Initial Clinical Experience of Cherenkov Imaging in External Beam Radiation Therapy Identifies Opportunities to Improve Treatment Delivery.

PURPOSE: The value of Cherenkov imaging as an on-patient, real-time, treatment delivery verification system was examined in a 64-patient cohort during routine radiation treatments in a single-center study. METHODS AND MATERIALS: Cherenkov cameras were mounted in treatment rooms and used to image patients during their standard radiation therapy regimen for various sites, predominantly for whole breast and total skin electron therapy. For most patients, multiple fractions were imaged, with some involving bolus or scintillators on the skin. Measures of repeatability were calculated with a mean distance to conformity (MDC) for breast irradiation images. RESULTS: In breast treatments, Cherenkov images identified fractions when treatment delivery resulted in dose on the contralateral breast, the arm, or the chin and found nonideal bolus positioning. In sarcoma treatments, safe positioning of the contralateral leg was monitored. For all 199 imaged breast treatment fields, the interfraction MDC was within 7 mm compared with the first day of treatment (with only 7.5% of treatments exceeding 3 mm), and all but 1 fell within 7 mm relative to the treatment plan. The value of imaging dose through clear bolus or quantifying surface dose with scintillator dots was examined. Cherenkov imaging also was able to assess field match lines in cerebral-spinal and breast irradiation with nodes. Treatment imaging of other anatomic sites confirmed the value of surface dose imaging more broadly. CONCLUSIONS: Daily radiation therapy can be imaged routinely via Cherenkov emissions. Both the real-time images and the posttreatment, cumulative images provide surrogate maps of surface dose delivery that can be used for incident discovery and/or continuous improvement in many delivery techniques. In this initial 64-patient cohort, we discovered 6 minor incidents using Cherenkov imaging; these otherwise would have gone undetected. In addition, imaging provides automated, quantitative metrics useful for determining the quality of radiation therapy delivery.

Effects of Therapeutic Intervention on Parentally Bereaved Children's Emotion Reactivity and Regulation 15 Years Later.

The Family Bereavement Program (FBP) is a family-based intervention for parentally bereaved children and surviving caregivers. Results are reported of a randomized controlled trial, examining intervention effects on emotional reactivity and regulation of young adults who participated in the program 15 years earlier. Participants (N = 152) completed four emotion challenge tasks: reactivity to negative images, detached reappraisal while viewing negative images, positive reappraisal while viewing negative images, and reengagement with positive images. Outcomes included cardiac interbeat interval (IBI), pre-ejection period (PEP), and respiratory sinus arrhythmia (RSA) as well as self-reported emotional experience and regulation effectiveness. Direct intervention effects and effects mediated through improved parenting were estimated. Several significant effects were observed in primary analyses; however, none remained significant after correction for familywise Type I error. Parenting mediated FBP effects on IBI during negative reactivity (b = 15.04), and on RSA during positive reengagement (b = 0.35); the latter effect was accounted for by changes in breathing. Intervention condition was a direct predictor of self-reported detached reappraisal effectiveness (b = 1.00). Intervention and gender interacted in predicting self-reported negative emotion during the negative reactivity (b = 1.04) and positive reappraisal tasks (b = 1.31) such that intervention-condition men reported more negative emotions during those tasks. Although these findings should be considered preliminary given the limited power of the corrected statistical tests, they suggest long-term effects of family intervention following the death of a parent on offspring's emotional reactivity and regulation ability that should be pursued further in future research.

Technical Note: A novel dosimeter improves total skin electron therapy surface dosimetry workflow.

