Pavel Kucera

Photodetection of early human bladder cancer based on the fluorescence of 5-aminolaevulinic acid hexylester-induced protoporphyrin IX: a pilot study

Exogenous administration of 5-aminolaevulinic acid (ALA) is becoming widely used to enhance the endogenous synthesis of protoporphyrin IX (PpIX) in photodynamic therapy (PDT) and fluorescence photodetection (PD). Recently, results have shown that the chemical modification of ALA into its more lipophilic esters circumvents limitations of ALA-induced PpIX like shallow penetration depth into deep tissue layers and inhomogeneous biodistribution and enhances the total PpIX formation. The present clinical pilot study assesses the feasibility and the advantages of a topical ALA ester-based fluorescence photodetection in the human bladder. In this preliminary study 5-aminolaevulinic acid hexylester (h-ALA) solutions, containing concentrations ranging from 4 to 16 mM, were applied intravesically to 25 patients. Effects of time and drug dose on the resulting PpIX fluorescence level were determined in vivo with an optical fibre-based spectrofluorometer. Neither local nor systemic side-effects were observed for the applied conditions. All conditions used yielded a preferential PpIX accumulation in the neoplastic tissue. Our clinical investigations indicate that with h-ALA a twofold increase of PpIX fluorescence intensity can be observed using 20-fold lower concentrations as compared to ALA.

OPTIMISATION OF THE FORMATION AND DISTRIBUTION OF PROTOPORPHYRIN IX IN THE UROTHELIUM: AN IN VITRO APPROACH

PURPOSE To optimize conditions for photodynamic detection (PDD) and photodynamic therapy (PDT) of bladder carcinoma, urothelial accumulation of protoporphyrin IX (PpIX) and conditions leading to cell photodestruction were studied. MATERIALS AND METHODS Porcine and human bladder mucosae were superfused with derivatives of 5-aminolevulinic acid (ALA). PpIX accumulation and distribution across the mucosa was studied by microspectrofluorometry. Cell viability and structural integrity were assessed by using vital dyes and microscopy. RESULTS ALA esters, especially hexyl-ALA, accelerated and regularized urothelial PpIX accumulation and allowed for necrosis upon illumination. CONCLUSIONS hexyl-ALA used at micromolar concentrations is the most efficient PpIX precursor for PDD and PDT.

Magnetic pill tracking: a novel non‐invasive tool for investigation of human digestive motility

Abstract  A new minimally invasive technique allowing for anatomical mapping and motility studies along the entire human digestive system is presented. The technique is based on continuous tracking of a small magnet progressing through the digestive tract. The coordinates of the magnet are calculated from signals recorded by 16 magnetic field sensors located over the abdomen. The magnet position, orientation and trajectory are displayed in real time. Ten young healthy volunteers were followed during 34 h. The technique was well tolerated and no complication was encountered. The information obtained was 3‐D configuration of the digestive tract and dynamics of the magnet displacement (velocity, transit time, length estimation, rhythms). In the same individual, repeated examination gave very reproducible results. The anatomical and physiological information obtained corresponded well to data from current methods and imaging. This simple, minimally invasive technique permits examination of the entire digestive tract and is suitable for both research and clinical studies. In combination with other methods, it may represent a useful tool for studies of GI motility with respect to normal and pathological conditions.

IN VITRO ENGINEERING OF HUMAN STRATIFIED UROTHELIUM: ANALYSIS OF ITS MORPHOLOGY AND FUNCTION

PURPOSE Gastric or intestinal patches, commonly used for reconstructive cystoplasty, may induce severe metabolic complications. The use of bladder tissues reconstructed in vitro could avoid these complications. We compared cellular differentiation and permeability characteristics of human native with in vitro cultured stratified urothelium. MATERIALS AND METHODS Human stratified urothelium was induced in vitro. Morphology was studied with light and electron microscopy and expression of key cellular proteins was assessed using immunohistochemistry. Permeability coefficients were determined by measuring water, urea, ammonia and proton fluxes across the urothelium. RESULTS As in native urothelium the stratified urothelial construct consisted of basal membrane and basal, intermediate and superficial cell layers. The apical membrane of superficial cells formed villi and glycocalices, and tight junctions and desmosomes were developed. Immunohistochemistry showed similarities and differences in the expression of cytokeratins, integrin and cellular adhesion proteins. In the cultured urothelium cytokeratin 20 and integrin subunits alpha6 and beta4 were absent, and symplekin was expressed diffusely in all layers. Uroplakins were clearly expressed in the superficial umbrella cells of the urothelial constructs, however, they were also present in intermediate and basal cells. Symplekin and uroplakins were expressed only in the superficial cells of native bladder tissue. The urothelial constructs showed excellent viability, and functionally their permeabilities for water, urea and ammonia were no different from those measured in native human urothelium. Proton permeability was even lower in the constructs compared to that of native urothelium. CONCLUSIONS Although the in vitro cultured human stratified urothelium did not show complete terminal differentiation of its superficial cells, it retained the same barrier characteristics against the principal urine components. These results indicate that such in vitro cultured urothelium, after being grown on a compliant degradable support or in coculture with smooth muscle cells, is suitable for reconstructive cystoplasty.

