Improved urethral fluorescence during low rectal surgery: a new dye and a new method.

BACKGROUND: The aim of this study was to demonstrate highlighting of the urethra during surgery through the use of two different methods: a new near-infrared fluorophore IRDye800BK, and indocyanine green (ICG) mixed with silicone. METHODS: Male cadavers from the department of anatomy at the University of Oxford were used to visualise the urethra during near-infrared fluorescence excitation. To assess IRDye800BK, a perineal incision was utilised after infiltrating the urethra directly with an IRDye800BK solution mixed with Instillagel. ICG-silicone was assessed when the urethra was purposely exposed as part of a simulated transanal total mesorectal dissection. ICG was previously mixed with ethanol and silicone and left to set in a Foley catheter. Fluorescence was visualised using an in-house manufactured fluorescence-enabled laparoscopic system. RESULTS: IRDye800BK demonstrated excellent penetration and visualisation of the urethra under fluorescence at an estimated tissue depth of 2 cm. An ICG-silicone catheter demonstrated excellent fluorescence without leaving any residual solution behind in the urethra after its removal. CONCLUSIONS: The newly described ICG-silicone method opens up the possibility of new technologies in this area of fluorescence guided surgery. IRDye800BK is a promising alternative to ICG in visualising the urethra using fluorescence imaging. Its greater depth of penetration may allow earlier detection of the urethra during surgery and prevent wrong plane surgery sooner.

Multislice CT in emergency room management of patients with chest pain and medium-low probability of acute coronary syndrome.

PURPOSE: The main cause of acute chest pain, which accounts for 6.5% of urgent medical examinations in emergency rooms in Italy, is acute coronary syndrome (ACS). We performed this prospective study to evaluate the diagnostic accuracy of a 16-channel computed tomography (CT) scanner with dedicated software in a group of patients with chest pain and medium to low risk of ACS. MATERIALS AND METHODS: This study involved a selected group of 31 patients reporting chest pain with a medium to low probability of ACS, defined on the basis of preliminary tests [electrocardiogram (ECG) and serum cardiac markers]. Coronary angiography, performed within 24 h of MSCT, was used as the gold standard. RESULTS: MSCT identified the presence of occlusions and significant (>50%) or nonsignificant stenoses in the main coronary segments, with a sensitivity of 65%, a specificity of 98.8%, a positive predictive value (PPV) of 81.2%, a negative predictive value (NPV) of 97.3% and an accuracy of 96.4%. Significant stenoses and occlusions were detected with a sensitivity of 71.4%, a specificity of 99.6%, a PPV of 93.7%, an NPV of 97.7% and an accuracy of 97.5%. CONCLUSIONS: Due to its high NPV, this technique can rule out significant stenoses or coronary occlusions provided that image quality is excellent. In patients with a medium to low coronary risk, MSCT is a more accurate indicator of the need for coronary angiography than is exercise stress testing, which is less expensive but has lower predictive values.

Thermodynamics of the high-affinity interaction of TCF4 with beta-catenin.

The formation of a complex between beta-catenin and members of the TCF/LEF family of high-mobility group proteins is a key regulatory event in the wnt-signaling pathway, essential for embryonal development as well as the growth of normal and malignant colon epithelium. We have characterized the binding of TCF4 to human beta-catenin by steady-state intrinsic fluorescence quenching experiments, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Binding studies in solution and in heterogeneous phase showed that TCF4 binds reversibly to beta-catenin with an affinity (KB) of 3(+/-1) 10(8) M(-1). Site-directed mutagenesis, together with calorimetric measurements, revealed that residue D16 in TCF4 plays a crucial role in high-affinity binding. Mutation of this residue to alanine resulted in a decrease of KB by two orders of magnitude as well as a significant reduction in binding enthalpy. Binding of TCF4 to beta-catenin gave rise to a large negative enthalpy change at 25 degrees C (-29.7 kcal/mol). Binding enthalpies were strongly temperature dependent, which resulted in the determination of a large heat capacity change upon binding of -1.5 kcal/(mol K). The molecular events that take place upon complex formation are discussed using the measured thermodynamic data together with the crystal structure of the beta-catenin arm repeat region/TCF complex.

