Cholinergic neurotransmission links solitary chemosensory cells to nasal inflammation.

Solitary chemosensory cells (SCCs) of the nasal cavity are specialized epithelial chemosensors that respond to irritants through the canonical taste transduction cascade involving Gα-gustducin and transient receptor potential melastatin 5. When stimulated, SCCs trigger peptidergic nociceptive (or pain) nerve fibers, causing an alteration of the respiratory rate indicative of trigeminal activation. Direct chemical excitation of trigeminal pain fibers by capsaicin evokes neurogenic inflammation in the surrounding epithelium. In the current study, we test whether activation of nasal SCCs can trigger similar local inflammatory responses, specifically mast cell degranulation and plasma leakage. The prototypical bitter compound, denatonium, a well-established activator of SCCs, caused significant inflammatory responses in WT mice but not mice with a genetic deletion of elements of the canonical taste transduction cascade, showing that activation of taste signaling components is sufficient to trigger local inflammation. Chemical ablation of peptidergic trigeminal fibers prevented the SCC-induced nasal inflammation, indicating that SCCs evoke inflammation only by neural activity and not by release of local inflammatory mediators. Additionally, blocking nicotinic, but not muscarinic, acetylcholine receptors prevents SCC-mediated neurogenic inflammation for both denatonium and the bacterial signaling molecule 3-oxo-C12-homoserine lactone, showing the necessity for cholinergic transmission. Finally, we show that the neurokinin 1 receptor for substance P is required for SCC-mediated inflammation, suggesting that release of substance P from nerve fibers triggers the inflammatory events. Taken together, these results show that SCCs use cholinergic neurotransmission to trigger peptidergic trigeminal nociceptors, which link SCCs to the neurogenic inflammatory pathway.

[Hyperthermic Isolated Limb Perfusion Combined with Tasonermin - a Perfusion Leakage Monitoring Technique]. / Hypertermická izolovaná perfuze koncetin v kombinaci s tasonerminem - technika monitorování úniku perfuzátu.

BACKGROUND: Hyperthermic isolated limb perfusion is used to treat irresectable extremity malignancies. It is based on the following principle - the perfusion of the extremity is isolated from systemic circulation and connected to an extra-corporal circuit via which a very high concentration of a chemotherapeutic agent is administered into the blood compartment of the extremity. In some cases, treatment efficiency can be improved using tasonermin (a TNF-α agent). By itself, tasonermin can cause severe health complications in patients if leakage into systemic circulation results in a level that exceeds the maximally tolerated dose. Therefore, it is important to monitor for leakage during the whole operation. METHOD: Leakage monitoring was performed by a nuclear medicine method based on the measurement of activity of a gamma-emitting radiotracer detected by a scintillation probe located over the heart. An amount of radiotracer that resulted in a basal level of measured signal was first administered into the systemic circuit followed by the administration of a second, one order of magnitude higher amount of radiotracer into the perfusion circuit. Leakage, when it occurred, increased the count rate detected over the heart, and the mathematical relation between leakage level and count rate increase was derived. RESULTS: In our department, the method was tested and optimized during isolated limb perfusion without using a TNF-α agent. Then, accreditation for the use of TNF-α was granted. Since then, the method has been used to monitor leakage in all cases of isolated limb perfusion with TNF-α. All isolated limb perfusion operations with TNF-α passed without complications. The radiation burden was almost negligible for both the patient and medical staff. CONCLUSION: The method described in this report represents a reliable method for perfusion leakage monitoring when using TNF-α in our department.Key words: perfusion - isolated limb - TNF-α - leakage - monitoring - nuclear medicine - radiopharmaceuticalsThe authors declare they have no potential confl icts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 16. 6. 2016Accepted: 21. 6. 2016.

Rapid-Steady-State-T1 signal modeling during contrast agent extravasation: toward tumor blood volume quantification without requiring the arterial input function.