Polymeric photosensitizer prodrugs for photodynamic therapy

A targeting strategy based on the selective enzyme‐mediated activation of polymeric photosensitizer prodrugs (PPP) within pathological tissue has led to the development of agents with the dual ability to detect and treat cancer. Herein, a detailed study of a simple model system for these prodrugs is described. We prepared “first‐generation” PPP by directly tethering the photosensitizer (PS) pheophorbide a to poly‐(l)‐lysine via epsilon amide links and observed that by increasing the number of PS on a polymer chain, energy transfer between PS units improved leading to better quenching efficiency. Fragmentation of the PPP backbone by trypsin digestion gave rise to a pronounced fluorescence increase and to more efficient generation of reactive oxygen species upon light irradiation. In vitro tests using the T‐24 bladder carcinoma cell line and ex vivo experiments using mouse intestines illustrated the remarkable and selective ability of these PPP to fluoresce and induce phototoxicity upon enzymatic activation. This work elucidated the basic physicochemical parameters, such as water solubility and quenching/activation behavior, required for the future elaboration of more adaptable “second‐generation” PPP, in which the PS is tethered to a proteolytically stable polymer backbone via enzyme‐specific peptide linkers. This polymer architecture offers great flexibility to tailor make the PPP to target any pathological tissue known to over‐express a specific enzyme.

Noninvasive Verification of Nasogastric Tube Placement Using a Magnet-Tracking System: A Pilot Study in Healthy Subjects

BACKGROUND Fluoroscopic verification of nasogastric (NG) feeding tube placement is inconvenient and involves radiation exposure. We tested whether the position of an NG tube can be assessed reliably by a recently introduced magnet-tracking system. METHODS A small permanent magnet was attached at the end of an NG tube and its position was monitored using an external sensor array connected to a computer. NG tube trajectory, spontaneous movements of the magnet, and its position relative to the lower esophageal sphincter (LES) and xiphisternum were assessed in 22 healthy subjects and compared with esophageal manometry. In 12 subjects, localization of the magnet was also compared with fluoroscopy. RESULTS Magnet-tracking displayed NG tube tip movement reproducibly as it moved vertically in the esophagus and then laterally into the stomach. Compared with manometry, the accuracy and sensitivity of magnet tracking for localization of the NG tube tip, above or below the diaphragm, were 100%. Compared with fluoroscopy, the accuracy of NG tube localization by magnet tracking was 100%. With the magnet in the stomach, but not in the esophagus or LES, low amplitude displacements at a frequency of 3 per minute, consistent with gastric slow wave activity, were observed. CONCLUSIONS Magnet tracking allows accurate, real-time, 3-dimensional localization of an NG tube with respect to anatomic landmarks. Recorded motor patterns are indicative of the position of the NG tube. Magnet tracking may be a useful tool for bedside placement of nasogastric and enteral feeding tubes.

Colonic movements in healthy subjects as monitored by a Magnet Tracking System

Abstract  The Magnet Tracking System (MTS) is a minimally‐invasive technique of continuous evaluation of gastrointestinal motility. In this study, MTS was used to analyse colonic propulsive dynamics and compare the transit of a magnetic pill with that of standard radio‐opaque markers. MTS monitors the progress in real time of a magnetic pill through the gut. Ten men and 10 women with regular daily bowel movements swallowed this pill and 10 radio‐opaque markers at 8 pm. Five hours of recordings were conducted during 2 following mornings. Origin, direction, amplitude and velocity of movements were analysed relative to space–time plots of the pill trajectory. Abdominal radiographs were taken to compare the progress of both pill and markers. The magnetic pill lay idle for 90% of its sojourn in the colon; its total retrograde displacement accounted for only 20% of its overall movement. Analysis of these movements showed a bimodal distribution of velocities: around 1.5 and 50 cm min–1, the latter being responsible for 2/3 of distance traversed. There were more movements overall and more mass movements in males. Net hourly forward progress was greater in the left than right colon, and greater in males. The position of the magnetic pill correlated well with the advancement of markers. MTS showed patterns and propulsion dynamics of colonic segments with as yet unmet precision. Detailed analysis of slow and fast patterns of colonic progress makes it possible to specify the motility of colonic segments, and any variability in gender. Such analysis opens up promising avenues in studies of motility disorders.