[Hypertonic saline solutions in resuscitation in hemorrhagic shock. An experimental study]. / Le soluzioni saline ipertoniche nel recupero dallo shock emorragico. Studio sperimentale.

The aim of the study was to evaluate the use of hypertonic solutions in restoring intravascular volume in a model of hemorrhagic shock. Eighteen pigs underwent general anesthesia and were instrumented with a carotid catheter to record mean arterial pressure (MAP), a pulmonary artery catheter for pulmonary arterial pressure (MPAP) and cardiac output (CO) monitoring and an electromagnetic flowmeter around the abdominal supraceliac aorta for aortic flow measurement (Vaor). Oxygen delivery (DO2) and oxygen consumption (VO2) data were calculated by standard formulas. The animals were hemorrhaged to a MAP of 45 mmHg, held for 1 hour. They were resuscitated during the following hour until the aortic flow regained its basal value, using three different solutions: normotonic saline (NS = NaCl 0.9%), hypertonic saline (HS = NaCl 7.5%), hypertonic saline added with dextran (HSDX = NaCl 7.5% + 6% dextran 70). An hour of autologous blood transfusion and a two hours follow-up concluded the experiment. Volumes infused were remarkably lower administering HS (13.70 +/- 1.44 ml/kg) and HSDX (9.11 +/- 1.20 ml/kg) compared to NS (90.32 +/- 24.83 ml/kg). MAP, CO and DO2 values resulted significantly higher in the HSDX animals, with lower MPAP levels. During the two hours follow-up only the animals reinfused with HSDX maintained hemodynamic and oxygen transport values at normal levels. We conclude that the administration of hypertonic saline solutions during hemorrhagic shock allows the saving of infusion volumes, thus diminishing the occurrence of interstitial edema formation. The adding of dextran to the solution prolongs the hemodynamic effects.

Agents combining thromboxane receptor antagonism with thromboxane synthase inhibition: [[[2-(1H-imidazol-1-yl)ethylidene]amino]oxy]alkanoic acids.

A new class of compounds combining thromboxane-A2 (TxA2) receptor antagonism and thromboxane synthase inhibition is described. A first series of (E)- and (Z)-[[[2-(1H-imidazol-1-yl)ethylidene]amino]oxy]pentanoic acids showed relevant thromboxane synthase inhibition associated with weak TxA2 receptor antagonism, while a series of (+/-)-(E)-[[[2-(1H-imidazol-1-yl)-3-phenylpropylidene]amino]oxy] pentanoic acids, structurally derived from the former, showed potent and well-balanced dual activity. Structural requirements for significant single and dual activity are discussed. Two close congeners of the latter series, (+/-)-(E)-5-[[[1-cyclohexyl-2-(1H-imidazol-1-yl)-3- phenylpropylidene]amino]oxy]pentanoic acid 23c and its p-fluorophenyl analog 23m, inhibited TxB2 production in vitro, in rat whole blood during clotting, with IC50 of 0.06 and 0.37 microM and antagonized the binding of [3H]SQ 29548 to washed human platelets, with IC50 of 0.08 and 0.02 microM, respectively. These two compounds were selected for further pharmacological evaluation and were shown to antagonize U46619-induced platelet aggregation in human platelet rich plasma with IC50 of 0.30 and 0.44 microM, respectively. They were both orally available, and in particular 23m caused a long lasting ex vivo TxA2 synthase inhibition in the fed rat. The levorotatory enantiomer of 23c, stereospecifically synthesized as a model compound, was found to be more potent than racemic 23c with regard to TxA2 receptor antagonism (IC50 = 0.04 microM) and equivalent to the latter with regard to TxA2 synthase inhibition. A molecular modeling study concerning the levorotatory enantiomer of 23c (S), TxA2, and representative TxA2 antagonists of different classes led to the definition of a putative pharmacophoric model for the TxA2 receptor ligands.

Endothelin-1-selective binding sites are downregulated by transforming growth factor-beta and upregulated by basic fibroblast growth factor in a vascular smooth muscle-derived cell line.