PURPOSE: This study demonstrates how to quantify the tumor blood volume fraction (BVf) using the dynamic Rapid-Steady-State-T1 (RSST1 )-MRI method despite contrast agent (CA) leakage and without arterial input function (AIF) determination. METHODS: For vasculature impermeable to CAs, the BVf is directly quantified from the RSST1 signal amplitude. In case of CA extravasation, we propose a two-compartment model to describe the dynamic RSST1 signal increase. We applied the mathematical model in a pilot-study on a RG2-glioma model to compare extravasation of two Gd-based CAs. The BVf quantification using the mathematical model in a C6-glioma model (n = 8) with the clinical CA Gd-DOTA was validated using a ΔR2 *-steady-state MRI method with an USPIO and by immunohistochemical staining of perfused vessels labeled with Hoechst-33342 dye in the same rats. RESULTS: BVf in tumor and in healthy brain tissues (0.034 ± 0.005 and 0.026 ± 0.004, respectively) derived from the dynamic RSST1 signal were confirmed by ΔR2 *-steady-state MRI (0.036 ± 0.003 and 0.027 ± 0.002, respectively, correlation coefficient rS = 0.74) and by histology (0.036 ± 0.003 and 0.025 ± 0.004 respectively, rS = 0.87). CONCLUSION: Straightforward tumor BVf quantification without AIF determination is demonstrated in presence of CA leakage. The method will facilitate angiogenesis assessment in longitudinal neuro-oncologic studies in particular when monitoring the response to antiangiogenic therapies.

Cooperative role of endogenous leucotrienes and platelet-activating factor in ischaemia-reperfusion-mediated tissue injury.

Insufficient oxygen delivery to organs leads to tissue dysfunction and cell death. Reperfusion, although vital to organ survival, initiates an inflammatory response that may both aggravate local tissue injury and elicit remote organ damage. Polymorphonuclear neutrophil (PMN) trafficking to remote organs following ischaemia/reperfusion (I/R) is associated with the release of lipid mediators, including leucotriene (LT) B4 , cysteinyl-LTs (CysLTs) and platelet-activating factor (PAF). Yet, their potentially cooperative role in regulating I/R-mediated inflammation has not been thoroughly assessed. The present study aimed to determine the cooperative role of lipid mediators in regulating PMN migration, tissue oedema and injury using selective receptor antagonists in selected models of I/R and dermal inflammation. Our results show that rabbits, pre-treated orally with BIIL 284 and/or WEB 2086 and MK-0571, were protected from remote tissue injury following I/R or dermal inflammation in an additive or synergistic manner when the animals were pre-treated with two drugs concomitantly. The functional selectivity of the antagonists towards their respective agonists was assessed in vitro, showing that neither BIIL 284 nor WEB 2086 prevented the inflammatory response to IL-8, C5a and zymosan-activated plasma stimulation. However, these agonists elicited LTB4 biosynthesis in isolated rabbit PMNs. Similarly, a cardioprotective effect of PAF and LTB4 receptor antagonists was shown following myocardial I/R in mice. Taken together, these results underscore the intricate involvement of LTB4 and PAF in each other's responses and provide further evidence that targeting both LTs and PAF receptors provides a much stronger anti-inflammatory effect, regulating PMN migration and oedema formation.

The cannabinoid receptor agonist WIN 55,212-2 inhibits antigen-induced plasma extravasation in guinea pig airways.

BACKGROUND: Although neurogenic inflammation of the airways via activation of C-fibers is thought to be important in the pathogenesis of asthma, the mechanisms regulating C-fiber activity remain uncertain. OBJECTIVE: The influence of a cannabinoid receptor agonist, WIN 55,212-2, on C-fiber activation in guinea pig airways was investigated, as was the mechanism by which cannabinoids regulate antigen-induced airway inflammation. METHODS: The inhibitory effect of WIN 55,212-2 on antigen-induced plasma extravasation was assessed in guinea pig tracheal tissues by photometric measurement of extravasated Evans blue dye after extraction with formamide. RESULTS: Pretreatment with WIN 55,212-2 (0.001, 0.01 or 0.1 mg/kg) significantly and dose-dependently reduced tracheal plasma extravasation induced by inhaling a 5% ovalbumin solution for 2 min after pretreatment with a neutral endopeptidedase inhibitor (phosphoramidon at 2.5 mg/kg i.v.). A cannabinoid CB2 receptor antagonist (SR144528) blunted the inhibitory effect of WIN 55,212-2, while a cannabinoid CB1 antagonist (SR141716A) did not. Pretreatment with a neurokinin-1 receptor antagonist (FK888) significantly reduced ovalbumin-induced extravasation of Evans blue dye. Pretreatment with the combination of WIN 55,212-2 and FK888 reduced antigen-induced plasma extravasation more markedly than FK888 alone. CONCLUSIONS: These findings suggest that WIN 55,212-2 inhibits C-fiber activation via the cannabinoid CB2 receptor and thus suppresses antigen-induced inflammation in guinea pig airways.

