all lines at 24 h with further increases at 48 h; resistant lines showed smaller increases than HeLa. Cytoplasmic phospholipase A(2) and fatty acid synthase levels were unchanged at 24 h and dropped at 48 h in HeLa; acyl-CoA synthetase1 was higher in Me180/C33A than in HeLa controls but did not increase significantly. The percentage of cells displaying lipid droplets increased significantly at 24 and 48 h in all lines; droplet size increased only in HeLa cells. Droplet number was >3-4× greater in apoptotic compared with mitotic-arrested cells. Apoptotic cells accumulate unsaturated fatty acids in large (relative to control) droplets; resistant lines accumulated smaller droplets with less triglycerides., (Copyright © 2011 Wiley Periodicals, Inc.)

Rapid induction of CD8(+) cytotoxic T lymphocyte (CTL) responses is critical to combat acute infection with intracellular pathogens. CD4(+) T cells help prime antigen-specific CTLs in secondary lymphoid organs after infection in the periphery. Although the frequency of naïve precursors is very low, the immune system is able to efficiently screen for cognate CTLs through mechanisms that are not well understood. Here we examine the role of CD4(+) T cells in early phases of the immune response. We show that CD4(+) T cells help optimal CTL expansion by facilitating entry of naïve polyclonal CD8(+) T cells into the draining lymph node (dLN) early after infection or immunization. CD4(+) T cells also facilitate input of naïve B cells into reactive LNs. Such "help" involves expansion of the arteriole feeding the dLN and enlargement of the dLN through activation of dendritic cells. In an antigen- and CD40-dependent manner, CD4(+) T cells activate dendritic cells to support naïve lymphocyte recruitment to the dLN. Our results reveal a previously unappreciated mode of CD4(+) T-cell help, whereby they increase the input of naïve lymphocytes to the relevant LN for efficient screening of cognate CD8(+) T cells.

The pseudomonad protein, carboxypeptidase G2 (CPG2), is a prodrug-activating enzyme utilized in the targeted chemotherapy strategies of antibody- and gene-directed enzyme prodrug therapy (ADEPT and GDEPT). We have developed a noninvasive imaging approach to monitor CPG2 activity in vivo that will facilitate the preclinical and clinical development of CPG2-based ADEPT and GDEPT strategies. Cleavage of the novel reporter probe, 3,5-difluorobenzoyl-L-glutamic acid (3,5-DFBGlu), by CPG2, in human colon adenocarcinoma WiDr xenografts engineered to stably express CPG2, was monitored using (19)F MRSI. The high signal-to-noise ratio afforded by the two MR-equivalent (19)F nuclei of 3,5-DFBGlu, and the 1.4 ppm (19)F chemical shift difference on CPG2-mediated cleavage, enabled the dynamics and quantification of the apparent pharmacokinetics of 3,5-DFBGlu and its CPG2-mediated cleavage in the tumor to be evaluated. In addition, the apparent rate of increase of 3,5-difluorobenzoic acid concentration could also provide a biomarker of CPG2 activity levels in tumors of patients undergoing CPG2-based therapies, as well as a biomarker of treatment response. The addition of in vivo reporter probes, such as 3,5-DFBGlu, to the armamentarium of prodrugs cleaved by CPG2 affords new applications for CPG2 as a gene reporter of transgene expression., (Copyright © 2010 John Wiley & Sons, Ltd.)