Endothelins (ETs) elicit in vivo and in vitro a potent vasoconstrictor activity after binding to high-affinity receptors on vascular smooth muscle cells (VSMC). A617 cells, a VSM-derived cell line, were used as an in vitro model system to study selected growth factors and cytokines involved in proliferative and/or inflammatory diseases of the vessel wall as possible regulators of the high-affinity binding capacity of ET-1 to the cells. Radioligand studies characterized the binding of ET-1 to the isopeptide selective ETA receptor subtype on A617 cells as a time- and temperature-dependent saturable process (Kd = 0.13 +/- 0.04 nM, Bmax = 49 +/- 7 fmol/10(6) cells). Pretreatment of A617 cells with basic fibroblast growth factor (bFGF), a mitogenic agent for vascular cells, resulted in a time- and dose-dependent increase in ET-1 binding capacity, whereas preexposure to transforming growth factor-beta (TGF-beta) induced a reduction of the Bmax for ET-1. Platelet-derived growth factor (PDGF), interleukin-6 (IL-6), tumor necrosis factor-alpha, and fetal bovine serum (FBS) pretreatments did not affect consequent ET-1 binding to A617 cells.

Pharmacological characterization of FCE 27262, a combined thromboxane synthase inhibitor and PGH2/TXA2 receptor antagonist.

The literature supports the hypothesis that the association of a thromboxane (TX)A2 synthase inhibitor and a PGH2/TXA2 receptor antagonist has a superior antithrombotic effect when compared to both aspirin and single agent alone; a compound endowed with the dual mechanism of action might therefore be of therapeutic value for the management of thrombotic disorders. FCE 27262, an imidazol-1-yl-ethylideneaminooxypentanoic acid, displaces in vitro the binding of [3H]SQ 29,548 to washed human platelets (IC50 = 6.0 +/- 0.6 x 10(-8) M) and antagonizes human platelet aggregation induced by U 46619 in PRP with an IC50 (95% confidence limits) of 4.5(3.3-5.1) x 10(-7) M. It also selectively antagonizes the isolated vessel contraction induced by U 46619. In the rat aorta the Kb (95% confidence limits) was 1.6(0.6-4.3) x 10(-7) M. Additionally it inhibits in vitro TXB2 production in rat and human whole blood, the IC50 being, respectively, 5.9(3.3-9.6) x 10(-8) M and 3.8(2.9-5.0) x 10(-8) M. When administered orally to fed rats it also inhibits ex vivo TXB2 production in whole blood during clotting, the ID50 being 0.62(0.4-0.8) mg/kg. Both in vitro and ex vivo the effect of FCE 27262 on TXA2 synthase was selective, the production of PGE2, the product of a different isomerase from the common precursors, PG-endoperoxides, being concomitantly enhanced. In a canine model of electrically-induced coronary thrombosis, FCE 27262 (1 mg/kg i.v.) inhibits ex vivo TXB2 synthesis (> 95%), antagonizes U 46619-induced platelet aggregation and prolongs occlusion time (controls: 72 +/- 8 min, FCE 27262: 215 +/- 38 min; p < 0.01). In the same model both aspirin (5 mg/kg i.v.) and a pure PGH2/TXA2 receptor antagonist (L 670596), at a dose giving a similar degree of TXA2 synthase inhibition and receptor blockade, respectively, are significantly less effective. Thus, FCE 27262 combines thromboxane synthase inhibition and PGH2/TXA2 receptor antagonism in one molecule, resulting in enhanced antithrombotic activity. FCE 27262 thus may be an appropriate pharmacological tool to test the therapeutic potential of the dual mechanism of action.

Inactivation of endothelin by polymorphonuclear leukocyte-derived lytic enzymes.