Pharmacological characterization of 3-[3-tert-butylsulfanyl-1-[4-(6-methoxy-pyridin-3-yl)-benzyl]-5-(pyridin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethyl-propionic acid (AM103), a novel selective 5-lipoxygenase-activating protein inhibitor that reduces acute and chronic inflammation.

Leukotrienes (LTs) are proinflammatory lipid mediators synthesized by the conversion of arachidonic acid (AA) to LTA(4) by the enzyme 5-lipoxygenase (5-LO) in the presence of 5-LO-activating protein (FLAP). 3-[3-tert-Butylsulfanyl-1-[4-(6-methoxy-pyridin-3-yl)-benzyl]-5-(pyridin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethyl-propionic acid (AM103) is a novel selective FLAP inhibitor in development for the treatment of respiratory conditions such as asthma. In a rat ex vivo whole-blood calcium ionophore-induced LTB(4) assay, AM103 (administered orally at 1 mg/kg) displayed >50% inhibition for up to 6 h with a calculated EC(50) of approximately 60 nM. When rat lung was challenged in vivo with calcium ionophore, AM103 inhibited LTB(4) and cysteinyl leukotriene (CysLT) production with ED(50) values of 0.8 and 1 mg/kg, respectively. In this model, the EC(50) derived from plasma AM103 was approximately 330 nM for inhibition of both LTB(4) and CysLT. In an acute inflammation setting, AM103 displayed dose-dependent inhibition of LTB(4), CysLT, and plasma protein extravasation induced by peritoneal zymosan injection. In a model of chronic lung inflammation using ovalbumin-primed and challenged BALB/c mice, AM103 reduced the concentrations of eosinophil peroxidase, CysLTs, and interleukin-5 in the bronchoalveolar lavage fluid. Finally, AM103 increased survival time in mice exposed to a lethal intravenous injection of platelet-activating factor. In summary, AM103 is a novel, potent and selective FLAP inhibitor that has excellent pharmacodynamic properties in vivo and is effective in animal models of acute and chronic inflammation and in a model of lethal shock.

Non-invasive imaging of barriers to drug delivery in tumors.

Solid tumors often develop high interstitial fluid pressure (IFP) as a result of increased water leakage and impaired lymphatic drainage, as well as changes in the extracellular matrix composition and elasticity. This high fluid pressure forms a barrier to drug delivery and hence, resistance to therapy. We have developed techniques based on contrast enhanced magnetic resonance imaging for mapping in tumors the vascular and transport parameters determining the delivery efficiency of blood borne substances. Sequential images are recorded during continuous infusion of a Gd-based contrast agent and analyzed according to a new physiological model, yielding maps of microvascular transfer constants, as well as outward convective interstitial transfer constants and steady state interstitial contrast agent concentrations both reflecting IFP distribution. We further demonstrated in non small cell human lung cancer xenografts the capability of our techniques to monitor in vivo collagenase induced increase in contrast agent delivery as a result of decreased IFP. These techniques can be applied to test drugs that affect angiogenesis and modulate interstitial fluid pressure and has the potential to be extended to cancer patients for assessing resistance to drug delivery.

Management of anthracycline extravasation injuries.