PURPOSE: The novel scintillator-based system described in this study is capable of accurately and remotely measuring surface dose during Total Skin Electron Therapy (TSET); this dosimeter does not require post-exposure processing or annealing and has been shown to be re-usable, resistant to radiation damage, have minimal impact on surface dose, and reduce chances of operator error compared to existing technologies e.g. optically stimulated luminescence detector (OSLD). The purpose of this study was to quantitatively analyze the workflow required to measure surface dose using this new scintillator dosimeter and compare it to that of standard OSLDs. METHODS: Disc-shaped scintillators were attached to a flat-faced phantom and a patient undergoing TSET. Light emission from these plastic discs was captured using a time-gated, intensified, camera during irradiation and converted to dose using an external calibration factor. Time required to complete each step (daily QA, dosimeter preparation, attachment, removal, registration, and readout) of the scintillator and OSLD surface dosimetry workflows was tracked. RESULTS: In phantoms, scintillators and OSLDs surface doses agreed within 3% for all data points. During patient imaging it was found that surface dose measured by OSLD and scintillator agreed within 5% and 3% for 35/35 and 32/35 dosimetry sites, respectively. The end-to-end time required to measure surface dose during phantom experiments for a single dosimeter was 78 and 202 sec for scintillator and OSL dosimeters, respectively. During patient treatment, surface dose was assessed at 7 different body locations by scintillator and OSL dosimeters in 386 and 754 sec, respectively. CONCLUSION: Scintillators have been shown to report dose nearly twice as fast as OSLDs with substantially less manual work and reduced chances of human error. Scintillator dose measurements are automatically saved to an electronic patient file and images contain a permanent record of the dose delivered during treatment.

Tracking tumor radiotherapy response in vivo with Cherenkov-excited luminescence ink imaging.

This study demonstrates remote imaging for in vivo detection of radiation-induced tumor microstructural changes by tracking the diffusive spread of injected intratumor UV excited tattoo ink using Cherenkov-excited luminescence imaging (CELI). Micro-liter quantities of luminescent tattoo ink with UV absorption and visible emission were injected at a depth of 2 mm into mouse tumors prior to receiving a high dose treatment of radiation. X-rays from a clinical linear accelerator were used to excite phosphorescent compounds within the tattoo ink through Cherenkov emission. The in vivo phosphorescence was detected using a time-gated intensified CMOS camera immediately after injection, and then again at varying time points after the ink had broken down with the apoptotic tumor cells. Ex vivo tumors were imaged post-mortem using hyperspectral cryo-fluorescence imaging to quantify necrosis and compared to Cherenkov-excited light imaging of diffusive ink spread measured in vivo. Imaging of untreated control mice showed that ink distributions remained constant after four days with less than 3% diffusive spread measured using full width at 20% max. For all mice, in vivo CELI measurements matched within 12% of the values estimated by the high-resolution ex vivo sliced luminescence imaging of the tumors. The tattoo ink spread in treated mice was found to correlate well with the nonperfusion necrotic core volume (R2 = 0.92) but not well with total tumor volume changes (R2 = 0.34). In vivo and ex vivo findings indicate that the diffusive spread of the injected tattoo ink can be related to radiation-induced necrosis, independent of total tumor volume change. Tracking the diffusive spread of the ink allows for distinguishing between an increase in tumor size due to new cellular growth and an increase in tumor size due to edema. Furthermore, the imaging resolution of CELI allows for in vivo tracking of subtle microenvironmental changes which occur earlier than tumor shrinkage and this offers the potential for novel, minimally invasive radiotherapy response assay without interrupting a singular clinical workflow.

Experimentally Observed Cherenkov Light Generation in the Eye During Radiation Therapy.

PURPOSE: Patients have reported sensations of seeing light flashes during radiation therapy, even with their eyes closed. These observations have been attributed to either direct excitation of retinal pigments or generation of Cherenkov light inside the eye. Both in vivo human and ex vivo animal eye imaging was used to confirm light intensity and spectra to determine its origin and overall observability. METHODS AND MATERIALS: A time-gated and intensified camera was used to capture light exiting the eye of a patient undergoing stereotactic radiosurgery in real time, thereby verifying the detectability of light through the pupil. These data were compared with follow-up mechanistic imaging of ex vivo animal eyes with thin radiation beams to evaluate emission spectra and signal intensity variation with anatomic depth. Angular dependency of light emission from the eye was also measured. RESULTS: Patient imaging showed that light generation in the eye during radiation therapy can be captured with a signal-to-noise ratio of 68. Irradiation of ex vivo eye samples confirmed that the spectrum matched that of Cherenkov emission and that signal intensity was largely homogeneous throughout the entire eye, from the cornea to the retina, with a slight maximum near 10 mm depth. Observation of the signal external to the eye was possible through the pupil from 0° to 90°, with a detected emission near 2500 photons per millisecond (during peak emission of the ON cycle of the pulsed delivery), which is over 2 orders of magnitude higher than the visible detection threshold. CONCLUSIONS: By quantifying the spectra and magnitude of the signal, we now have direct experimental observations that Cherenkov light is generated in the eye during radiation therapy and can contribute to perceived light flashes. Furthermore, this technique can be used to further study and measure phosphenes in the radiation therapy clinic.