Objective: The purpose of this study was to compare apparent diffusion coefficients, metabolic ratios, and vascularity values within histologically defined prostate tumors with those in nontumor tissue to determine which functional parameter or combination of parameters is best for differentiating tumor from nontumor tissue., Subjects and Methods: Twenty patients due for prostatectomy underwent endorectal MRI at 1.5 T. Transverse T2-weighted, diffusion-weighted, 2D chemical shift, and dynamic contrast-enhanced images were acquired. After prostatectomy, the gland was sectioned transversely. Fresh slices and stained whole-mount sections with histologically defined tumor outlines were photographed. The tumor outlines were mapped onto images, and the apparent diffusion coefficient (ADC), choline-to-citrate (Cho/cit) ratio, and vascularity of the histologically defined tumor, normal peripheral zone, and central gland were quantitatively measured. Area under the receiver operating characteristics (ROC) curve (A(z)) was used to determine the sensitivity and specificity of parameter combinations in cancer detection., Results: In tumor regions larger than 1 cm(2), the Cho/cit ratio was higher in tumor than in nontumor tissue (p < 0.001), in the peripheral zone alone (p = 0.007), and in the central gland alone (p = 0.005). ADC was lower and tumor vascularity greater in tumor than in nontumor tissue (ADC, p = 0.003; initial area under the gadolinium plasma concentration-time curve [initial gadolinium AUC], p = 0.012; forward rate constant [K(trans)], p = 0.011; return rate constant [k(ep)], p = 0.036). No single parameter had a significantly greater A(z) (ADC, 0.71; Cho/cit ratio, 0.79; initial gadolinium AUC, 0.60; K(trans), 0.62; k(ep), 0.65). Pairs of parameters, however, did increase A(z): ADC and initial gadolinium AUC (A(z) = 0.94) versus ADC (p = 0.001) and initial gadolinium AUC (p < 0.001); ADC and Cho/cit ratio (A(z) = 0.94) versus ADC (p = 0.001) and Cho/cit ratio (not significant); and Cho/cit ratio and initial gadolinium AUC (A(z) = 0.88) versus Cho/cit ratio (not significant) and initial gadolinium AUC (p < 0.001). All three functional techniques together had an A(z) of 0.95, showing no further improvement., Conclusion: The combination of two functional parameters is associated with significant improvement in prostate cancer detection over use of any parameter alone. Use of a third parameter does not increase the rate of detection.

Carboxypeptidase G2 (CPG2) is a bacterial enzyme that is currently employed in a range of targeted cancer chemotherapy strategies such as gene-directed enzyme prodrug therapy (GDEPT). Employing dynamic nuclear polarization (DNP) and natural abundance (13)C magnetic resonance spectroscopy (MRS), we observed the CPG2-mediated conversion of a novel hyperpolarized reporter probe 3,5-difluorobenzoyl-L-glutamic acid (3,5-DFBGlu) to 3,5-difluorobenzoic acid (3,5-DFBA) and L-glutamic acid (L-Glu) in vitro. Isotopic labeling of the relevant nuclei with (13)C in 3,5-DFBGlu or related substrates will yield a further factor of 100 increase in the signal-to-noise. We discuss the feasibility of translating these experiments to generate metabolic images of CPG2 activity in vivo., ((c) 2009 Wiley-Liss, Inc.)

Choline metabolites are widely studied in cancer research as biomarkers of malignancy and as indicators of therapeutic response. However, endogenous phosphocholine levels are determined by a number of processes that confound the interpretation of these measurements, including membrane transport rates and a series of enzyme catalysed reactions in the Kennedy pathway. Employing a dynamic (31)P NMR assay that is specific to choline kinase (ChoK) we have measured the rates of this enzyme reaction in cell lysates of MDA-MB-231 breast, PC-3 prostate and HeLa cervical cancer cells and in solutions of purified human ChoK. The rates are sensitive to inhibition by hemicholinium-3 (HC-3), a competitive ChoK inhibitor, and to N-[2-bromocinnamyl(amino)ethyl]-5-isoquinolinesulphonamide (H-89), an agent commercialized as a specific cyclic-AMP-dependent protein kinase A (PKA) inhibitor.