OBJECTIVE: To review the evidence for the management of anthracycline extravasation and determine the optimal treatment of such injuries. DATA SOURCES: A search of MEDLINE (1966-February 2007) and International Pharmaceutical Abstracts (1970-February 2007) was performed using the search terms anthracyclines and extravasation. DATA SYNTHESIS: Extravasation of anthracyclines can have devastating effects. After infiltration of these drugs into the interstitial tissue, damage may range from mild erythema and pain to severe tissue necrosis. Many agents have been studied in the management of these injuries; however, few have demonstrated efficacy and treatment remains controversial. Nonpharmacologic modalities shown to limit extravasation injuries include local tissue cooling and elevation of the affected area. Corticosteroids, sodium bicarbonate, hyaluronidase, hyperbaric oxygen, heparin fractions, alpha-tocopherol, N-acetylcysteine, and granulocyte macrophage-colony stimulating factor have all either been shown to be ineffective or have limited data supporting their use. Topical dimethyl sulfoxide (DMSO) has been shown in prospective studies to limit the course of extravasation injuries. Dexrazoxane has been shown in animal models and case reports to be useful in the management of anthracycline extravasation. Two recent prospective clinical trials examining intravenous dexrazoxane 1000 mg/m2 within 6 hours of extravasation, 1000 mg/m2 24 hours after extravasation, and 500 mg/m2 48 hours after extravasation injuries add to the data supporting the use of this agent in such injuries. Of the 54 patients enrolled, surgery-requiring necrosis was avoided in 98.2%. CONCLUSIONS: The optimal treatment of anthracycline extravasation includes local tissue cooling, elevation of the afflicted extremity, dexrazoxane administration, and possibly topical DMSO. Many other drugs have been investigated; however, due to a lack of data, they cannot be recommended for the management of anthracycline extravasation.

Characterizing extravascular fluid transport of macromolecules in the tumor interstitium by magnetic resonance imaging.

Noninvasive imaging techniques to image and characterize delivery and transport of macromolecules through the extracellular matrix (ECM) and supporting stroma of a tumor are necessary to develop treatments that alter the porosity and integrity of the ECM for improved delivery of therapeutic agents and to understand factors which influence and control delivery, movement, and clearance of macromolecules. In this study, a noninvasive imaging technique was developed to characterize the delivery as well as interstitial transport of a macromolecular agent, albumin-GdDTPA, in the MCF-7 human breast cancer model in vivo, using magnetic resonance imaging. The transport parameters derived included vascular volume, permeability surface area product, macromolecular fluid exudate volume, and drainage and pooling rates. Immunohistochemical staining for the lymphatic endothelial marker LYVE-1 was done to determine the contribution of lymphatics to the macromolecular drainage. Distinct pooling and draining regions were detected in the tumors using magnetic resonance imaging. A few lymphatic vessels positively stained for LYVE-1 were also detected although these were primarily collapsed and tenuous suggesting that lymphatic drainage played a minimal role, and that the bulk of drainage was due to convective transport through the ECM in this tumor model.

Hemoglobin extravasation in the brain of rats exchange-transfused with hemoglobin-based oxygen carriers.

Haemoglobin (Hb)-based oxygen carriers are under consideration as oxygen therapeutics. Their effect on apoptosis is critical, because the onset of pro-apoptotic pathways may lead to tissue damage. MP4OX, a polyethylene glycol-conjugated human Hb preserves the baseline level of neuron apoptosis with respect to sham. Here we develop a method for measuring Hb extravasation in brain. We exchange transfused rats by haemorrhaging 50% of their blood with simultaneous, isovolemic replacement with Hextend (negative control), MP4OX, or αα-cross-linked Hb. Animals were sacrificed 2 h after transfusion, brain tissue was harvested and processed for double-staining immunofluorescence, whereby Hb ? chain and NeuN (a neuron protein) were stained and quantitated. Whereas Hextend did not induce Hb extravasation, in both MP4OX and ??Hb brains Hb molecules were detected outside neurons. The level of extravasated Hb chains was > 3-fold higher in Hb compared to MP4OX. Western blot analysis revealed that the expression levels of protein related to redox imbalance (e.g., Nrf2, iNOS and ERK phosphorylation) were higher in ααHb than MP4OX. In conclusions, higher Hb extravasation in ααHb than MP4OX induces redox imbalance, which causes higher anti-oxidant response. Whereas Nrf2 response may be considered protective, iNOS response appears damaging.

Comparison of extraarticular leakage values of radiopharmaceuticals used for radionuclide synovectomy.