Oxidative and Glycogenolytic Capacities within the Developing Chick Heart

Cardiac morphogenesis and function are known to depend on both aerobic and anaerobic energy-producing pathways. However, the relative contribution of mitochondrial oxidation and glycogenolysis, as well as the determining factors of oxygen demand in the distinct chambers of the embryonic heart, remains to be investigated. Spontaneously beating hearts isolated from stage 11, 20, and 24HH chick embryos were maintained in vitro under controlled metabolic conditions. O2 uptake and glycogenolytic rate were determined in atrium, ventricle, and conotruncus in the absence or presence of glucose. Oxidative capacity ranged from 0.2 to 0.5 nmol O2/(h·μg protein), did not depend on exogenous glucose, and was the highest in atria at stage 20HH. However, the highest reserves of oxidative capacity, assessed by mitochondrial uncoupling, were found at the youngest stage and in conotruncus, representing 75 to 130% of the control values. At stage 24HH, glycogenolysis in glucose-free medium was 0.22, 0.17, and 0.04 nmol glucose U(h·μg protein) in atrium, ventricle, and conotruncus, respectively. Mechanical loading of the ventricle increased its oxidative capacity by 62% without altering glycogenolysis or lactate production. Blockade of glycolysis by iodoacetate suppressed lactate production but modified neither O2 nor glycogen consumption in substrate-free medium. These findings indicate that atrium is the cardiac chamber that best utilizes its oxidative and glycogenolytic capacities and that ventricular wall stretch represents an early and major determinant of the O2 uptake. Moreover, the fact that O2 and glycogen consumptions were not affected by inhibition of glyceraldehyde-3-phosphate dehydrogenase provides indirect evidence for an active glycerol-phosphate shuttle in the embryonic cardiomyocytes.

Origins of motility patterns in isolated arterially perfused rat intestine

BACKGROUND/AIMS Assessment of the neuromuscular control of small intestinal motility and movement of luminal contents is hampered in vivo by measurement techniques and in vitro by tissue viability. The aim of this study was to establish the structural and functional integrity of an isolated segment of rat ileum and characterize its motility. METHODS Segments of rat ileum were perfused arterially with oxygenated fluorocarbon and luminally with saline. Oral and aboral pressures were correlated with conformational changes detected by concurrent video imaging. RESULTS Light and electron microscopy showed no neuromuscular abnormalities after experiments, and acetylcholine-induced pressure amplitudes were unchanged during experiments. Under basal conditions, low-frequency contractions showing constant frequency (0.27/min) and amplitudes (oral, 17 hPa; aboral, 15 hPa) corresponded to luminally occlusive aborally propagated contractions, which were eliminated by tetrodotoxin. High-frequency contractions with a constant frequency (27/min) were also seen; their basal amplitude (0.3 hPa) increased immediately before and after low-frequency contractions and after tetrodotoxin. Tetrodotoxin also increased basal intestinal tone. CONCLUSIONS An isolated, arterially perfused segment of rat ileum retains structural and functional integrity. It shows low-frequency propulsive contractions, controlled by the enteric nervous system, and myogenic high-frequency contractions, probably subject to tonic neural inhibition.

Mdx myotubes have normal excitability but show reduced contraction–relaxation dynamics

The pathogenesis of Duchenne muscular dystrophy (DMD), characterised by lack of the cytoskeletal protein dystrophin, is not completely understood. An early event in the degenerative process of DMD muscle could be a rise in cytosolic calcium concentration. In order to investigate whether this leads to alterations of contractile behaviour, we studied the excitability and contractile properties of cultured myotubes from control (C57BL/10) and mdx mice, an animal model for DMD. The myotubes were stimulated electrically and their motion was recorded photometrically. No significant differences were found between control and mdx myotubes with respect to the following parameters: chronaxy and rheobase (0.33 ± 0.03 ms and 23 ± 4 V vs. 0.39 ± 0.07 ms and 22 ± 2 V for C57 and mdx myotubes, respectively), tetanisation frequency (a similar distribution pattern was found between 5 and 30 Hz), fatigue during tetanus (found in 35% of both types of myotubes) and post-tetanic contracture. In contrast, contraction and relaxation times were longer (P < 0.005) in mdx (36 ± 2 and 142 ± 13 ms, respectively) than in control myotubes (26 ± 1 and 85 ± 9 ms, respectively). Together with our earlier findings, these results suggest a decreased capacity for calcium removal in mdx cells leading, in particular, to alterations of muscle relaxation.