The aim of this study was to determine the metabolic changes in the transition from pre-invasive to invasive cervical cancer using high-resolution magic angle spinning (HR-MAS) MRS. Biopsy specimens were obtained from women with histologically normal cervix (n = 5), cervical intraepithelial neoplasia (CIN; mild, n = 5; moderate/severe, n = 40), and invasive cancer (n = 23). (1)H HR-MAS MRS data were acquired using a Bruker Avance 11.74 T spectrometer (Carr-Purcell-Meiboom-Gill sequence; TR = 4.8 s; TE = 135 ms; 512 scans; 41 min acquisition). (31)P HR-MAS spectra were obtained from the normal subjects and cancer patients only (as acetic acid applied before tissue sampling in patients with CIN impaired spectral quality) using a (1)H-decoupled pulse-acquire sequence (TR = 2.82 s; 2048 scans; 96 min acquisition). Peak assignments were based on values reported in the literature. Peak areas were measured using the AMARES algorithm. Estimated metabolite concentrations were compared between patient diagnostic categories and tissue histology using independent samples t tests. Comparisons based on patient category at diagnosis showed significantly higher estimated concentrations of choline (P = 0.0001) and phosphocholine (P = 0.002) in tissue from patients with cancer than from patients with high-grade dyskaryosis, but no differences between non-cancer groups. Division by histology of the sample also showed increases in choline (P = 0.002) and phosphocholine (P = 0.002) in cancer compared with high-grade CIN tissue. Phosphoethanolamine was increased in cancer compared with normal tissue (P = 0.0001). Estimated concentrations of alanine (P = 0.01) and creatine (P = 0.008) were significantly reduced in normal tissue from cancer patients compared with normal tissue from non-cancer patients. The estimated concentration of choline was significantly increased in CIN tissue from cancer patients compared with CIN tissue from non-cancer patients (P = 0.0001). Estimated concentrations of choline-containing metabolites increased from pre-invasive to invasive cervical cancer. Concurrent metabolite depletion occurs in normal tissue adjacent to cancer tissue., (Copyright (c) 2008 John Wiley & Sons, Ltd.)

Objective: The objective of our study was to establish the sensitivity and specificity for prostate cancer detection using a combined 1H MR spectroscopy and diffusion-weighted MRI approach., Subjects and Methods: Forty-two men (mean age +/- SD, 69.3 +/- 4.7 years) with prostate cancer were studied using endorectal T2-weighted imaging, 2D chemical shift imaging (CSI), and isotropic apparent diffusion coefficient (ADC) maps. Regions of interest (ROIs) were drawn around the entire gland, central gland, and peripheral zone tumor, diagnostically defined as low signal intensity on T2-weighted images within a sextant that was biopsy-positive for tumor. Lack of susceptibility artifact on a gradient-echo B0 map through the slice selected for CSI and no high signal intensity on external array T1-weighted images confirmed the absence of significant hemorrhage after biopsy. CSI voxels were classified as nonmalignant or as tumor (ROI included > or = 30% or > or = 70% tumor). Choline-citrate (Cho/Cit) ratios and average ADCs were calculated for every voxel. A plot of Cho/Cit ratios versus ADCs yielded a line of best separation of tumor voxels from nonmalignant voxels. Receiver operating characteristic (ROC) curves were plotted for Cho/Cit ratios alone, ADCs alone, and a combination of the two., Results: The Cho/Cit ratios were significantly higher (p < 0.001) and the ADCs were significantly lower (p < 0.006) in tumor-containing voxels than in non-tumor-containing voxels. When voxels containing 30% or more tumor were considered positive, the area under the ROC curves using combined MR spectroscopy and ADC (0.81) was similar to that of Cho/Cit alone (0.79) and better than ADC alone (0.66). When voxels containing 70% or more tumor were considered positive and cutoffs to achieve a 90%-or-greater sensitivity chosen, a combination of Cho/Cit and ADC achieved a significant improvement in specificity compared with Cho/Cit alone (p < 0.0001) or ADC alone (p < 0.0001)., Conclusion: When voxels containing > or = 70% tumor are considered positive, the combined use of MR spectroscopy and diffusion-weighted MRI increases the specificity for prostate cancer detection while retaining the sensitivity compared with MR spectroscopy alone or diffusion-weighted MRI alone.

Double resonance techniques such as INEPT (insensitive nuclei enhanced by polarization transfer) and JCP (J cross-polarization) have previously been applied in vitro to enhance the SNR of low-sensitivity nuclei and detect altered metabolism, for example, with 13C magnetic resonance spectroscopy (MRS), where the 1H-13C scalar couplings are of the order of 130 Hz. The aim of the present study was to investigate the potential advantage of using JCP for the detection of phosphomonoesters (PME) and phosphodiesters (PDE) with 31P MRS in vivo. These metabolites are involved in membrane metabolism and their concentration is altered in tumors and other pathologies. JCP has been implemented and compared with INEPT and pulse-and-acquire in vivo both in unlocalized and in localized spectra in order to select the optimum method for in vivo applications for PME and PDE detection. The results suggest that JCP can give up to 20% more signal in the PME region and up to 70% more signal in the PDE region, with 20 to 70% lower power deposition than INEPT. Such enhancement could be used to reduce the measurement times for equivalent signal-to-noise ratios. The JCP sequence is, however, slightly more sensitive than INEPT to RF field inhomogeneities, as predicted from theory., ((c) 2005 Wiley-Liss, Inc.)