OBJECTIVES: Radionuclide synovectomy is a reliable therapy in patients with chronic synovitis. However, radiation doses delivered to non-target organ systems due to leakage of radioactive material from the articular cavity are an important disadvantage of this procedure. In this study we compared extraarticular leakage values of the 3 commonly used radiopharmaceuticals; 90Y-citrate, 90Y-silicate and 186Re-sulfide colloid. MATERIALS AND METHODS: Thirty-five patients with persistent synovitis were enrolled in the study. Twenty-two hemophilic, 8 rheumatoid arthritis and 5 patients with pigmented villonodular synovitis were studied. 90Y labeled silicate and citrate were used for knee joints and 186Re-sulfide for intermediate sized joints. Radiocolloid leakage values were evaluated using a gamma camera with 20% window centered over the bremsstrahlung photopeak of 90Y and a respective window over the 137 keV photopeak of 186Re. Regions of interest were drawn over the injection site, the regional lymph nodes and the background areas. Leakage of radiocolloid was calculated by dividing the counts/pixel in the regional lymph node area to the counts/pixel in the injection site. RESULTS: No visible leakage was observed. The median leakage values calculated for 90Y-citrate, 90Y-silicate and 186Re-sulfide were found as 1.9%, 2.4% and 2.7%, respectively. The difference between the variability of leakage values was not statistically significant (p > 0.05). CONCLUSION: There was no significant difference in terms of extraarticular leakage between 9Y-citrate, 9Y-silicate and 186Re-sulfide radiocolloids.

Pharmacokinetic analysis of the perivascular distribution of bifunctional antibodies and haptens: comparison with experimental data.

A mathematical model is developed to describe the concentration profiles around individual tumor blood vessels for two-step approaches to cancer treatment. The model incorporates plasma pharmacokinetics, interstitial diffusion, reversible binding between antibody and hapten and between antibody and tumor-associated antigens, and physiological parameters to evaluate present experimental approaches and to suggest new guidelines for the effective use of two-step approaches. Results show considerable interaction between the binding kinetics, initial drug doses, and antigen density, with optimal parameter ranges depending on the desired goal: treatment or detection. The hapten concentration in tumors was found to be nonuniform because of specific binding to antibodies. While binding of the hapten to the bifunctional antibody is necessary for improved retention, too large a binding affinity may lead to very poor penetration of the hapten into regions far away from blood vessels. The time delay between antibody and hapten injection was found to be an important parameter. Longer time delays were found to be advantageous, subject to constraints such as internalization of the antibody and tumor growth during treatment. A proper combination of initial doses for the two species was also seen to be crucial for maximum effectiveness. Comparison of the model with the experimental data of Le Doussal et al. (Cancer Res., 51: 6650-6655, 1991) and Stickney et al. (Cancer Res., 50: 3445-3452, 1990) suggests two novel, yet testable, hypotheses: (a) the early pharmacokinetics of low molecular weight agents can have an important effect on later concentrations using two-step approaches; and (b) metabolism may play an important role in reducing concentrations in the tumor and tumor:plasma concentration ratios. These results should help in the effective design of two-step strategies.

Dynamic application of microprojection arrays to skin induces circulating protein extravasation for enhanced biomarker capture and detection.

Surface modified microprojection arrays are a needle-free alternative to capture circulating biomarkers from the skin in vivo for diagnosis. The concentration and turnover of biomarkers in the interstitial fluid, however, may limit the amount of biomarker that can be accessed by microprojection arrays and ultimately their capture efficiency. Here we report that microprojection array insertion induces protein extravasation from blood vessels and increases the concentration of biomarkers in skin, which can synergistically improve biomarker capture. Regions of blood vessels in skin were identified in the upper dermis and subcutaneous tissue by multi-photon microscopy. Insertion of microprojection array designs with varying projection length (40-190 µm), density (5000-20,408 proj.cm(-2)) and array size (4-36 mm(2)) did not affect the degree of extravasation. Furthermore, the location of extravasated protein did not correlate with projection penetration to these highly vascularised regions, suggesting extravasation was not caused by direct puncture of blood vessels. Biomarker extravasation was also induced by dynamic application of flat control surfaces, and varied with the impact velocity, further supporting this conclusion. The extravasated protein distribution correlated well with regions of high mechanical stress generated during insertion, quantified by finite element models. Using this approach to induce extravasation prior to microprojection array-based biomarker capture, anti-influenza IgG was captured within a 2 min application time, demonstrating that extravasation can lead to rapid biomarker sampling and significantly improved microprojection array capture efficiency. These results have broad implications for the development of transdermal devices that deliver to and sample from the skin.