Improvements to an optical scintillator imaging-based tissue dosimetry system.

Previous work has shown that capturing optical emission from plastic discs attached directly to the skin can be a viable means to accurately measure surface dose during total skin electron therapy. This method can provide accurate dosimetric information rapidly and remotely without the need for postprocessing. The objective of this study was to: (1) improve the robustness and usability of the scintillators and (2) enhance sensitivity of the optical imaging system to improve scintillator emission detection as related to tissue surface dose. Baseline measurements of scintillator optical output were obtained by attaching the plastic discs to a flat tissue phantom and simultaneously irradiating and imaging them. Impact on underlying surface dose was evaluated by placing the discs on-top of the active element of an ionization chamber. A protective coating and adhesive backing were added to allow easier logistical use, and they were also subjected to disinfection procedures, while verifying that these changes did not affect the linearity of response with dose. The camera was modified such that the peak of detector quantum efficiency better overlapped with the emission spectra of the scintillating discs. Patient imaging was carried out and surface dose measurements were captured by the updated camera and compared to those produced by optically stimulated luminescence detectors (OSLD). The updated camera was able to measure surface dose with < 3 % difference compared to OSLD­Cherenkov emission from the patient was suppressed and scintillation detection was enhanced by 25 × and 7 × , respectively. Improved scintillators increase underlying surface dose on average by 5.2 ± 0.1 % and light output decreased by 2.6 ± 0.3 % . Disinfection had < 0.02 % change on scintillator light output. The enhanced sensitivity of the imaging system to scintillator optical emission spectrum can now enable a reduction in physical dimensions of the dosimeters without loss in ability to detect light output.

Technical Note: Quality assurance and relative dosimetry testing of a 60 Co total body irradiator using optical imaging.

PURPOSE: The aim of this study was to create an optical imaging-based system for quality assurance (QA) testing of a dedicated Co-60 total body irradiation (TBI) machine. Our goal is to streamline the QA process by minimizing the amount time necessary for tests such as verification of dose rate and field homogeneity. METHODS: Plastic scintillating rods were placed directly on the patient treatment couch of a dedicated TBI 60 Co irradiator. A tripod-mounted intensified camera was placed directly adjacent to the couch. Images were acquired over a 30-s period once the cobalt source was fully exposed. Real-time image filtering was used; cumulative images were flatfield corrected as well as background and darkfield subtracted. Scintillators were used to measure light-radiation field correspondence, dose rate, field homogeneity, and symmetry. Dose rate effects were measured by modifying the height of the treatment couch and scintillator response was compared to ionization chamber (IC) measurements. Optically stimulated luminesce detector (OSLD) used as reference dosimeters during field symmetry and homogeneity testing. RESULTS: The scintillator-based system accurately reported changes in dose rate. When comparing normalized output values for IC vs scintillators over a range of source-to-surface distances, a linear relationship (R2  = 0.99) was observed. Normalized scintillator signal matched OSLD measurements with <1.5% difference during field homogeneity and symmetry testing. Beam symmetry across both axes of the field was within 2%. The light field was found to correspond to 90 ± 3% of the isodose maximum along the longitudinal and latitudinal axis, respectively. Scintillator imaging output results using a single image stack requiring no postexposure processing (needed for OSLD) or repeat manual measurements (needed for IC). CONCLUSION: Imaging of scintillation light emission from plastic rods is a viable and efficient method for carrying out TBI 60 Co irradiator QA. We have shown that this technique can accurately measure field homogeneity, symmetry, light-radiation field correspondence, and dose rate effects.

Assessment of imaging Cherenkov and scintillation signals in head and neck radiotherapy.