Biliary excretion is a significant component in the metabolism of many drugs, but remains difficult to detect and characterise non-invasively. A previous publication recently described the detection of metabolites of ifosfamide in gall bladder in a guinea pig model using in vivo 1H-decoupled 31P 3-D magnetic resonance spectroscopic imaging and a clinical 1.5 T MR scanner.. Here high-resolution 31P magnetic resonance spectroscopy (MRS) of extracted bile identifies peaks as parent ifosfamide (1.19+/-1.47 mM; mean+/-sd), carboxyifosfamide (2.04+/-1.04 mM) and a major contribution from a previously unreported peak at 16.0 ppm (4.05+/-2.38 mM). The unknown resonance was identified using liquid chromatography-mass spectrometry (LCMS) as the glutathione conjugate of ifosfamide (MW=531). This was confirmed by analysing products from the reaction of glutathione with ifosfamide using LCMS and MRS. These results demonstrate how combined in vivo and analytical MRS, together with mass spectrometry, can help identify visceral routes of drug metabolism, thereby aiding understanding of +/-drug disposition and mechanisms of action and toxicity. In particular, the distribution of ifosfamide and its metabolites into bile may be related to oxazophosphorine-related cholecystitis reported in patients.

Physical diminishes with ageing, but little is known of how the microvascular supply to skeletal muscle fibres is affected. To test the hypothesis that ageing alters blood flow control, we investigated network architecture and vasomotor responses of arterioles in the gluteus maximus muscle of young (2-3 months), adult (12-14 months) and old (18-20 months) C57BL6 male mice (n = 83) (Young, Adult and Old, respectively). Microvascular casts revealed that the total number, length and surface area of arteriolar segments (diameter, 10-50 microm) were not significantly different across age-groups. However, for arterioles with diameter of 30 microm, tortuosity and branch angles increased with age (P < 0.05). In anaesthetized mice, second-order (2A) distributing arterioles had similar resting (17 +/- 1 microm) and maximal (37 +/- 1 microm) diameters and similar responsiveness to cumulative (10(-10)-10(-4) M) superfusion of acetylcholine or phenylephrine. With superfusate oxygen level raised from 0 to 21%, 2A arteriolar constriction in Young (11 +/- 1 microm) was greater (P < 0.05) than Adult and Old (5 +/- 1 microm). Observed 1 mm upstream from microiontophoresis of ACh (1 microA, 1 s), conducted vasodilatation was 10 +/- 1 microm in Young, 17 +/- 1 microm in Adult and 6 +/- 1 microm in Old (P < 0.05). With muscle contractions (2, 4 and 8 Hz; 30 s) arteriolar diameter increased similarly across age-groups (6 +/- 1, 11 +/- 1 and 18 +/- 1 microm, respectively). Muscle mass and active tension were similar across age-groups yet postcontraction vasodilatation recovered more rapidly in Old versus Adult and Young (P < 0.05). With arteriolar network architecture maintained during ageing, the impairment in conducted vasodilatation and attenuation of postcontraction vasodilatation may compromise exercise tolerance.

Intermolecular multiple-quantum coherences (iMQCs) have been reported to offer a sensitivity to sample structure at a specific user-defined length scale down to the order of 10 microm. When assessing this novel contrast mechanism in controlled phantom experiments, we have observed three different mechanisms whereby residual single-quantum coherences (SQCs) arising from intense high spatial frequencies, stimulated echoes and strong spatially encoding gradients can produce significant changes in signal contrast at particular length scales. These changes which only appear when components arising from SQCs and iMQCs are both present in the detected signal, are similar to changes previously attributed to iMQCs alone. We demonstrate each mechanism by which these residual SQCs arise and describe methods for their suppression.