Prediction of extravasation in pelvic fracture using coagulation biomarkers.

PURPOSE: To evaluate the usefulness of coagulation biomarkers, which are easy and quick to analyze in emergency settings, for prediction of arterial extravasation due to pelvic fracture. PATIENTS AND METHODS: The medical records of pelvic fracture patients transferred to the emergency department of Gunma University Hospital between December 2009 and May 2015 were reviewed. Patients were divided into two groups, those with (Extra(+)) and without (Extra(-)) arterial extravasation on enhanced CT or angiography. Levels of fibrin degradation products (FDP), D-dimer, fibrinogen, the ratio of FDP to fibrinogen, the ratio of D-dimer to fibrinogen, systolic blood pressure, heart rate, the Glasgow Coma Scale, pH, base excess, hemoglobin and lactate levels, the pattern of pelvic injury, and injury severity score were measured at hospital admission, and compared between the two groups. Parameters with a significant difference between the two groups were used to construct receiver operating characteristic (ROC) curves. RESULTS: The study included 29 patients with pelvic fracture. FDP, D-dimer, the ratio of FDP to fibrinogen and the ratio of D-dimer to fibrinogen were the most useful parameters for predicting arterial extravasation due to pelvic fracture. FDP, D-dimer, the ratio of FDP to fibrinogen, the ratio of D-dimer to fibrinogen, and hemoglobin and lactate levels were significantly higher in the Extra(+) group than in the Extra(-) group (FDP, 354.8µg/mL [median] versus 96.6µg/mL; D-dimer, 122.3µg/mL versus 42.1µg/mL; the ratio of FDP to fibrinogen, 3.39 versus 0.42; the ratio of D-dimer to fibrinogen, 1.14 versus 0.18; hemoglobin, 10.5g/dL versus 13.5g/dL; lactate, 3.5mmol/L versus 1.7mmol/L). The area under the ROC curves for FDP, D-dimer, the ratio of FDP to fibrinogen, the ratio of D-dimer to fibrinogen, hemoglobin and lactate levels were 0.900, 0.882, 0.918, 0.900, 0.815 and 0.765, respectively. CONCLUSION: Coagulation biomarkers, and hemoglobin and lactate levels could be useful to predict the existence of arterial extravasation due to pelvic fracture. The ratio of FDP to fibrinogen and the ratio of D-dimer to fibrinogen were the most accurate markers. Coagulation biomarkers may enable more rapid and specific treatment for pelvic fracture.

The vesiculo-vacuolar organelle (VVO): a distinct endothelial cell structure that provides a transcellular pathway for macromolecular extravasation.

The vesiculo-vacuolar organelle (VVO) is a recently described organelle found in the cytoplasm of endothelial cells that line tumor microvessels and normal venules. VVOs are grape-like clusters of interconnecting uncoated vesicles and vacuoles, bounded by trilaminar unit membranes, that span the entire thickness of vascular endothelium, thereby providing a potential trans-endothelial connection between the vascular lumen and the extravascular space. Macromolecular tracers preferentially cross hyperpermeable tumor microvessels through VVOs. The present investigation was undertaken to elucidate further the ultrastructure and function of VVOs in a murine ovarian carcinoma (MOT) and in normal venules. Morphometry revealed that VVOs were enormous cytoplasmic structures (median area, 0.12-0.14 microns2 in single electron micrographs). Moreover, the individual vesicles and vacuoles that comprised VVOs were on average substantially larger than capillary caveolae and followed a non-normal distribution that was skewed to the right. Specimen tilting provided conclusive evidence that individual VVO vesicles and vacuoles communicated with each other and with the endothelial cells' plasma membranes by stomata, some of which were closed by diaphragms composed of a single membrane. Studies with two tracers, ferritin (FE, diameter approximately 11 nm) and horseradish peroxidase (HRP, diameter approximately 5 nm), revealed that passage of macromolecules through VVOs was regulated at the level of stomatal diaphragms, thereby demonstrating a mechanism for controlling the passage of macromolecules across endothelial cells. Thus, compared with tumor microvessels, little circulating FE and HRP entered the VVOs of normal venular endothelium because stomata joining vesicles and vacuoles to each other and to the lumen and ablumen were closed. VVOs and their component vesicles/vacuoles were readily distinguished from endosomal organelles such as coated vesicles and multivesicular bodies, which also accumulated FE and HRP. Our findings indicate that VVOs provide a major pathway for the extravasation of circulating macromolecules across endothelia taller than capillary endothelium and suggest that upregulated VVO function accounts for the well-known hyperpermeability of tumor blood vessels.