The goal of this study was to test the utility of time-gated optical imaging of head and neck (HN) radiotherapy treatments to measure surface dosimetry in real-time and inform possible interfraction replanning decisions. The benefit of both Cherenkov and scintillator imaging in HN treatments is direct daily feedback on dose, with no change to the clinical workflow. Emission from treatment materials was characterized by measuring radioluminescence spectra during irradiation and comparing emission intensities relative to Cherenkov emission produced in phantoms and scintillation from small plastic targets. HN treatment plans were delivered to a phantom with bolus and mask present to measure impact on signal quality. Interfraction superficial tumor reduction was simulated on a HN phantom, and cumulative Cherenkov images were analyzed in the region of interest (ROI). HN human patient treatment was imaged through the mask and compared with the dose distribution calculated by the treatment planning system. The relative intensity of radioluminescence from the mask was found to be within 30% of the Cherenkov emission intensity from tissue-colored clay. A strong linear relationship between normalized cumulative Cherenkov intensity and tumor size was established ([Formula: see text]). The presence of a mask above a scintillator ROI was found to decrease mean pixel intensity by >40% and increase distribution spread. Cherenkov imaging through mask material is shown to have potential for surface field verification and tracking of superficial anatomy changes between treatment fractions. Imaging of scintillating targets provides a direct imaging of surface dose on the patient and through transparent bolus material. The first imaging of a patient receiving HN radiotherapy was achieved with a signal map which qualitatively matches the surface dose plan.

Characterization of a non-contact imaging scintillator-based dosimetry system for total skin electron therapy.

Surface dosimetry is required for ensuring effective administration of total skin electron therapy (TSET); however, its use is often reduced due to the time consuming and complex nature of acquisition. A new surface dose imaging technique was characterized in this study and found to provide accurate, rapid and remote measurement of surface doses without the need for post-exposure processing. Disc-shaped plastic scintillators (1 mm thick × 15 mm [Formula: see text]) were chosen as optimal-sized samples and designed to attach to a flat-faced phantom for irradiation using electron beams. Scintillator dosimeter response to radiation damage, dose rate, and temperature were studied. The effect of varying scintillator diameter and thickness on light output was evaluated. Furthermore, the scintillator emission spectra and impact of dosimeter thickness on surface dose were also quantified. Since the scintillators were custom-machined, dosimeter-to-dosimeter variation was tested. Scintillator surface dose measurements were compared to those obtained by optically stimulated luminescence dosimeters (OSLD). Light output from scintillator dosimeters evaluated in this study was insensitive to radiation damage, temperature, and dose rate. Maximum wavelength of emission was found to be 422 nm. Dose reported by scintillators was linearly related to that from OSLDs. Build-up from placement of scintillators and OSLDs had a similar effect on surface dose (4.9% increase). Variation among scintillator dosimeters was found to be 0.3 ± 0.2%. Scintillator light output increased linearly with dosimeter thickness (~1.9 × /mm). All dosimeter diameters tested were able to accurately measure surface dose. Scintillator dosimeters can potentially improve surface dosimetry-associated workflow for TSET in the radiation oncology clinic. Since scintillator data output can be automatically recorded to a patient medical record, the chances of human error in reading out and recording surface dose are minimized.

Improving treatment geometries in total skin electron therapy: Experimental investigation of linac angles and floor scatter dose contributions using Cherenkov imaging.