Purpose: To perform a Phase I study of SR-4554, a fluorinated 2-nitroimidazole noninvasive probe of tumor hypoxia detected by (19)F magnetic resonance spectroscopy (MRS)., Experimental Design: SR-4554 administration, on days 1 and 8, was followed by plasma sampling for pharmacokinetic studies and by three MRS studies performed over 24 h on days 8 and 9. Unlocalized MR spectra were acquired from tumor (10- or 16-cm dual resonant 1H/19F surface coil; 1.5 T Siemens Vision MR system; 2048 transients acquired over 34 min; 1.28-ms adiabatic pulse; repetition time, 1 s). Plasma drug concentrations were measured with a validated high-performance liquid chromatography method. Noncompartmental pharmacokinetic analysis was performed., Results: Eight patients underwent pharmacokinetic studies, receiving doses of SR-4554 of 400-1600 mg/m(2). Peak plasma concentrations increased linearly with the SR-4554 dose (r(2) = 0.80; P = 0.0002). The plasma elimination half-life was relatively short (mean +/- SD, 3.28 +/- 0.59 h), and plasma clearance was quite rapid (mean +/- SD, 12.8 +/- 3.3 liters/h). Urinary recovery was generally high. SR-4554 was well tolerated. A single patient experienced dose-limiting toxicity (nausea and vomiting) at 1600 mg/m(2). The maximum tolerated dose was 1400 mg/m(2). SR-4554 was detected spectroscopically in tumors immediately after infusion at doses of 400-1600 mg/m(2). At the highest dose (1600 mg/m(2)), SR-4554 was detectable in tumor at 8 h, but not at 27 h., Conclusions: SR-4554 has plasma pharmacokinetic and toxicity profiles suitable for use as a hypoxia probe. It can be detected in tumors by unlocalized MRS. Additional clinical studies are warranted.

Several (31)P MRS studies in tumors in vivo have shown that levels of phosphocholine (PC) and other phosphomonoesters (PME) and phosphodiesters (PDE) are useful prognostic or early-response indicators. To improve sensitivity for such measurements, four polarization transfer (PT) sequences (insensitive nuclei enhanced by PT (INEPT), distortionless enhancement by PT (DEPT), reverse-INEPT, and heteronuclear single-quantum coherence (HSQC)) were assessed theoretically and experimentally. The presence of homonuclear ((1)H-(1)H) and heteronuclear ((31)P-(1)H) couplings of similar magnitude makes theoretical analysis very sensitive to precise model parameters, especially for the (1)H-detected sequences. The (1)H-(1)H coupling causes the splitting of (1)H peaks, and hence reduces the proton spectral resolution. This effect and a 50% signal loss from gradient-enhanced water suppression negate the usual advantages of (1)H-detection. Among the PT methods, INEPT gave the higher signal enhancement. However, T(2) losses during the long echo times (TEs) required by the weak coupling limited the resulting signals from PC., (Copyright 2003 Wiley-Liss, Inc.)

Histamine increases the permeability of capillaries and venules but little is known of its precapillary actions on the control of tissue perfusion. Using gene ablation and pharmacological interventions, we tested whether histamine could increase muscle blood flow through stimulating nitric oxide (NO) release from microvascular endothelium. Vasomotor responses to topical histamine were investigated in second-order arterioles in the superfused cremaster muscle of anesthetized C57BL6 mice and null platelet endothelial cell adhesion molecule-1 (PECAM-1-/-) and null endothelial NO synthase (eNOS-/-) mice aged 8-12 wk. Neither resting (17 +/- 1 microm) nor maximum diameters (36 +/- 2 microm) were different between groups, nor was the constrictor response (approximately 5 +/- 1 microm) to elevating superfusate oxygen from 0 to 21%. For arterioles of C57BL6 and PECAM-1-/- mice, cumulative addition of histamine to the superfusate produced vasodilation (1 nM-1 microM; peak response, 9 +/- 1 microm) and then vasoconstriction (10-100 microM; peak response, 12 +/- 2 microm). In eNOS-/- mice, histamine produced only vasoconstriction. In C57BL6 and PECAM-1-/- mice, vasodilation was abolished with Nomega-nitro-l-arginine (30 microM); in all mice, vasoconstriction was abolished with nifedipine (1 microM). Vasomotor responses were eliminated with pyrilamine (1 microM; H1 receptor antagonist) yet remained intact with cimetidine (1 microM; H2 receptor antagonist). These findings illustrate that the biphasic vasomotor response of mouse cremaster arterioles to histamine is mediated through H1 receptors on endothelium (NO-dependent vasodilation) as well as smooth muscle (Ca2+ entry and constriction). Thus histamine can increase as well as decrease muscle blood flow, according to local concentration. However, when NO production is compromised, only vasoconstriction and flow reduction occur.