A pitfall of FDG-PET image interpretation: accumulation of FDG in the dependent area of the urinary bladder after bladder irrigation--the usefulness of the prone position.

In F-18 FDG PET studies, retrograde irrigation of the urinary bladder is usually used to reduce the interference with physiological urinary accumulation of F-18 FDG in patients with possible pelvic lesions. A 34-year-old female who had recently been diagnosed with cervical cancer had an F-18 FDG-PET scan performed for whole-body evaluation. The bladder was irrigated with physiological saline and filled with 200 mL of irrigation fluid through a 3-way balloon catheter inserted before the scan. Two areas with high FDG accumulation were noted in the posterior pelvis. Tumor invasion or metastasis could not be ruled out. Additional focal imaging of the pelvis was performed with the patient in the prone position. The lesion on the left side of the patient was still noted, whereas the lesion on the right had disappeared, which proved that it was a false-positive lesion. Great caution is required when assessing imaging results to avoid misdiagnosis in patients after bladder irrigation.

Vagal modulation of spinal nicotine-induced inhibition of the inflammatory response mediated by descending antinociceptive controls.

Noxious stimuli activate neuroendocrine axes, inhibiting inflammation, an effect that is powerfully attenuated by ongoing activity in subdiaphragmatic vagal afferents. To evaluate whether this inhibitory effect of vagal afferent activity is mediated by descending antinociceptive control, we tested whether antagonizing descending antinociceptive controls: (i) enhances the inhibition of inflammation produced by spinal nicotine (which stimulates central terminals of nociceptors) and (ii) occludes the enhancing effect of subdiaphragmatic vagotomy, in the rat. Spinal intrathecal co-administration of the alpha-adrenergic receptor antagonist phentolamine and the non-selective opioid receptor antagonist naloxone, and acute subdiaphragmatic vagotomy each produced enhancement, with similar magnitude, of nicotine-induced inhibition of plasma extravasation, produced by the potent inflammatory mediator, bradykinin. The combination of subdiaphragmatic vagotomy and intrathecal receptor antagonists, however, produced no further enhancement compared to each treatment alone. These findings support the suggestion that activity in descending antinociceptive controls modulates noxious stimulus-induced inhibition of inflammation and the vagal modulation of noxious stimulus-induced inhibition of inflammation is mediated by descending antinociceptive controls.

Breakdown of the blood-retinal barrier in a model of retinal neovascularization.

Breakdown in the blood-retinal barrier occurs in retinal neovascularization in a number of diseases. To study the anatomic basis of this breakdown, we examined retinal neovascularization induced by injection of 250,000 homologous fibroblasts into the vitreous cavity of pigmented rabbits. Neovascularization is evident by electron microscopy in this model 3 days after fibroblast injection. Fluorescein angiography followed by intravenous horseradish peroxidase (HRP) injection was performed prior to enucleation on 2, 3, 5, 7, and 14 days after fibroblast injection. Fluorescein leakage from retinal vessels occurs early (at day 1) and persists as the neovascularization progresses. The leakage in the early stages is concentrated near puckers from the medullary wings. In the later stages, fluorescein leakage is most prominent in the developing tips of the new vessels. Horseradish peroxidase was not observed to leak from the lumen of new vessels. "Gaps" or separations in the endothelial cell junctions were not observed in developing vessels. The breakdown of the blood-retinal barrier in this model of retinal neovascularization is therefore selective, (ie, fluorescein leaks but not HRP) and it is not due to gaps or fenestrations between endothelial cells in developing vessels.

Characterization of tachykinin receptors mediating plasma extravasation and vasodilatation in normal and acutely inflamed knee joints of the rat.