PURPOSE: The purpose of this study was to identify the optimal treatment geometry for total skin electron therapy (TSET) using a new optimization metric from Cherenkov image analysis, and to investigate the sensitivity of the Cherenkov imaging method to floor scatter effects in this unique treatment setup. METHODS: Cherenkov imaging using an intensified charge coupled device (ICCD) was employed to measure the relative surface dose distribution as a 2D image in the total skin electron treatment plane. A 1.2 m × 2.2 m × 1 cm white polyethylene sheet was placed vertically at a source to surface distance (SSD) of 300 cm, and irradiated with 6 MeV high dose rate TSET beams. The linear accelerator coordinate system used stipulates 0° is the bottom of the gantry arc, and progresses counterclockwise so that gantry angle 270° produces a horizontal beam orthogonal to the treatment plane. First, all unique pairs of treatment beams were analyzed to determine the performance of the currently recommended symmetric treatment angles (±20° from the horizontal), compared to treatment geometries unconstrained to upholding gantry angle symmetry. This was performed on two medical linear accelerators (linacs). Second, the extent of the floor scatter contributions to measured surface dose at the extended SSD required for TSET were imaged using three gantry angles of incidence: 270° (horizontal), 253° (-17°), and 240° (-30°). Images of the surface dose profile at each angle were compared to the standard concrete floor when steel plates, polyvinyl chloride (PVC), and solid water were placed on the ground at the base of the treatment plane. Postprocessing of these images allowed for comparison of floor material-based scatter profiles with previously published simulation results. RESULTS: Analysis of the symmetric treatment geometry (270 ± 20°) and the identified optimal treatment geometry (270 + 23° and 270 - 17°) showed a 16% increase in the 90% isodose area for the latter field pair on the first linac. The optimal asymmetric pair for the second linac (270 + 25° and 270 - 17°) provided a 52% increase in the 90% isodose area when compared to the symmetric geometry. Difference images between Cherenkov images captured with test materials (steel, PVC, and solid water) and the control (concrete floor) demonstrated relative changes in the two-dimensional (2D) dose profile over a 1 × 1.9 m region of interest (ROI) that were consistent with published simulation data. Qualitative observation of the residual images demonstrates localized increases and decreases with respect to the change in floor material and gantry angle. The most significant changes occurred when the beam was most directly impinging the floor (gantry angle 240°, horizontal -30°), where the PVC floor material decreased scatter dose by 1-3% in 7.2% of the total ROI area, and the steel plate increased scatter dose by 1-3% in 7.0% of the total ROI area. CONCLUSIONS: An updated Cherenkov imaging method identified asymmetric, machine-dependent TSET field angle pairs that provided much larger 90% isodose areas than the commonly adopted symmetric geometry suggested by Task Group 30 Report 23. A novel demonstration of scatter dose Cherenkov imaging in the TSET field was established.

Time-gated scintillator imaging for real-time optical surface dosimetry in total skin electron therapy.

The purpose of this study was to measure surface dose by remote time-gated imaging of plastic scintillators. A novel technique for time-gated, intensified camera imaging of scintillator emission was demonstrated, and key parameters influencing the signal were analyzed, including distance, angle and thickness. A set of scintillator samples was calibrated by using thermo-luminescence detector response as reference. Examples of use in total skin electron therapy are described. The data showed excellent room light rejection (signal-to-noise ratio of scintillation SNR ≈ 470), ideal scintillation dose response linearity, and 2% dose rate error. Individual sample scintillation response varied by 7% due to sample preparation. Inverse square distance dependence correction and lens throughput error (8% per meter) correction were needed. At scintillator-to-source angle and observation angle <50°, the radiant energy fluence error was smaller than 1%. The achieved standard error of the scintillator cumulative dose measurement compared to the TLD dose was 5%. The results from this proof-of-concept study documented the first use of small scintillator targets for remote surface dosimetry in ambient room lighting. The measured dose accuracy renders our method to be comparable to thermo-luminescent detector dosimetry, with the ultimate realization of accuracy likely to be better than shown here. Once optimized, this approach to remote dosimetry may substantially reduce the time and effort required for surface dosimetry.

Label-free imaging of atherosclerotic plaques using third-harmonic generation microscopy.

Multiphoton microscopy using laser sources in the mid-infrared range (MIR, 1,300 nm and 1,700 nm) was used to image atherosclerotic plaques from murine and human samples. Third harmonic generation (THG) from atherosclerotic plaques revealed morphological details of cellular and extracellular lipid deposits. Simultaneous nonlinear optical signals from the same laser source, including second harmonic generation and endogenous fluorescence, resulted in label-free images of various layers within the diseased vessel wall. The THG signal adds an endogenous contrast mechanism with a practical degree of specificity for atherosclerotic plaques that complements current nonlinear optical methods for the investigation of cardiovascular disease. Our use of whole-mount tissue and backward scattered epi-detection suggests THG could potentially be used in the future as a clinical tool.

Overexpression of transforming growth factor ß1 in malignant prostate cells is partly caused by a runaway of TGF-ß1 auto-induction mediated through a defective recruitment of protein phosphatase 2A by TGF-ß type I receptor.