MRS has considerable potential for the measurement of drug pharmacokinetics in vivo. In this study single- and double-resonance (31)P MRS was used to investigate the biodistribution, pharmacokinetics, and metabolism of ifosfamide following administration of 500 mg/kg ifosfamide in guinea pigs. (1)H-decoupling was found to nearly double the signal of detected peaks. However, in contrast to studies of ifosfamide in solution, the polarization transfer sequence gave no further signal enhancements. This was attributed to significantly reduced relaxation times in vivo. Chemical shift imaging (CSI) measurements showed that significant proportions of ifosfamide-related (31)P MRS signals arose from the liver, as expected, but also from the gall bladder, which was not predicted from the current literature. Signals were observed within 5 min of the end of administration. The halflife in liver was approximately 74 min, whereas in gall bladder there was no measurable signal decay during the 2.5-hr studies. High-resolution (31)P MRS of bile showed that the "ifosfamide" peak in vivo consists of at least two compounds. The lower-concentration peak is ifosfamide, and an investigation is under way to identify the higher-concentration peak. Other peaks observed in bile are tentatively assigned to carboxy-ifosfamide and dechloroethyl-ifosfamide. Overall, (1)H-decoupled (31)P MRS has proved to be a useful tool for investigating the metabolism of ifosfamide in vivo., (Copyright 2003 Wiley-Liss, Inc.)

Background and Purpose: Dahl salt-sensitive rats fed an 8.7% NaCl diet exhibited hypertensive encephalopathy and developed seizures associated with areas of blood-brain barrier (BBB) disruption without brain ischemia. The incidence of hemorrhagic stroke was low (7/47). We tested the hypothesis that a defect in cerebral blood flow (CBF) autoregulation under hypertensive conditions preceded hypertensive encephalopathy., Methods: Brain ischemia and BBB disruption were assessed with the use of tetrazolium red staining and Evans blue dye extravasation, respectively. Myogenic constriction to pressure was measured in isolated middle cerebral arteries (MCAs) with a pressure myograph. CBF autoregulation was assessed with the use of laser-Doppler techniques., Results: Asymptomatic rats fed 8.7% NaCl had MCAs that developed an age-related attenuation in their ability to constrict to pressure, which was amplified in rats exhibiting hypertensive encephalopathy. The MCAs of rats with hemorrhagic stroke lost this function and developed large degrees of basal tone. The majority (4/6) of asymptomatic rats fed high salt for longer than 3 weeks exhibited a linear relationship between CBF and blood pressure. The characteristics of CBF regulation were consistent with the possible absence of autoregulation coupled with cerebrovascular vasoconstriction., Conclusions: Both MCA pressure-dependent constriction and CBF autoregulation in the MCA perfusion domain were lost before the development of hypertensive encephalopathy or hemorrhagic stroke. These defects could contribute to the development of BBB disruption during hypertension. Cerebrovascular vasoconstriction in the absence of CBF autoregulation may protect the brain from excessive overperfusion during hypertension and could account for the low incidence of cerebral hemorrhage in this model.

This brief review considers to what extent Magnetic Resonance Spectroscopy (MRS) can play a role in monitoring early tumour response with examples of preclinical studies and selected clinical studies in tumours of children and young adults. An early non-invasive indicator of tumour response to therapy would provide useful information regarding the effectiveness of therapy. This might be a relevant prognostic factor in new patients and in phase II studies could facilitate recommendations at an early stage as to whether to continue treatment. This review suggests that several markers and ratios are emerging as potential prognostic markers, but larger prospective studies are needed before translating this into clinical practice.