1. Inflammatory actions of tachykinins in normal rat knee joints were compared with those of animals with acutely inflamed joints induced by intra-articular injection of 2% carrageenan. Plasma protein extravasation in rat knee joints, measured by protein micro-turbidimetry, was induced by intra-articular perfusion of selective tachykinin receptor agonists. Changes in joint blood flow, measured by laser Doppler perfusion imaging, were produced by topical applications of selective tachykinin receptor agonists to the joint capsule. 2. Carrageenan-injected rat knee joints showed significantly higher (P < 0.001) basal plasma extravasation (56 +/- 4 micrograms ml-1, n = 5) than normal rat knee joints (10 +/- 4 micrograms ml-1, n = 6). Intra-articular perfusion of the selective neurokinin1 (NK1) receptor agonist [Sar9, Met(O2)11]-substance P (0.8 nmol min-1) for 60 min elevated the basal plasma extravasation to 90 +/- 17 micrograms ml-1 (n = 6, P < 0.001) in normal joints, and to 150 +/- 14 micrograms ml-1 (n = 5, P < 0.001) in inflamed joints. Perfusion of the selective NK1 receptor antagonist N2-[(4R)-4-hydroxy-1-(1-methyl-1H- indol-3-yl)carbonyl-L-prolyl]-N-methyl-N-phenylmethyl-3-(2-naphthyl)- L-alaninamide (FK888; 0.8 nmol min-1) for 20 min followed by co-perfusion with the NK1 receptor agonist (0.8 nmol min-1) produced complete inhibition of the NK1 receptor agonist-induced plasma extravasation in the two groups of animals (for both groups; n = 3, P < 0.001). 3. Intra-articular perfusion of the selective NK receptor agonist [Nle10]-neurokinin A4-10 (0.8 nmol min-1) and the selective NK3 receptor agonist [MePhe7]-neurokinin B (0.8 nmol min1) produced no increase in plasma extravasation in normal or in inflamed rat knee joints (n = 4 and 11, P > 0.05). 4. Topical bolus applications of the NK1 receptor agonist [Sar9, Met(O2)11]-substance P onto normal joint capsules produced dose-dependent vasodilatation expressed as a voltage increase from control level. The maximum increase in blood flow was 2.05-0.21 V from a basal voltage of 3.42 +/- 0.07 V (n = 13, P < 0.001). To a much lesser extent, administration of the NK2 receptor agonist [Nle10]-neurokinin A4-10 also produced dose-dependent vasodilatation with maximum increase of 0.46 +/- 0.08 V from a basal level of 3.38 +/- 0.1 V (n = 7, P < 0.01). Animals with acutely inflamed joints showed enhanced vasodilator responses to the NK1 and NK2 receptor agonists (for both: P vs non-inflamed joints < 0.001). Thus, the NK1 and NK2 receptor agonists produced maximum increases of 2.56 +/- 0.19 V (basal level = 5.84 +/- 0.07 V; n = 7, P < 0.001) and 1.97 +/- 0.26 V (basal level = 6.31 +/- 0.23 V; n = 11, P < 0.001), respectively. The NK3 receptor agonist [MePhe7]-neurokinin B produced no change in blood flow in normal or in inflamed rat knee joints (n = 7 and 5, P > 0.05). 5. Bolus administration of the NK1 receptor antagonist FK888 (10 pmol) alone followed 5 min later by another dose of 10 pmol FK888 (i.e. total dose of 2 x 10 pmol) applied together with the NK1 receptor selective agonist [Sar9, Met(O2)11]-substance P produced partial, but significant inhibition of the NK1 receptor agonist-induced vasodilatation in both normal (maximum response reduced by 51.9 +/- 5.4%; n = 6, P < 0.001) and inflamed rat knee joints (maximum response reduced by 49.3 +/- 6.1%; n = 5, P < 0.001). The NK2 receptor agonist [Nle10]-neurokinin A4-10-induced vasodilator responses in inflamed joints were not affected by this treatment (n = 6, P > 0.05). However, with two higher doses of FK888 (both 1 nmol), the NK1 and the NK2 receptor agonist-induced vasodilator responses were abolished in the two groups of animals (n = 6-8, P < 0.005). 6. Administration of two doses of the selective NK2 receptor antagonist (S)-N-methyl-N-[4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl) -butyl]benzamide (SR48968;...