OBJECTIVES: To elucidate the mechanism of transforming growth factor (TGF)-ß1 overexpression in prostate cancer cells. METHODS: Malignant (PC3, DU145) and benign (RWPE1, BPH1) prostate epithelial cells were used. Phosphatase activity was measured using a commercial kit. Recruitment of the regulatory subunit, Bα, of protein phosphatase 2A (PP2A-Bα) by TGF-ß type I receptor (TßRI) was monitored by coimmunoprecipitation. Blockade of TGF-ß1 signaling in cells was accomplished either by using TGF-ß-neutralizing monoclonal antibody or by transduction of a dominant negative TGF-ß type II receptor retroviral vector. RESULTS: Basal levels of TGF-ß1 in malignant cells were significantly higher than those in benign cells. Blockade of TGF-ß signaling resulted in a significant decrease in TGF-ß1 expression in malignant cells, but not in benign cells. Upon TGF-ß1 treatment (10 ng/mL), TGF-ß1 expression was increased in malignant cells, but not in benign cells. This differential TGF-ß1 auto-induction between benign and malignant cells correlated with differential activation of extracellular signal-regulated kinase (ERK). Following TGF-ß1 treatment, the activity of serine/threonine phosphatase and recruitment of PP2A-Bα by TßRI increased in benign cells, but not in malignant cells. Inhibition of PP2A in benign cells resulted in an increase in ERK activation and in TGF-ß1 auto-induction after TGF-ß1 (10 ng/mL) treatment. CONCLUSIONS: These results suggest that TGF-ß1 overexpression in malignant cells is caused, at least in part, by a runaway of TGF-ß1 auto-induction through ERK activation because of a defective recruitment of PP2A-Bα by TßRI.

Effect of tecarfarin, a novel vitamin K epoxide reductase inhibitor, on coagulation in beagle dogs.

BACKGROUND AND PURPOSE: Tecarfarin (ATI-5923) is a novel vitamin K epoxide reductase inhibitor that is metabolized by esterase (mainly human carboxylesterase 2) to a single major metabolite, ATI-5900, in rats, dogs and humans. Tecarfarin is not significantly metabolized by CYP450 enzymes. The objective of this study was to test and compare the efficacy of tecarfarin with that of warfarin, when administered either intravenously or once a day orally, to produce stable anticoagulation in beagle dogs. EXPERIMENTAL APPROACH: Effects on coagulation were assessed by measuring the activity levels of Factor VII and Factor X and thromboplastin-induced coagulation times, reported as prothrombin time (PT). KEY RESULTS: Continuous intravenous infusions and oral administration of tecarfarin and warfarin caused a dose-dependent decrease in activity of Factor VII and Factor X, and associated increase in PT. Intravenous fresh frozen canine plasma or subcutaneous vitamin K(1) treatment reversed the anticoagulant effects of orally administered tecarfarin. Consistent with the inhibitory effects of amiodarone on CYP2C9, co-administration of amiodarone significantly increased the anticoagulation effect of warfarin and plasma warfarin concentrations. In contrast, amiodarone had no effect on the anticoagulation induced by tecarfarin or tecarfarin plasma concentrations in this model. CONCLUSIONS AND IMPLICATIONS: Overall, the data presented herein indicate that tecarfarin, via a vitamin K-dependent mechanism, causes changes in key parameters of haemostasis in beagle dogs that are consistent with effective anticoagulation. Compared to warfarin it has a decreased potential to interact metabolically with drugs that inhibit CYP450 enzymes and, therefore, may offer an improved safety profile for patients.

17Beta-estradiol at low concentrations acts through distinct pathways in normal versus benign prostatic hyperplasia-derived prostate stromal cells.

The aim of this study was to identify differential responses to low concentrations of 17beta-estradiol (E2) in primary stromal cell cultures derived from either normal organ donors or benign prostatic hyperplasia or hypertrophy (BPH) specimens. Furthermore, we sought to identify the potential mechanism of E2 action in these cell types, through either a genomic or nongenomic mechanism. We initially treated stromal cells derived from five normal prostates or five BPH specimens with low concentrations of E2 (0.001-1.0 nM) and analyzed their growth response. To determine whether genomic or nongenomic pathways were involved, we performed studies using specific estrogen receptor antagonists to confirm transcriptional activity or MAPK inhibitors to confirm the involvement of rapid signaling. Results of these studies revealed a fundamental difference in the mechanism of the response to E2. In normal cells, we found that a nongenomic, rapid E2 signaling pathway is predominantly involved, mediated by G protein-coupled receptor-30 and the subsequent activation of ERK1/2. In BPH-derived prostate stromal cells, a genomic pathway is predominantly involved because the addition of ICI 182780 was sufficient to abrogate any estrogenic effects. In conclusion, prostate stromal cells respond to far lower concentrations of E2 than previously recognized or examined, and this response is mediated through two distinct mechanisms, depending on its origin. This may provide the basis for new insights into the causes of, and possible treatments for, BPH.