Cultured bovine aortic endothelial cells (BAEC) released endothelin-1 (ET-1) in the culture medium in a time-dependent fashion. Coincubation of fMLP-activated human polymorphonuclear leukocytes (PMN) with BAEC caused a fast (maximal activity was reached within 15 minutes) and cell number-dependent disappearance of ET-1 from the medium. This effect was direct to ET-1, because it was also present when PMN were incubated with the synthetic peptide in the absence of BAEC. PMN-dependent disappearance of ET-1 was associated with loss of constrictor activity on isolated rabbit aorta. PMN-released products were responsible for ET-1 degrading activity, because supernatants of activated PMN were equally effective as the intact cells. Resting PMN, in the same time frame, were uneffective. Eglin C, a potent blocker of PMN-derived elastase and cathepsin G, reversed the ET-1 inhibitory activity of fMLP-stimulated PMN and of their supernatant. Direct addition of elastase and cathepsin G to synthetic ET-1 destroyed its immunoreactivity and this effect was blocked by eglin C. High-performance liquid chromatography (HPLC) analysis supported the hypothesis that ET-1 degradation by PMN was due to enzymatic proteolysis. These data provide evidence that activated PMN are able to degrade ET-1 through the release of proteases. Because physiologic concentrations of PMN can destroy high amounts (up to 100 nmol/L) of ET-1 within a few minutes, we propose that this mechanism of ET-1 inactivation has biologic relevance.

Effects of racemic, S- and R-indobufen on cyclooxygenase and lipoxygenase activities in human whole blood.

Racemic indobufen inhibits human platelet aggregation by reducing thromboxane (TX) A2 biosynthesis. In order to ascertain which of the two optical isomers is responsible for its pharmacological activity, we compared the effects of racemic (SR +/-), S(+) enantiomer and R(-) enantiomer indobufen on cyclooxygenase and 5-lipoxygenase activities by assessing the biosynthesis of TXB2, prostaglandin (PG) E2 and leukotriene (LT) B4 in human whole blood stimulated with the Ca2+ ionophore A23187. Racemic indobufen caused a dose-dependent inhibition of TXB2 and PGE2 production (IC50: 0.53 +/- 0.06 and 0.34 +/- 0.02 micrograms/ml, respectively; mean +/- S.D., n = 4). S-Indobufen was approximately 2-fold more potent than the racemate in inhibiting the synthesis of cyclooxygenase products. R-Indobufen affected the same enzyme but only at considerably higher concentrations (IC50: 53 +/- 8 micrograms/ml, n = 3). Serum LTB4 concentrations were significantly reduced only at indobufen concentrations greater than 50 micrograms/ml. In conclusion, indobufen is a selective inhibitor of the cyclooxygenase activity of platelet PGG/H synthase in a concentration range corresponding to the therapeutic plasma levels in man. This inhibitory effect is largely due to the S isomer of the drug.

[Potentials of echography in the diagnosis of gallbladder carcinoma]. / Possibilità dell'ecografia nella diagnosi del carcinoma colecistico.

A study was carried out on 27 patients (22 women and 5 men) suffering from gallbladder carcinoma confirmed through surgery or autopsy. The examinations were performed with a real-time scanner and 3.5 MHz transducer. Lately, higher resolution transducers (5 MHz and 7.5 MHz) have been employed which have proven more effective due to their improved resolution. The importance is stressed of scanning patients in different positions in order to allow stones and echogenic material to redistribute inside the gallbladder. A correct diagnosis was obtained in 20/27 cases. Bile duct metastases were correctly evaluated by US in 13/13 cases; liver metastases in 25/27 patients. US proved unreliable in those cases where lymph nodes were involved (0/4). Six patients underwent scanning few months, or even years, before the onset of the symptoms. Three patients presented with fundic tumors, whose wall thickening and shadowing of the external wall had been incorrectly evaluated. In the other 3 cases the gallbladder was filled with stones and had thick and constricted walls. There was only a case of invisible carcinoma. Gallbladder carcinoma has often an unfavorable prognosis due to its being diagnosed in an advanced stage. The prognosis of such patients can be improved through an earlier US diagnosis only. The first step in this direction seems to be the identification of the precursor lesions.

Risk factors for intubation injury of the larynx.

The endolaryngeal structures are subjected to insult from prolonged endotracheal intubation. Factors that may exacerbate this injury include intubation technique, duration of intubation, tube geometry and constitution, frequency of reintubation, and patient-related factors such as concomitant medical diseases. The contribution of underlying medical disease to laryngeal intubation injury was studied prospectively by sequential endoscopy from the time of tracheotomy. Diabetes mellitus, congestive heart failure, and a history of stroke or tuberculosis increased the likelihood of severe laryngeal injury. The association of these disorders with severe laryngeal injury should lead to consideration of earlier tracheotomy in such patients.