CCR5 blockade modulates inflammation and alloimmunity in primates.

Pharmacologic antagonism of CCR5, a chemokine receptor expressed on macrophages and activated T cells, is an effective antiviral therapy in patients with macrophage-tropic HIV infection, but its efficacy in modulating inflammation and immunity is only just beginning to be investigated. In this regard, the recruitment of CCR5-bearing cells into clinical allografts is a hallmark of acute rejection and may anticipate chronic rejection, whereas conventionally immunosuppressed renal transplant patients homozygous for a nonfunctional Delta32 CCR5 receptor rarely exhibit late graft loss. Therefore, we explored the effects of a potent, highly selective CCR5 antagonist, Merck's compound 167 (CMPD 167), in an established cynomolgus monkey cardiac allograft model. Although perioperative stress responses (fever, diminished activity) and the recruitment of CCR5-bearing leukocytes into the graft were markedly attenuated, anti-CCR5 monotherapy only marginally prolonged allograft survival. In contrast, relative to cyclosporine A monotherapy, CMPD 167 with cyclosporine A delayed alloantibody production, suppressed cardiac allograft vasculopathy, and tended to further prolong graft survival. CCR5 therefore represents an attractive therapeutic target for attenuating postsurgical stress responses and favorably modulating pathogenic alloimmunity in primates, including man.

Double-label expression studies of prostacyclin synthase, thromboxane synthase and COX isoforms in normal aortic endothelium.

We have performed double-label immunofluorescence microscopy studies to evaluate the extent of co-localization of prostacyclin synthase (PGIS) and thromboxane synthase (TXS) with cyclooxygenase (COX)-1 and COX-2 in normal aortic endothelium. In dogs, COX-2 expression was found to be restricted to small foci of endothelial cells while COX-1, PGIS and TXS were widely distributed throughout the endothelium. Quantification of the total cross-sectioned aortic endothelium revealed a 6- to 7-fold greater expression of COX-1 relative to COX-2 (55 vs. 8%) and greater co-distribution of PGIS with COX-1 compared to COX-2 (19 vs. 3%). These results are in contrast to the extensive co-localization of PGIS and COX-2 in bronchiolar epithelium. In rat and human aortas, immunofluorescence studies also showed significant COX-1 and PGIS co-localization in the endothelium. Only minor focal COX-2 expression was detected in rat endothelium, similar to the dog, while COX-2 was not detected in human specimens. Inhibition studies in rats showed that selective COX-1 inhibition caused a marked reduction of 6-keto-PGF(1alpha) and TXB(2) aortic tissue levels, while COX-2 inhibition had no significant effect, providing further evidence for a functionally larger contribution of COX-1 to the synthesis of prostacyclin and thromboxane in aortic tissue. The data suggest a major role for COX-1 in the production of both prostacyclin and thromboxane in normal aortic tissue. The extensive co-localization of PGIS and COX-2 in the lung also indicates significant tissue differences in the co-expression patterns of these two enzymes.

Sphingosine-1-phosphate agonists increase macrophage homing, lymphocyte contacts, and endothelial junctional complex formation in murine lymph nodes.

The sphingosine-1-phosphate (S1P) receptor agonist, phosphorylated FTY720 (FTY-P), causes lymphopenia, lymphocyte sequestration in mesenteric lymph nodes (MLNs), and immunosuppression. Using multiple techniques to analyze MLN cells harvested from mice treated with S1P receptor agonists, we saw a redistribution of lymphocytes out of nodal sinuses and an expansion of follicles. Although changes in circulating monocytes were not observed with overnight exposure to FTY720, we saw a significant increase in S1P receptor 1 (S1P1)-expressing CD68+ macrophages in subcapsular sinuses of FTY-P-treated MLNs. This was confirmed by quantitative analysis of F4/80+ cells in MLN suspensions. The sinus volume and number of S1P1-positive cells within sinuses were also increased by FTY-P. High endothelial venules and lymphatic endothelium expressed high levels of S1P1, and treatment with FTY-P resulted in intense staining and colocalization of CD31, beta-catenin, and zona occludens 1 in junctions between sinus cells. Transmission electron microscopy showed that FTY-P greatly reduced lymphocyte microvilli and increased cell-cell contacts in the parenchyma. Immunoelectron microscopy revealed that intranodal lymphocytes lacked surface expression of S1P1, whereas S1P1 was evident on the surface and within the cytoplasm of macrophages, endothelial cells, and stromal cells. This subcellular pattern of intranodal receptor distribution was unchanged by treatment with FTY-P. We conclude that S1P1 agonists have profound effects on macrophages and endothelial cells, in addition to inducing lymphopenia.

Cytomegalovirus prophylaxis and treatment after hematopoietic stem cell transplantation in Canada: a description of current practices and comparison with Centers for Disease Control/Infectious Diseases Society of America/American Society for Blood and Marrow Transplantation guideline recommendations.

Prevention and management of cytomegalovirus (CMV) disease after hematopoietic stem cell transplantation (HSCT) is critically important, but clinical practices have historically been heterogeneous. The Centers for Disease Control (CDC), as part of a larger clinical practice guideline initiative, has published evidence-based recommendations, but their effect on clinical practice has not been assessed. A survey was sent to all Canadian HSCT program directors to describe current practices. Current practices were subsequently compared with CDC guideline recommendations and with a Canadian survey published before the CDC guidelines. When current practices did not conform to guideline recommendations, a literature review was performed to determine whether current practices were supported by evidence published after the CDC guidelines. The survey response rate was 100%. Variability in practices was observed in several aspects of patient care, including (1) indications for CMV-negative blood products, (2) surveillance tests used to detect CMV infection, (3) duration of surveillance testing, (4) duration of maintenance treatment after preemptive therapy was initiated, and (5) treatment of CMV disease. Overall adherence to guideline recommendations was good, especially when they were supported by high-quality data. However, deviations from guideline recommendations were observed: (1) most Canadian allogeneic programs used shorter courses of preemptive therapy; (2) prevention measures aimed at late CMV disease were not systematically applied at most allogeneic programs; and (3) most autologous HSCT programs did not administer preemptive therapy even in high-risk recipients. When deviations occurred, recent evidence supported current practices in some instances (shorter courses of preemptive therapy) but not in others (absence of strategies to prevent late CMV disease). Compared with practices in Canada before publication of the CDC guidelines, a higher proportion of programs used CDC-recommended surveillance tests and treatment for CMV infection. Current practices in Canada remain heterogeneous. Discrepancies between current practices and CDC guideline recommendations occurred in situations either in which practices had changed in response to recently published data or in which evidence supporting a recommendation was poor. These results suggest an urgent need for the development of well-designed clinical trials. Incorporation of recent data into updated guidelines may be appropriate.

A small molecule very late antigen-4 antagonist can inhibit ovalbumin-induced lung inflammation.

A nonpeptidyl small molecule antagonist, compound A, to nonactivated very late antigen-4 (VLA4) was examined in lung inflammation induced by a single dose of ovalbumin challenge. Compound A presented a good pharmacokinetic property, when given intratracheally, and the blood cells from such pharmacokinetic study showed good receptor occupancy of the compound for approximately 8 hours. Compound A was then tested in an ovalbumin-induced airway inflammation model by intranasal or intravenous route of administration. There was a dose-dependent inhibition of eosinophilia in the bronchiolar lavage fluid, when compound A was given intranasally but not when it was given intravenously. For comparison, antibody to VLA4 and another compound, BIO1211, which reacts only with activated VLA4, were examined in this system. Immunohistochemical analyses of the lung tissue substantiated the findings in the bronchiolar lavage fluid. Specific staining of the major basic protein of eosinophils showed peribronchiolar infiltration of eosinophils. Some of these eosinophils were also positive for nitrotyrosine, suggesting activation of eosinophils in the lung interstitium. There was deposition of major basic protein and nitrotyrosine at the base of the perivascular endothelium, indicative of degranulation of eosinophils in the area. After intranasal treatment with compound A, eosinophils in the lungs and their activation products were substantially decreased, documenting its effectiveness in inhibiting lung inflammation.